I. Purpose
The purpose of the study is to identify an unknown microorganism using multiple microbiology lab techniques. Through this process I will gain knowledge on how to perform these techniques as well as the importance of these tests on identifying unknown microorganisms. This is significant as the goal of this course is to familiarize ourselves with the common microbiology tests as well as the microorganisms we encounter in our daily activities.
II. Materials
Media:
TSA
TSB
Thioglycolate
Motility Gel
Agar
Simmon’s citrate agar
Nutrient broth
MR-VP broth
Nitrate broth
Starch Agar plate
Skim milk agar plate
Urea slant
Phenylalanine agar
SIM
Spirit Blue Agar
Dnase Agar
Nutrient Gelatin
Mineral Oil
Carbohydrate
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Broth (Glucose, Lactose, Mannito finish Tryptone Broth Indicators and Reagents: Methyl Red Barrit’s Reagent KOH Hydrogen Peroxide Sulfanilic Acid Dimethyl-Aplha-Apthylamine Zinc Oxide Ferric Chloride Iodine Cultures: Escherichia coli Proteus vulgaris Staphylococcus aureus Klebsiella pneumoniae Bacillus cereus Listeria greyii Pseudomonas aeruginosa Enterobacteria aerogenes Bacillus megatarium Stains: Gram: Crystal Violet, Gram’s Iodine, Safranin, Alcohol, Water Spore: Malachite Green, Safranin, Water Capsule: India Ink, Crystal Violet, Water Acid-Fast: Basic Fuchsin, Acid-Alcohol, Methylene Blue Equipment: Microscope Spectrophotometer Loop Needle Bunsen Burner Durham Tubes Cuvettes Ice Water Bath III. Methods Constants: Sterile technique was used for all procedure. All incubations were kept at 38 degrees Celsius. All cultures will be incubated unless otherwise stated Smear prep technique and heat fixation was used in all stains unless otherwise specified. All inoculations in liquid media are done with a loop. All inoculations are in solid media are done with a needle. Durham tubes are used to measure gas, the tube must contain at least 10% gas for a + reading. All inoculations are done with the organism in question unless otherwise stated. All stains are heat fixed unless otherwise specified Maintaining a viable culture: To ensure that our unknown organism did not become contaminated during the process of tests it was necessary it was necessary to perform a gram stain each day we entered the lab. Also to maintain a viable culture we were required to make a reserve culture each day we entered lab. To make a reserve culture we simply transferred the organism from our “working” TSA slant to our “reserve” TSA slant and maintained it at our organism’s optimal temperature for growth. Morphological Tests: Grams Stain: Several tests we performed to determine the morphology of our microorganism. The first likely most important was the Gram stain. This stain is used to determine if the unknown is Gram positive or Gram negative. In doing this we can eliminate bacteria based off the content of their cell wall and outer membrane. When performing a Gram stain one must first place the organism on the slide using a loop if coming from liquid media or a needle if coming from solid media. The organism is then heat fixed and flooded with violet blue, after this the violet blue is then washed away with water and Grams iodine is added and later washed away with alcohol. Gram-positive bacteria will retain the violet blue stain and appear as purple under the microscope; Gram-negative bacteria will not. The slide is then flooded with BLANK washed away. Gram-negative bacteria will retain this stain and appear pink under the microscope. The details of this procedure are listed in Brown, Microbiological Applications p. 108. Mixed Gram Stain: This test was used to determine to size of our unknown.
To perform this test we first did a Gram stain on our organism to determine if it was gram-positive or gram-negative. After this we performed a mixed Gram stain by incorporating our organism with a known bacteria that stained opposite of unknown. We were given the size of the known bacteria and performed a comparative analysis under the microscope to determine the size of our unknown. In my case the control was a gram-negative bacteria Escherichia coli.
Acid-fast Stain: The Acid-fast stain is used to determine whether are unknown contains a high amount of mycolic acid in its cell wall. Those organisms that are acid fast will retain the basic fuschin stain while those who are not will have this stain washed away by ethanol and subsequently stained blue with methyl blue in the final step of this stain. The details of this procedure are listed in Brown, Microbiological Applications p. 115.
Spore Stain: The spore stain is used to determine if the unknown organism produces endospores as way of protecting itself. This stain is useful for detecting bacteria in the genus Bacillus and Clostridia. The endospores are highly resistance to staining a simple stain is not usual in visualizing them. Heat must be used as a mordant to drive in the malachite green stain. The endospore will then appear green. The details of this procedure are listed in Brown, Microbiological Applications p.
111. Capsule Stain: The capsule stain is utilized to determine if the unknown organism contains an extracelluar layer called a glycocalyx. The glycocalyx plays a part in pathogenicity for certain microorganisms and therefore is an important test. A heat smear cannot be performed on the capsule stain as it may shrink the capsule and as a result cannot be visualized under the microscope. In a capsule stain nigrosin along with the organism is drug across the slide. The slide is then washed with copper sulfate and can be visualized. The details of this procedure are listed in Brown, Microbiological Applications p. 101. Motility Stab: We will utilize this test to determine if our unknown is motile or in otherwise have flagella. To perform this test will inoculate our motility media with a needle stab into the media. This media is special as it contains a terminal electron acceptor called TTC. As the organism grows and respires the TTC will cause the media to turn red this will give a better view of motility. To determine if the organism is motile we just examine the media for spreading of the organism. If the organism is spread out in the media it is motile. If the organism stays along the stab line it is immotile. The details of this procedure are listed in Brown, Microbiological Applications p. 121. Cultural Characteristics: Temperature: To determine the optimal growth temperature for our organism we simply made 3 nutrient agar slants and stored them at three separate temperatures 25, 30 and 37 degrees Celsius. Oxygen requirements: Fluid Thioglycollate medium is used to test the oxygen requirements of our unknown. The details of this procedure are listed in Brown, Microbiological Applications p. 193. Where the organism grows in the media is dependent on how and if they utilize oxygen. If there is growth at the top of the media it is classified as aerobic. If growth is at the bottom of the tube it is anaerobe. If growth is seen throughout tube it is a facultative anaerobe. If growth is seen slightly below the surface media it is considered a microaerophile. The aerobic control was Pseudomonas aeruginosa and the facultative control was Escherichia coli. Colony characteristics: To determine the colony characteristics i.e. size, shape, margins and elevation. We simply inoculate a nutrient agar temperature, incubate overnight and examine the plate the next day. A descriptive chart of characteristics can be found in Brown, Microbiological Applications p. 260. Pattern of Growth in Broth & Slant: These experiments are used to determine how the organism looks on a macroscopic level. It may be useful to know this information in order distinguish between microorganisms when viewing them with the naked eye. To perform these tests we simply inoculate the broth or slant with our microorganism, incubate the media and view the media to determine their characteristics in both media. A list of descriptive characteristics can be found in Physiological characteristics Gelatin Stab: The gelatin stab is performed to determine if our unknown produces protease, enzymes that break down proteins. The details of this procedure are listed in Brown, Microbiological Applications p. 259. To perform this test we inoculate our nutrient agar with a needle stab. We then incubate our inoculated media and look for liquefaction of the media. Liquefaction of the media is a positive sign of the organism producing proteases. O/F Glucose: The O/F Glucose test is used to determine if the unknown microorganism uses fermentation as a source of energy production. The details of this procedure are listed in Brown, Microbiological Applications p. 264. Two O/F Glucose tubes are inoculated with our organism and set to incubate overnight. One tube contains mineral oil above the media to set up an anaerobic environment conducive to fermentation. The other has no oil to set up an aerobic environment conducive to oxidation. If the organism cannot ferment glucose the media in the anaerobic environment will stay green and the media in the aerobic environment will turn yellow. If the organism can perform both oxidation and fermentation both tubes will change to yellow. If the organism does not use sugar as an energy source both tubes will remain green. The oxidative control is Escherichia coli. The fermentative control is Pseudomonas aeruginosa. Carbohydrate Fermentation: Now that we have found out if our microorganism can use fermentation as an energy source. It is important to identify which sugars the organism can ferment as different types of bacteria ferment specific sugars. This test will also indicate if the organism is capable of breaking down proteins. The use of Durham tubes to measure gas production during fermentation is also another way to distinguish between bacterial species. The procedure for performing this test can be found in Brown, Microbiological Applications p. 26, Although we will be inoculating 5 sugars instead of 3. To start we will inoculate five sugar broths that also include Durham tubes and phenol red as an indicator. The five sugars we will inoculate are glucose, lactose, sucrose, mannitol and trehalose. Tubes are incubated; readings are taken at 24 hours and 48 hours. If the organism can ferment the sugar it will produce acid and the broth will turn yellow. If the organism is capable of breaking down peptides it will create an alkaline environment and the broth will turn pink. Our control in this experiment is Escherichia coli. Mixed-Acid fermentation: We will use this test to determine if our unknown is a mixed-acid fermenter. This is important in differentiating some gram-negative intestinal bacteria (Brown, 2012, p. 267). The procedure for performing this test can be found in Brown, Microbiological Applications p.267. We will first inoculate a tube containing MR-VP glucose and let it incubate overnight. After we will add the indicator methyl red to the media, if the media changes to red this is an indication that our organism is a mixed acid fermenter. Our positive control is Escherichia coli while or negative control is Enterobacter aerogenes. Butanediol Fermentation: This test will determine if our organism ferments glucose to butanediol. The procedure for performing this test can be found in Brown, Microbiological Applications p. 268. In this test we will inoculate a tube filled with MR-VP and allow it to incubate overnight. After incubation Barrit’s Reagent is added to the tube. If butanediol is present the reagent will oxidize it to acetonin and media will turn red. The positive control is Escherichi coli while the negative control is Enterobacter aerogenes. Citrate Test: The test is performed to determine if our organism as the ability to utilize citrate as a sole source of carbon. The procedure for performing this test can be found in Brown, Microbiological Applications p. 268. We will inoculate the Simmon’s citrate agar with our organism. This media contains the pH indicator bromophenol blue and ammonia salts. If the organism can utilize citrate it will grow, using the ammonia salts as a source of carbon in turn releasing ammonia into the media. The release of ammonia turns the media more alkaline and causes it to turn blue. The control in this experiment is Enterobacter aerogenes. Catalase Test: This test will be used to determine if our organism grows by aerobic respiration. If it does it will produce an enzyme that breaks down the hydrogen peroxide produced during aerobic respiration. Break down of hydrogen peroxide ensure that no harm will come to the organism from this molecule. Strict anaerobes and aero tolerant organism lack this enzyme and therefore cannot grown in the presence of hydrogen peroxide. This test then tells us that we are dealing with aerobes or anaerobes. The procedure for performing this test can be found in Brown, Microbiological Applications p. 270. The media we are inoculating is a TSA plate. If the unknown can make catalase bubbles will be created upon addition of hydrogen peroxide. Our control is Staphylococcus aureus. Nitrate Reduction: This test will determine if our microorganism has the ability to use nitrate as terminal electron acceptor in a type of anaerobic respiration called nitrate respiration. (Brown, p. 270) This reaction is considered to be a reduction. The procedure for performing this test can be found in Brown, Microbiological Applications p.270. This test will require us to inoculate two nitrate broth cultures with Durham tubes. To assay these cultures for the reduction of nitrate sulfanilic acid will be added along with dimethyl-alpha-naphthylamine. If nitrate reduction occurred the broth will turn. If there is no reaction we must add another to reagent to make sure that some other type of reduced nitrogen was not produced. To do this we will add Zinc metal test will result in the broth turning red while no color change connotes a negative test. The positive control is Escherichia coli. Amylase: This test will determine if our organism produces amylase an enzyme that breaks down starch into several different sugars that the organism will use for energy. The procedure for performing this test can be found in Brown, Microbiological Applications p. 275. To begin we will inoculate an agar plate containing starch with our microorganism. We will then add iodine to the plate; iodine turns plates containing starch black. If our organism produces amylase and utilizes starch there will be a clearing where the organism is. Our control is Bacillus megatarium. Dnase: This test will determine if our organism produces DNase an enzymes that hydrolyzes DNA molecules. The media we will be using for this experiment is a nutrient agar that contains DNA and methyl green. Methyl green a cation binds to the negatively charged DNA backbone ("DNase Test," n.d.). If the organism can produce DNase the bond between methyl green and DNA will break and clearing will appear in the media around the organism. The control is Staphylococcus aureus. Caseniase: This test will determine if our organism produces Caseinase a protease that breaks down the Caseinase protein by hydrolysis. This is the predominant protein in milk and causes it to have its white color. (Brown, 2012, p. 275) The procedure for performing this test can be found in Brown, Microbiological Applications p. 275. Our experiment begins with an inoculation of a skim milk agar plate. If the organism produces Caseinase there will be a clearing in the agar plate where the Casein has been broken down. Our control is Bacillus megatarium. Lipase: In this experiment we determine if our organism has the ability to breakdown fats by producing the enzyme lipase. Lipase breaks down lipids by hydrolysis, removing fatty acid tales from glycerol. The procedure for performing this test can be found in Brown, Microbiological Applications p. 275. This experiment begins with the inoculation of spirit blue agar plate that contains an animal glyceride call tributyrin. When this is broken by lipase the pH of the media is lowered and the media in that area turns dark blue. This is an indication that the unknown organism produces lipase. The control is Bacillus megatarium. Urea hydrolysis: This experiment will determine if the unknown produces urease an enzyme that breaks down urea into carbon dioxide and ammonia. The test is useful in distinguishing between different types of gram-negative bacteria. The procedure for performing this test can be found in Brown, Microbiological Applications p. 275. To start of we will inoculate a medium that contains urea, a buffer and a pH indicator methyl red. When the urea is hydrolyzed by urease ammonia is produced and the media become more alkaline causing the media to turn red. This is due the pH indicator mentioned in the previous sentence. A red color change is conducive to a urease-producing organism being present in that medium. Our control is Proteus vulgaris. Phenylalanine: This test will determine if our unknown contains phenylalanine deaminase an enzyme the removes the amine group from phenylalanine. This creates phenylpyruvic acid and ammonia. Certain gram-negative bacteria have the ability to produce this enzyme. The procedure for performing this test can be found in Brown, Microbiological Applications p. 275. To start off we will inoculate a regular TSA slant. After this we will add ferric chloride to the culture this will turn green in the presence of phenylpyruvic acid. This in turn is an indication that the microorganism produces phenylalanine deaminase. Our control is Proteus vulgaris. SIM medium: SIM medium is a multiple test medium in the identification of our unknown we will use to test for the presence of hydrogen sulfide and well as tryptophanase. Both tests will be described in this section. Some bacteria contain to the ability to degrade the amino acid cysteine. This is accomplished by the enzyme cysteine desulfurase. The procedure for performing this test can be found in Brown, Microbiological Applications p. 280. We start by inoculating the SIM media. This medium contains iron salts, which react with hydrogen sulfide and produce a black precipitate. Therefore if our organism has the ability to produce cysteine desulfurase we will have a black precipitate in our media. The control is Escherichia coli. The second test will be conducting in the SIM media is the indole test. In this experiment we will determine if our unknown bacteria has the ability to degrade the amino acid tryptophan using tryptophanase. Degradation of tryptophan produces indole, ammonia, and pyruvic acid. The procedure for performing this test can be found in Brown, Microbiological Applications p. 276. The addition of Kovac’s reagent to media will turn the top layer of the media red in the presence of indole. Therefore if this red layer is produced we can conclude that our unknown produces tryptophanase. The control is Proteus vulgaris. Decarboxylase: In this experiment we will test to see if our organism has the ability to produce the enzyme decarboxylase. This enzyme has the ability to remove the carboxyl group from an amino acid. To start we will inoculate three nutrient broths containing pH indicators bromocresol purple this indicator turns purple at alkaline pH and cresol red, which turns yellow at acidic pH. The three broths each contain a single amino acid: lysine, arginine, or ornithine. This will tell us specifically what decarboxylase the organism produces, as each decarboxylase is specific to a certain amino acid. The positive indication for decarboxylase is the media changing color to purple. The control is Enterobacteria aerogenes. IV. Conclusion and Discussion Performance of the test listed above revealed that my unknown organism was Staphylococcus epidermidis. Several tests that we performed were essential in correctly identifying the unknown bacteria. The first was the Gram stain with the stain I was able to identify my organism as being gram-positive. The next important identifier was the shape of the organism the gram stain revealed that it was coccus in shape. After performing the catalase test and getting a positive result, I was able to narrow my organism down to three genus’s Micrococcus, Planococcus and Staphylococcus. The next in line was the motility test in which I found my organism was immotile. This narrowed my organism down to either Staphylococcus or Micrococcus. In the next test, which was for oxygen requirement, I found that my organism was a facultative anaerobe. I was then able to determine the genus of my species, which isde Staphylococcus. The final two test that helped me determine the species of my organism were hemolysis test and the fermentation test. My organism performed gamma hemolysis and tested negative for mannitol fermentation. From these results I concluded that my organism was Staphylococcus epidermidis. In route of determining what my unknown organism, I ran into one problem. The first oxygen requirement test revealed that my organism was aerobic. After performing several other tests and narrowing down my organism I found this to be incorrect. The reason for this is that although it is facultative it grows best in aerobic environments (Bukhari, 2004, p. 1). This explains my previous results. Staphylococcus epidermidis resides on the skin and mucosa of humans therefore it is part of our body’s normal flora. It is an opportunistic pathogen, which means it requires a break in the body’s innate immune system. It is one of the leading pathogens in causing nosocomial infections. Staphylococcus epidermidis accounts for a large percentage of Staphylococci on the human body. It is said that up to 24 species of this organism are present on the host at one time (Bukhari, 2004, p. 1). Works Cited Brown, A. E. (2012). Benson's Microbiological Applications. New York, NY: McGraw Hill. Bukhari, M. (2004, September 27). Staphylococcus epidermidis. Retrieved July 28, 2015, from http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20 epidermidis /sepidermidis.html Decarboxylation Test. (n.d.). Retrieved July 28, 2015, from http://www.austincc.edu/microbugz/decarboxylation_test.php DNase Test. (n.d.). Retrieved July 28, 2015, from http://www.austincc.edu/microbugz/dnase_test.php
Upon receiving the unknown Microorganism (M.O.) #16, I prepared a slide by cleaning and drying it. Then, using a bottle of water I placed sterile drop of water on the slide and used an inoculating loop, flame sterilized, I took a small sample of the unknown growth in my agar slant and smeared it onto the slide in a dime sized circle and then heat fixed it for ten minutes. After ten minutes had passed, I collected the ingredients needed to perform a gram stain. I got the primary stain, crystal violet, and flooded my smear for sixty seconds, and then rinsed the color off with water until the water ran clear. I then flooded the smear with the mordant, grams iodine, and let that sit on the slide for sixty seconds as well. I then rinsed the grams iodine off with water and applied alcohol to the smear to decolorize the cells; however I made sure not to over decolorize and only put enough drops on the smear till the purple ran clear. I then rinsed the slide with water and flooded the smear with safranin the counter stain and let it sit for sixty seconds and then rinsed the color off with water. I blo...
The unknown bacterium that was handed out by the professor labeled “E19” was an irregular and raised shaped bacteria with a smooth texture and it had a white creamy color. The slant growth pattern was filiform and there was a turbid growth in the broth. After all the tests were complete and the results were compared the unknown bacterium was defined as Shigella sonnei. The results that narrowed it down the most were the gram stain, the lactose fermentation test, the citrate utilization test and the indole test. The results for each of the tests performed are listed in Table 1.1 below.
In this lab project, the microbiology students were given 2 unknown bacteria in a mixed broth each broth being numbered. The goal of this project is to determine the species of bacteria in the broth. They had to separate and isolate the bacteria from the mixed broth and ran numerous tests to identify the unknown bacteria. The significance of identifying an unknown bacteria is in a clinical setting. Determining the exact bacteria in order to prescribe the right treatment for the patient. This project is significant for a microbiology students because it gives necessary skills to them for future careers relating to clinical and research work.
The name to this lab is identification of unknown species. To know the species from unknown culture, our instructor handover test tube labeled with different number to all students each student with different number. I got test tube labeled #15 culture, which was assigned our instructor. We need to use different media and reagents to work safely and correctly. Before started to working we need to know how to do Gram Stain technique and biochemical testing to determine the name of the unknown species. We used 18 media with 15 reagents, to determine unknown species, which was provided by the Eastfield Dallas community College’s science department. All the methods to determine the “Unknown Lab” were in the book written by Tammy Oliver, Ph.D.
After incubation, we checked the broth for the production gas and a color change from red to yellow which would indicate the presence of an acid. In order to perform a gram stain, we obtained a colony that we had grown on the TSA slant. Then we gathered one test tube containing tryptone broth, one containing citrate slant, and the last two containing MVRP broths. For the MVRP broths, one test tube should be labeled with MR and the next tube should be labeled VP. Next, we inoculated each test tube with the coliforms that we had confirmed as containing negative
The unknown identification laboratory test is made for students to guide themselves into the microbiology laboratory. Students will use the producers, techniques and experiment that they have been used and learned in other pervious classes. During previous laboratory test, many bacteria have been tested and experimented in finding the results of what bacteria was truly growing. There are so many bacteria in our surroundings many of them can be found all around us and in our environment. Some of the bacteria have been tested and are known to help patients when they are sick, they can treat diseases, certain foods, and make antibiotics and many other things. Thought-out this laboratory
In the experiment, five periods are done to accomplish the goal of identification of the unknown microorganism. The first period aims to isolate the genomic DNA from the unknown microorganism using a
In order to preform this experiment you will need; cotton swabs, agar plates, microscope, unused slides, oil immersion, nigrosin, and crystal violet. The first task we must do is use the cotton swabs and swab an item out side of the laboratory, that has the capability of containing either yeast, bacteria, and mold. My lab partners and I chose to swab one of our group members cell phone. Once we swabbed the phone with the cotton swab, we then each had a plate of agar. To start the process of the transfer of the microorganisms you will need to label the agar plates and state where the plate is going to be located after you run your cotton swab over the plate of agar. One group member placed it in the 37C incubator and the others placed it in the cabinet to be stored at room temperature.
In the late 1800s, Hans Christian Gram developed the gram staining procedure. Gram staining is a valuable diagnostic tool used in the clinical and research world. The gram stain is a method used to determine the identification of unknown bacteria. (BIO215, 2017)
The mystery microbe T4 first results were from my gram stain that showed a purple color, rod shape bacteria, that demonstrated the stain was positive. The next test preformed was spore stain, it was positive for spores by taking up on the counterstain that appeared green. The capsule stain was negative the microbes did not have clear halos around them. Next up was the Acid-fast stain that appeared purple/bluish color meaning it was negative.
Most of the results from the science experiments conducted did not occur like they were expected. Most of the results were false negative or false positive due to a human error within preparation of the bacteria slides. For example, the simple stain results for the Corynebacterium xerosis bacteria were inconclusive because they appeared to be very tiny circular dots instead of their usual rod shape. There is a possibility that it could have been the result of overheating the bacteria during the heat fixing process. This also could have been the result of leaving the crystal violet dye on for too long allowing the dye to dry up on the slide. The next example of an experiment’s results gone wrong is apparent in the Gram staining experiment. The
Gram staining is the most essential and all around used staining technique in bacteriology lab. Gram staining was firstly presented by Christian Gram in 1883. This method is utilized to distinguish between gram positive and gram negative bacteria which have consistent differences in their cell wall. Gram positive bacteria stain blue-purple and gram negative bacteria stain pink-red. There are a few objectives as to why we ought to do this experiment; to gain knowledge of the differences between gram positive and gram negative bacteria, develop the lab skill, and be more acquainted with the gram staining procedure. There are also several different materials that we use in this experiment such as absolute methanol, Gram Crystal Violet, Gram Decolorizer,
The gram stain reaction of L. interrogans is gram negative. To stain this bacteria you must use a relatively new staining technique termed immunofluorescence which uses a ultraviolet light to identify its staining. You can also use an older technique that is very time consuming, which is silver staining.
Gram staining can also be called Gram’s Method because of the biologist it was named
The identification of the bacterial unknown was determined through a series of tests using differential media and a gram stain. These tests revealed information about the motility, the metabolism, and the enzymes of the unknown microorganism. The most basic technique for all tests is called the aseptic technique. This technique is “to prevent contamination of the sample” (Leboffe and Pierce, 2010). This is the first technique taught to students in the lab.