...In this experiment we were able to use the technique of Gram staining to differentiate two types of bacteria that were unknown. Gram staining is a method that is used by many scientists in order to identify unknown bacteria’s via the unique physical and chemical characteristics of their cell walls. In a gram test a bacteria can either be gram positive or gram negative. The Gram-positive bacteria have an intricate set of amino sugars which we call peptidoglycan that form a thick cell wall around the plasma membrane. On the other hand, Gram-negative bacteria have an uncomplicated cell wall which thus has less peptidoglycan. The main source of identification within the Gram test is the fact that Gram-positive bacteria appear blue-purple while Gram-negative bacteria appear pink (Gram Stain). Bacteria can come in one of three different shapes; spirilla (spiral-shaped), cocci (round-shaped), and bacilli (rod-shaped). In order to be able to see these shapes a bacteria must be stained or dyed. I hypothesis that we will have one Gram-positive culture and one Gram-negative culture. (Lab Manual) In order to prepare this lab experiment we performed five steps. The first step in preparing this experiment was to heat fix a bacteria culture sample which in return stopped the sample from coming off the slide during the experiment. In order to perform this first step we first turned on a Bunsen burner and set the flames to a blue color which was used to sterilize an inoculating loop. Next, a...
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...to isolate our particular bacteria. Daily Notes Day 1 – Chose unknown bacteria #70, which is a broth culture. It is cloudy and yellow in color, with sediment gathered at the bottom. It has a strong odor, and I question whether I have smelled it before. The first test I’m doing is a Gram stain. Doing 3 slides – all air dried and heat fixed. Also streaking 2 plates today: TSA plate, and 2nd plate will be based on the results of the Gram stain. Either: MSA= isolates S. aureus, inhibits G-, EMB= fecal coliforms, G- growth (selective isolation G- rods), MAC= promotes G- growth. I am worried that I won’t be able to achieve isolation of separate colonies from the broth culture when I streak the plates. Viewed results of Gram stain with microscope under 100x oil immersion. Results of 1st Gram stain were inconclusive, so staining again with another slide. Many more cells on this slide. 2nd Gram stain = G- bacilli (rods), confirmed with Claudia. Decide to streak 2nd plate = MAC since I believe I have a G- bacteria. Day 2 – Achieved isolation on both TSA and MAC agar’s. Definite growth on MAC means I do have a G- bacteria. Will now perform a 2nd Gram stain from TSA plate to confirm Gram status; confirmed that it is G- . After looking at the chart, decided to try to narrow down options by doing an oxidase test. There are 6 oxidase positive bacteria, and 8 oxidase negative bacteria. Oxidase test results = negative. Inoculated a TSA slant today before leaving. Day 3...
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...so as to know how it can be treated, to knowing the correct microorganism to be used for making certain foods or antibiotics. This study was done by applying all the methods that have been learned so far in the microbiology laboratory class for the identification of an unknown bacterium. MEDIA LIST Unknown Bacterium #5 Mannitol Salt Agar Plate DNA Agar Plate Blood Agar Plate RESULTS/ DATA The Unknown #5 had the following morphology after Gram staining and observed under a brightfiled microscope: purple color, spherical shape and clustered like grape. After determining that it was a Gram positive staphylococcus, it was inoculated on a MSA plate, Blood Agar, DNA agar and Catalase test was also performed to help figure out the staphylococcus type. The Table below lists all of the biochemical tests, their purpose and results. TEST PURPOSE REAGENTS OBSERVATIONS RESULTS Gram Stain To determine the Gram reaction of the bacterium Crystal violet, Iodine, Alcohol, Safranin Purple grape cluster arrangement cocci Gram Positive coccus Mannitol Salt Agar To determine the if the bacterium can tolerate high saline levels and grow If the bacterium can ferment mannitol None There was a full growth and the mannitol turned to yellow (acidic) Positive Blood Agar To determine if the bacterium can hemolysis blood None Metallic gray, Creamy – golden color ß-hemolysis Positive DNA Agar To determine if bacterium has DNase and can hydrolyze DNA None Clearing...
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...Microbiology Final exam Study reference 1. Gram Stain * Verify if bacteria are present or not. * Controls – positive (purple) – S.aureus * negative ( red/pink) – E.coli 2. Endospore Staine Positive controller – B. magneterium Green spore- Positive Pink (vegetative ) – Negative 3. Acid fast Positive control – M. smeagmatis Blue – negative Pink /red- positive 4. Motility Positive control - P.vulgaris A. non motile is negative test B. motile is a positive test 5. Carbohydrates fermentation (test for gram -) ( glucose , manitol, lactose) Positive control – ecoli (yellow) Red- no color change – negative test Yellow – color change – positive test 6. Mae Conkey’s Agar Ecoli – positive control Selective for negative gram stain Differential for organism that could ferment lactase Growth pink – positive No growth – negative 7. Gelatin Hydrolysis ( Gelatinase) Positive control – P.aeruginosa Liquid –positive test Solid - Negative test 8. Blood agar Positive control – S. aureus A. Betahemolysis B. alphahemolysis C. gammahemolysis 9. FTM *broth – O2 relationship with 10. MRVP (mix acid fermentation) MR- methyl red (PH lower than 4.4) Positive control – e.coli red color- positive test no red – negative test 11. Voges Proskauer ( acetone) –precursor for those who fermented butane Positive control- E. aerogenosa Red color – positive test No color...
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...most commonly used technique was gram staining. This technique was used to determine if bacteria is present. Then, it had to be determined if the organism was gram positive or gram negative. During the staining procedure there is a primary stain of crystal violet, the mordant gram iodine, a decolorizer acetone alcohol, and a counterstain of safranin. After the steps are completed the gram positive organism remains purple and the gram negative bacteria shows as a reddish-pinkish organism. Gram staining came about by Hans Christian Gram. He was trying to see cocci in the lungs of individuals who lost their lives to pneumonia. Once Gram gathered a collection of materials to complete a stain, he figured out a way to view the bacteria in the lungs. The stain of crystal violet retained to the people who were infected with pneumonia. He realized that his stain worked with envisioning various types of bacteria pertaining to “cocci of supportive arthritis following scarlet fever”. Because Gram finally discovered how to distinguish between a positive bacteria organism from a negative bacteria organism, many doctors are able to figure out ones illness at an early stage, once a doctor does so they might be able to use...
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...Process and Techniques of Finding Unknown J Abstract: As part of a focal study within Microbiology, many scientists have endeavored to identify the many species and types of microbes found in different environments. In an effort to categorize, understand and distinguish one microbe from another, scientists developed tests to show unique characteristics of microbes. This experiment enlists these tests, such as PCR, Simple and Gram staining, anaerobic growth tests, IMViC, Catalase, Oxidase, selective and differential media to identify an unknown microorganism. The Unknown organism studied was labeled “J” and found to be a gram negative, rod shaped bacteria that does not produce endospores. The selective and differential agars produced no growth on the MSA agar plate showing that the bacteria did not favor a salty environment of the Mannitol salts and showed an acidic by product in the selective and differential media of MacConkey’s Agar. The bacteria showed to metabolize sugars but did not produce any gaseous byproducts. After 16s rRNA was processed and run through a PCR, electricphoresis was used to run the RNA out on a gel in order to sequence the RNA which was then compared in a database. Furthermore, the Unknown J did not produce or metabolize starch. The bacteria did not react with the Citrate, KEY oxidase. When compared to other known bacteria tested, unknown J proved to be Escherichia coli. Introduction: Identifying bacterial causes for certain diseases that have...
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...Bacteria can be found in plants and animals, in all habitats, on all surfaces and even in our bodies. To investigate the type bacteria existing in different locations, we took a culture from the inside of a mouth and the surface of a tabletop and let the bacteria grow in agar plates for one week. Separate agar plates were prepared for each of the two locations that also contained a small amount of two different cleaning solutions, Lysol and Green disinfectants, to observe which product worked better at killing bacteria. Both agar plates contained white and yellow bacteria growths. The bacteria in the agar plates was not evenly distributed throughout the surface, but rather was clustered in small areas. This was most like due to uneven distribution of the sample. It could also be attributed to the fact that bacteria grow in colonies. The plates with cultures from site 1 had slightly less bacteria coverage than the coverage from site 2. Although, this was really only the case for one of the cultures from site 2 while the other culture from the same site grew the same amount as the two cultures from site 1. It was observed that no bacteria grew on the portion of the agar nutrient where cleaning product was applied. It should be noted that the very minimal amount of bacteria that grew in the agar nutrient made it difficult to determine if this result was due to the effectiveness of the disinfectants. For this reason as well it is unclear which cleaning product out...
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...Discussion a) Gram Staining Escherichia coli is a Gram-negative rod- shaped bacterium. Initially the bacterium is stained purple by the primary stain, crystal violet. The alcohol which is used as a decolourising agent washes away the outer membrane and allows the crystal violet complex to wash out from the thin peptidoglycan, making the cell colourless. When the counterstain, safranin is applied, they appear pink. Staphylococcus aureus is a Gram-positive bacterium and appear in cocci grape-like clusters. They have a very thick peptidoglycan layer that can retain the purple stain from crystal violet. The decolourising solution has no effect on this bacterium besides a slight drying effect on the wall, The pink counterstain does bind the cells but it is not seen because covered by a darker purple colour primary stain. Bacillus subtilis are Gram-positive rod shaped bacteria that appear as small chains or single cells. They have thick peptidoglycan layer that can retain the purple stain from crystal violet. Saccharomyces cerevisiae are circular which occur singly or in a group. They do not respond to gram staining due to their cell wall that consists of glucan and mannoproteins. They usually bind to the original dye, look blue but they are not Gram-positive. Thus, they are Gram non-reactive. Water isolates found in rod shaped and they are Gram-negative bacteria that stains pink after safranin applied due to the thin peptidoglycan layer. b) Acid fast staining Mycobacterium...
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...over a long period of time, an orderly succession of microorganism communities takes place. In this experiment, me and my partner will work together to observe the environmental conditions, and pattern of succession that takes place in plain whole milk, over a period of eight days. Lactobacillus and streptococcus both survive pasteurization. These two ferment lactose to lactic acid, thus dropping in pH level. This acidic environment causes casein to curdle up. We expect to see yeasts and fungi, because they grow well in acidic environment. METHOD We recorded pH using pH paper of day 0, day 1, day 2, day 4, day 6, and day 8. Than we observed the consistency, odor, and color of each. Than we made slides using the gram stain method for each day. Which is to take a thin smear on the slide, and then heat fix it. Than place crystal violet on it for a minute. Rinse with water. Than cover it with iodine for one minute. Rinse again with water. Than decolorize with alcohol with 10-20 seconds. Rinse again with water. Than cover it with safranin for two minutes. Then rinse with water and gently blot dry. After repeating this method for each day, place under...
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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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...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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...Biochemical tests are often used to determine the identity of bacteria and differentiate between its species. There are numerous types of tests meant to detect key characteristics such as morphology, stain, motility, fermentation pathways used, oxygen requirements, enzymes present, and redox tests used. By conducting these tests with aseptic techniques, one can usually narrow down an unknown bacterium to its family; with the help of dichotomous keys and Bergey’s Manual of Determinative Bacteriology, particular genus and species can be identified. The first tests commonly conducted on a bacterium also give the broadest results. Initial tests employ differential staining and normally include gram, capsule, and endospore stains. All three indicate...
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...In biology, in order to purify biological molecules, a common process called chromatography is often used. This process involves the separation of the large particles in the solution from the smaller particles. This is instrumental in filtering the solution, but without specialty scientific instruments, there is no way to immediately know the concentration of the, now separated, solution. However, there are multiple processes that are utilized to find unknown concentrations of a solution. One of these, known as the Bradford method, allows for an accurate, fairly simple, and timely assessment of these unknown concentrations by using a dye called the Coomassie Brilliant Blue G-250. The samples that contain this dye are run through a spectrophotometer, which reads the absorbance levels of the solution. Generally, the darker the solution is, the higher the concentration will be. This experiment utilizes both chromatography and the Bradford method to determine the concentrations of various protein samples. In this experiment, we worked with proteins over the course of two weeks in order to learn how to purify proteins in a solution by using chromatography and how to calculate the protein concentration in that solution. In the first week, protein samples were collected using chromatography, which is a process that is used to purify biological molecules. Two phases make up the chromatography process: the mobile phase, which consists of the solvent and molecules that are going to be...
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...on very tight. Next, the broth was incubated on a slant, at 37 °C. The slant had surface growth that was echinulate. There was also a smooth texture, with an accumulation of white growth on the bottom of the slant. There was not any apparent branching or dots. After that, a streak plate was inoculated. The streak plate had a round configuration of white clumps that was diffusing outward. The growth was slightly raised, with a convex shape. It was noted that there was also an abundance of growth. After observation, the media testing began. The first test was a gram stain. The gram stain showed purple cocci in chains. As a result, it was concluded that the bacterium was gram-positive, based on the fact that, gram-positive bacteria will stain purple due to their thick peptidoglycan layer (Figure 1). If the result had shown a red/pink bacterium that would prove that the bacterium was gram-negative. Gram-negative bacteria contain a thin peptidoglycan layer, with no teichoic acid, surrounded by an outer membrane. It was also determined that the bacterium had cocci morphology because it was circular. If it had a rod shape, it would have been bacilli morphology. The second test that was performed was a catalase test. The catalase test result was negative. There were not any bubbles, on neither the solid nor the broth. The lack of bubbles meant that H2O2 was not produced during aerobic respiration, and that it was not an oxidizing agent. Additionally, it meant that...
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...Discussion In order to figure out what family an organism belongs to all the way down to the species, a scientist will perform many tests. Each test done will help to eliminate other bacteria. Once a scientist knows the species, the right steps can be taken to help out people if they get contaminated with the bacteria. When I received my unknown bacteria, I started of doing a Gram Stain. This test revealed to me that I had a Gram negative bacilli in a random arrangement. This step is very important to figure out what type of family the bacteria belongs to. Gram negative bacteria have a thin peptidoglycan layer and a waxy outer layer. This is important to know when it comes down to giving antibiotics. Most bacteria are susceptible to antibiotics...
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