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MicroBiology- MLT1
LabPaq / Published by: Hands-On Labs, Inc.

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A Laboratory Manual of Small-Scale Experiments for the Independent Study of
Microbiology
50-0222-MB-01
LabPaq® is a registered trademark of Hands-On Labs, Inc. (HOL). The LabPaq referenced in this manual is produced by Hands-On Labs, Inc. which holds and reserves all copyrights on the intellectual properties associated with the LabPaq’s unique design, assembly, and learning experiences. The laboratory manual included with a LabPaq is intended for the sole use by that
LabPaq’s original purchaser and may not be reused without a LabPaq or by others without the specific written consent of HOL. No portion of any LabPaq manual’s materials may be reproduced, transmitted or distributed to others in any manner, nor may be downloaded to any public or privately shared systems or servers without the express written consent of HOL. No changes may be made in any LabPaq materials without the express written consent of HOL. HOL has invested years of research and development into these materials, reserves all rights related to them, and retains the right to impose substantial penalties for any misuse.
Published by:

Hands-On Labs, Inc.
3880 S. Windermere St.
Englewood, CO 80110
Phone: Denver Area: 303-679-6252
Toll-free, Long-distance: 866-206-0773

www.LabPaq.com
E-mail: info@LabPaq.com
Printed in the United States of America.
The experiments in this manual have been and may be conducted in a regular formal laboratory or classroom setting with the users providing their own equipment and supplies. However, this manual was especially written for the benefit of the independent study of students who do not have convenient access to such facilities. It allows them to perform college and advanced high school level experiments at home or elsewhere by using a LabPaq, a collection of experimental equipment and supplies specifically packaged to accompany this manual.
Use of this manual and authorization to perform any of its experiments is expressly conditioned upon the user reading, understanding and agreeing to fully abide by all the safety precautions contained herein.
Although the author and publisher have exhaustively researched many sources to ensure the accuracy and completeness of the information contained in this manual, we assume no responsibility for errors, inaccuracies, omissions or any other inconsistency herein. Any slight of people, organizations, materials, or products is unintentional.

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Table of Contents
5

Important Information to Help Students with the Study of Microbiology

Experiments
50 Observing Bacteria and Blood
72

Bacterial Morphology

84

Aseptic Technique & Culturing Microbes

102

Isolation of Individual Colonies

125

Differential Staining

136

Methyl Red Voges-Proskauer Test

147

Motility Testing

158

Carbohydrate Fermentation Testing

169 Osmosis
186

Antibiotic Sensitivity

199

Fomite Transmission

208

Microbes in the Environment

217 Fungi

Appendix
231

Preparation of Cultures

234

Preparation of Disinfecting Solution

236

Final Cleanup Instructions

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Introduction

Important Information to Help Students with the Study of Microbiology
Welcome to the study of microbiology. Don’t be afraid of taking this course. By the end of the semester you will be really proud of yourself and will wonder why you were ever afraid of the m-word, microbiology! After their first microbiology class, most students say they thoroughly enjoyed it, learned a lot of useful information for their lives, and only regret not having studied it sooner.
Microbiology is not some “mystery” science only comprehendible by eggheads. Microbiology is simply the study of microscopic living organisms. It will be easier for you to understand the world we live in and to make the multitude of personal and global decisions that affect our lives and our planet after you have learned about the characteristics of life around you and how organisms change and interact with each other, with the environment, and with you. Plus, having microbiology credits on your transcript will certainly be impressive, and your microbiology knowledge may create some unique job opportunities for you.
This lab manual of microbiology experiments was designed to accompany any entry level college or advanced high school level microbiology course. It can be used by all students, regardless of the laboratory facilities available to them. Its experiments have been and continue to be successfully performed in regular microbiology laboratories. With the special LabPaq experiments can be performed at home by independent-study students or at small learning centers that do not have formal laboratories. Throughout the manual there are references about campus-based and independent study, but all of the information and references herein are equally relevant to both types of students.

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Introduction
How to Study Microbiology
Microbiology is not the easiest subject to learn, but neither is it the hardest. As in any other class, if you responsibly apply yourself, conscientiously read your text, and thoughtfully complete your assignments, you will learn the material. Here are some basic hints for effectively studying microbiology - or any other subject - either on or off campus.
Plan to Study: You must schedule a specific time and establish a specific place in which to seriously, without interruptions or distractions, devote yourself to your studies. Think of studying like you would think of a job, except that now your job is to learn. Jobs have specific times and places in which to get your work done, and studying should be no different. Just as television, friends, and other distractions are not permitted on a job; you should not permit them to interfere with your studies. You cannot learn when you are distracted. If you want to do something well, you must be serious about it. Only after you’ve finished your studies should you allow time for distractions.
Get in the Right Frame of Mind: Think positively about yourself and what you are doing. Give yourself a pat on the back for being a serious student and put yourself in a positive frame of mind to enjoy what you are about to learn. Then get to work! Organize any materials and equipment you will need in advance so you don’t have to interrupt your thoughts to find them later. Look over your syllabus and any other instructions to know exactly what your assignment is and what you need to do. Review in your mind what you have already learned. Is there anything that you aren’t sure about? Write it down as a formal question, then go back over previous materials to try to answer it yourself. If you haven’t figured out the answer after a reasonable amount of time and effort, move on. The question will develop inside your mind and the answer will probably present itself as you continue your studies. If not, at least the question is already written down so you can discuss it later with your instructor.
Be Active with the Material: Learning is reinforced by relevant activity. When studying feel free to talk to yourself, scribble notes, draw pictures, pace out a problem, tap out a formula, etc.
The more active things you do with study materials, the better you will learn. Have highlighters, pencils, and note pads handy. Highlight important data, read it out loud, and make notes. If there is a concept you are having problems with, stand up and pace while you think it through. See the action taking place in your mind. Throughout your day try to recall things you have learned, incorporate them into your conversations, and teach them to friends. These activities will help to imprint the related information in your brain and move you from simple knowledge to true understanding of the subject matter.
Do the Work and Think about What You are Doing: Sure, there are times when you might get away with taking a shortcut in your studies, but in doing so you will probably shortchange yourself. The things we really learn are the things we discover ourselves. That is why we don’t learn as much from simple lectures or when someone gives us the answers. And when you have an assignment, don’t just go through the motions. Enjoy your work, think about what you are doing, be curious, examine your results, and consider the implications of your findings. These “critical thinking” techniques will improve and enrich your learning process. When you complete your assignments independently and thoroughly you will have gained knowledge and you will be proud of yourself.

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Introduction
How to Study Microbiology Independently
There is no denying that learning through any method of independent study is a lot different than learning through classes held in traditional classrooms. A great deal of personal motivation and discipline is needed to succeed in a course of independent study where there are no instructors or fellow students to give you structure and feedback. But these problems are not insurmountable and meeting the challenges of independent study can provide a great deal of personal satisfaction.
The key to successful independent study is in having a personal study plan and the personal discipline to stick to that plan.
Properly Use Your Learning Tools: The basic tools for telecourses, web courses and other distance-learning methods are often similar and normally consist of computer software or videos, textbooks, and study guides. Double check with your course administrator or syllabus to make sure you acquire all the materials you will need. These items are usually obtained from your campus bookstore, library, or via the Internet. Your area’s public and educational television channels may even broadcast course lectures and videos. If you choose to do your laboratory experimentation independently, you will need the special equipment and supplies described in this lab manual and contained in its companion LabPaq. The LabPaq can be purchased on the Internet at www.
LabPaq.com.
For each study session, first work through the appropriate sections of your course materials. These basically serve as a substitute for classroom lectures and demonstrations. Take notes as you would in a regular classroom. Actively work with any computer and/or text materials, carefully review your study guide, and complete all related assignments. If you do not feel confident about the material covered, repeat these steps until you do. It’s a good idea to review your previous work before proceeding to a new section. This reinforces what you previously learned and prepares you to absorb new information. Experimentation is the very last thing done in each study session and it will only be really meaningful if you have first absorbed the text materials that it demonstrates.
Plan to Study: A regular microbiology course with a laboratory component will require you to spend around 15 hours a week studying and completing your assignments. Remember, microbiology is normally a 5-credit hour course! To really learn new material there is a generally accepted 3-to1 rule that states that at least 3 hours of class and study time are required each week for each hour of course credit taken. This rule applies equally to independent study and regular classroom courses. On campus, microbiology students are in class for 4 hours and in the laboratory for
2 to 3 hours each week. Then they still need at least 8 hours to read their text and complete assignments. Knowing approximately how much time you need will help you to formulate a study plan at the beginning of the course and then stick with it.
Schedule Your Time Wisely: The more often you interact with study materials and call them to mind, the more likely you are to reinforce and retain the information. Thus, it is much better to study in several short blocks of time rather than in one long, mind-numbing session. Accordingly, you should schedule several study periods throughout the week, or better yet, study a little each day. Please do not try to do all of your study work on the weekends! You will just burn yourself out, you won’t really learn much, and you will probably end up feeling miserable about yourself and microbiology. Wise scheduling can prevent such unpleasantness and frustration.

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Introduction
Choose the Right Place for Your Home Laboratory
If you are experimenting at home, the best place to perform your micro- and small-scale microbiology experiments is in an uncluttered room that has these important features:
●●

a door that can be closed to keep out pets and children,

●●

a window or door that can be opened for fresh air ventilation and fume exhaust,

●●

a source of running water for fire suppression and cleanup,

●●

a counter or table-top work surface, and

●●

a heat source such as a stove top, hot dish, or Bunsen burner.

The kitchen usually meets all these requirements, but you must make sure you clean your work area well both before and after experimentation. This will keep foodstuff from contaminating your experiment and your experiment materials from contaminating your food. Sometimes a bathroom makes a good laboratory, but it can be rather cramped and subject to a lot of interruptions.
Review the “Basic Safety” section of this manual to help you select the best location for your home-lab and to make sure it is adequately equipped.

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Introduction
Organization of the Lab Manual
Before proceeding with the experiments you need to know what is expected of you. To find out, please thoroughly read and understand all the various sections of this manual.
Laboratory Notes: Like all serious scientists you will record formal notes detailing your activities, observations, and findings for each experiment. These notes will reinforce your learning experiences and knowledge of microbiology. Plus, they will give your instructional supervisor a basis for evaluating your work. The “Laboratory Notes” section of this manual explains exactly how your lab notes should be organized and prepared.
Required Equipment and Supplies: This manual also contains a list of the basic equipment and supplies needed to perform all the experiments. Students performing these experiments in a non-lab setting must obtain the “LabPaq” specifically designed to accompany this manual.
It includes all the equipment, materials, and chemicals needed to perform these experiments, except for some items usually found in the average home or obtainable in local stores. At the beginning of each experiment there is a “Materials” section that states exactly which items the student provides and which items are found in the LabPaq. Review this list carefully to make sure you have all these items on hand before you begin the experiment. It is assumed that campusbased students will have all the needed equipment and supplies in their laboratories and that the instructors will supply required materials and chemicals in the concentrations indicated.
Laboratory Techniques: While these techniques primarily apply to full-scale experiments in formal laboratories, knowledge of them and their related equipment is helpful to the basic understanding of microbiology and may also be applicable to your work with micro- and smallscale experimentation.
Basic Safety and Micro-scale Safety Reinforcement: The use of this lab manual and the LabPaq, plus authorization to perform their experiments, are expressly conditioned upon the user reading, understanding and agreeing to abide by all the safety rules and precautions noted. Additional terms authorizing use of the LabPaq are contained in its purchase agreement. These safety sections are relevant to both laboratory and non-laboratory experimentation. They describe potential hazards plus the basic safety equipment and safety procedures designed to avoid such hazards. The Basic
Safety and Micro-scale Safety Reinforcement sections are the most important sections of this lab manual and should always be reviewed before starting each new experiment.
Experiments: All experimental materials and procedures are fully detailed in the laboratory manual for each experiment. Chemicals and supplies unique for a specific experiment are contained in a bag labeled with the experiment number.

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Introduction
How to Perform an Experiment
Although each experiment is different, the process for preparing, performing, and recording all the experiments is essentially the same.
Review Basic Safety: Before beginning reread the safety sections, try to foresee potential hazards, and take appropriate steps to prevent problems.
Read through the Entire Experiment before You Start: Knowing what you are going to do before you do it will help you to be more effective and efficient.
Organize Your Work Space, Equipment, and Materials: It is hard to organize your thoughts in a disorganized environment. Assemble all required equipment and supplies before you begin working. These steps will also facilitate safety.
Outline Your Lab Notes: Outline the information needed for your lab notes and set up required data tables. This makes it much easier to concentrate on your experiment. Then simply enter your observations and results as they occur.
Perform the Experiment According to Instructions: Follow exactly all directions in a step-by-step format. This is not the time to be creative. DO NOT attempt to improvise your own procedures!
Think About What You Are Doing: Stop and give yourself time to reflect on what has happened in your experiment. What changes occurred? Why? What do they mean? How do they relate to the real world? This step can be the most fun and often creates “light bulb” experiences of understanding. Complete Your Lab Notes and Answer Required Questions: If you have properly followed all the above steps, this concluding step will be easy.
Clean-up: Blot any minute quantities of unused chemicals with a paper towel or flush them down the sink with generous amounts of water. Discard waste in your normal trash. Always clean your equipment immediately after use or residue may harden and be difficult to remove later. Return equipment and supplies to their proper place, and if working at home with a LabPaq, store it out of the reach of children and pets.

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Introduction
Estimated Time Requirements for Each Experiment
Note: These estimates are provided to help you plan and schedule your time. They are given per individual lab performed separately and do not consider time and step savings possible when several labs are grouped together. Of course, these are only estimates and your actual time requirements may differ.
Experiment No. / Title

Preparation

Experimenting

Incubation

After Incubation

None

3 - 4 hours

None

None

None

3 - 4 hours

None

None

None

1 - 2 hours

24 - 48 hours

Less than 1 hour

3 - 4 hours

24 - 48 hours

Less than 1 hour

3 - 4 hours

24 - 48 hours

None

Less than 1 hour

48 - 72 hours

1 hour

Less than 1 hour

24 - 48 hours

Less than 1 hour

Less than 1 hour

12 - 24 hours

Less than 1 hour

Less than 1 hour

24 - 72 hours

Less than 1 hour

24 - 48 hrs. ahead

1 hour

24 - 72 hours

1 hour

None

1 - 2 hours

24 - 72 hours

Less than 1 hour

None

1 - 3 hours

24 - 72 hours

Less than 1 hour

EXPERIMENT 1:
Observing Bacteria & Blood
EXPERIMENT 2:
Bacterial Morphology
EXPERIMENT 3:
Aseptic Techniques & Culturing
Microbes
EXPERIMENT 4:
Isolation of Individual Colonies
EXPERIMENT 5:
Differential Staining

None-use Exp. 3 cultures 30 minutes
24 - 48 hours ahead

EXPERIMENT 6:
Methyl Red

30 minutes

Voges-Proskauer Test

24 - 48 hours ahead

EXPERIMENT 7:
Motility Testing

30 minutes
24 - 48 hours ahead

EXPERIMENT 8:
Carbohydrate

30 minutes

Fermentation Testing

24 - 48 hrs. ahead

EXPERIMENT 9:
Osmosis
EXPERIMENT 10:
Antibiotic Sensitivity

30 minutes
24 - 48 hrs. ahead
30 minutes

EXPERIMENT 11:
Fomite Transmission
EXPERIMENT 12:
Microbes in the Environment
EXPERIMENT 13:
Fungi

24 hour intervals
None
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Less than 1 hour

12

Up to 1 week

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2 - 3 hours

Introduction
Preparation of Solid Media
Microbiological media are used to grow microbes for study and experimentation. Most bacteria collected in the environment will not be harmful. However, once an isolated microbe multiplies by millions in a broth tube or petri dish it can become more of a hazard. Be sure to protect open cuts with rubber gloves and never ingest or breathe in growing bacteria. Keep growing petri dishes taped closed until your experiment is done. Then you should safely destroy the bacteria colonies using bleach.
Microbiological media may be prepared as either liquid or as a solid media. When a solid medium is prepared, a corresponding liquid broth is solidified by the addition of agar to the broth. Agar is a polysaccharide found in the cell walls of some algae. It is inert and degraded by very few microorganisms. In addition, the fact it melts at around 100oC and solidifies at approximately
45oC-50oC makes it an ideal solidifying agent for microbiological media.

PROCEDURES
Preparation of Solid Media
1. Disinfect your work area with a 10%-bleach solution.
2. Place the test tube rack into a pan of water and place your tubes of agar into the rack. The agar will melt more easily if the water level is above or at the level of the agar. If your pan is not deep enough to bring the water above the level of the agar you will need to shake the tubes during the melting process to mix the melted and unmelted portion of the agar.
3. Place the pan on the stove top and bring to a boil. Once the water begins to boil, the agar should melt within 10 to 15 minutes. Remember, if your water level is below the level of the agar you will need to shake the tubes to mix the unmelted agar into the melting agar. Be careful as the heating tubes will be hot!
4. Once the agar media is melted, remove the pan from the heat but do not remove the tubes from the hot water.
5. Allow the water to cool until the tubes are cool enough to handle but the agar media is still liquid (50° - 60°C).
6. Label the bottom of two petri dishes (per tube) with the type of medium you are using (in this
LabPaq you will use nutrient or MRS agar).
7. Using aseptic handling techniques pour the liquid agar from the 18-mL tube into the bottom of the labeled petri dishes. If you are preparing both types of medium, be careful to pour each medium into the correctly labeled dish. Pour enough to cover the bottom of each dish 1/8”1/4” thick (approximately 9mL so each 18-mL tube will make two dishes). Cover each dish with its lid immediately.
8. When all the dishes are poured, cover them with a paper towel to help prevent contamination and allow them to cool and solidify.
9. The agar dishes are done when solid. You may store the cooled dishes in a zip baggie in the refrigerator for later use or use them immediately.

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Introduction
Preparation of Cultures
Culture tubes should remain lidded while incubating. Do not open them once inoculated unless under aseptic conditions and to perform a necessary experimental step.
Saccharomyces cervicae: Add 1/2 teaspoon dry Saccharomyces cervicae (active dry yeast envelope) to 1/8 cup warm water (you can use a sample cup or any household cup) and gently swirl to mix. Set the culture aside to activate for at least 10 minutes. Stir to mix prior to using.
Escherichia coli:
10. Remove the tube labeled: Broth, Nutrient - 5 mL in Glass tube, from culture media bag #2 from the refrigerator and allow it to come to room temperature..
11. Moisten a paper towel with a small amount of alcohol and wipe the work area down.
12. Once the nutrient broth media is at room temperature:
●●

Remove the numbered E-coli culture tube from the cultures bag and remove its cap. Set the cap upside down to avoid contamination.

●●

Uncap the nutrient broth; set its cap upside down to avoid contaminating it while the broth is open.

●●

Use sterile techniques and draw 0.25mL of the nutrient broth into a sterile graduated pipet. NOTE: To sterilize the pipet draw a small amount of 70% alcohol into the bulb, and then expel it into a sink. Remove any excess alcohol by forcefully swinging the pipet in a downward arch several times to ensure that the pipet is dry before drawing up the nutrient broth. Add the broth to the vial containing the lyophilized E-coli pellet. Recap the
E-coli vial and shake to mix until the pellet has dissolved in the broth. Note that the vial should be about one-half full to allow for shaking and mixing the pellet.

●●

Once the pellet has dissolved, use the same sterile pipet to draw up the E. coli solution and expel it into the original tube of nutrient broth. Recap the broth. NOTE: If the pipet has become contaminated, simply draw a small amount of 70% alcohol into the bulb and then expel it into a sink. Remove any excess alcohol by forcefully swinging the pipet in a downward arch several times to ensure that the pipet is dry before drawing up the E. coli solution. Recap the nutrient broth and incubate the now E-coli inoculated tube of nutrient broth at 37°C.
The culture should show active growth between 24 to 48 hours; it can be left as a liquid culture or plated out. Most freeze dried cultures will grow within a few days however some may exhibit a prolonged lag period and should be given twice the normal incubation period before discarding as non-viable.
Refer to Experiment 3 for a description of indicators of growth.
●●

Lactobacillus acidophilus: Remove a tube of MRS broth from the refrigerator and allow it to come to room temperature. Aseptically transfer a portion of a tablet of L. acidophilus into the tube of media. Allow the tube to set, swirling periodically, as the tablet dissolves. There will be a significant amount of sediment in the bottom of the tube. Mark the level of the sediment with a marker, pencil, or pen. Incubate the inoculated tube at 37°C. The culture should show active growth between 24 to 48 hours. Refer to Experiment 3 for a description of indicators of growth. L. acidophilus often sediments as it grows. An increase (above the sediment line

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Introduction you marked on the tube) in the sediment is an indication of growth. Swirl the tube to mix the organisms back into the broth prior to use.
●●

Staphylococcus epidermidis: You can culture S. epidermidis as a liquid or solid culture.
Because you are inoculating from an environmental source (your skin), your sample may contain bacteria other than S. epidermidis. Thus, broth cultures derived directly from sampling may not be pure cultures of S. epidermidis. With the exception of Experiments 3 and 4 (#3 establishes a broth culture and #4 uses it to establish a pure culture), use the dish culture method to ensure you are using a pure sample for your experiment.

●●

Broth cultures of S. epidermidis: Without contaminating the cotton tip, cut the length of the swab such that it will fit entirely into a capped test tube. Dampen the cotton tip sterile swab with distilled water and rub it vigorously on your skin. Do not try to obtain a bacterial culture soon after washing your skin. Additionally choose an area that is not as likely to have been scrubbed as recently (the inside of the elbow or back of the knee is generally a good site).
Do not obtain a sample from any bodily orifice (mouth, nose, etc.) as you are not likely to culture the desired microbe (Staphylococcus epidermidis). Using aseptic technique, place the swab into a tube of nutrient media, label the tube accordingly. Incubate the inoculated tube at 37°C. The culture should show active growth between 24 to 48 hours. Refer to Experiment
3 for a description of indicators of growth.

●●

Dish cultures of S. epidermidis: Use a sterile swab to obtain a sample of S. epidermidis from your skin described in the generation of a broth culture. Rub the swab lightly on the surface of one dish of nutrient agar to inoculate it with S. epidermidis. As the swab may not contain a high number of bacteria, be sure to rub all sides of the swab on the dish to transfer as many individual bacterium as possible. Incubate the dish at 37°C for 24 to 48 hours. The S. epidermidis culture was not a pure culture (derived from a single organism) and will most likely contain colonies from several different organisms. You will need to identify and select a colony. Staphylococci produce round, raised, opaque colonies, 1 – 2 mm in diameter. S. epidermidis colonies are white in color. Below is a picture of S. epidermidis grown on blood agar.
As the sample is of human origin, it potentially contains bacteria that can act as opportunistic pathogens. Do not select or use any colony that does not appear to be S. epidermidis. If your dish contains colonies other than S. epidermidis, soak it in a 10%-bleach solution and discard.
Do not attempt to save the dish for use in future experiments!

You can either use the S. epidermidis colonies directly or amplify growth in a broth culture. If you choose to amplify into nutrient broth, 24 hours beginning the experiment, choose a S. epidermidis colony from the incubated dish and aseptically transfer the colony using an inoculation loop into a tube of nutrient media. Be sure to mix the broth gently to disburse the clumped bacteria into the

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Introduction broth. Incubate the tube at 37°C for an additional 24 hours.

Microbiology Safety
●●

Any microbe can be hazardous. While the majority of microorganisms are not pathogenic to humans and have never been shown to cause illness, under unusual circumstances a few microorganisms that are not normally pathogenic can act as pathogens. These are called opportunistic pathogens. Treat all microorganisms—especially unknown cultures such as from skin swabs or environmental samples—as if they were pathogenic. A student who has a compromised immune system or has had a recent extended illness is at higher risk for opportunistic infections. Do not attempt to swab your throat or nasal passages when sampling for S. epidermidis. You are not likely to culture the correct organism. Additionally, you are more likely to culture an opportunistic pathogen from these areas!

●●

Sterilize equipment and materials. All materials, media, tubes, dishes, loops, needles, pipets, and other items used for culturing microorganisms should be sterilized. Most of the materials and media you will be using are commercially sterilized products. You will be given instruction for sterilization with either flame or with a 10%-bleach solution for items that are not sterilized or that will be reused.

●●

Disinfect work areas before and after use. Use a disinfectant, such as a 10%-bleach solution to wipe down benches and work areas both before and after working with cultures. Also be aware of the possible dangers of the disinfectant. Bleach, if spilled, can ruin your clothing and can be dangerous if splashed into the eyes. Students should work where a sink is located to facilitate immediate rinsing if bleach is splashed or spilled.

●●

Wash your hands. Use an antibacterial soap to wash your hands before and after working with microorganisms. Non-antibacterial soap will remove surface bacteria and can be used if antibacterial soap is not available. Gloves should be worn as an extra protection.

●●

Never pipet by mouth. Use pipet bulbs or pipet devices for the aspiration and dispensing of liquid cultures.

●●

Do not eat or drink while working with microorganisms. Never eat or drink while working with microorganisms. Keep your fingers out of your mouth, and wash your hands before and after the laboratory activity. Cover any cuts on your hands with a bandage. Gloves should be worn as an extra protection.

●●

Label everything clearly. All cultures, chemicals, disinfectants, and media should be clearly and securely labeled with their names and dates.

●●

Disinfect all waste material. All items to be discarded after an experiment, such as culture tubes, culture dishes, swabs, and gloves, should be covered with a 10%-bleach solution and allowed to soak for at least 1 to 2 hours. After soaking, the materials can be rinsed and disposed of by regular means.

●●

Clean up spills with care. Cover any spills or broken culture tubes with a 10%-bleach solution; then cover with paper towels. After allowing the spill to sit with the disinfectant, carefully clean up and place the materials in a bag for disposal. If you are cleaning up broken glass, place the materials in a puncture-proof container (such as a milk carton), and label the container
“broken glass” before placing in the trash. Wash the area again with disinfectant. Never pick up glass fragments with your fingers or stick your fingers into the culture itself. Instead, use a www.LabPaq.com 38

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Introduction brush and dustpan.
●●

Be certain to dispose of cultures properly. Liquid cultures should have bleach added to them
(to create a solution that is approximately 10% bleach) and allowed to set for a minimum of one hour before disposal. The deactivated samples can be discarded in the sink. Be sure to flush with plenty of water to remove any bleach residue. Petri dishes or any solid culture material should be soaked in a 10%-bleach solution for a minimum of one hour. They can then be bagged and discarded in the trash.

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Introduction
Basic Safety Guidelines
This section contains vital information that must be thoroughly read and completely understood before a student begins to perform experiments.
PREVENT INJURIES AND ACCIDENTS! Science experimentation is fun, but does involve potential hazards which must be acknowledged to be avoided. To safely conduct science experiments, students must first learn and then always follow basic safety procedures. Although there are certainly not as many safety hazards in experimenting with physics and geology as there are in chemistry and biology, safety risks exist in all science experimentation and science students need to be aware of safety issues relevant to all the disciplines. Thus, the following safety procedures review is relevant to all students regardless of their field of study.
While this manual tries to include all relevant safety issues, not every potential danger can be foreseen as each experiment involves slightly different safety considerations. Thus, students must always act responsibly, learn to recognize potential dangers, and always take appropriate precautions. Regardless of whether a student will be working in a campus or home laboratory setting, it is extremely important that he or she knows how to anticipate and avoid possible hazards and to be safety conscious at all times.
BASIC SAFETY PROCEDURES: Science experimentation often involves using toxic chemicals, flammable substances, breakable items, and other potentially dangerous materials and equipment. All of these things can cause injury and even death if not properly handled. These basic safety procedures apply when working in a campus or home laboratory.
●●

Because eyesight is precious and eyes are vulnerable to chemical spills and splashes, to shattered rocks and glass, and to floating and flying objects,
»»

●●

Students must always wear eye protecting safety goggles when experimenting

Because toxic chemicals and foreign matter may enter the body through digestion,
»»
»»

Students must always wash their hands before leaving their laboratory

»»
●●

Drinking and eating are always forbidden in laboratory areas

Students must always clean their lab area after experimentation

Because toxic substances may enter the body through the skin and lungs,
»»
»»

Students must never “directly” inhale chemicals

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Students should wear long-sleeved shirts, pants, and enclosed shoes when in their lab area »»
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The laboratory area must always have adequate ventilation

Students must wear gloves and aprons when appropriate

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Introduction
»»
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Students should always wear snug fitting clothing (preferably old)

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Students should always tie or pin back long hair,

Students should never wear dangling jewelry or objects

Because a laboratory area contains various fire hazards,
»»

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Smoking is always forbidden in laboratory areas

Because chemical experimentation involves numerous potential hazards,
»»
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Students must never leave a burning flame or reaction unattended

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Students must specifically follow all safety instructions

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Students must never perform any unauthorized experiments

»»

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Students must know how to locate and use basic safety equipment

Students must always properly store equipment and supplies and ensure these are out of the reach of small children and pets

Because science equipment and supplies often include breakable glass and sharp items that pose potential risks for cuts and scratches and small items as well as dangerous chemicals that could cause death or injury if consumed,
»»
»»

Students must keep science equipment and supplies stored out of the reach of pets and small children

»»

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Students must carefully handle all science equipment and supplies

Students must ensure pets and small children will not enter their lab area while they are experimenting Because science experimentation may require students to climb, push, pull, spin, and whirl
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»»

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Students should undertake these activities cautiously and with consideration for people, property, and objects that could be impacted
Students must ensure any stool, chair, or ladder used to climb is sturdy and take ample precautions to prevent falls

Because students’ best safety tools are their own minds and intellectual ability
»»

Students must always preview each experiment, and carefully think about what safety precautions need to be taken to perform the experiment safely

BASIC SAFETY EQUIPMENT: The following pieces of basic safety equipment are found in all campus laboratories. Informal and home laboratories may not have or need all of these items, but simple substitutes can usually be made or found. Students should know their exact location

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Introduction and proper use.
SAFETY GOGGLES - There is no substitute for this important piece of safety equipment! Spills and splashes do occur, and eyes can very easily be damaged if they come in contact with laboratory chemicals, shattered glass, swinging objects, and flying rock chips. While normal eyeglasses do provide some protection, these items can still enter the eyes from the side. Safety goggles cup around all sides of the eyes to provide the most protection and can be worn over normal eyeglasses if required.
EYEWASH STATION - All laboratories should have safety equipment to wash chemicals from the eyes. A formal eyewash station looks like a water fountain with two faucets directed up at spaces to match the space between the eyes. In case of an accident, the victim’s head is placed between the faucets while the eyelids are held open so the faucets can flush water into the eye sockets and wash away the chemicals. In an informal laboratory, a hand-held shower wand can be substituted for an eyewash station. After the eyes are thoroughly washed, a physician should be consulted promptly. FIRE EXTINGUISHER - There are several types of fire extinguishers, at least one of which should be found in all types of laboratories. Students should familiarize themselves with and know how to use the particular type of fire extinguisher in their laboratory. At a minimum, home laboratories should have a bucket of water and a large pot of sand or dirt available to smother fires.
FIRE BLANKET - This is a tightly woven fabric used to smother and extinguish a fire. It can cover a fire area or be wrapped around a victim who has caught on fire.
SAFETY SHOWER - This shower is used in formal laboratories to put out fires or douse people who have caught on fire or suffered a large chemical spill. A hand-held shower wand is the best substituted for a safety shower in a home laboratory.
FIRST-AID KIT - This kit of basic first-aid supplies is used for the emergency treatment of injuries and should be found in both formal and informal laboratories. It should be always well stocked and easily accessible.
SPILL CONTAINMENT KIT - This kit consists of absorbent material that can be ringed around a spilled chemical to keep it contained until the spill can be neutralized. The kit may simply be a bucket full of sand or other absorbent material such as kitty litter.
FUME HOOD - This is a hooded area containing an exhaust fan that expels noxious fumes from the laboratory. Experiments that might produce dangerous or unpleasant vapors are conducted under this hood. In an informal laboratory such experiments should be conducted only with ample ventilation and near open windows or doors. If a kitchen is used for a home laboratory, the exhaust fan above the stove substitutes nicely for a fume hood.
POTENTIAL LABORATORY HAZARDS: Recognizing and respecting potential hazards is the first step toward preventing accidents. Please appreciate the grave dangers the following laboratory hazards represent. Work to avoid these dangers and consider how to respond properly in the event of an accident.
FIRES: The open flame of a Bunsen burner or any heating source combined, even momentarily, with inattention may result in a loose sleeve, loose hair, or some unnoticed item catching fire.
Except for water, most solvents including toluene, alcohols, acetones, ethers, and acetates which

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Introduction are highly flammable and should never be used near an open flame. As a general rule NEVER
LEAVE AN OPEN FLAME OR REACTION UNATTENDED. In case of fire, use a fire extinguisher, fire blanket and/or safety shower.
CHEMICAL SPILLS: Flesh burns may result if acids, bases, or other caustic chemicals are spilled and come in contact with skin. Flush the exposed skin with a gentle flow of water for several minutes at a sink or safety shower. Acid spills should be neutralized with simple baking soda, sodium bicarbonate. If eye contact is involved use the eyewash station or its substitute. Use the spill containment kit until the spill is neutralized. To better protect the body from chemical spills, wear long-sleeved shirts, full-length pants, and enclosed shoes, not sandals, when in the laboratory.
ACID SPLATTER: When water is added to concentrated acid the solution becomes very hot and may splatter acid on the user. Splattering is less likely to occur if acid is slowly added to the water:
Remember this AAA rule: Always Add Acid to water, NEVER add water to acid.
GLASS TUBING HAZARDS: Never force a piece of glass tubing into a stopper hole. The glass may snap and the jagged edges can cause a serious cut. Before inserting glass tubing into a rubber or cork stopper hole be sure the hole is the proper size. Lubricate the end of the glass tubing with glycerol or soap, and then while grasping it with a heavy glove or towel, gently but firmly twist the tubing into the hole. Treat any cuts with appropriate first-aid.
HEATED TEST TUBE SPLATTER: Splattering and eruptions can occur when solutions are heated in a test tube. Thus, you should never point a heated test tube toward anyone. To minimize this danger direct the flame toward the top, rather than the bottom, of the solution in a test tube.
Gently agitate the tube over the flame to heat the contents evenly.
SHATTERED GLASSWARE: Graduated cylinders, volumetric flasks and certain other pieces of glassware are NOT designed to be heated. If heated, they are likely to shatter and cause injuries.
Always ensure you are using heatproof glass before applying it to a heat source. Special caution should always be taken when working with any type of laboratory glassware.
INHALATION OF FUMES: To avoid inhaling dangerous fumes, partially fill your lungs with air and, while standing slightly back from the fumes, use your hand to waft the odors gently toward your nose and then lightly sniff the fumes in a controlled fashion. NEVER INHALE FUMES DIRECTLY!
Treat inhalation problems with fresh air and consult a physician if the problem appears serious.
INGESTION OF CHEMICALS: Virtually all the chemicals found in a laboratory are potentially toxic.
To avoid ingesting dangerous chemicals, never taste, eat, or drink anything while in the laboratory.
All laboratories, especially those in home kitchens, should always be thoroughly cleaned after experimentation to avoid this hazard. In the event of any chemical ingestion immediately consult a physician.
HORSEPLAY: A laboratory full of potentially dangerous chemicals and equipment is a place for serious work, not for horseplay! Fooling around in the laboratory is just an invitation for an accident. VERY IMPORTANT CAUTION FOR WOMEN: If you are pregnant or could be pregnant, you should seek advice from your personal physician before doing any type of science experimentation.
If you or anyone accidentally consumes or otherwise comes into contact with something that is

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Introduction
MSDS: Material Safety Data Sheets
A Material Safety Data Sheet (MSDS) is designed to provide chemical, physical, health, and safety information on chemical reagents and supplies. An important skill in the safe use of chemicals is being able to read an MSDS. It provides information about how to handle store, transport, use, and dispose of chemicals in a safe manner.
MSDS also provide workers and emergency personnel with the proper procedures for handling and working with chemical substances. While there is no standard format for an MSDS, they all provide basic information about physical data (melting point, boiling point, flash point, etc.), toxicity, health effects, first aid procedures, chemical reactivity, safe storage, safe disposal, protective equipment required, and spill cleanup procedures. An MSDS is required to be readily available at any business where any type of chemical is used. Even day-care centers and grocery stores need MSDS for their cleaning supplies.
It is important to know how to read and understand the MSDS. They are normally designed and written in the following sections:
Section 1: Product Identification (Chemical Name and Trade Names)
Section 2: Hazardous Ingredients (Components and Percentages)
Section 3: Physical Data (Boiling point, density, solubility in water, appearance, color, etc.)
Section 4: Fire and Explosion Data (Flash point, extinguisher media, special fire fighting procedures, and unusual fire and explosion hazards)
Section 5: Health Hazard Data (Exposure limits, effects of overexposure, emergency and first aid procedures) Section 6: Reactivity Data (Stability, conditions to avoid, incompatible materials, etc.)
Section 7: Spill or Leak Procedures (Steps to take to control and clean up spills and leaks, and waste disposal methods)
Section 8: Control Measures (Respiratory protection, ventilation, protection for eyes or skin, or other needed protective equipment)
Section 9: Special Precautions (How to handle and store, steps to take in a spill, disposal methods, and other precautions)
Summary: The MSDS is a tool that is available to employers and workers for making decisions about chemicals. The least hazardous chemical should be selected for use whenever possible, and procedures for storing, using, and disposing of chemicals should be written and communicated to workers.
View MSDS information at www.hazard.com/msds/index.php. You can also find a link to MSDS information at www.LabPaq.com. If there is ever a problem or question about the proper handling of any chemical, seek information from one of these sources.

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LabPaq by
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Experiments

Experiment

Bacterial Morphology: TASK 11
Cynthia Alonzo, M.S.
Version 42-0240-00-01
Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will observe various bacterial morphologies using prepared slides. They will prepare live culture smears of Saccharomyces cerevisiae and cheek cells, and view these specimens under a microscope using direct and indirect staining techniques. Students will also learn how to prepare disinfectants and use them to decontaminate working surfaces.

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Bacterial Morphology

Objectives●
● Observe
● Learn

bacterial morphologies

about direct and indirect staining techniques

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Bacterial Morphology

discussion and review
The size, shape, and arrangement of bacteria and other microbes are the result of their genes, a defining characteristic called morphology. Bacteria come in a variety of sizes and shapes and new ones are discovered all the time. Nature loves variety in its life forms. The most common bacterial shapes are rods, cocci, and spiral. However, within each of these groups are hundreds of unique variations. Rods may be long, short, thick, thin, have rounded or pointed ends, or be thicker at one end than the other. Cocci may be large, small, or oval-shaped to various degrees.
Spiral-shaped bacteria may be fat, thin, loosely spiraled or tightly spiraled.
The group associations of microbes, both in liquid and on solid medium, are also defining. Bacteria may exist as single cells or in a common grouping such as chains, uneven clusters, pairs, tetrads, octads, or other packets. Bacteria may exist as masses embedded within a capsule. A description of the physical qualities – form and structure – of bacteria constitutes its individual morphology and is an identifying quality of the specific bacteria. There are square bacteria, star-shaped bacteria, stalked bacteria, budding bacteria that grow in net-like arrangements, and many other morphologies. When observing bacteria, describe as many of these characteristics as possible.
In this experiment, bacterial morphology will be examined by:
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Observing living, unstained organisms

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Observing killed, stained organisms

Because bacteria are almost colorless and show little contrast with the broth in which they are suspended, they are difficult to observe when unstained. Staining microorganisms allows you to:
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See greater contrast between the organism and the background

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Differentiate various morphological types by shape, arrangement, gram reaction, etc.

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Observe certain structures such as flagella, capsules, endospores, etc.

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Bacterial Morphology

Exercise 1: Viewing Prepared Slides of Common Bacterial Shapes
PROCEDURE
1. View the prepared slides for bacterial morphology. Record your observations.
2. Use each morphological type as a comparative tool for the remainder of the exercise.

Spiral bacteria – 100x magnification

Bacillus – 100x magnification bacillus

Spiral bacteria – 400x magnification

Bacillus – 400x magnification

Bacillus – 1000x oil immersion bacillus
Spiral bacteria – 1000x oil immersion
Figure 1: Morphological Examples

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Experiment

Yeast – 100x wet-mount

Bacterial Morphology

Cheek smear – 40x wet-mount
Cheek smear – 100x wet-mount
Figure 2: Wet-Mount Samples

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Experiment

Bacterial Morphology

Exercise 3: Direct Staining
In order to understand how staining works, it will be helpful to know a little about the physical and chemical nature of stains. Stains are generally salts in which one of the ions is colored. A salt is a compound composed of a positively charged ion and a negatively charged ion. For example, the dye methylene blue, is actually the salt methylene blue chloride. Methylene blue chloride dissociates in water into a positively charged methylene blue ion which is blue in color and a negatively charged chloride ion which is colorless.
Dyes or stains may be divided into two groups: basic and acidic. If the chromophore or colored portion of the dye resides in the positive ion, it is called a basic dye – methylene blue, crystal violet, and safranin. If the chromophore is in the negatively charged ion, it is called an acidic dye
– India ink, nigrosin, and Congo red.
Because of their chemical nature, the cytoplasm of all bacterial cells have a slight negative charge when growing in a medium of near neutral pH. Therefore, when using a basic dye, the positively charged chromophore of the stain combines with the negatively charged bacterial cytoplasm – opposite charges attract – and the organism becomes directly stained. An acidic dye, due to its chemical nature, reacts differently. Because the chromophore of the dye is on the negative ion, it will not readily combine with the negatively charged bacterial cytoplasm – like charges repel.
Instead, it forms a deposit around the organism, leaving the organism itself colorless. Since the organism is seen indirectly, this type of staining is called indirect or negative and is used to get a more accurate view of bacterial sizes, shapes, and arrangements.

Before direct staining bacteria, the organism must be fixed to the glass slide. If the preparation is not fixed, the organisms will be washed off the slide during staining. Simple methods to fix a slide include air-drying and heat-fixing. The organisms are heat-fixed by passing an air-dried smear of the organism through flame. The heat coagulates the organism’s proteins causing the bacteria to stick to the slide.

procedure
1.

View the prepared yeast, cheek cell and plaque slides stained with crystal violet.

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Experiment

Bacterial Morphology

Cheek Smear – 10x direct stain

Yeast – 100x direct stain

Plaque smear – 100x direct stain Cheek smear – 100x direct stain
Figure 3: Direct Stain Examples

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Bacterial Morphology

Exercise 4: Indirect Staining
In negative staining, the negatively charged color portion of the acidic dye is repelled by the negatively charged bacterial cell. Therefore the background will be stained and the cell will remain colorless. Indirect, or negative staining, does not require heat-fixing; thus, it is less likely to create abnormal cellular images or staining artifacts. Congo red is a common negative stain used in this exercise. 1. View the prepared yeast, cheek cell and plaque slides stained with Congo red.

Cheek smear – 100x indirect stain

Cheek smear – 40x indirect stain

Plaque smear – 100x indirect stain

Yeast – 100x indirect stain

Figure 4: Indirect Stain Examples

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C

Experiment

Aseptic Technique & culturing
Microbes: TASK 2
Cynthia Alonzo, M.S.
Version 42-0239-00-01
Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will use aseptic techniques to transfer cultures, including Lactobacillus acidophilus and
Staphylococcus epidermidis. They will learn about culture media and how to distinguish various types of microbial growth. Students will also learn about variable conditions that are required for microbial growth, including oxygen levels and temperature.

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Aseptic technique and culturing microbes

Objectives
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Learn and employ aseptic technique

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Become familiar with basic requirements of microbial growth

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Learn the basic forms of culture media

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Become familiar with methods used to control microbial growth

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Aseptic technique and culturing microbes

materials
Materials
Student provides

LabPaq provides

Qty
Item Description
1 Small cardboard box or Styrofoam cooler
(optional)
1 Desk lamp or heating pad (optional)
1 Aluminum foil (optional)
1 10%-bleach solution
1 Paper towels
1 S. epidermidis sample
1
1
2
1
1
1
1
1
1

Gloves, Disposable (1 pair)
Thermometer-in-cardboard-tube
Candles (flame source)
Test-tube-rack-6x21-mm
Broth, MRS - 9 mL in Glass Tube
Broth, Nutrient - 5 mL in Glass Tube
Lactobacillus acidophilus - capsule in Bag 2"x 3"
Swab, Sterile (pkg of 2)
Mask, Face with Earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Aseptic technique and culturing microbes

discussion and review
Controlling microbial growth is necessary in many practical situations. Significant advances in agriculture, medicine, and food science have been made through the study of this area of microbiology. Control of growth refers to the prevention of growth of microorganisms. This control is affected in two basic ways: by killing microorganisms or by inhibiting the growth of microorganisms. Control of growth usually involves the use of physical or chemical agents which either kill or prevent the growth of microorganisms. Agents that kill cells are called cidal agents; agents that inhibit the growth of cells without killing them are called static agents. Thus the term bactericidal refers to killing bacteria, and bacteriostatic refers to inhibiting the growth of bacterial cells. A bactericide kills bacteria; a fungicide kills fungi, and so on.
Sterilization is the complete destruction or elimination of all viable organisms in or on an object.
There are no degrees of sterilization; an object is either sterile or it is not. Sterilization procedures involve the physical removal of cells or the use of heat, radiation, or chemicals.

Methods of Killing Microbes
Heat is the most important and widely used method of killing microbes. For sterilization always consider the type of heat, the time of application, and the temperature to ensure the destruction of all microorganisms. Endospores of bacteria are considered the most thermoduric of all cells, so their destruction guarantees sterility.
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Incineration burns organisms and physically destroys them. This method is used for needles, inoculating wires, glassware, etc.

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Boiling at 100oC for 30 minutes kills almost all endospores. Very long or intermittent boiling is required to kill endospores and sterilize a solution.

Note: For the purpose of purifying drinking water, boiling at 100oC for five minutes is probably adequate. However, there have been some reports that Giardia cysts can survive this process.
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Autoclaving (steam under pressure or pressure cooker) at 121oC for 15 minutes (15lbs/in2 pressure) is good for sterilizing almost anything; however, autoclaving will denature or destroy heat-labile substances.

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Dry heat (hot air oven) at 160oC for 2 hours or 170oC for 1 hour is used for glassware, metal, and objects that will not melt.

You can refer to Table 1: Recommended Use of Heat to Control Bacterial Growth in the Lab Report
Assitant to find further information on using heat with bacteria.

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Experiment

Treatment
Incineration
Boiling

Aseptic technique and culturing microbes

Table 1: Recommended Use of Heat to Control Bacterial Growth
Temperature
Effectiveness
Incineration vaporizes any organic material on
>500oC
nonflammable surfaces but may destroy many substances in the process.
30 minutes of boiling kills microbial pathogens o 100 C and vegetative forms of bacteria but may not kill bacterial endospores.

Intermittent boiling

100oC

Three 30-minute intervals of boiling followed by periods of cooling kills bacterial endospores.

Autoclaving kills all forms of life including
Autoclave and pressure
121oC/15 minutes bacterial endospores. The item being sterilized cooker (steam under at 15# pressure must be maintained at the effective temperature pressure) for the full time.
Dry heat is used for materials that must remain dry and which are not destroyed at o temperatures between 121oC and 170oC. The
Dry heat (hot air oven) 160 C/2 hours method is good for glassware and metal, but not plastic or rubber items.
The effects are the same as above. Note that increasing the temperature by 10o shortens the
Dry heat (hot air oven) 170oC/1 hour sterilizing time by 50%.
Pasteurization kills most vegetative bacterial
Pasteurization (batch o 63 C/30 minutes cells including pathogens such as streptococci, method) staphylococci, and Mycobacterium tuberculosis.
The effect on bacterial cells is similar to the batch method. For milk, this method is more
Pasteurization (flash
72oC/15 seconds method) conducive to the industry and has fewer undesirable effects on quality or taste.

Irradiation usually destroys or distorts nucleic acids. Ultraviolet light is generally used to sterilize the surfaces of objects, although x-rays and microwaves can be useful.
Filtration involves the physical exclusion and removal of all cells in a liquid or gas, and is especially important to sterilize solutions which would be denatured by heat (antibiotics, injectable drugs, vitamins, etc.).
Toxic chemicals and gas such as formaldehyde, glutaraldehyde, and ethylene oxide can kill all forms of life in a specialized gas chamber.

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Aseptic technique and culturing microbes

In natural environments, microorganisms usually exist as mixed populations. However, if we are to study, characterize, and identify microorganisms, we must have the organisms in the form of a pure culture. A pure culture is one in which all the organisms are descendants of the same organism. Techniques for obtaining pure cultures from a mixed population will be described in the Isolation of Individual Colonies experiment.
To culture microorganisms we must have a sterile, nutrient-containing medium in which to grow the organisms. Anything in or on which we grow microorganisms is termed a medium. A medium is usually sterilized by heating it to a temperature at which all contaminating microorganisms are destroyed. Finally, in working with microorganisms, we must have a method of transferring growing organisms, called the inoculum, to a sterile medium without introducing any unwanted, outside contaminants. This method of preventing unwanted microorganisms from gaining access is termed aseptic technique.
The first step of aseptic technique is awareness – awareness that microbes are found on virtually every surface and in the air itself. Take care to minimize the culture’s exposure to environmental microbes. This is not as difficult as it may seem. Using gloves prevents contamination of the culture with the bacteria on our skin. Using a mask when handling cultures prevents contamination of the culture from microbes contained in our breath and minimizes the air currents our breath causes towards the culture tube. Think of how your breath affects a nearby candle. Take care not to touch caps or tube tops to counter tops or other surfaces. Use both disinfectant and a flame source to remove potential contaminants and to prevent further possible contamination.

Aseptically Inoculating a Broth Medium
1. Put on gloves and a mask and disinfect the work area.
2. Place the sample source and the target source (tube of sterile medium) in front of you. A primary goal of aseptic transfer is to avoid the possibility of contamination, so it is important to minimize the time the samples are exposed.
3. Pick up the instrument you are using to inoculate your new culture, such as a sterile swab, in one hand, taking care not to touch the microbe containing area.
4. Pick up the target medium tube in the other hand, and remove the cap with the hand holding the inoculation instrument. Do not to touch the inner surface of the cap. Keep the cap in your hand; do not set the cap down on the counter.

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Figure 1: Uncapping Medium Tube with Inoculation Instrument
5. Light a candle. Then run the top of the tube through the tip of the flame (flame the lip). The flame will sterilize the lip of the tube, and the heat will create an updraft which takes air contaminants away from the tube entrance.

Figure 2: Top of the Tube in the Flame
6. Quickly transfer the bacterial sample to the tube.

Figure 3: Transferring Bacterial Sample

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7. Use the candle flame to sterilize the top of the tube again to eliminate potential contamination and replace the cap.

Figure 4: Sterilizing the Tube
8. Disinfect your work area.

Forms of Culture Media
Nutrient Broth is a liquid medium. A typical nutrient broth medium, such as Trypticase soy broth, contains substrates for microbial growth such as pancreatic digest of casein, pancreatic digest of soybean meal, sodium chloride, and water. After incubation, growth, the development of many cells from a few cells, may be observed as one or a combination of three forms:
●●

Pellicle: A mass of organisms floats in or on top of the broth. Smaller masses or clumps of organisms that are dispersed throughout the broth form an even pattern called flocculent.

Figure 5: Pellicle Form

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Aseptic technique and culturing microbes

Turbidity: The organisms appear as a general cloudiness throughout the broth.

Figure 6: Turbidity Form
●●

Sediment: A mass of organisms appears as a deposit at the bottom of the tube.

Figure 7: Sediment Form

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Aseptic technique and culturing microbes

Agar slant tubes are tubes containing a nutrient medium plus a solidifying agent, called agar. The medium has been allowed to solidify at an angle in order to generate a flat inoculating surface.

Figure 8: Slant Tube
Stab tubes, called deeps, are tubes of hardened agar medium which are inoculated by stabbing the inoculum into the agar.

Figure 9: Stab Tube

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Agar dishes are sterile Petri dishes aseptically filled with a melted sterile agar medium and allowed to solidify. Dishes are much less confining than slant and stab tubes and are commonly used when culturing, separating, and counting microorganisms.

Figure 10: Agar Dishes

Requirements for Microbial Growth
Oxygen: Microorganisms show a great deal of variation in their requirements for gaseous oxygen.
Most microorganisms can be placed in one of the following groups.
●●

Obligate aerobes are organisms that grow only in the presence of oxygen. They obtain energy from aerobic respiration.

●●

Microaerophiles are organisms that require a low concentration of oxygen for growth. They obtain energy from aerobic respiration.

●●

Obligate anaerobes are organisms that grow only without oxygen; oxygen inhibits or kills them. They obtain energy from anaerobic respiration or fermentation.

●●

Aerotolerant anaerobes, like obligate anaerobes, cannot use oxygen for growth, but they tolerate oxygen fairly well. They obtain energy from fermentation.

●●

Facultative anaerobes are organisms that grow with or without oxygen, but generally better with oxygen. They obtain energy from aerobic respiration, anaerobic respiration, and fermentation. Most bacteria are facultative anaerobes.

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Temperature: Microorganisms are divided into groups based on their preferred ranges of temperature. ●●

Psychrophiles are cold-loving bacteria. Their optimum growth temperature is between -5°C and 15°C. They are usually found in the Arctic and Antarctic regions and in streams fed by glaciers. ●●

Mesophiles are bacteria that grow best at moderate temperatures. Their optimum growth temperature is between 25°C and 45°C. Most bacteria are mesophilic and include common soil bacteria and bacteria that live in and on the body.

●●

Thermophiles are heat-loving bacteria. Their optimum growth temperature is between 45°C and 70°C. They are commonly found in hot springs and compost heaps.

●●

Hyperthermophiles are bacteria that grow at very high temperatures. Their optimum growth temperature is between 70°C and 110°C. They are usually members of the Archaea and are found growing near hydrothermal vents at great depths in the ocean.

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Exercise: Culturing Microbes
Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic.
Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

PROCEDURES
Pre-Experiment Preparation: Find an incubation site or construct an incubator at least 24 hours in advance of the experiment to allow for time to monitor temperatures.

Part I: Set Up Incubation Site
Each bacterium has an optimal temperature at which it grows best. You can estimate the optimal growth temperature by considering the bacteria’s natural environment. Through the course of this experiment series, you will be culturing microbes from various sources, but most will fall into two main categories:
●●

Microbes from the environment that grow best at room temperature

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Microbes from our bodies that grow best at physiological or body temperature

You will need to establish sites that you can use to incubate both types of organisms. The sites should be free from draft and maintained at a consistent temperature. You will need to incubate your samples for 24 – 72 hour periods, so the site should be out of the way and free from interference. Use the thermometer to test the temperature of various areas in your home.
Some household areas that often closely approximate body temperature are the tops of water heaters or refrigerators. If you do not have access to these types of areas, use a desktop lamp or heating pad as a heat source to construct an incubator. Each lamp or pad is a bit different, so use the thermometer and test how far from the bulb or pad to keep the samples to keep them at
35°C–37°C (physiological temperature).

To construct an incubator:
1. Use a small box that is tall enough to hold the test tube rack with the broth tubes upright (6 inches minimum) and wide enough to set agar filled Petri dishes.
It is best if the box has a lid to reduce air drafts and help maintain a consistent temperature.
However, you can line a piece of cardboard with foil to lie across the top of the box in place of a lid.
Alternately, use a small Styrofoam cooler in place of a box. Cut the lid in half to allow space to direct the lamp into the box.

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2. Line the interior of the box with foil. If available, line pieces of Styrofoam with foil to fit in the box and provide greater insulation.

Figure 11: Foil Lined Box
3. Cut a small hole in the side of the box to fit a thermometer for monitoring temperature. Place the hole so the bulb of the thermometer will be at the same level as the cultures.

Figure 12: Thermometer Hole

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4. When using a desk lamp as the heating source, place the lamp so the light is aimed into the opening of the box. If the box has a lid, cut a hole in the lid and aim the lamp bulb through the hole.

Figure 13: Lamp Heat Source
5. Insert the thermometer into the box and monitor the temperature to determine the optimum distance to keep the lamp bulb. Monitor the temperature at different times of the day to ensure the temperature remains stable as environmental temperatures change.
6. When using a heating pad as the heating source, place the heating pad in the bottom of the box. Then, place a towel or folded paper towels on top of the heating pad. Insert the thermometer and monitor the temperature to determine both the optimal setting for the heating pad and the amount of padding to put between the pad and the samples to achieve the appropriate temperature. Monitor the temperature at different times of the day to ensure the temperature remains stable as environmental temperatures change.

Part II: Determine Medium Type
Two types of media will be used to grow the microbial specimens: nutrient medium and MRS medium. Though both media are available in liquid broth and solidified agar form, this experiment will use the liquid broth. Nutrient medium is the standard growth medium used for culturing most microbes. It consists of heat-stable digestive products of proteins (called peptones) and beef extract. These ingredients provide amino acids, minerals, and other nutrients used by a wide variety of bacteria for growth.
The MRS culture medium contains polysorbate, acetate, magnesium, and manganese which are known as a rich nutrient base and act as special growth factors for lactobacilli. The MRS medium will be used to culture Lactobacillus acidophilus. L. acidophilus will not grow sufficiently in nutrient media. Label the tubes carefully so the MRS medium will be easily identified for experiments using L. acidophilus. Remember, L. acidophilus needs to be cultured in MRS medium to grow!

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Part III: Generate Microbial Cultures
1. Disinfect the work area.
2. Use the ASEPTIC TRANSFER TECHNIQUE described above to generate liquid broth cultures of
L. acidophilus, and S. epidermidis.
3. S. epidermidis – Refer to the Preparation of Cultures section in the Appendix.
a. Label a tube of Nutrient Broth “S. epidermidis.”
b. Use a sterile swab to obtain a sample of bacteria from your skin.
c. Aseptically transfer the swab into the tube of sterile media.
d. Incubate the culture tube at 37°C for 24 – 72 hours.
4. L. acidophilus. – Refer to the Preparation of Cultures section in the Appendix.
a. Label a tube of MRS broth “L. acidophilus.”
b. Taking care not to touch the contents, open a capsule of L. acidophilus.
c. Aseptically transfer the contents of the capsule into the sterile MRS media.
d. Incubate the culture tube at 37°C for 24 – 72 hours.

Part IV: Observe Your Microbial Cultures
1. Observe the organisms after 24 hours and again after 48 hours to assess the growth pattern of each tube. Record your macroscopic observations.
Note: If there is no observable growth after 48 hours, allow the tubes to incubate an additional
24 hours.

Figure 14: Broth Growth Patterns
2. Prepare wet-mount slides of both the S. epidermidis and L. acidophilus cultures.
3. Prepare direct stained slides of both the S. epidermidis and L. acidophilus cultures.
4. Observe the slides microscopically at both 40X and 100X oil immersion magnification. Record the results.
5. Store both cultures in the refrigerator for use in future experiments.
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Experiment

Isolation of Individual colonies: TASK 12
Cynthia Alonzo, M.S.
Version 42-0245-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will learn about two types of culture growth media and colony morphology. Students will use several isolation techniques, including the pour plate method, the dilution method, and the streak plate method to prepare pure cultures. They will also learn how to maintain stock cultures.

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Objectives
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Become familiar with subtypes of culture media and the uses for each

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Learn and employ the streak and pour dish techniques

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Generate a pure culture of a specific organism

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materials
Materials
Student provides

LabPaq provides

Qty
1
1
1
1
1
1
1
1
1
11
2
1
6
1
1
1
1
1
4
1
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1

Item Description
Distilled water
Paper towels
10%-bleach or 70% alcohol solution
Zip bag
Pan to heat agar
Isopropyl alcohol (rubbing alcohol)
Cultures: S. epidermidis and L. acidophilus
Gloves, Disposable
Pencil, marking
Petri dish, 60 mm
Candles (flame source)
Thermometer-in-cardboard-tube
Test Tube(6), 16 x 125 mm in Bubble Bag
Test tube holder
Test-tube-rack-6x21-mm
Pipet Graduated Small (5 mL)
Baker’s Yeast Packet – Saccharomyces cerevisiae
Agar, MRS - 18 mL in Glass Tube
Agar, Nutrient - 18 mL in Glass Tube
Broth, Nutrient - 5 mL in Glass Tube
Inoculation Loop, Plastic
Mask with Earloops (11) in Bag 5" x 8"

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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discussion and review
Bacteria are everywhere! They are on bench tops, in water, soil, and food, on your skin, and in your ears, nose, throat, and intestinal tract (normal flora). The diversity of bacteria present in our environment and on and in our bodies is incredible. When trying to study bacteria from the environment, we quickly discover that bacteria usually exist in mixed populations. It is only in very rare situations that bacteria occur as a single species.
However, to study the cultural, morphological, and physiological characteristics of an individual species, we must separate the organism from other species normally found in its habitat by creating a pure culture of the microorganism. A pure culture is defined as a population containing only a single species or strain of bacteria. Contamination means more than one species is present in a culture that is supposed to be pure. Contamination does not imply that the contaminating organism is harmful. It simply means the contaminating organism is unwanted in the culture being isolated and studied.
Petri dishes or plates are covered dishes used to culture microorganisms. The sterile Petri dish is filled with a solidified nutrient medium.

Media Composition and Function
In addition to its physical state (liquid or solid), microbiological media are categorized by composition and/or function.
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Chemically Defined or Synthetic Media: In a synthetic medium, the exact amount of pure chemicals used to formulate the medium is known.

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Complex Media: A complex medium is composed of a mixture of proteins and extracts in which the exact amount of a particular amino acid, sugar, or other nutrient is not known.

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Enrichment Media: An enrichment medium contains some important growth factor (vitamin, amino acid, blood component, or carbon source) necessary for the growth of fastidious organisms. The MRS medium used in the Aseptic Technique & Culturing Microbes experiment is an enriched medium due to the presence of growth factors that encourage Lactobacillus acidophilus growth.

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Selective Media: Selective media allow for the selection of particular microorganisms that may be present in a mixed culture. Selective media usually contain a component that enhances the growth of the desired organism or inhibits the growth of competing organisms.

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Differential Media: Differential media allow for the separation of organisms based on some observable change in the appearance of the medium or by an observable effect on the microbe. Any single medium may be a combination of the previous categories. For example, Mannitol Salt
Agar (MSA), used for the isolation and identification of Staphylococcus, is a complex, selective, and differential medium. MSA contains NaCl, mannitol (a simple sugar), pancreatic digest of soy bean meal, potassium phosphate, and phenol red (a pH indicator). The presence of the pancreatic digest in the medium makes it a complex medium, because the exact composition of the pancreatic digest is not known. The relatively high concentration of salt in the medium

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is designed to inhibit many organisms and to select salt tolerant organisms. This makes MSA a selective medium. Staphylococcus is commonly found on the human skin – a salty environment due to sweat. Finally, the inclusion of mannitol and phenol red makes MSA a differential medium.
Staphylococci metabolize mannitol and produce acid as a waste product. This acid lowers the pH of the agar in the immediate vicinity of the organism. Phenol red changes color from red to yellow when the pH falls. Thus mannitol fermenting organisms can be differentiated from other organisms because the area around colonies of mannitol fermenters changes color from red to yellow as the organisms grow.
Microbiological media may be prepared as either liquid broth or solid medium. When a solid medium is prepared, the corresponding broth is solidified by the addition of agar. Agar is a gelatin type substance that is extracted from red-purple marine algae. Though it is possible to use standard gelatin, agar is preferred because it is stronger than gelatin and will not be degraded (eaten) by the bacteria. Agar is a solid gel at room temperature and melts at approximately 85°C. A particularly useful feature of agar is that while it melts at 85°C, it does not solidify until it cools to 32oC-40°C.
This allows the agar to remain in liquid form long enough and at a cool enough temperature to be managed. Agar is added to liquid nutrient medium, generally in a final concentration of 1%-2%, to obtain a solid culture medium.

Colony Morphology
To obtain a pure culture, it is necessary to separate individual cells of a particular microbe. This requires the use of a solid medium that provides a surface for the individual cells to be separated and isolated from the other microbial cells that may be present in the original sample.
A colony is a visible mass of microorganisms growing on a solid medium. A colony is considered to form from reproduction of a single cell. Thus, all the members of a colony are descendents from that original cell.
The colonies of different types of bacteria will have a distinct appearance. The visual characteristics of a colony (shape, size, pigmentation, etc.) are referred to as the colony morphology and can be used to identify bacteria. Bergey's Manual of Determinative Bacteriology, a standard resource used by many microbiologists, describes the majority of bacterial species identified by scientists so far. Bergey’s Manual provides descriptions for the colony morphologies of each bacterial species.
Though there are many identifiable characteristics that a colony may posses, there are six main criteria that comprise a standard morphology:
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Shape: What is the basic formation of the colony? Is it circular, irregular, or filamentous?

Figure 1: Shape Characteristics

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Isolation of Individual Colonies

Elevation: What is the cross-sectional form of the colony when viewed from the side? Is it flat, raised, or convex?

Figure 2: Elevation Characteristics
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Margin: How does the edge of the colony appear when magnified? Is it smooth, lobed, or curled? Figure 3: Margin Characteristics
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Surface: What is the appearance of the surface of the colony? Is it glistening, rough, or dull?

Figure 4: Surface Characteristics

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Pigmentation: Is the colony colored? Is it white, cream colored, pink, etc.?

Figure 5: Pigmentation Characteristics
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Opacity: Is the colony transparent, opaque, translucent, or iridescent?

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There are three additional characteristics that are sometimes used for identification but should be examined only in a controlled setting such as in a laboratory containment hood. These characteristics are consistency, emulsifiability, and odor.

Pure Cultures and Microbial Enumeration
Several different methods for getting a pure culture from a mixed culture are available including streak plate, pour plate, and dilution to extinction. All of these techniques depend on the physical isolation of a single bacterial cell on or in a solid medium. These cells give rise to isolated pure colonies of the bacteria.
In addition to the isolation of pure colonies, diluting to extinction also allows for the enumeration or determination of the number of organisms present in the original culture or sample. Microbial enumeration is routinely used in public health. Public safety officials test food, milk, or water and calculate the number of microbial pathogens present to determine if these products are safe for human consumption. Microbial counting techniques are also used to determine the number of microbes present in a given culture in commercial or scientific settings. For example, if the number of bacteria present in a fermentation culture is known, it is then possible to calculate the amount of fermentation product (such as insulin) that can be harvested from that population.
Several methods can be used to determine the number of microbes in a given sample. Viable counts include cells that can be cultured or are metabolically active. Total counts include all cells present, including dead or inactive cells. Direct methods count actual cells or colonies; indirect methods estimate the number of cells present based on the measurement of an indicator such as light absorption. Some of the more commonly used techniques are to measure the optical density of the population using a spectrophotometer, directly count the microorganisms using a hemocytometer, or serial dilute the bacteria and plate the diluted bacteria on a medium that supports the growth of the micro-organisms.

Optical Density: Spectroscopy Enumeration Method
Optical density is an indirect method of determining the cell concentration in a bacterial culture.
Bacterial cells absorb light well at the wavelength of 686 nm when grown in standard media. A spectrophotometer is used to measure the amount of light at a wavelength of 686 nm that is transmitted through a bacterial culture. Because the bacteria absorb the light of that wavelength, the amount of light transmitted through the culture, rather than absorbed by it, is inversely proportional to the number of bacteria present in the sample. The more bacteria present, the less light that will transmit through the sample.

Figure 6: Optical Density

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Measurement of light transmitted through the culture can be used to determine the number of bacteria present by graphing absorbance against known bacterial counts to obtain a standard curve. Figure 7: Light Absorbance
The measurements can also be converted to Optical Density (OD) which is a quantitative method of describing the cellular mass of a culture. The measurements obtained through spectrophotometer readings are considered total count measurements because they include all cells present, both viable and nonviable.

Aseptically Inoculating from a Liquid Culture
When working with bacterial cultures, it is essential to use proper aseptic culture techniques.
Remember, aseptic techniques are the precautionary measures used to avoid contamination of cultures and manipulate microorganisms to prevent contamination by undesirable organisms.
Aseptic techniques not only protect a laboratory culture from becoming contaminated, but also protect the experimenter and the environment from becoming contaminated by the microorganisms. 1. Disinfect the inoculating loop. Never lay the loop down once it is disinfected or it may become contaminated. To disinfect the plastic inoculation loop, swish the loop in a 10%-bleach solution or 70%-alcohol solution for about 10 seconds. Then rinse the loop with distilled water and allow it to completely air dry before using. Do not use a flame for disinfecting the plastic loop.
2. Hold the culture tube in one hand and the inoculating loop in the other hand as if it were a pencil. 3. Remove the cap of the culture tube with the little finger of your loop hand. Never lay the cap down or it may become contaminated.
4. Light a candle and briefly flame the lip of the culture tube.
5. Keeping the culture tube at an angle, insert the inoculating loop into the tube and remove a loop full of inoculum.

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6. Flame the lip of the culture tube again. Then replace the cap.
7. Pick up the sterile tube of medium. For the purposes of this experiment, you will be inoculating sterile agar medium.
8. Briefly flame the lip of the tube.
9. Place the loop full of inoculum into the medium. Withdraw the loop, but do not lay the loop down! 10. Flame the lip of the tube again.
11. Disinfect the inoculation loop.

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Exercise 1: Isolation Using the Pour Plate Method
Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic.
Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

procedure
Part I: Preparation of Solid Media
1. Disinfect the work area.
2. Melt the agar tubes. Refer to the Preparation of Solid Media section in the Introduction for further instruction.
Note: When you remove melted agar tubes from the water bath, they cool rapidly. Agar will solidify at about 45oC. If the agar solidifies, boil the tubes again to re-melt. It is time consuming and inconvenient if the agar deeps solidify in the tube before they are inoculated.
If the agar solidifies after inoculation but before it is poured into Petri dishes, re-melt the agar to kill the inoculated organisms. Under these circumstances, there is nothing to do but start over.
The key to successful pour plates is to be well organized and work quickly.
3. Leave the 18 mL tube of MRS agar in hot water (50°C) for use in Part II.
4. Use the marking pencil to label the bottom of one Petri dish S. epidermidis. Pour one half (9 mL) of the contents of a tube of nutrient agar into the S. epidermidis Petri dish and the other half into the bottom of an unmarked Petri dish. Cover the dishes and allow them to solidify for use in Part IV.
5. Pour the remaining melted nutrient agar into the unmarked Petri dishes (half a tube per dish).
Cover the dishes and allow them to solidify for use in Part III.
Note: There will be one extra nutrient agar dish. Store the dish in the refrigerator for use in the
Antibiotic Sensitivity experiment. Invert the dish in a zip bag to protect it from contamination and dry-out. Part II: Isolation Using the Pour Plate Method
The pour plate technique, sometimes called the loop dilution method, involves the successive transfer (serial dilution) of bacteria from the original culture to a series of tubes of liquefied agar.
A loop of the original culture is transferred to a tube of liquid agar and mixed. As a result of this transfer, the concentration of bacteria in the tube is lower than the concentration in the original culture – in effect, a dilution of the original culture. A loop of material from the first tube of liquefied agar is then transferred to a second tube, effecting an additional dilution of the bacterial

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culture. The process is repeated for a third tube of agar. Following inoculation of the tubes of liquid agar, the contents of each tube are poured into separate Petri dishes. After incubation, one of the dishes should have a low enough concentration of microbes to allow separation and isolation of individual colonies.
1. Disinfect the work area.
2. Label the bottom surface of three sterile Petri dishes L. acidophilus #1, #2, and #3, respectively.

Figure 8: Part II Pour Plates
3. Disinfect three test tubes by submerging them in boiling water for 5 minutes. The tubes will be hot, so use tongs or tweezers to lift them out of the water. Be careful not to contaminate the tubes by touching their lips or interiors. When the tubes are cool, label them to match the
L. acidophilus Petri dishes.
4. Divide the liquid MRS agar into the three test tubes marked L. acidophilus. If the agar has begun to solidify, reheat it until it is fully melted. Set the test tubes of agar in the hot water to prevent them from solidifying.
5. After ensuring the tubes of agar are cool enough not to kill the bacterial culture but are still fully liquid, use aseptic techniques to inoculate the tube labeled L. acidophilus #1 with one loop full of the saved L. acidophilus culture. Gently mix and return the tube to the hot water.
6. Inoculate L. acidophilus #2 with one loop full of the bacteria media mix from tube #1. Gently mix and return the tubes to the hot water.
7. Inoculate L. acidophilus #3 with one loop full of the bacteria media mix from tube #2. Gently mix and return the tubes to the hot water.

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Figure 9: Inoculated Test Tubes
8. Pour the contents of L. acidophilus #1 into the corresponding Petri dish and cover the dish immediately. Repeat for L. acidophilus #2 and #3.
9. Allow the agar to solidify at room temperature.

Figure 10: Inoculated Petri Dishes
10. Incubate the dishes in an inverted position for 24–72 hours at 35oC–37oC.
11. Examine the dishes for isolated colonies. Record the appearance of each dish.
12. Store the culture in the refrigerator for use in future experiments.
13. Soak the Petri dishes in a 10%-bleach solution for 1 hour and then discard them.
14. Soak the test tubes in a 10%-bleach solution for 1 hour and then discard the contents. Clean and rinse the test tubes for future use.

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Exercise 3: Isolation by the Streak Plate Method procedure In the streaking procedure, a disinfected loop or sterile swab is used to obtain a microbial culture.
The inoculating instrument is then streaked lightly over an agar surface. On the initial section of the streak, many microorganisms are deposited, resulting in confluent (solid) growth, which is growth over the entire surface of the streaked area. However, because the loop is sterilized or disinfected between streaking different sections, or zones, fewer and fewer microorganisms are deposited as the streaking progresses. Finally, only an occasional microorganism is deposited, because the streaking process dilutes the sample placed in the initial section.

Figure 16: Streak Pattern
During incubation, the isolated microbes multiply, giving rise to individually isolated colonies in the lightest inoculated areas. Colonies appear as piles of material on the agar surface, and they come in a variety of shapes, sizes and textures which are characteristic of individual microorganisms.
For example, if a single Escherichia coli cell is deposited on a nutrient agar dish and incubated at
37°C, the cell and its progeny will divide every 30–40 minutes. In 10–12 hours, the colony will have reached a population of one million, and a pinpoint colony will be visible. To obtain good results with this technique, the agar surface should be smooth, moist, and free of contamination.
However, excessive moisture from the condensation of water, derived from the initial cooling of the hot sterile medium, can collect on the inside of the lid and sides of the dish. If the water drops onto the agar surface, spreading and merging of colonies can occur. Always invert the dishes after streaking them and when incubating them.

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1. Disinfect the work area.
2. Use the nutrient agar dish labeled S. epidermidis from Exercise 1.
3. Use aseptic techniques to obtain a loop full of the saved liquid S. epidermidis culture.
4. Streak the inoculum into the first quadrant as shown in Figure 16.
5. Disinfect the inoculation loop. Do not obtain a new inoculum. Instead, use the disinfected inoculation loop to streak several times through Quadrant 1 to pick up some organisms on the loop. Then streak from Quadrant 1 to Quadrant 2 as shown in Figure 16.
6. Repeat the procedure for Quadrants 3 and 4, respectively. Be sure to disinfect the inoculation loop between each quadrant.
7. Disinfect the inoculation loop.
8. Cover the dish, invert it, and incubate it for 48–72 hours at 35oC–37oC.
9. Identify an S. epidermidis colony. The S. epidermidis culture was not a pure culture (derived from a single organism) and will most likely contain colonies from several different organisms.
Staphylococci produce round, raised, opaque colonies 1–2 mm in diameter. S. epidermidis colonies are white in color.

Figure 17: S. Epidermidis

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Exercise 4: Stock Cultures
Procedure
When a particular organism is going to be used more than once, create a stock culture of that organism. A stock culture is maintained for as long as the organism is needed. The use of stock cultures is beneficial for several reasons including:
●●

They maintain consistency by ensuring the same strain of organism is used.

●●

They save time and money by eliminating the need to recreate the same culture.

In the following steps you will make a stock culture of S. epidermidis to use in the remaining experiments. 1. Label a tube of nutrient broth S. epidermidis Stock Culture.
2. Aseptically transfer an S. epidermidis colony from your Petri dish into the nutrient broth. The culture that grows in the broth will be a pure culture because it originated from only a single colony, which originated from a single organism.
3. Incubate the stock culture for 24–48 hours to establish the culture. Then store the culture in a zip bag in the refrigerator for use in future experiments. You may also store dish cultures in a similar manner.
4. Mix 1 tablespoon of bleach into the original S. epidermidis culture and let it stand for at least
30 minutes to ensure all organisms have been destroyed. Then discard the contents.
5. Soak the Petri dishes in a 10%-bleach solution for 1 hour and then discard them.
6. Disinfect the work area.

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Differential Staining: TASK 6
Cynthia Alonzo, M.S.
Version 42-0242-00-01
Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will use Gram’s stain techniques to differentiate between types of bacteria and explore the difference between Gram-positive and Gramnegative bacteria. Students will explore what properties differentiate microorganisms including
Escherichia coli, Staphylococcus epidermidis,
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Objectives
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Understand and employ differential staining techniques

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Describe the differences between Gram-negative and Gram-positive bacteria

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materials
Materials
LabPaq provides

Qty
1

Item Description
Prepared slide images.

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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discussion and review
There are several staining methods used routinely with bacteria. These methods are generally classified as either simple, nonspecific, or differential (specific). Simple stains will react with all microbes in an identical fashion. They are used solely for increasing contrast, so an organism’s morphology, size, and arrangement can be determined. Differential stains provide varying results depending on the organism being treated. These results are often helpful in identifying the microbe. This exercise will focus on one of the most commonly used differential stains – the
Gram’s stain.
The Gram’s stain is the most widely used staining procedure in bacteriology. It is called a differential stain because it differentiates between Gram-positive and Gram-negative bacteria. Bacteria that stain purple are termed Gram-positive. Those that stain pink are termed Gram-negative.

Figure 1: Gram-Positive (left); Gram-Negative (right)
Gram-positive and Gram-negative bacteria stain differently because of fundamental differences in the structure of their cell walls. The bacterial cell wall serves to give the organism its size and shape as well as to prevent osmotic lysis. The material in the bacterial cell wall that confers rigidity is peptidoglycan. The Gram-positive cell wall appears thick and consists of numerous interconnecting layers of peptidoglycan. Also interwoven in the cell wall of Gram-positive bacteria are teichoic acids. Generally, 60%-90% of the Gram-positive cell wall is peptidoglycan.

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Figure 2: Gram-Positive Peptidoglycan
The Gram-negative cell wall contains a much thinner section of peptidoglycan – only two or three layers thick. This section is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, and lipoprotein. Only 10% - 20% of the Gram-negative cell wall is peptidoglycan.

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Figure 3: Gram-Negative Peptidoglycan

Gram Staining Procedure:
1. The bacteria are first stained with the basic dye, crystal violet. Both Gram-positive and Gramnegative bacteria become directly stained and appear purple.
2. Then the bacteria are treated with Gram's iodine solution. The iodine acts as a MORDANT.
The solution helps retain the stain by forming an insoluble crystal violet-iodine complex.
Both Gram-positive and Gram-negative bacteria remain purple.
3. Then the bacteria are treated with Gram's decolorizer, a mixture of ethyl alcohol and acetone.
This is the differential step. Gram-positive bacteria retain the crystal violet-iodine complex in their thick peptidoglycan layer. The complex washes out of the thinner peptidoglycan layer of
Gram-negative bacteria which become decolorized.
4. Finally, the bacteria are treated with the counterstain, safranin. Because the Gram-positive bacteria are already stained purple, they are not affected by the counterstain. The Gramnegative bacteria are colorless and become directly stained pink by the safranin. Consequently, the Gram-positive bacteria appear purple and the Gram-negative bacteria appear pink.

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Exercise 1: Differential Staining
PROCEDURE
1. View slide images of Gram stained E. coli, Staphylococcus, S. cerevisiae and
Lactobacillus.

Figure 1: E. coli Gram Stain

Figure 3: Staphylococcus aureus Gram Stain

Figure 2: Lactobacillus Gram Stain

Figure 4: Yeast Gram Stain

R

Experiment

Methyl red Voges-Proskauer
Test: TASK 1
Cynthia Alonzo, M.S.
Version 42-0246-00-01
Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will learn how different biochemical tests, including methyl red, Voges-Proskauer, and catalase, are used to differentiate microorganisms. Students will test Escherichia coli and Staphylococcus epidermidis with these biochemicals to analyze the bacteria’s use of various sugars and different biochemical pathways.

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Methyl Red Voges-Proskauer Test

Objectives
●●

Become familiar with and perform the MR-VP biochemical test

●●

Learn some variations in how different organisms metabolize glucose

●●

Become familiar with and perform the catalase biochemical test

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Methyl Red Voges-Proskauer Test

materials
Materials
Student provides

LabPaq provides

Qty
Item Description
1 Hydrogen peroxide
1 10%-bleach solution
1 Paper towels
1 Stock culture: S. epidermidis
1 Saved culture: E. coli
1 Gloves packages - 11 pairs
2 Candles (flame source)
1 Test Tube(6), 16 x 125 mm in Bubble Bag
1 Test-tube-rack-6x21-mm
1 Pipet, Long Thin Stem
2
1
1
1
1
1

Broth, MR-VP - 5 mL in Glass Tube
Barritt's A Reagent - 3 mL in Pipet
Barritt's B Reagent - 3 mL in Pipet
Methyl Red Reagent, 0.1% - 1 mL in Pipet
Inoculation Loop, Plastic
Mask with Earloops (11) in Bag 5” x 8”

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Methyl Red Voges-Proskauer Test

discussion and review
Different bacteria may have similar morphologies or produce colonies that are indistinguishable from those of other types of bacteria. Staining techniques provide additional opportunities to gather information such as bacterial morphology, cell wall composition, and the presence of capsules, flagella, or endospores. However, visual examination, both macroscopic and microscopic is often not enough to identify a specific bacterial species. In such cases, we must rely on biochemical characteristics to differentiate between organisms.
All organisms utilize a vast array of biochemical pathways to perform metabolic functions. Each pathway consists of a series of chemical reactions. Specialized proteins called enzymes are used to catalyze these reactions. Many enzymes require dietary minerals, vitamins, and other cofactors in order to function properly. As you can imagine, each pathway can be quite elaborate and require many different proteins, minerals, vitamins, or other molecules. The metabolic processes used by a cell are similar from organism to organism. However, the specific pathway or molecules used in the pathway can and do vary. The specific pathways or molecules used by a specific organism comprise its biochemical profile or “fingerprint” and can be used to identify a particular species.
Microbiologists have developed series of biochemical tests that use the biochemical profile of a particular microbe to differentiate between even closely related species.

Figure 1: Biochemical Test Series
There are many types of commonly used biochemical tests that test for either the presence of a particular enzyme or for a byproduct or end product of a particular pathway. The tests can be done individually or as a series. There are a number of commercially produced test strips that combine tests designed to identify specific groups of organisms. Each strip is a collection of mini chambers, each of which contains the reagents necessary to test for a specific biochemical characteristic. The strip is designed so that a microbe of interest can be inoculated into a groove or tube that carries the microbe into each chamber without intermixing. Once inoculated, the strip is incubated and the results of the tests can be observed. The interpretation of positive and

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negative test results allows for identification of the bacteria to the species level. Additionally, newer methods of testing identify organisms using other characteristics, such as DNA sequence or reaction to monoclonal antibodies.
In this experiment, you will perform three common biochemical tests: the Methyl Red test, the
Voges-Proskauer test, and the catalase test.

Methyl Red Test
The Methyl Red (MR) test is used to identify bacteria based on their pattern of glucose metabolism.
Most bacteria that ferment glucose produce pyruvic acid as an early step in metabolism; however, not all bacteria metabolize pyruvic acid like other acids, such as lactic acid and formic acids.
Methyl Red broth contains glucose, peptone, and a phosphate buffer. Bacteria that produce mixed-acids as an end product of glucose fermentation overwhelm the buffer in the broth and cause a decrease in pH. Bacteria that utilize other fermentation pathways and produce other, non-acidic end products do not cause a drop in the pH of the broth.

Figure 2: MR Results
After incubation, Methyl Red, a pH indicator, is added to the broth. Methyl Red turns red when the pH is below 4.4; yellow when the pH is above 6.0; and orange when the pH is between 4.4 and 6. A positive Methyl Red test result, indicating the production of stable acidic end products, is evidenced when the incubated broth turns red. A yellow color is a negative result indicating acidic end products were not produced. If the incubated tube turns orange, the result is inconclusive.
It is likely that the bacteria are producing acidic products but not in large enough quantities to overwhelm the phosphate buffer in the broth. In these cases, the tube should be incubated for an additional 24 hours to see if more acid is produced.

Voges-Proskauer Test
The Voges-Proskauer (VP) test is an assay for the presence of acetyl methyl carbinol (acetoin).
Acetoin can be produced as an intermediate product in the fermentation of pyruvate to
2,3-Butanediol. It can also be produced by some organisms that ferment glucose to form unstable acid products which can be converted to acetoin. After incubation of the organism in the MRVP broth, Barritt’s Reagent A (a-napthol) and B (40% KOH) are added. The reagents react with

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Methyl Red Voges-Proskauer Test

acetoin, creating a maroon band at the top of the broth. The appearance of the band is a positive
VP result, indicating the production of acetoin.

Figure 3: VP Results

Catalase Test
Hydrogen peroxide is a harmful byproduct of many normal metabolic processes. To prevent damage, hydrogen peroxide must be quickly converted into other, less dangerous substances. Like many other organisms, microorganisms may produce enzymes which neutralize toxic forms of oxygen such as hydrogen peroxide. One such enzyme is catalase, which facilitates the breakdown of hydrogen peroxide into water and molecular oxygen.
2 H2O2 → 2 H2O + O2
Microbes which produce catalase will bubble when placed into hydrogen peroxide, as the enzyme speeds the decomposition of the hydrogen peroxide to water and gaseous oxygen.

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Methyl Red Voges-Proskauer Test

Exercise 1: Methyl Red-Voges Proskauer Tests
Procedure
Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic.
Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.
The MR and VP tests use the same base broth as the medium. The MR-VP broth contains peptone, buffers, and glucose. Because they use the same broth, the tests are usually done together.
Pre-Experiment Preparation: Place the saved E. coli culture and the S. epidermidis stock culture in an incubator 12–24 hours prior to the start of the experiment.
1. Disinfect the work area.
2. Remove the tubes of MR-VP broth from the refrigerator and allow them to come to room temperature. 3. Label the MR-VP broth tubes E. coli and S. epidermidis.
4. Use aseptic techniques to inoculate each MR-VP broth tube with the corresponding organism.

Figure 5: MP-VP Test Tubes

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Methyl Red Voges-Proskauer Test

5. Incubate the tubes for 48 hours at 35°C–37°C.
6. Allow the reagents to warm to room temperature.
7. Sterilize and label two test tubes E. coli and two test tubes S. epidermidis.
8. Transfer half (2.5 mL) of the incubated MR-VP broth labeled E. coli into each of the corresponding test tubes. Repeat for the broth labeled S. epidermidis.
9. Choose one tube for each organism for the Methyl Red test and label it accordingly. Use a pipet to add six to eight drops of Methyl Red reagent to each of the tubes. If the test is positive, the red-pink color of acid presence from glucose use will appear within seconds.
10. Use the remaining tubes for the Voges-Proskauer test. Add 12 drops of Barritt’s A Reagent to each tube and mix gently.
11. Add four drops of Barritt’s B Reagent to each tube. Shake the tube gently for 30 seconds. The broth must be exposed to oxygen for a color reaction to occur.
12. Allow the tubes to stand for 30 minutes before interpreting.

Figure 6: MR Test
Note: The reagents must be added in the correct order and in the correct amounts. The tubes must sit undisturbed and open to the air (no cap) for at least 30–45 minutes as the light pink color intensifies at the top of the tube. Do not shake the tube after sitting it down for the waiting period. Do not read test results more than one hour after adding the reagents.
13. Record the results.
14. Soak the test tubes in a 10%-bleach solution for 1 hour and then discard the contents. Clean and rinse the test tubes for future use.

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Experiment

Motility Testing: TASK 3
Cynthia Alonzo, M.S.
Version 42-0248-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will explore flagellar structure and other arrangements common to microbes. They will use motility test agar tubes to determine whether
Escherichia coli and Staphylococcus epidermidis are motile. www.LabPaq.com

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Motility Testing

Objectives
●●

Learn flagellar structure and arrangements common in microbes

●●

Use direct observation and testing to determine if a given microbe is motile

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Motility Testing

materials
Materials
Student provides

LabPaq provides

Qty
1
1
1
1

1
2
1
1

Item Description
Paperclip
10%-bleach solution
Paper towels
Cultures: E. coli and S. epidermidis

Gloves, Disposable
Agar, 0.4% Motility Test Agar - 8 mL in Glass
Tube Inoculation Loop, Plastic
Mask, Face with Earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Motility Testing

discussion and review
Many bacteria are capable of motility – the ability to move under their own power. Most motile bacteria propel themselves by special organelles termed flagella. The bacterial flagellum is a noncontractile, semi-rigid, helical tube composed of protein and anchors to the bacterial cytoplasmic membrane and cell wall by means of disk-like structures. The rotation of the inner disk causes the flagellum to act much like a propeller.

Figure 1: Flagellum
Bacterial motility constitutes unicellular behavior. In other words, motile bacteria are capable of a behavior called taxis. Taxis is a motile response to an environmental stimulus and functions to keep bacteria in an optimum environment. The arrangement of the flagella about the bacterium is of use in classification and identification. The following flagellar arrangements may be found:
●●

Monotrichous: a single flagellum at one pole of the cell

●●

Lophotrichous: two or more flagella at one or both poles of the cell

●●

Amphitrichous: a single flagellum at both poles of the cell

●●

Peritrichous: a cell completely surrounded by flagella

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Motility Testing

Figure 2: Flagellar Arrangements
One group of bacteria, the spirochetes, has internally located axial filaments or endoflagella.
Axial filaments wrap around the spirochete from both ends toward the middle. Axial filaments are located above the peptidoglycan cell wall but underneath the outer membrane or sheath.
You cannot directly observe flagella on most microbes with a standard light microscope in a wetmount.
To detect bacterial motility, we can use any of three methods: direct observation, motility media, and flagella staining. In this experiment, we will use two methods:
●●

Direct observation: An actively growing, young broth culture can be used for observation of motility. Motile organisms can be seen as they move among each other in separate directions.
However, direct observation of motility can sometimes be difficult. Cultures, particularly older cultures, may be a mix of both active and inactive members, and wet-mounts containing a larger percentage of inactive microbes can make motility difficult to observe. In very active cultures, the opposite problem may exist. When using a microscope, the field of view encompasses a very small area, and fast moving microbes may leave the viewable area before they can be clearly observed.

●●

Motility test medium: Semi-solid Motility Test medium is used to detect motility. The agar concentration (0.4%) is sufficient to form a soft gel without hindering motility. When a nonmotile organism is stabbed into Motility Test medium, growth occurs only along the line of inoculation. Growth along the stab line is very sharp and defined. When motile organisms are stabbed into the soft agar, they swim away from the stab line. Growth occurs throughout the tube and is not concentrated along the line of inoculation. Growth along the stab line appears cloud-like as it moves away from the stab.

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Motility Testing

Figure 3: Motile Test Medium

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Motility Testing

Exercise 1: Motility Testing
Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic.
Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

Procedure
Pre-Experiment Preparation: Place the saved cultures of E. coli and S. epidermidis in an incubator 12–24 hours prior to the start of the experiment.

Part II: Motility Tubes
1a. Disinfect the work area.
1b. Label each of the Motility Test medium tubes (0.4% agar) E. coli and S. epidermidis.
2. Straighten a paperclip to serve as an inoculating needle. Then disinfect the inoculating needle by soaking it in a 10%-bleach solution.
3. Use aseptic techniques to open both the E. coli motility medium tube and the E. coli culture.
4. Insert the inoculating needle directly into the E. coli culture. Then insert the inoculating needle straight down the center of the motility medium tube. Withdraw the inoculating needle along the same path as the entry without disturbing the agar.
5. Use aseptic techniques to close the tubes.
6. Disinfect the inoculation needle by returning it to the 10%-bleach solution.
7. Repeat the inoculation steps for S. epidermidis.
8. Incubate the motility medium tubes at 35oC–37oC for 24–48 hours.
9. After incubation, observe the tubes and record any observations of motility.
10. Soak the motility tubes in a 10%-bleach solution for 1 hour and then discard the contents.
Clean and rinse the test tubes for future use.
11. You will not be using the E. coli culture for future experiments. Mix 1 tablespoon of bleach into the E. coli culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents.

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Motility Testing

12. Soak the test tubes in a 10%-bleach solution for 1 hour and then discard the contents. Clean and rinse the test tubes for future use.
13. Store the S. epidermidis culture in the refrigerator for use in future experiments.
14. Disinfect the work area.

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C

Experiment

carbohydrate Fermentation
Testing: TASK 4
Cynthia Alonzo, M.S.
Version 42-0241-00-01
Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will explore the use of phenol red to indicate changes in the pH of fermented sugars. They will use
Durham test tubes to test for the production of carbon dioxide during fermentation of the carbohydrates fructose, glucose, and mannitol. Students will test
Staphylococcus epidermidis and Saccharomyces cerevisiae to establish a fermentation profile.

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Carbohydrate Fermentation Testing

Objectives
●●

Generate a fermentation profile for specific organisms

●●

Learn how biochemical tests are used and employ a chemical indicator

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Experiment

Carbohydrate Fermentation Testing

materials
Materials
Student provides

LabPaq provides

Qty
Item Description
1 10%-bleach solution
1 Microscope
1 Immersion Oil
1 Paper towels
1 Stock culture: S. epidermidis
1 Gloves packages - 11 pairs
1 Test-tube-rack-6x21-mm
2 Candles (flame source)

6
6
1
1
1
1
1
1

Broth, Phenol Red - 9 mL in Glass Tube
Durham Tube, Glass, 6 x 50 mm in Bag 2"x3"
Baker’s Yeast Packet – Saccharomyces cerevisiae
Fructose Powder, 0.2 g, in Vial
Glucose Powder - 0.2 g, in Vial
Mannitol Powder - 0.2 g, in Vial
Inoculation Loop, Plastic
Mask, Face with Earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Carbohydrate Fermentation Testing

discussion and review
Fermentation is a metabolic process that allows the production of Adenosine triphosphate (ATP) without the need for oxygen. During fermentation, the final electron acceptor is an organic molecule rather than oxygen. There are many different kinds of carbohydrates that may serve as substrates for fermentation. Not all bacteria can utilize all of the possible fermentable carbohydrates. The ability or inability of a particular species to ferment a particular carbohydrate depends on the presence of the enzymes needed for a particular fermentation pathway.
Because the DNA of an organism codes for which particular enzymes the organism contains, the presence or lack of a particular enzyme is decided at the genetic level and varies by species.
More variety exists among those bacteria that can ferment a particular carbohydrate; a variety of fermentation end products and byproducts can be produced depending on what enzymes are used at later stages of the fermentation pathway. Bacteria may produce acidic, neutral, alcoholic, or gaseous end products. These differences in fermentation pathways can be used as a diagnostic tool. To identify a particular species based on fermentation, a series of tests is used to generate a fermentation profile for an organism. These profiles are unique to particular species and are used in the identification of the bacteria, particularly Gram-negative enteric (gut) bacteria.
For each carbohydrate tested, the following questions can be answered:
●●

Can the organism ferment the particular carbohydrate?

●●

If so, does it produce acidic end- or byproducts?

If so, does it produce gaseous products?
In a fermentation series, each medium has a single fermentable carbohydrate added to a peptone broth. Phenol red is also added as a pH indicator. Phenol red will turn yellow below pH 6.8 and a dark pinkish-red above pH 7.4. If the organism metabolizes the carbohydrate, subsequent acid production will result in lowered pH. If the organism does not ferment the carbohydrate, the pH may remain neutral. If the organism does not ferment the carbohydrate, but utilizes the peptone, accumulation of the ammonia as a byproduct will raise the pH. A small tube, called a Durham tube, is inverted and placed in each larger test tube of liquid medium. The inverted tube is able to trap any gas products produced by fermentation.

Interpretation
Acid: (yellow) Acid production produces a color change from red to yellow, indicating the organism is capable of metabolizing the sugar in the tube.

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Carbohydrate Fermentation Testing

Figure 1: Acid Production
Acid, Gas: (yellow plus gas bubble) Fermentation of the sugar is indicated by a color change to yellow. Gas is trapped in the Durham tube, replacing the medium in the tube. A bubble indicates gas production.

Figure 2: Acid With Gas Production
Negative: Negative fermentation can be indicated two ways:
●●

No color change: The sugar was not utilized by the organism.

●●

Dark, pinkish-red color change: The darker color indicates an alkaline or basic metabolic product, which is due to utilization of the peptone instead of sugar. When the tube is read within 48 hours, the darker-red color indicates negative fermentation, although the result is usually recorded as alkaline.

Figure 3: Negative Fermentation

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Carbohydrate Fermentation Testing

A carbohydrate profile should be written using the following notation:
●●

A = Acid production

●●

AG = Both acid and gas production

●●

- = Negative fermentation

For example, organism X could have a carbohydrate profile of:
Glucose AG
Sucrose A
Fructose –
Maltose A
Galactose AG
Lactose –

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Carbohydrate Fermentation Testing

Exercise 1: Carbohydrate Fermentation Testing
Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic.
Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter-free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

procudure
Pre-Experiment Preparation: Place the stock culture of S. epidermidis in an incubator 12–24 hours prior to the start of the experiment. Prepare an S. cerevisiae culture in accordance with the
Preparation of Cultures section in the Appendix.

Part I: Fermentation Tube Preparation
1. Label three phenol red tubes S. cerevisiae #1, #2, and #3. Label the other three tubes S. epidermidis #1, #2, and #3.

Figure 4: Labeled Phenol Red Tubes
2. Using aseptic techniques, divide the glucose powder between the two tubes labeled #1.
Divide the fructose powder between the two tubes labeled #2. Divide the mannitol powder between the two tubes labeled #3.
3. Sterilize each Durham tube and aseptically place one in each tube of phenol red. Slide the tubes into the broth so that the open end of the Durham tube is at the bottom of the broth tube. www.LabPaq.com

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Carbohydrate Fermentation Testing

Figure 5: Durham Tube
4. Tilt each phenol red tube so the Durham tube fills with broth. If an air bubble forms in the tube, turn the broth tube upside down to allow the bubble to escape the tube. It may be necessary to shake the tube slightly to dislodge the bubbles.
5. Use aseptic technique to inoculate each tube with the corresponding organism.
6. Incubate the tubes at 35°C–37°C for 12 hours. Record your observations. Do not let the cultures incubate for more than 24 hours to avoid inaccurate results.

Note: Although a microbe may use a particular sugar as its primary nutrient, when the microbe runs out of sugar, it will attack protein or other nutrients. When microbes use proteins, they produce alkaline by-products, and the medium can change colors as a result of the pH indicator added to detect acid production. . If sugar tests are extended for more than a day, there is the possibility that the color will have changed, and the test results will appear negative rather than positive. 7. Mix 1 tablespoon of bleach into the yeast culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents.
8. Soak the tubes in a 10%-bleach solution for 1 hour and then discard the contents. Clean and rinse the tubes for future use.
9. Store the S. epidermidis culture in the refrigerator for use in future experiments.
10. Disinfect the work area.

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O

Experiment

osmosis: TASK 5
Cynthia Alonzo, M.S.
Version 42-0250-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will learn about osmosis by studying the effect on a shelled egg submersed in corn syrup.
They will study the effect of various sodium chloride concentrations on the growth of Staphylococcus epidermidis and Saccharomyces cerevisiae. Students will learn about permeability of membranes and define solution, solvent, hydrophilic, hydrophobic, hypotonic, and isotonic solutions.

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Osmosis

Objectives
●●

Learn the basic principles of osmosis

●●

Learn and test for the effects osmotic changes have on microbes

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Experiment

Osmosis

materials
Materials
Student provides

LabPaq provides

Qty
Item Description
1 10%-beach solution
1 Paper towels
1
Black marker
1 Stock culture: S. epidermidis

1
1
1
1
2
4

Gloves, Disposable
Ruler, Metric
Cup, Plastic, 9 oz Tall
Test-tube-rack-6x21-mm
Candles (flame source)
Pipet, Long Thin Stem

2
2
2
1
1
1

Broth, Nutrient with 1% NaCl - 10 mL in Glass Tube
Broth, Nutrient with 7% NaCl - 10 mL in Glass Tube
Broth, Nutrient with 15% NaCl - 10 mL in Glass Tube
Baker's Yeast Packet - Saccharomyces cerevisiae
Inoculation Loop, Plastic
Mask, Face with Earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Osmosis

discussion and review
Diffusion is the process by which molecules spread from areas of high concentration to areas of low concentration. Osmosis is a specific type of diffusion; it is the diffusion of water across a membrane. During osmosis, water molecules move across a membrane from an area of higher water concentration (lower solute concentration) to lower water concentration (higher solute concentration). No energy is required. To understand osmosis, we must understand what is meant by a solution. A solution consists of a solute dissolved in a solvent. In terms of osmosis, solute refers to all the molecules or ions dissolved in the water (the solvent). When a solute such as sugar dissolves in water, it forms weak hydrogen bonds with water molecules. While free, unbound water molecules are small enough to pass through membrane pores, water molecules bound to solutes are not. Therefore, the higher the solute concentration, the lower the concentration of free water molecules capable of passing through the membrane.

Figure 1: Osmosis
This process takes on special significance when considered in living systems. Cellular membranes are selectively or semi-permeable, meaning they allow some molecules to pass through while preventing the passage of others. For example, water and oxygen can move freely across the cell's membrane while larger molecules and ions cannot. Diffusion and osmosis are important mechanisms used by the cell to control the movement of molecules in and out of the cell.
It is the structure of the membrane that allows selective permeability to occur. Membranes are composed primarily of phospholipid molecules. Phospholipids have a hydrophilic (water loving) head and a hydrophobic (water fearing) tail. When in an environment that contains water, the molecules group together with the hydrophilic heads outward, toward the water. The grouping forms a double layer of phospholipids in which the hydrophobic tails are protected from the water in the center of the two layers of hydrophilic heads. This configuration of phospholipids is called a lipid bilayer and is the foundation of cellular membranes. Proteins and other molecules can be included in the bilayer and perform a variety of roles based on the type of cellular membrane.

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Osmosis

Figure 2: Lipid Bilayer
A cell can find itself in one of three environments: isotonic, hypertonic, or hypotonic. The prefixes iso-, hyper-, and hypo- refer to the solute concentration.
●●

Isotonic: An environment where both the water and solute concentration are the same inside and outside of the cell. Water goes into and out of the cell at an equal rate.

Figure 3: Isotonic Environment

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●●

Osmosis

Hypertonic: If a hypertonic environment, the water concentration is greater inside the cell, while the solute concentration is higher outside the cell. Water goes out of the cell.

Figure 4: Hypertonic Environment
●●

Hypotonic: In a hypotonic environment, the water concentration is greater outside the cell, and the solute concentration is higher inside. Water goes into the cell.

Figure 5: Hypotonic Environment
Osmotic pressure is the force on a semi-permeable membrane caused by a difference in the amounts of solutes between solutions separated by that membrane. This force can have a significant effect on cells.

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When a cell is in a hypertonic environment – one that has more solute molecules than the cell – water will leave the area of higher water concentration (the cell) and flow into the environment.
As a result the cell looses water and shrinks or shrivels.

Figure 6: Cell in Hypertonic Environment
If the outside environment is hypotonic to the cell and has less solute molecules than the cell, water will flow into the cell from the environment. This can cause the cell to swell and burst.

Figure 7: Cell in Hypotonic Environment
Cells that are in an isotonic environment where the levels of solute are the same both inside and outside of the cell have an equal flow of water in and out of the cell. The osmotic pressure on both sides of the cell membrane is balanced.

Figure 8: Cell in Isotonic Environment

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Bacteria and other microbes are open to the environment and are faced with variations in osmotic pressure on a continual basis. How they react to or protect themselves from these changes determines their ability to survive in a given environment. Most bacteria require an isotonic environment or slightly hypotonic environment for optimum growth. However, there are microbes that have adapted to live in a variety of osmotic conditions. The most common group is the halophiles, or salt loving bacteria. These bacteria can survive and even thrive in environments in which the salt concentrations are hypertonic. Another common group, the osmophiles, can live in environments that are higher in sugar.
Another way to look at a microorganism’s relationship with the water in its environment is by how available water is to the organism. The presence of water in a solution or substance is referred to as its water activity (aw). Water activity is affected by the amount of solutes present. The higher the level of solutes, the lower the water activity of a solution. The aw of pure water is 1.00. The addition of solutes such as salt or sugar molecules lowers the aw. The following list gives the aw of some common substances:
●●

Pure Water: 1.00

●●

Human Blood: 0.99

●●

Seawater: 0.98

●●

Maple Syrup: 0.90

●●

Water from the Great Salt Lake: 0.75

Microorganisms are very sensitive to changes in water activity. Most bacteria cannot grow in environments in which the aw is below 0.91. This factor has been utilized extensively in the preservation of food. The aw of foods are most generally lowered by dehydration. Food can be dehydrated by either the evaporation of water (drying the food) or by the addition of a solute, such as salt or sugar, which creates a hypertonic environment that draws water from the food.
The list in Table 1: aw for Common Foods, in the Lab Report Assistant, shows the aw of some common preserved foods. aw 0.95
0.91
0.87
0.80
0.75
0.65
0.60

Table 1: aw for Common Foods

Food Examples
Highly perishable foods such as milk, cooked sausages, breads
Cheeses such as cheddar or provolone, cured meat
Fermented sausage, sponge cakes, dry cheeses, margarine
Most fruit juice concentrates, condensed milk, syrup, flour
Jam, marmalade, glace fruits, marzipan, marshmallows
Rolled oats, jelly, molasses, nuts
Dried fruits, caramel, toffee, honey

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Exercise 1
Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic.
Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter-free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

Procedures
Pre-Experiment Preparation: Place the saved stock culture of S. epidermidis in the incubator for
12–24 hours prior to the start of the experiment. Prepare an S. cerevisiae culture in accordance with the Preparation of Cultures section in the Appendix. ●

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Exercise 2: Effects of Salt Concentration on Bacterial
Growth
PROCEDURES
Before beginning, set up a data table similar to the Data Table 2 in the Lab Report Assistant section. 1. Disinfect the work area.
2. Label the two 1% NaCl broth tubes S. cerevisiae #1 and S. epidermidis #1.
3. Label the two 7% NaCl broth tubes S. cerevisiae #2 and S. epidermidis #2.
4. Label the two 15% NaCl broth tubes S. cerevisiae #3 and S. epidermidis #3.
5. Use aseptic techniques to inoculate each tube with the corresponding organism.

Figure 11: NaCl Broth Tubes
6. Incubate the tubes at 35oC–37oC for 24 to 72 hours.
7. Observe the tubes for the presence or absence of growth , and rate each tube as follows:
●●

Significant growth

●●

Moderate growth

●●

Minimal growth

●●

No growth

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8. Mix 1 tablespoon of bleach into the yeast culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents.
9. Soak the test tubes in a 10%-bleach solution for 1 hour and then discard the contents. Clean and rinse the test tubes for future use.
10. Store the S. epidermidis culture in the refrigerator for use in future experiments.

Figure 12: Effects of NaCl on aw
Use the data in Figure 12 to complete a Data Table similar to Data Table 2: aw Results, in the Lab
Report Assistant.

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Antibiotic Sensitivity: TASK 10
Cynthia Alonzo, M.S.
Version 42-0238-00-01
Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will use the Kirby-Bauer method to test the sensitivity of Staphylococcus epidermidis to the antibiotics gentamicin, novobiacin, and penicillin.
Students will learn about the various types of antibiotics and how they affect bacteria. Students will learn the most common mechanisms through which bacteria become resistant to antimicrobial agents. www.LabPaq.com

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Objectives
●●

Understand the basic principles of antimicrobial therapy

●●

Become familiar with the phenomenon of antibiotic resistance

●●

Become familiar with and employ an antibiotic sensitivity test

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antibiotic sensitivity

materials
Materials
Student provides

LabPaq provides

Qty
1
1
1
1
1
1
1
1
1
1
1
1
1
1

Item Description
Distilled water
10%-bleach Solution
Paper towels
Culture: S. epidermidis
Prepared nutrient agar dish
Gloves packages - 11 pairs
Ruler, Metric
Tweezers, plastic
Pencil, marking
Antibiotic Disk - Gentamicin in Bag 2"x 3"
Antibiotic Disk - Novobiacin in Bag 2"x 3"
Antibiotic Disk - Penicillin in Bag 2"x 3"
Sterile Swabs - 2 per Pack
Mask with Ear loops (11) in Bag 5" x 8"

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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antibiotic sensitivity

discussion and review
Antimicrobial therapy is the use of chemicals to inhibit or kill microorganisms in or on the host.
Drug therapy is based on selective toxicity, which means the agent used must inhibit or kill the microorganism in question without seriously harming the host.
In order to be selectively toxic, a therapeutic agent must interact with some microbial function or microbial structure that is either not present or is substantially different from that of the host. For example, in treating infections caused by prokaryotic bacteria, the agent may inhibit peptidoglycan synthesis or alter bacterial (prokaryotic) ribosomes. Human cells do not contain peptidoglycan and possess eukaryotic ribosomes. Therefore, the drug shows little if any effect on the host (selective toxicity).
Eukaryotic microorganisms, on the other hand, have structures and functions more closely related to those of the host. As a result, the variety of agents selectively effective against eukaryotic microorganisms such as fungi and protozoans is small when compared to the number available against prokaryotes. Also keep in mind that viruses are not cells and, therefore, lack the structures and functions altered by antibiotics, so antibiotics are not effective against viruses.
There are two general classes of antimicrobial agents based on origin:
●●

Antibiotics: Substances produced as metabolic products of one microorganism which inhibit or kill other microorganisms.

●●

Antimicrobial chemicals: Chemicals synthesized in the laboratory which can be used therapeutically on microorganisms.

Today the distinction between the two classes is not as clear, because many antibiotics are extensively modified in the laboratory (semi-synthetic) or even synthesized without the help of microorganisms. Most of the major groups of antibiotics were discovered prior to 1955, and most antibiotic advances since then have come about by modifying the older forms. In fact, only three major groups of microorganisms have yielded useful antibiotics: the actinomycetes (filamentous, branching soil bacteria such as Streptomyces), bacteria of the genus Bacillus, and the saprophytic molds Penicillium and Cephalosporium.
To produce antibiotics, manufacturers inoculate large quantities of medium with carefully selected strains of the appropriate species of antibiotic-producing microorganism. After incubation, the drug is extracted from the medium and purified. Its activity is standardized, and it is put into a form suitable for administration.
Some antimicrobial agents (penicillins, cephalosporins, streptomycin, and neomycin) are cidal in action: they kill microorganisms. Other antimicrobial agents (tetracyclines, gentamicin, and sulfonamides) are static in action: they inhibit microbial growth long enough for the body's own defenses to remove the organisms.
Antimicrobial agents also vary in their spectrum. Drugs that are effective against a variety of both Gram-positive and Gram-negative bacteria are said to be broad spectrum (tetracycline, streptomycin, cephalosporins, ampicillin, and sulfonamides). Those effective against just Gram-

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positive bacteria, just Gram-negative bacteria, or only a few species are termed narrow spectrum
(penicillin G, clindamycin, and gentamicin).
If a choice is available, a narrow spectrum is preferable since it will cause less destruction to the body's normal flora. In fact, indiscriminate use of broad spectrum antibiotics can lead to superinfection by opportunistic microorganisms, such as Candida (yeast infections) and Clostridium difficile (antibiotic-associated ulcerative colitis), when the body's normal flora is destroyed. Other dangers from indiscriminate use of antimicrobial chemotherapeutic agents include drug toxicity, allergic reactions to the drug, and selection for resistant strains of microorganisms.
Following are examples of commonly used antimicrobial agents arranged according to their modes of action:
●●

Antimicrobial agents that inhibit peptidoglycan synthesis: Inhibition of peptidoglycan synthesis in actively-dividing bacteria results in osmotic lysis. These include penicillins, cephalosporins, carbapenems, monobactems, carbacephem, vancomycin, and bacitracin.

●●

Antimicrobial agents that alter the cytoplasmic membrane: Alteration of the cytoplasmic membrane of microorganisms results in leakage of cellular materials. These include polymyxin
B, amphotericin B, nystatin, and imidazoles.

●●

Antimicrobial agents that inhibit protein synthesis: These agents prevent bacteria from synthesizing structural proteins and enzymes. These include rifampins, streptomycin, kanamycin, tetracycline, minocycline, doxycycline, and gentamicin.

●●

Antimicrobial agents that interfere with DNA synthesis: These agents inhibit one or more enzymes in the DNA synthesis pathway. These include norfloxacin, ciprofloxacin, sulfonamides, and metronidazole.

A common problem in antimicrobial therapy is the development of resistant strains of bacteria.
Most bacteria become resistant to antimicrobial agents by one or more of the following mechanisms: ●●

Producing enzymes which detoxify or inactivate the antibiotic such as penicillinase and other beta-lactamases. ●●

Altering the target site in the bacterium to reduce or block binding of the antibiotic, which produces a slightly altered ribosomal subunit that still functions but to which the drug cannot bind. ●●

Preventing transport of the antimicrobial agent into the bacterium, which produces an altered cytoplasmic membrane or outer membrane.

●●

Developing an alternate metabolic pathway to bypass the metabolic step being blocked by the antimicrobial agent and overcome drugs that resemble substrates and tie up bacterial enzymes. ●●

Increasing the production of a certain bacterial enzyme, which overcomes drugs that resemble substrates and ties up bacterial selection of antibiotic resistant pathogens at the site of infection – indirect selection enzymes.

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These changes in the bacterium that enable it to resist the antimicrobial agent occur naturally as a result of mutation or genetic recombination of the DNA in the nucleoid, or as a result of obtaining plasmids from other bacteria. Exposure to the antimicrobial agent then selects for these resistant strains of organism.
The spread of antibiotic resistance in pathogenic bacteria is due to both direct selection and indirect selection. Direct selection refers to the selection of antibiotic-resistant normal floras within an individual any time an antibiotic is given. At a later date, these resistant normal floras may transfer resistance genes to pathogens that enter the body. In addition, these resistant normal flora may be transmitted from person to person through such means as the fecal-oral route or through respiratory secretions. The direct selection process can be significantly accelerated by both the improper use and overuse of antibiotics.
For some microorganisms, susceptibility to antimicrobial agents is predictable. However, for many microorganisms there is no reliable way of predicting which antimicrobial agent will be effective in a given case. This is especially true with the emergence of many antibiotic-resistant strains of bacteria. Consequently, antibiotic susceptibility testing is often essential in order to determine which antimicrobial agent to use against a specific strain of bacterium.
Several tests may be used to tell a physician which antimicrobial agent is most likely to combat a specific pathogen.
●●

Tube dilution test: In this test, a series of culture tubes are prepared, each containing a liquid medium and a different concentration of an antimicrobial agent. The tubes are inoculated with the test organism and incubated. After incubation, the tubes are examined for turbidity
(growth). The lowest concentration of antimicrobial agent capable of preventing growth of the test organism is the Minimum Inhibitory Concentration (MIC).

●●

The Minimum Bactericidal Concentration (MBC) is determined by subculturing tubes showing no turbidity into tubes containing medium but no antimicrobial agent. MBC is the lowest concentration of the antimicrobial agent that results in no growth (turbidity) of the subcultures. These tests, however, are rather time-consuming and expensive to perform.

●●

The Kirby-Bauer test (agar diffusion test): The Kirby-Bauer disc diffusion method is commonly used in clinical labs to determine antimicrobial susceptibility. In this test, the in vitro response of bacteria to a standardized antibiotic-containing disc is correlated with the clinical response of patients given that drug.

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Figure 1: Agar Diffusion Test
In the development of this method, a single high-potency disc of each chosen chemotherapeutic agent was used. Zones of growth inhibition surrounding each type of disc were correlated with the minimum inhibitory concentrations of each antimicrobial agent (as determined by the tube dilution test). The MIC for each agent was then compared to the usually-attained blood level in the patient with adequate dosage. As a result, the categories of Resistant,
Intermediate, and Sensitive were established.

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Exercise 1: Antibiotic Sensitivity
Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic.
Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter-free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

procedures
Pre-Experiment Preparation: Place the stock culture of S. epidermidis in an incubator 12–24 hours prior to the start of the experiment.

Part I: Kirby-Bauer Test
1. Disinfect the work area.
2. Use the extra nutrient agar dish prepared in the Isolation of Individual Colonies experiment.
Using a sterile swab, thoroughly coat the surface of the agar with liquid S. epidermidis. Do not leave any un-swabbed areas on the agar dish.
3. After swabbing the dish, turn it 90o and repeat the swabbing process. It is not necessary to re-moisten the swab.
4. Run the swab around the circumference of the dish. Then soak the swab in the 10%-bleach solution and discard it. Let the dish dry upright for 5 minutes to allow the S. epidermidis culture to absorb completely.
5. Using a marking pencil, divide the outside bottom surface of the dish into three triangular segments similar to Figure 2.

Figure 2: Petri Dish Segments

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antibiotic sensitivity

6. Label the first section novobiocin; the second penicillin, and the third gentamicin.
7. Wash the tweezers with detergent, rinse well, and shake dry. Use the tweezers to transfer the gentamicin antibiotic disk to its corresponding section on the surface of the agar. Transfer the novobiocin and penicillin disks to their appropriate sections on the agar.
8. Lightly touch each disc with the tweezers to ensure each is in good contact with the agar surface. 9. Incubate the agar dish upside down at 35oC–37oC for 24–48 hours.
10. You will not need the S. epidermidis stock culture for future experiments. Mix 1 tablespoon of bleach into the stock culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents.

Part II: Data Interpretation
To interpret the results:
1. Place the metric ruler across the zone of inhibition at the widest diameter and measure from one edge of the zone to the other. Note: Holding the dish up to the light may help.
a. The disc diameter will be part of the measurement.
b. If there is no zone at all, record the measurement as 0, even though the disc itself is approximately 7 mm.

Figure 3: Zone of Inhibition
2. Record the zone diameter in millimeters.
3. Locate the zone on the following Antibiotic Susceptibility Zone: Diameter Interpretation chart to determine if S. epidermidis is sensitive, resistant, or intermediate.
4. Soak the dish in 10%-bleach solution for 1 hour and then discard it.

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Fomite Transmission: TASK 8
Cynthia Alonzo, M.S.
Version 42-0243-00-01
Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will learn modes of pathogen transmission and identify potential fomite transmission sites in the environment. They will collect microbial samples from various sources and then grow the microorganisms on agar plates. Students will study the morphology of the growth and count the different colonies.

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Fomite Transmission

Objectives
●●

Recognize modes of pathogen transmission

●●

Identify and test sites of potential fomite transmission in the environment

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Fomite Transmission

materials
Materials
Student provides

LabPaq provides

Qty
1
1
1
1
1
2
1
8
1

Item Description
Distilled water
10%-bleach solution
Paper towels
Gloves, Disposable
Pencil, marking
Petri dish, 60 mm
Agar, Nutrient - 18 mL in Glass Tube
Sterile Swabs - 2 per Pack
Mask, Face with Earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Fomite Transmission

discussion and review
The world is teeming with microorganisms, many of which are harmless to humans but some of which are not. To cause an infection, pathogenic organisms need to gain access to a susceptible human body. The spread of infection requires three elements:
●●

a source of infecting microorganisms

●●

a means of transmission for the microorganism

●●

a susceptible host

To prevent the spread of infection, it is necessary to eliminate at least one of these elements.

Source of Infecting Microorganisms
Some infections, called endogenous infections, are caused by the microorganisms that are already present on or in the human body. Other infections, called exogenous infections, are caused by microorganisms from the external environment.
Organisms that cause exogenous infections usually have a preferred portal of entry like the gastrointestinal and respiratory tracts. The intact skin and mucous membranes lining the respiratory, gastrointestinal, and genitourinary tract provide a protective barrier against these organisms. If this barrier is damaged or penetrated, the organisms can potentially gain entry to the body.

Susceptible Hosts
Whether or not a particular microorganism infects a person depends on the balance between the power of the organism to cause disease and the power of the body to resist it. A variety of circumstances may increase the risk of infection, including:
●●

compromised immune status

●●

age of host (the very young and very old are at higher risk)

●●

stress

●●

overall health

●●

pre-existing injury

Transmission of Microorganisms
To cause an infection, pathogens have to be transferred from a reservoir or source to a susceptible host. Microorganisms can be transmitted via several routes; for example, vertical transmission occurs when a pathogen is passed from mother to child across the placental barrier. One of the most common routes of transmission occurs from person to person and is known as horizontal transmission. The horizontal spread of organisms occurs by contact transmission, which involves direct or indirect contact with the reservoir or source.

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Fomite Transmission

●●

Direct contact refers to close contact that results in exposure to skin and body secretions.
Organisms can be transmitted from one part of a person’s body, such as the skin or an infected wound, to another part of the body or to another individual.

●●

Droplet transmission, the transmission of infectious agents in droplets from respiratory secretions by coughing, sneezing or talking, is another form of contact transmission. Pathogens that are transmitted in this way are the cold and influenza viruses and the bacteria responsible for tuberculosis.

●●

Indirect contact occurs when organisms from an infected host or other reservoir are transmitted to a susceptible host via an inanimate object or fomite. In homes, hospitals, and public environments, fomites, which can become contaminated and act as sources of infection, include clothing, bedding, door knobs, counters, sinks, faucets, and medical equipment.

Fomites are one of the most common ways that people, children in particular, are exposed to pathogens. Pathogens that can be spread by droplet transmission or direct contact transmission often do so by means of fomites as well. Yet, many people have never heard of fomites, and do not give much thought to the very objects that, when exposed to pathogens, pose a risk of infection to countless individuals (e.g., cutting boards, kitchen sponges, toothbrushes, door handles, faucet handles, shopping carts, etc.) Germs can survive on fomites for minutes, hours, or even days in some cases.
Some diseases commonly spread by fomite transmission include the common cold, cold sores, conjunctivitis, influenza, meningitis, pinworms, diarrhea, and strep infections. In health care settings, the risk of pathogen exposure is far worse. There are likely to be a greater number of pathogens (many of which cause serious illnesses) present in the setting as well as antibioticresistant bacteria. Potential fomites and pathogens are just about everywhere!
Understanding fomite transmission can provide the opportunity to disrupt the spread of infection.
Taking the time to disinfect potential fomites can be a powerful tool in controlling the spread of pathogens. Too few people recognize that simple hand washing is perhaps the easiest, cheapest, and most effective way to guard against viral and bacterial infections!

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Fomite Transmission

Exercise 1: Fomite Transmission
Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic.
Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter-free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

Procedure
Pre-Experiment Preparation: Prepare two agar dishes prior to the start of the experiment. Refer to the Preparation of Solid Media section in the Introduction for further instruction.
1. Use a marking pencil to divide the bottoms of two prepared Petri dishes into quadrants. Label the quadrants #1 through #8.

Figure 1: Labeled Petri Dishes
2. Identify eight areas in your home or environment that you feel may be a source of fomite transmission. Then form a hypothesis regarding the type and amount of microbial growth you expect to see.
3. Moisten a sterile swab with distilled water and rub it vigorously on the first potential fomite.
4. Inoculate Quadrant #1 by swabbing the area of the dish with the sterile swab. Be careful not to contaminate the other quadrants.
5. Repeat the steps for each of the remaining seven potential fomite sites.

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Figure 2: Inoculated Dishes
6. Incubate the dishes upside down at room temperature for 24–72 hours.
7. Evaluate the dishes for the number and type of colonies in each quadrant. Prepare a data table similar to Data Table 1 in the Lab Report Assistant to record your results.
8. Soak the Petri dishes in a 10%-bleach solution for 1 hour and then discard them.

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Microbes in the
Environment: TASK 9
Cynthia Alonzo, M.S.
Version 42-0247-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will identify environmental sources of microbes, learn about microbial adaptability and scientific significance, and classify microorganisms.
Students will grow microorganisms obtained from soil, water, and air in order to view the vast variety of microorganisms found within these environments.

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Microbes in the Environment

Objectives
The student will have the opportunity to:
●●

Gain an appreciation for the adaptability and importance of microbes.

●●

Identify environmental sources of microbes.

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Microbes in the Environment

materials
Materials
Student provides

LabPaq provides

Qty
1
1
1
1
3
1
4
2
2
1

Item Description
Distilled water
10%-bleach solution
Paper towels
Samples: Soil and water
Cup, Plastic, 9 oz Tall
Gloves, Disposable
Petri dish, 60 mm
Pipet, Long Thin Stem
Agar, Nutrient - 18 mL in Glass Tube
Mask, Face with earloops

Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq.

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Microbes in the Environment

discussion and review
The many and varied metabolic activities of microbes assure they participate in chemical reactions in almost every environment on earth. They require an energy producing system to sustain life and nutrients, including liquid water, in order to grow and reproduce. Since microbes have been present on Earth longer than other organisms, they have evolved the ability to thrive in almost any environment that meets these minimal criteria.
Microorganisms are classified as either heterotrophs which derive energy from preexisting organic matter; or autotrophs which derive energy from one of two sources, light (photosynthesis) or the oxidation of reduced molecules. Oxidizable molecules may be organic or a variety of inorganic molecules such as sulfur, iron, hydrogen, carbon monoxide, ammonia, or even a combination of organic/inorganic molecules. Autotrophs and heterotrophs can be further divided into the following four subcategories:
●●

Photoautotrophs: Use light as an energy source and CO2 as a carbon source

●●

Photoheterotrophs: Use light as an energy source and reduced organic compounds as a carbon source

●●

Chemoautotrophs: Use inorganic chemicals as an energy source and CO2 as a principal carbon source ●●

Chemoheterotrophs: Use organic compounds as an energy source as well as a principal carbon source

Microorganisms can reproduce by doubling in approximately 20 minutes or by dividing only once in 100 years. In most natural environments, such as soil or lakes, the average generation time is approximately one day. Microbes are estimated to comprise one-third or more of Earth's biomass.
On average, bacteria are found in concentrations of up to 106 cells/mL of surface water, and up to
109 cells/mL of soil or sediment.
Microbes have a significant impact on the natural world including:
●●

Production of oxygen: Almost all of the production of oxygen by bacteria on Earth today occurs in the oceans by the cyanobacteria (blue-green algae).

●●

Soil fertility maintenance: Decomposition releases mineral nutrients such as potassium and nitrogen from dead organic matter, making it available for primary producers to use.
Primary production of organic material would not be possible without the recycling of mineral nutrients. Decomposition also produces CO2 and CH4 that is released into the atmosphere.

●●

Nitrogen fixation: Bacteria are the only organisms capable of removing N2 gas from the atmosphere and fixing it into a useable nitrogen form (NH3).

●●

Base of ocean food chain: Plankton are the most numerous organisms in Earth’s oceans and include the protists, algae, and phytoplankton that comprise the basis of the marine food chain. www.LabPaq.com

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Microbes in the Environment

Exercise 1: Microbes in the Environment
Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic.
Therefore be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter-free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

procedure
Pre-Experiment Preparation: Prepare four Petri dishes with agar prior to the start of the experiment. Refer to the Preparation of Solid Media section in the Introduction for further instruction. Part I: Microbes in the Air
Before beginning, set up a data table similar to the Data Table 1: Environmental Colony Formation in the Lab Report Assistant section.
1. Label the bottom of three prepared dishes: air, water, and soil. Set aside the dishes labeled soil and water for Parts II and III. You will have an extra dish if you wish to test an additional environmental source!

Figure 1: Labeled Petri Dishes
2. Choose a location in your home and leave the agar dish labeled air uncovered for 1–2 hours.
3. Close the dish and incubate it upside down at room temperature for 24–72 hours.
4. Observe the dish and count the number and types of colonies. Record the results in Data
Table 1: Environmental Colony Formation, in the Lab Report Assistant section.
5. Soak the dish in a 10%-bleach solution for 1 hour and then discard it.

Part II: Microbes in the Water
1. Choose an environmental site to collect a water sample, such as a pond, puddle, birdbath, or

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Microbes in the Environment

stream.
2. Use a sample cup to collect the water sample. Stir the water to mix any sediment or edge bacteria before sampling.
3. With a pipet, inoculate the agar dish labeled water with the water sample. Use only enough water to cover the top surface of the dish (approximately 4 drops).
4. Cover the dish and let it sit for 30 minutes to ensure the water soaks into the agar.
5. Incubate the dish upside down at room temperature for 24–72 hours.
6. Observe the dish and count the number and types of colonies. Record the results in Data
Table 1: Environmental Colony Formation, in the Lab Report Assistant section.
7. Soak the dish in a 10%-bleach solution for 1 hour and then discard it.

Part III: Microbes in the Soil
1. Choose an environmental site to collect a soil sample. Then use a new sample cup to collect a soil sample.
2. Pour distilled water into the cup, so the water sits just above the soil level. Mix the water and soil well.
3. Let the sample sit until the soil settles to the bottom of the cup.
4. With a pipet, collect a sample of the water layer on top of the soil and inoculate the agar dish labeled soil. Use only enough water to cover the top surface of the dish (approximately
4 drops).
5. Cover the dish and let it sit for 30 minutes to ensure the water soaks into the agar.
6. Incubate the dish upside down at room temperature for 24–72 hours.
7. Observe the dish and count the number and types of colonies. Record the results in Data
Table 1: Environmental Colony Formation, in the Lab Report Assistant section.
8. Soak the dish in a 10%-bleach solution for 1 hour and then discard it.

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Experiment

Fungi: TASK 7
Cynthia Alonzo, M.S.
Version 42-0244-00-01

Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise.
Experiment Summary:
Students will learn about the classifications of fungi as well as identify primary fungal structures and morphologies. They will grow fungi on various foods and then identify basic macroscopic and microscopic features. www.LabPaq.com

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Experiment

Fungi

Objectives
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Learn the identifying features of common groups of mold and yeast

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Become familiar with different classifications of fungi

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Identify primary fungal structures and morphologies

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EXPERIMENT 13: Fungi

Objectives: To learn identifying features of common groups of mold and yeast,
To become familiar with different reproductive strategies in fungi, and
To identify primary fungal structures and morphologies.

Discussion and Review: Fungi are eukaryotic organisms and include the yeasts, molds, and fleshy fungi. Yeasts are microscopic, unicellular fungi; molds are multinucleated, filamentous fungi (such as mildews, rusts, and common household molds); the fleshy fungi include mushrooms and puffballs. All fungi are chemoheterotrophs, requiring organic compounds for both an energy and carbon source, which obtain nutrients by absorbing them from their environment. Most live off of decaying organic material and are termed saprophytes. Some are parasitic, getting their nutrients from living plants or animals.

The study of fungi is termed mycology and the diseases caused by fungi are called mycotic infections or mycoses.

In general, fungi are beneficial to humans. They are involved in the decay of dead plants and animals (resulting in the recycling of nutrients in nature), the manufacturing of various industrial and food products, the production of many common antibiotics, and may be eaten themselves for food. Some fungi, however, damage wood and fabrics, spoil foods, and cause a variety of plant and animal diseases, including human infections. YEASTS: Yeasts are unicellular, oval or spherical fungi which increase in number asexually by a process termed budding. A bud forms on the outer surface of a parent

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cell, the nucleus divides with one nucleus entering the forming bud, and cell wall material is laid down between the parent cell and the bud. Usually the bud breaks away to become a new daughter cell but sometimes, as in the case of the yeast Candida, the buds remain attached forming fragile branching filaments called pseudohyphae.
Yeasts are facultative anaerobes and can therefore obtain energy by both aerobic respiration and anaerobic fermentation. Most yeasts are nonpathogenic and some are of great value in industrial fermentations. For example, Saccharomyces species are used for both baking and brewing.
The yeast Candida is normal flora of the gastrointestinal tract and is also frequently found on the skin and on the mucous membranes of the mouth and vagina. Candida is normally held in check in the body by normal immune defenses and normal flora bacteria. Therefore, they may become opportunistic pathogens and overgrow an area if the host becomes immunosuppressed or is given broad spectrum antibiotics that destroy the normal bacterial flora.
Any infection caused by the yeast Candida is termed candidiasis. The most common forms of candidiases are oral mucocutaneous candidiasis (thrush), vaginitis, onychomycosis (infection of the nails), and dermatitis (diaper rash and other infections of moist skin). However, antibiotic therapy, cytotoxic and immunosuppressive drugs, and immunosuppressive diseases such as diabetes, leukemias, and AIDS can enable Candida to cause severe opportunistic systemic infections involving the skin, lungs, heart, and other organs. In fact, Candida now accounts for 10% of the cases of septicemia. Candidiasis of the esophagus, trachea, bronchi, or lungs, in conjunction with a positive HIV antibody test, is one of the indicator diseases for AIDS.
MOLDS: Molds are multinucleated, filamentous fungi composed of hyphae. A hypha is a branching, tubular structure from 2 - 10 µm in diameter and is usually divided into celllike units by crosswalls called septa. The total mass of hyphae is termed a mycelium.
The portion of the mycelium that anchors the mold and absorbs nutrients is called the vegetative mycelium; the portion that produces asexual reproductive spores is termed the aerial mycelium.

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Molds possess a rigid polysaccharide cell wall composed mostly of chitin and, like all fungi, are eukaryotic. Molds reproduce primarily by means of asexual reproductive spores such as conidiospores, sporangiospores, and arthrospores. These spores are disseminated by air, water, animals, or objects. Upon landing on a suitable environment the spores germinate and produce new hyphae. Molds may also reproduce by means of sexual spores such as ascospores and zygospores, but this is not common.

The form and manner in which the spores are produced, along with the appearance of the hyphae and mycelium, provide the main criteria for identifying and classifying molds.
The two most common types of asexual reproductive spores produced by molds are conidiospores and sporangiospores.
Conidiospores are borne externally in chains on an aerial hypha called a Conidiophore.

Sporangiospores are produced within a sac or sporangium on an aerial hypha called a sporangiophore. ←← Penicillium and Aspergillus are examples of molds that produce conidiospores. Penicillium is one of the most common household molds and is a frequent food contaminant. The conidiospores of Penicillium usually appear grey, green or blue and are produced in chains on finger-like projections called sterigmata. 105

Aspergillus is another common contaminant.
Although usually nonpathogenic, it may become opportunistic in the respiratory tract of a compromised host and, in certain foods, can produce mycotoxins.



The conidiospores of Aspergillus appear brown to black and are produced in chains on the surface of a ball-like structure called a vesicle. ← ←

← ← Rhizopus is an example of a mold that produces sporangiospores. Although usually nonpathogenic, it sometimes causes opportunistic wound and respiratory infections in the compromised host.

The sporangiospores of Rhizopus appear brown or blackand are found within sacs called sporangia. Anchoring structures called rhizoids are also produced on the vegetative hyphae.




Rhizopus can also reproduce sexually. During sexual reproduction hyphal tips of a (+) and (-) mating type join together and their nuclei fuse to form a sexual spore called a zygospore. This gives rise to a new sporangium producing sporangiospores having
DNA that is a recombination of the two parent strain's DNA.

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Experiment

Fungi

Exercise 1: Growing Fungi
Warning: Because this experiment involves the culturing of microorganisms from a human or environmental source, it is possible that unknown microbes have been incorporated into the sample. Treat any culture that may contain an unknown organism as potentially pathogenic.
Therefore, be certain to wear gloves and a mask when handling cultures to protect yourself from unintended exposure. When you have completed the experiment, dispose of the used mask and gloves. Handle your liquid cultures carefully and maintain an organized, clutter free work space to prevent spills. Additionally, use and store your cultures and materials out of the reach of children, other individuals, and pets.

procedure
Part I: Growing Fungal Cultures

Please note that step 1 was intentionally omitted as it was not needed for completion of this experiment. 1. Before beginning, set up a data table similar to the Data Table 1: Microbial Growth Observations in the Lab Report Assistant section.
2. Gather six food items to use as substrate. Bread without preservatives and fruit are good growth media for fungus.
3. Place each substrate into a plastic baggie and add a small amount of water. The substrate should be moist but not wet.
4. Close the baggies, but do not seal them airtight. If using zip baggies, poke a few air holes into the bag or zip the bag only partially shut.
5. Use a marker to label each baggie with the date and name of the substrate.

Figure 6: Examples of substrates in baggies
6. Choose a location in your home that is dark and warm. Leave the baggies in this location to incubate. www.LabPaq.com

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Fungi

7. Observe the substrates after 72 hours. Record the observations in Data Table 1.
8. Continue observing the substrates at 24-hour intervals for a total of 7 days to monitor microbial growth. Growth should be visible in 3–7 days. Record the observations in Data Table 1.
9. At the end of the incubation period, note what has grown on the substrates and how many different colonies you identify.

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Appendix

Preparation of Cultures
Culture tubes should remain lidded while incubating. Do not open them once inoculated unless under aseptic conditions and to perform a necessary experimental step.
1. Saccharomyces cerevisiae: Add 1/2 teaspoon dry Saccharomyces cerevisiae (active dry yeast envelope) to 1/8 cup warm water (you can use a sample cup or any household cup) and gently swirl to mix. Set the culture aside to activate for at least 10 minutes. Stir to mix prior to using.
2. Escherichia coli:
a. Remove the tube labeled: Broth, Nutrient - 5 mL in Glass tube, from culture media bag #2 from the refrigerator and allow it to come to room temperature.
b. Moisten a paper towel with a small amount of alcohol and wipe the work area down.
c. Once the nutrient broth media is at room temperature:
i. Remove the numbered E. coli culture tube from the cultures bag and remove its cap.
Set the cap upside down to avoid contamination. ii. Uncap the nutrient broth; set its cap upside down to avoid contaminating it while the broth is open. iii. Use sterile techniques and draw 0.25 mL of the nutrient broth into a sterile pipet.
NOTE: To sterilize the pipet draw a small amount of 70% alcohol into the bulb and then expel it into a sink. Remove any excess alcohol by forcefully swinging the pipet in a downward arch several times to ensure that the pipet is dry before drawing up the nutrient broth. Add the broth to the vial containing the lyophilized E. coli pellet. Recap the
E. coli vial and shake to mix until the pellet has dissolved in the broth. Note that the vial should be about one-half full to allow for shaking and mixing the pellet. iv. Once the pellet has dissolved, use the same sterile pipet to draw up the E. coli solution and expel it into the original tube of nutrient broth. Recap the broth. NOTE: If the pipet has become contaminated, simply draw a small amount of 70% alcohol into the bulb, and then expel it into a sink. Remove any excess alcohol by forcefully swinging the pipet in a downward arch several times to ensure that the pipet is dry before drawing up the E. coli solution.
d. Recap the nutrient broth and incubate the now E. coli inoculated tube of nutrient broth at 37°C. The culture should show active growth between 24 to 48 hours; it can be left as a liquid culture or plated out. Most freeze dried cultures will grow within a few days however some may exhibit a prolonged lag period and should be given twice the normal incubation period before discarding as non-viable.

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3. Lactobacillus acidophilis: Remove a tube of MRS broth from the refrigerator and allow it to come to room temperature. Aseptically transfer a portion of a capsule of L. acidopholis into the tube of media. To do so, sterilize your work area with alcohol and allow to dry. Carefully open the capsule and divide the contents between the two capsule halves. Set one half aside on the sterile work area and get the test tube of MRS broth. Open the tube and flame the top. Allow the tube to cool for a few seconds before transferring the contents of half of the capsule to the test tube. Carefully swirl the tube to remove any powder from the sides, then flame the top and close the tube. Close the capsule and set aside in case you need to start a new culture. Allow the tube to set, swirling periodically, as the powder dissolves. There will be a significant amount of sediment in the bottom of the tube. Mark the level of the sediment with a marker pencil or pen. Incubate the inoculated tube at 37°C. The culture should show active growth between 24 to 48 hours. Refer to Experiment 3 for a description of indicators of growth. L. acidopholis often sediments as it grows. An increase (above the sediment line you marked on the tube) in the sediment is an indication of growth. Swirl the tube to mix the organisms back into the broth prior to use.
4. Staphylococcus epidermidis: You can culture S. epidermidis as a liquid or solid culture.
Because you are inoculating from an environmental source (your skin) your sample may contain bacteria other than S. epidermidis. Thus, broth cultures derived directly from sampling may not be pure cultures of S. epidermidis. With the exception of Experiments 3 and 4 (#3 establishes a broth culture and #4 uses it to establish a pure culture), use the dish culture method to ensure you are using a pure sample for your experiment.
5. Broth cultures of S. epidermidis: Without contaminating the cotton tip, cut the length of swab such that it will fit entirely into a capped test tube. Dampen the cotton tip sterile swab with distilled water and rub it vigorously on your skin. Do not try to obtain a bacterial culture soon after washing your skin. Additionally, choose an area that is not as likely to have been scrubbed recently (the inside of the elbow or back of the knee is generally a good site). Do not obtain a sample from any bodily orifice (mouth, nose, etc.) as you are not likely to culture the desired microbe (Staphyloccocus epidermidis). Using aseptic technique, place the swab into a tube of nutrient media, label the tube accordingly. Incubate the inoculated tube at
37°C. The culture should show active growth between 24 to 48 hours. Refer to Experiment 3 for a description of indicators of growth.
6. Dish cultures of S. epidermidis: Use a sterile swab to obtain a sample of S. epidermidis from your skin described in the generation of a broth culture. Rub the swab lightly on the surface of one dish of nutrient agar to inoculate it with S. epidermidis. As the swab may not contain a high number of bacteria, be sure to rub all sides of the swab on the dish to transfer as many individual bacterium as possible. Incubate the dish at 37°C for 24 to 48 hours. The S. epidermidis culture was not a pure culture (derived from a single organism) and will most likely contain colonies from several different organisms. You will need to identify and select a colony. Staphylococci produce round, raised, opaque colonies, 1 – 2 mm in diameter. S. epidermidis colonies are white in color. Below is a picture of S. epidermidis grown on blood agar.

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As the sample is of human origin, it potentially contains bacteria that can act as opportunistic pathogens. Do not select or use any colony that does not appear to be S. epidermidis. If your dish contains colonies other than S. epidermidis, soak it in a 10%-bleach solution and discard.
Do not attempt to save the dish for use in future experiments!

You can either use the S. epidermidis colonies directly or amplify growth in a broth culture. If you choose to amplify into nutrient broth, 24 hours beginning the experiment, choose a S. epidermidis colony from the incubated dish and aseptically transfer the colony using an inoculation loop into a tube of nutrient media. Be sure to mix the broth gently to disburse the clumped bacteria into the broth. Incubate the tube at 37°C for an additional 24 hours.

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Preparation of Disinfecting Solution
When working with live organisms, always disinfect your work area and tools prior to use. Soak experiment tools in disinfectant for 30 minutes, and then rinse the tools with distilled water to remove any chemical residue. Both alcohol and bleach are good choices for disinfectants.
However, due to the dilution factor when disinfectant solution is added to broth cultures, use undiluted bleach when disposing of cultures.
When mixed with water, alcohol is an effective disinfectant. The water prevents organism cells from dehydrating and allows the alcohol component to enter the cell and denature the cellular proteins. 70% alcohol mixtures are capable of killing most bacteria within 5 minutes of exposure.
The primary disadvantages of using 70% alcohol as a disinfectant are that it is ineffective against spores and has limited effectiveness against many viruses. Alcohol is also flammable and should not be used near a flame source. Rubbing alcohol, which is a 70% isopropyl alcohol solution, is readily available at most drug stores and is safe for contact with the skin.
Bleach is also a strong and effective disinfectant. Its active ingredient, sodium hypochlorite, denatures protein in micro-organisms and is effective in killing bacteria, fungus, and viruses.
Household bleach works quickly and is widely available at a low cost. Exercise caution when using bleach as bleach irritates mucous membranes, the skin, and the airway. Bleach also decomposes under heat or light and reacts readily with other chemicals. Improper use of bleach can reduce its effectiveness in disinfection and can be harmful to your health. Bleach solutions begin to lose effectiveness after 2 hours, so you will need to make a fresh solution for each experiment.

Diluted Bleach Solution Preparation
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A 10%-bleach solution is one part bleach to every nine parts water. For a spray bottle that holds 100 mL, add 10 mL liquid bleach to 90 mL of water.

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Keep windows open when using bleach to ensure good ventilation.

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Take care not to splash or inhale fumes when using bleach. The fumes irritate mucous membranes, the skin, and the airways. Wear gloves and an apron to protect your skin and clothes when preparing and using bleach solutions.

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Use cold water for dilution. Hot water can release some of the chlorine in the bleach as a gas, and the fumes can irritate your respiratory system.

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Spray your work surface thoroughly with the bleach solution and wipe it down with paper towels before and after every experiment.

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Precautions
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Avoid using bleach on metals, wool, nylon, silk, dyed fabric, and painted surfaces.

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Avoid touching the eyes. If bleach gets into the eyes, immediately rinse the eyes with water and continue rinsing for at least 15 minutes. Consult a doctor if needed.

●●

Bleach should not be used or mixed with other household detergents. Mixtures reduce the bleach’s effectiveness in disinfection and may cause harmful chemical reactions (e.g., a toxic gas is produced when bleach is mixed with ammonia or acidic detergents). Chemical reactions could result in accidents and injuries. If necessary for disinfection, use detergents first and then rinse thoroughly with water before rinsing with diluted bleach.

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Undiluted bleach liberates a toxic gas when exposed to sunlight, so it should be stored in a cool and shaded place out of reach of children and pets.

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Final Cleanup Instructions
Congratulations on completing your science course’s lab assignments! We hope you had a great science learning experience and that what you’ve learned in this course will serve you well in the future. Studying science at a distance and performing laboratory experiments independently are certainly not easy tasks, so you should be very proud of your accomplishments.
Since LabPaqs often contain potentially dangerous items, it is important that you perform a final cleanup to properly dispose of any leftover chemicals, specimens, and unused materials. Please take a few minutes to protect others from possible harm and yourself from future liability by complying with these final cleanup instructions.
While you may wish to sell your used LabPaq, this is not advisable and would be unfair to a potential purchaser. It is unlikely that a new student trying to utilize a used LabPaq would have adequate quantities or sufficiently fresh chemicals and supplies to properly perform all the experiments and to have an effective learning experience. Further, it is doubtful that adequate safety information would be passed on to a new student in the same way it was presented to you. This is a significant concern and one of the reasons why a new user would not be covered by LabPaq’s insurance. Instead, you would be responsible for any problems experienced by a new user. Chemical Disposal
●●

Due to the minute quantities, low concentrations, and diluted and/or neutralized chemicals used in LabPaqs, it is generally sufficient to blot up any remaining chemicals with paper towels and dispose of them in a trash bin or flush remaining chemicals down a drain with copious amounts of water. Empty dispensing pipets and bottles can be placed in a normal trash bin.

●●

These disposal methods are well within acceptable levels of the waste disposal guidelines defined for the vast majority of state and community solid and wastewater regulations. However, since regulations can vary in some communities, if you have any doubts or concerns, you should check with your area authorities to confirm compliance with local regulations and/or if assistance with disposal is desired.

Specimen and Supply Disposal
●●

To prepare any used dissection specimens for normal garbage disposal, wrap them in news or waste paper and seal them in a plastic bag before placing them in a securely covered trash container that will prevent children and animals from accessing the contents.

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●●

Non chemical supplies can also be discarded with household garbage, but should first be wrapped in news or waste paper. Place such items in a securely covered trash container that will prevent children and animals from accessing the contents.

Lab Equipment
●●

Many students choose to keep the durable science equipment included with their LabPaq as most of these items may have future utility or be used for future science exploration.
However, take care to store any dangerous items, especially dissection knives and breakable glass, out of the reach of children.

●●

Please do not return items to LabPaq as we are unable to resell items or issue any refunds.

Best wishes for a happy and successful future!
The LabPaq Team

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...system the crime rate is not reducing. Contrast in the police reporting and actual crime report is another issue. Proper prosecution is not observed when a crime is committed. Due to differences in data quality a crime rate can not be feasible work to reduce the rate of crime. Difference between micro- and macro theories: Differences between micro and macro level theories exist. To identify a theory whether it is macro or micro level is to check what the theory predicts. Focus of the micro-level theory is on the individual interaction. Individual characteristics can be explained by the interactions of people within an environment. These are described as epidemiology. Epidemiology here is concerned with the overall crime rates. Example of this kind of theory is the relationship between adult children and their parents. Macro-level theory is most extended and focuses on the social problems, social conditions and social processes. For example; how the old people’s status is affected by industrialization. Micro level theories are called role theories while macro level theories include age stratification theory. Macro theory shows the criminal behavior of the crimes across the world. Micro-level, or individual-level theories “link individual characteristics to the Probability that an individual will engage in criminal behaviors. (Bernard & Snipes 1996, p.335). Crime States: Crimes consists of following states. Crime can be measured from the intensity and nature of the......

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...Skip to NavigationSkip to Content TermPaperWarehouse.com - Free Term Papers, Essays and Research DocumentsThe Research Paper Factory JoinSearchBrowseSaved Papers Search Criminology In: Social Issues Criminology Task 1: How would you define criminology? Criminology is a social science; its main aim is to research crime and individuals who commit crime, while also looking at the criminal justice system in the hope that this information can be transformed into policies that will be effective in handling, or even eliminating crime. Although it is a specialty, it's not a single discipline. It combines the efforts of sociologists, psychologists, psychiatry, biology, law and statistics. It produces findings that can support, judges, prosecutors, lawyers, probation officers, and prison officials, giving them a better understanding of crime and criminals, and to develop improved and more appropriate sentences and treatments for criminal behaviour. Criminology centres its attention on the criminal as a person, his or hers behaviour, and what has led him or her to a life of crime. It also looks at society's reaction towards breaking laws. Task 2: Explain the difference between macro and micro theories used by Criminologists. Macro theory and Micro theory are both detailed theories that pay close attention to different aspects of crime and criminal behaviour. The Macro theory of crime and criminal behaviour explains the larger scale of crime across the......

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