...In 1884, Hans Christian Gram developed the Gram staining method when trying to discover the cause of pneumonia. While looking at lung tissues, he discovered that the staining method was adhering better to some bacteria than others. (Allen , Anderson , Nester , & Salm , 2016) This led to the discovery that two different types of bacteria were causing the pneumonia. By discovering that the staining method showed two different types of bacteria, the technique became the Gram Staining method. The technique allowed for the bacteria to either show results for gram positive or gram negative. To determine the different characteristics between Gram Positive and Gram-Negative, we start by using the differential stain that is the gram stain, The gram stain helps differentiate between bacteria’s by identify what color they would become. The Gram positive would stain blue or purple in color where the negative results would be pink to red in color. These colors allow scientist to categorize based off the cell wall of the bacteria. If a bacterium is determined to be a gram positive the cell wall will have a thicker layer of peptidoglycan outside the plasma membrane compared to the negative bacteria will have a thinner peptidoglycan layer between the plasma membrane...
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...Task 4 A. Describe the differences between gram-positive and gram-negative cell walls. The difference is the outer casing of the bacteria. Gram positive cell wall consists of a smooth and thick wall. A gram positive bacteria will have a thick layer of peptidoglycan (a sugar-protein shell) that the stain can penetrate and teichoic acids. In this case the lactobacillus and staphylococcus are gram positive. A gram negative cell wall is wavy and much thinner and has a couple of layers of peptidoglycan. This is enclosed by an outer membrane made of phospholipids other substances. The outer membrane prevents the initial stain from penetrating. The Escherichia coli has a gram negative cell walls dye (Levinson, 2014). B. Explain what causes gram-negative bacteria to stain pink. Gram-negative bacteria are bacteria that do not retain the crystal violet dye in the Gram stain procedure. The crystal violet dye is washed by acetone and counter stain stick on its cell wall. The cells appear pink because of the color of the counterstain (safranin) (Levinson, 2014). In this case, it applied to the Escherichia coli bacteria since the thinner peptidoglycan layer in their cell wall did not allow for the stain to retain. C. Explain what causes gram-positive bacteria to stain purple. The iodine binds the crystal violet stain in the cell wall preventing counter stain from sticking onto the wall...
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...collecting a sputum sample from a patient that is suspected or may be infected with a bacterium from one of the following genera: Escherichia, Mycoplasma or Bacillus; Each bacteria listed should be isolated by utilizing each of the various staining techniques. The best staining techniques to use is the Gram stain or the Acid- fast stain due to the fact that they both will provide a lot of information in detail regarding the bacteria being studied. It is very important to be observant of how each bacterium obtained reacts to each stain, and how the results obtained will lead us in a developing diagnosis. The first technique being used is the Gram stain. Gram stain is probably one of the most common used staining procedures used in the field of microbiology. It is one of the differential stains that are used to characterize bacteria in one of the two groups: either gram positive or gram negative bacteria. Bacteria prepared for the Gram stain is a heated fixed smear that is covered with a crystal violet. Because the purple stain impart its color to all cells. After a short period of time, the purple dye is then washed off, and the smear is then covered with iodine, a mordant. When the iodine is then washed off, both the gram-positive and gram-negative bacteria appear dark violet in color or purple. The next, process is the slide is then washed with alcohol or an alcohol- acetone solution. This solution is a decolorizing agent, which removes the purple dye from the cells of some...
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...that can distinguish them. There are also staining processes that can identify each. Let’s get started. The genera Bacillus is a gram positive rod shaped bacterium. When viewed under a microscope Bacillus can be seen as a single organism or in chains. It forms endospores that are resistant to heat, cold, radiation and disinfectants. Bacillus is also aerobic, it needs oxygen to grow. Bacillus can be the cause of infections including abcesses, wound, burn and ear infections, endocardistist, meningitis, ophthalmitis, osteomyelitis, peritonitis and respiratory and urinart tract infections. The genera Escherichia is a gram-negative rod shaped bacterium. It does not form endospores. They are anerobic and can survive with or without oxygen. Escherichia can normally be found in the lower intestines. This can be the cause of gastroenteritis and urinary tract infections. The genera Mycoplasma are also rod shaped, like Bacillus and Escherichia. Mycoplasma lack a cell wall which makes staining difficult. It is also so small it makes it undetectable with a normal light microscope. This can be the cause of lung infections and urogenital infections. Infections caused by Mycoplasma are not easily treated by antibiotics. A way to distinguish these genera is by staining. This is a procedure developed by Hans Christian Joachim Graham, a Danish bacteriologistis. This process became known as Gram’s stain or the Gram stain. Gram staining is the coloring of microorganisms with dye...
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...Name ___________________________________________(5 pts.) Chap 3 study Questions - You will learn and understand more if you complete these questions within 1 -2 days after lecture! Must use textbook or think it through yourself to answer some questions. What is the range of individual cell length in micrometers? 1) What do the following abbreviations stand for? m = mm= nm = 2) How many micrometers in a millimeter? How many millimeters in a meter? How many nanometers in a micrometer? To convert from one unit of measure to another, use an equivalency statement: Starting units (equivalency statement) = ending units 1) write the starting units with number 2) write the ending units 3) For the equivalency statement, put the ending units on top and the starting units at the bottom as shown below. This way the starting units cancel out and you are left with the ending units. Starting units (ending units/starting units) = ending units 4) Ask yourself which is the smaller unit: starting or ending? How many of the smaller units make up the larger unit? The unit that is smaller will be 1000 or 100 etc. The unit that is bigger will be 1. 5) Multiply to determine answer. Example: 6.8 m == ? nm Step 1 &2: 6.8 m (equivalency statement) = ____ nm Step 3: 6.8 m ( nm ) = _____ nm m Step 4: 6.8 m ( 1000 nm ) = _____ nm 1 m Step 5: 6800 nm 3) Convert the following (SHOW YOUR WORK to receive credit...
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...by one. I would start with Bacillus. They are rod shaped bacterium which form endospores. Their movement is propelled by flagella. Bacilli are gram positive, but because if forms endospores, I would do the Endospore staining. Endospores are resistant to stain and relatively uncommon in bacterial cells. You start with a heat fixed smear. Fixing a slide kills the microorganism. A thin film of the microorganism is put on the slid and allowed to dry. Once dried, it is then fixed by either being passed through the flame of a Bunson burner several times or covered with methyl alcohol for 1 minute. Malachite green is applied and the smear is heated to steaming for 5 minutes, helping the stain penetrate the endospore wall. It is then washed with water for 30 seconds. This removes the stain from the cell except for the endospore wall. Safranin is applied as a counterstain to stain portions of the cell other than the endospore. In the smear, the endospores appear green with the rest of the cell red or pink. If there no endospores in the smear, it would eliminate bacillus as a contributing factor. Escherichia, commonly called E-coli, are also rod shaped microorganisms, and are gram negative. A gram stain is a differential stain developed Danish bacteriologist Hans Christian Gram. You start with a heat fixed smear using the procedure stated above. After they are fixed, it is then covered with crystal violet (or basic purple dye). After a short time, the dye is washed off and the...
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...2014 ! ! ! ! Gram Staining Gram staining is the single most important and universally used staining technique in the laboratory. The name comes from it’s inventor Hans Christian Gram who developed the technique in 1882 (published 1884). Gram staining is almost always the first step in identifying a bacteria. The process is a complex and differential staining procedure with a series of staining and decolorization steps. Bacteria are differentiated by their cell wall composition. Gram positive cells have thick cell walls containing peptidoglycan which stain purple with Crystal Violet (or substitute). Gram negative cells with thin layers of peptidoglycan and high lipid content stain pink or red. The procedure is not used in Eukaryotic cells as they do not contain peptidoglycan. There are four basic steps to Gram staining a sample. The initial step is applying a primary stain to the heat fixed sample followed by Grams iodine as a mordant. The sample is rinsed with alcohol or acetone and finally counterstained with Safranin. The results, as mentioned above, yield purple (Gram positive) or pink/red (Gram negative) structures which differentiate the bacteria into classes. The process can also be indeterminate or variable which is beyond the scope of this paper. Gram Staining is a remarkable method of determining bacteria type and beginning to narrow down infectious etiology in medical application. In the proper context and setting, the use of Gram staining can have great implications...
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...Westchester Community College to observe the different types of micro bacteria growing on campus. Several different locations on the second floor of the Science Building were sampled as well as samples that were collected from a living organism. The samples were given a nutrient source and incubated for a period of one week. The samples were then observed for growth; slides were created, stained and then studied to determine morphology of organisms. Materials - Sterile cotton swab - Water - Beaker - Slides - 1 Blood Agar plate and 2 Nutrient Agar plates - A Microscope - Emersion oil - Incubator - Refrigerator - Loop - Slide Holder (Clothes pin) - Bunsen burner - Flint - Gram Staining Agents (Crystal Violet, Iodine, Decolorizer, Safranin) - Wax pencil (for documentation) Procedures Collection of Environmental Samples - Sterile cotton swab was taken and wet with water - A 1” sample was taken of the specific locations listed in the table - The applicator was then placed back into the paper container for transport back to the lab. This was done to preserve the integrity of the specimen - The sample was then smeared onto one half of an agar plate. The two samples from the biological specimen were smeared onto a blood agar plate and the specimens taken from the environment were placed on a nutrient agar plate. - The cotton applicator was then disposed of The agar plates were placed in the incubator where they stayed for one week. After incubation the agar...
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...that may be infected with a bacterium from one of the following genera: Bacillus, Escherichia, or Mycoplasma. In a Microsoft Word document, describe the important anatomical differences among these three genera (i.e., those features that could be used to differentiate among them) and the staining protocols you would use to identify which genera is causing the patient’s infection. When discussing each staining protocol, it is important not only to mention the steps involved, but also how each step and each protocol would identify or eliminate each of these genera as a suspect. You may refer to other scientific resources, but they should be in addition to and not in place of the Module 2 resources. Bacillus: Rod Shaped Bacteria, plural bacilli, Divide only across their short axis, most appear in single rods. Diplocacilli appear in pairs after divison, and streptobacilli occur in chains. Some bacilli look like straws, others are tapered and look like cigars and some are oval and look similar to cocci so they are called coccobacilli. Genera Bacillus contains bacteria such as Bacillus anthracis the bacteria that causes anthrax. Bacillus cells often form long, twisted chains of cells. Gram POSITIVE, can be aerobic or anaerobic. Prokaryotes, Has a cell wall Mycoplasma: Lack a cell wall Unaffected by many common antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. May be parasitic or saprotrophic, can cause pneumonia and...
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...collecting a sputum sample from a patient that is suspected or may be infected with a bacterium from one of the following genera: Escherichia, Mycoplasma or Bacillus; Each bacteria listed should be isolated by utilizing each of the various staining techniques. The best staining techniques to use is the Gram stain or the Acid- fast stain due to the fact that they both will provide a lot of information in detail regarding the bacteria being studied. It is very important to be observant of how each bacterium obtained reacts to each stain, and how the results obtained will lead us in a developing diagnosis. The first technique being used is the Gram stain. Gram stain is probably one of the most common used staining procedures used in the field of microbiology. It is one of the differential stains that are used to characterize bacteria in one of the two groups: either gram positive or gram negative bacteria. Bacteria prepared for the Gram stain is a heated fixed smear that is covered with a crystal violet. Because the purple stain impart its color to all cells. After a short period of time, the purple dye is then washed off, and the smear is then covered with iodine, a mordant. When the iodine is then washed off, both the gram-positive and gram-negative bacteria appear dark violet in color or purple. The next, process is the slide is then washed with alcohol or an alcohol- acetone solution. This solution is a decolorizing agent, which removes the purple dye from the cells of some...
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...Acid-Fast Staining Acid-fast stain is a differential stain. More so, it is a physical property of certain bacteria, particularly a bacteria’s resistance to decolorization by certain acids during staining procedures. Most bacteria are decolorized by the use of acid-alcohol. However, the families of Mycobacteriaceae, Nocardiceae, Gordoniaceae, Dietziaceae, and Tsukamurellacea are acid fast. In 1882 acid fast staining was developed by Paul Ehrlich and was later successfully modified by Ziehl And Neelsen. Ehrlich first discovered that Mycobacterium tuberculosis, which is the main causing agent of tuberculosis, retained the primary stain given even after washing it with an acid-alcohol mixture. Acid fastness in the property of certain bacteria (mycobacterium), their structures (spores) or protozoa forms; resist the decolorization by weak mineral acids following staining by the use of an intense dye. The most commonly known acid fast staining technique is the Ziehl-Neelsen technique. The cell walls of acid-fast organisms contain a wax like lipid called mycolic acid. As a result the cell wall is relatively impermeable to ordinary staining techniques. They can be stained by aniline dyes using drastic measures such as application of heat and phenol. The heat softens the wax in the cell wall, allowing the stain to enter, which is a basic fuchsin. The fuchsin dye is more soluble in phenol than in water or alcohol. Phenol in turn is more soluble in lipids or waxes, thus...
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...Antibiotic Resistance and Gram Staining of Unknown Bacterial Samples Microbiology Lab July 1, 2013 Abstract: In this final laboratory assignment, each student was given an agar plate. Each plate was swiped with a pure culture bacteria sample. Where the sample came from and what kind of bacteria was unknown to the students. We were asked to observe the colony morphology on our plates and to perform the antibiotic sensitivity/resistance test. The plates were put into a 37° incubator oven. The next day the plates were taken out of the oven and we calculated the zone of inhibition. We then performed a gram stain of our bacteria and analyzed it under a microscope. We were then able to assess what kind of bacteria we had and if it was gram positive or negative. Introduction: Simple staining (the use of a single stain) allows a microbiologist to observe the morphology (shape) and arrangement of bacteria. In order to classify bacteria into different groups a differential staining procedure must be done. A differential stain involves the use of two or more stains. Depending on the components of the bacterial cell wall or outer layers, the bacteria will either retain the primary stain or have the primary stain removed in a decolorizing step and then retain the secondary stain. The gram stain is the most common differential stain used in the microbiology laboratory to categorize bacteria. The primary stain is the cationic dye Crystal violet and the secondary...
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...previous the microbiology laboratory class. Identifying the unknown bacterium was determined by separating and differentiating possible bacteria based on specific biochemical characteristics. The differential tests used to identify the unknown cultures were Gram staining, oxidase, indole test, urea test, and casein test. MATERIALS AND METHODS: The unknown bacteria were given out by the lab instructor. Each student chose their own unknown bacteria according to the number. All methods have been practiced to ensure proper procedure identifying bacteria have been applied to this unknown. Procedures were followed as stated in the course laboratory manual provided by the instructor, unless otherwise noted. Each test performed identified was used to determine the specifics and identify the unknown bacterium. All of the following tests were performed on this unknown on February 09, 2008. Some of the tests required a follow-up right after the next lab. The first procedure that needed to be accomplished was to streak the unknown out on a Trypticase Soy Agar plate, using the T streak method described in the lab manual. The unknown culture was inoculated in the Tryptic Soy Broth tube, a TSA plate agar, and a TSA slant. This procedure is needed to be done in order to test...
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...In Microbiology Lab, we learn the scientific method of streaking, staining, microscopy, aseptic culture, and selective and differential tests. These tests will help me identify my Unknown Bacteria. The first procedure I did was the isolation streak of my unknown out on a Trypticase Soy Agar. The purpose of Trypticase Soy Agar is to provide enough nutrients of the microorganism to grow. After I finish my isolation streak, my plate were incubated and grown. Once I got my plate to look at my results and I realize I started my streaking pattern wrong. Because of the way I started my streaking pattern I wasn’t able to see my own results. However, I did my own research of how my plate should have look like if I had did my streaking right. With an educated guess of my unknown species, the results I found for the plate have colonies agar that are tan- pigment, convex, circular, and smooth. The cells are also motile rods. The colonies can be observed under Ultraviolet Light. The second procedure was to perform a Gram stain. The purpose of the Gram stained method is to differentiate between a Gram positive and Gram negative organisms and also learning about the cellular morphology and arrangements. I carefully prepare a slide smear on three slides and then started the Gram stain procedure. Once the procedure was done, all three of my slides had a pink stain which automatically figured that my unknown bacteria was Gram negative. Then I examine the finished slide under a microscope. When...
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...Microbiology lab study guide Exam 1 Know step by step instructions on use of microscope to view a specimen (image remains in focus even when changing objective lens – parfocal) * Bright field- Dark image against bright background. * Dark field- Field surrounding the specimen appears black while object is brightly illuminated. * Phase contrast- Specimens appear as various levels of “darks” against a bright background. * Fluorescence- fluorescence dye is used which emits fluorescence under UV light.. * Total Magnification = Magnification by the objective lens X Magnification by ocular lens. * FOCUS MICROSCOPE PROPERLY by starting with scanning lens and moving up each objective, using scope’s parfocal property Know step by step instructions for ocular micrometer calibration using the stage micrometer * Calibration = Stage Micrometer /Ocular Micrometer * Stage Micrometer: mm convert to um * Ocular Micrometer: 0 – 100 Ocular Units * EX: 0.2mm X 1,000 um = 200 um * 1mm * Placed stage micrometer superimposed by the ocular micrometer. * First left line of the ocular micrometer aligned with one of the marks on the stage micrometer * EX 25 of ocular micrometer on 8 major lines of the stage micrometer are perfectly aligned= 800 µm ÷ 25 ocular units = 32 um/OU * Record more than one measurement * Calibrate each magnification * Know the different parts of the microscope...
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