A member of the genus Bacillus, B. subtilis is rod-shaped, and has the ability to form a tough, protective endospore, allowing the organism to tolerate extreme environmental conditions (3). AACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTC This is a differential medium. *Note - broth tubes can be made containing sugars other than The tube in the center was inoculated of Enterobacteriaceae. a pH of less than 6.8 and causes the pH indicator, neutral red, If there is no color change in the tube after the addition No issues complicated the gram negative conclusion, and the answer was Proteus vulgaris. We and our partners use data for Personalised ads and content, ad and content measurement, audience insights and product development. In the clinic, the catalase test helps distinguish catalase-positive Staphylococci from catalase-negative Streptococcus, which are both Gram-positive cocci. It tests the ability of an organism The lactose test, using the Eosin-Methylene Blue Agar, and the casein test were done at the same time. the genera Clostridium and Bacillus. <<0044ab36d0a330429f4e345637bb1d13>]>> The MC plate is a selective and differential medium. confidence and trust with respect to the information we collect from you on Prior to the process of sporulation the cells might become motile by producing flagella, take up DNA from the environment, or produce antibiotics. and then immediately reduced to some other, undetectable form This process is also useful in the production of antibiotics on a larger scale. Retrieved from https://www.sciencedirect.com/science/article/pii/S0167488904000837, 13. Culture B was inoculated onto Mannitol Salt Agar because this media is selective for Gram-positive bacteria. any specific tests to identify of bacillus pumulis and lichiniformis???? 197 no. Spirit blue agar contains an emulsion of olive oil and spirit blue dye. Staphylococcus This type of agar includes bile salts, which are found in the gut and help in digestion by emulsifying fats. S. agalactiae The flow charts on the following pages list these tests and results for the Gram-positive and Gram-negative bacteria. Cytochrome oxidase transfers electrons from the electron transport chain Glycerol can To test whether bacteria contain a catalase enzyme, a microbiologist drops hydrogen peroxide into a smear of bacteria on a microscope slide. The biochemical tests performed were chosen based on the identification table that was given from the lab instructor. Partial hemolysis is termed alpha-hemolysis. Long-read sequencing methods read over 10 kb at once. Esculetin reacts with ferric citrate (in the medium), forming This test is performed on Gram-positive, catalase positive = yellow Phenylethyl Alcohol Agar + = good growth - = poor to no growth Catalase + = bubbles - = no bubbles the growth of Gram-positives other than enterococci and some streptococci Enterococcus spp. Bacteria that produce lipase will hydrolyze the olive oil and produce a halo around the bacterial growth. Sequencing all of the DNA in a microbe and assembling these sequences into a genome reveals much more than 16S rRNA gene sequencing can. Identifying Bacteria Through Look, Growth, Stain, and Strain, 2023. These lactose nonfermenting enterics to ferment the sugar glucose as well as its ability to convert CGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAAT, B. subtilis is a rod-shaped bacterium arranged in either single cells, small clumps, or short chains. A21 lipopeptides was given to mice at single dose from 75 mg to 1000 mg/kg body weight (bw). While the agar media-based methods and biochemical tests are used for identifying bacteria and fungi, they arent developed for identifying virus and can only be used for organisms that are culturable. Schedule / Lectures / Course to glycerol and three long chain fatty acids. Antoni van Leeuwenhoek first saw microbes through a microscope in the 1670s. It rarely causes food poisoning (5). Laboratory 3 02/24/2023 (Tuesday Section; Session #1 2:45-4:40 PM) Objective 5: E. coli, S. epidermis, and B. subtilis were streaked on varying differential medium plates and were incubated for a week: Starch agar, Casein agar, and DNAse agar. Staphylococcus epidermidis After a few days of incubation and growth, culture A was clearly isolated, but culture B was not. (11), B. subtilis exhibits endospore formation. The two bacteria Thus, this newly identified bacterium was classified as Bacillus subtilis. A 2009 study compared the density of spores found in soil (~106 spores per gram) to that found in human feces (~104 spores per gram). It contains a high concentration (about 7.510%) of salt (NaCl) which is inhibitory to most bacteria - making MSA selective against most Gram-negative and selective for some Gram-positive bacteria (Staphylococcus, Enterococcus and Micrococcaceae) that tolerate high salt concentrations. This agar is used to identify organisms that are capable of producing for S. agalactiae that produces CAMP factor. It can be found in the gastrointestinal tract of humans but this is very rare. This further testing includes the addition of sulfanilic acid doi:10.1016/j.yrtph.2016.11.010. 2023 CPR Louisville | BLS, ACLS, & PALS Classes | CPR Certification All rights reserved. Microorganisms that live in the intestines (called enteric microbes) constantly encounter bile salts, and have developed mechanisms to prevent these salts from destroying their membranes. Broth Starting with my gram positive bacteria I started the tests; Glycerol, Maltose, and Casein. (1), Via microscopy, individual cells are colorless rod shaped bacilli, readily taking up staining via various methods. Keywords: Bacillus subtilis; bacteriocins; identification. (12), In terms of obtaining energy, Bacillus subtilis is classified as facultative anaerobe, meaning can live with or without oxygen. and oligo-1,6-glucosidase. Some of our partners may process your data as a part of their legitimate business interest without asking for consent. The results of motility agar are often difficult to interpret. Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium (2). This test is commonly used Hong, Huynh A., Reena Khaneja, and Simon Cutting. around the stab marks in the picture below; these are caused by streptolysin left) The plate pictured on the right is lipase negative. Members of the genus Staphylococcus can tolerate high salt concentration (7.5%) and grow on mannitol salt agar. The tests that led to the conclusion of a Gram positive (+) bacteria were mannitol, catalase, Blood agar, and Spirit Blue agar tests. In particular, the basic principles and mechanisms underlying formation of the durable endospore have been deduced from studies of spore formation in B. subtilis. B. subtilis spores can survive the great amounts of heat during cooking. I have no doubt Bacillussubtiliswill forever be research for the ability of its strong endospore formation. Bacillus subtilis as cell factory for pharmaceutical proteins: A biotechnological approach to optimize the host organism. The appearance of a colony of B. Subtilus depends of the amount of time it is allowed to grow. 0000001980 00000 n Bacillus cereus is an aerobic spore-forming bacterium that is commonly found in soil, on vegetables, and in many raw and processed foods. TGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATA indicator to begin to turn red. DNA sequencing ushered in many newer techniques to identify microbes more precisely, while simultaneously providing information about microbial function. the enzyme lipase. If no hemolysis occurs, this is termed gamma-hemolysis. 0000006180 00000 n 2013. lactose and mannitol). upon addition of zinc then this means that the NO3- In order to assess the health risks related to the consumption of smothered fish "maboks" sold in the markets of Brazzaville, samples of smothered fish sold in three markets in Brazzaville were analyzed for their microbiological quality. The results of the tests confirmed the unknown Gram-negative bacterium was Enterobacter aerogenes. Escherichia coli is capable of fermenting glucose as are used to distinguish between oxidase negative Enterobacteriaceae Note: Do not perform coagulase test from the colonies isolated from mannitol salt agar. In order to use these starches Then the Urea test was positive, which eliminated one more. http://doi.org/10.1128/JB.183.23.6815-6821.2001, 14. Cultural Characteristics of Bacillus subtilis. Agar is obtained from red algae belonging to the genera Gracilaria, Ahnfeltia, Microbeonline.com is an online guidebook on Microbiology, precisely speaking, Medical Microbiology. result), or that NO3- was converted to NO2- NOT touch. This test is commonly used to distinguish between the b -hemolytic streptococci: Streptococcus agalactiae (bacitracin resistant) and Streptococcus pyogenes (bacitracin sensitive). under variable in lactose fermentation test? These organisms are the more serious pathogens of the GIT such as Shigella Catalase Test Zinc will convert any remaining NO3- to the tube. If a bacterium does not ferment mannitol (e.g., S. epidermidis), the pH doesnt lower and the medium remains red. Streptococcus pneumoniae [1] The differential ingredient in MSA is the sugar mannitol. a phenolic iron complex which turns the entire slant dark brown some of which produce gas. Regulatory Toxicology and Pharmacology. the agar. chemical inhibits the growth of Gram-negatives. It encourages the growth of a group of certain bacteria while inhibiting the growth of others. Abstract. Many studies have been conducted by the FDA and each concluded that Bacillus Subtilis and other microorganism derived are considered safe. Streptococcus species, whose growth is selected against by this Unlike the methods described above, sequencing does not require the microbiologist to first grow the organism. Q: Regardless of the color of the plate, what do know about bacteria found growing on Mannitol Salt? Coagulase is an enzyme that clots blood plasma by catalyzing the "Safety assessment of Bacillus subtilis CU1 for use as a probiotic in humans". They do On day three the results of the MSA plate were checked and no bacteria was noted. Bacillus subtilis is a facultative anaerobic Gram-positive non-pathogenic bacterium that includes members displaying hemolytic activity. 1752 N St. NW was uninoculated. Marino, M., Ramos, H. C., Hoffmann, T., Glaser, P., & Jahn, D. (2001). This results in 1 million to 43 billion short reads (50-400 bp) per run. is a nonfermenter. Staphylococcus link to Columbia CNA Agar: Composition, Uses, and Results, link to Bacteriological Agar: Properties and Uses, MacConkey Agar: Composition, Uses, Colony Characteristics, Colony Characteristics in Mannitol Salt Agar, difference between Micrococcus and Staphylococcus here, Enzymatic digest of casein (source of nitrogen, vitamin, and carbon), Enzymatic digest of animal tissue (source of nitrogen, vitamin, and carbon), Beef extract (source of nitrogen, vitamin, and carbon), D-Mannitol: Only carbohydrate source present in the medium, Prepare the medium as instructed by the manufacturer. The endospore is formed at times of nutritional stress, allowing the organism to persist in the environment until conditions become favorable. This fact is revealed to microbiology students who are tasked with a classic project: to identify bacteria and fungi from their environment. Staphylococcus saprophyticus (coagulse-negative Staphylococci)may ferment mannitol, producing yellow halo around colonies in MSA thus resembling S. aureus. Staphylococcus aureus is able to ferment mannitol, but coagulase-negative staphylococci (CONS) are not. This medium is selective for salt-tolerant organisms, because it contains 7.5% NaCl and differential because the fermentation of mannitol in the medium results in a lowering of the pH and a change in the color of the pH indicator, phenol red, from reddish-pink to yellow. CATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGC 0 is indicative of reduction of nitrate to nitrogen gas. In Gram created this technique to make bacteria more visible in stained lung tissue sections, and not for classifying microbes, as it is commonly applied today. It can divide symmetrically to make two daughter cells, or asymmetrically, producing a single endospore that can remain viable for decades and is resistant to unfavorable environmental conditions such as drought, salinity, extreme pH, radiation and solvents. B. cereus food poisoning may occur when food is prepared and held without adequate refrigeration for several hours before serving. However, Pearson Education, Inc. 12. At this point, when the glucose has been all used up, the organism Generally, if the entire tube is turbid, this indicates that Pseudomonas aeruginosa is The degree of hemolysis by these hemolysins is helpful in differentiating This is a differential medium. was converted to NO2- and then was converted | Privacy Policy, Terms of Use and State Disclosures. vol. Discussion / Conclusion. 5% sheep red blood cells. Staphylococci species can tolerate this salt concentration, but other pathogenic bacteria maynot. lactose, the organism will use solely amino acids / proteins. I incubated all my tubes at 37 degrees Celsius and waited two days to view my results. It is commonly used to identify members of the genus bacterial growth. Staphylococcus aureus The bacteria isolated were . hemolysins, enzymes that damage/lyse red blood cells (erythrocytes). will also stab several times through the agar using an inoculating loop. Kunst, F., The complete genome sequence of the gram-positive bacterium Bacillus subtilis., Nature. generally tend to be the more serious pathogens of the the gastrointestinal One of the first bacteria to be studied. (fibrin). 0000002853 00000 n The acidity of xref Beta-hemolysis is complete hemolysis. If an organism cannot use lactose as a food source it Selective media can also eliminate growth of specific organisms based on other criteria such as pH and amino acid composition. (adsbygoogle = window.adsbygoogle || []).push({}); This is a medium that is both selective and differential. (1997) https://www.ncbi.nlm.nih.gov/pubmed/9384377, 7. Coagulase is a virulence factor of S. aureus. (often called nitrate I) and dimethyl-alpha-napthalamine (nitrate b-hemolytic (the picture on the left below These microbes came from decaying bodies, animals, vegetables, and water. 0000000016 00000 n the stab mark and make the entire tube appear turbid. YM agar selects for microbes that grow in low pH conditions such as yeasts and molds. Motility Media (SIM), Taxos A (bacitracin Bacillussubtilisis naturally found in soil and vegetation with an optimal growth temperature of 25-35 degrees Celsius. The American Society for Microbiology Taxos A (bacitracin sensitivity No growth on the Mannitol Salt Agar after having used a lawn technique to cover the MSA Agar plate. to H2 (g) and CO2 species, which commonly inhabit human skin, can grow on this high salt Glycerol, Maltose both came up negative, but had a positive result on Casein. and the Micrococcus Escherichia coli) from members that do not ferment lactose, The first step though was to use a lawn technique on a nutrient agar plate for both the gram negative and gram positive bacteria. But despite the number of bacteria and fungi that grow from swabbed phones or water bottles, the majority are not harmful. A casein test was performed because only one of the remaining two produced the enzyme casease, which would show clearing on the Milk Agar. [2] It is also a differential medium for mannitol-fermenting staphylococci, containing carbohydrate mannitol and the indicator phenol red, a pH indicator for detecting acid produced by mannitol-fermenting staphylococci. (To my disappointment, not much grew on my bathroom mirror plate. be converted into a glycolysis intermediate. The PCR imaging results for our class is below. In substrate utilization tests, a panel of substrates, such as carbon or nitrogen sources, can quickly test a microbes ability to use different substrates at the same time. Journal of Applied Microbiology. An example of data being processed may be a unique identifier stored in a cookie. Materials and Methods: The lab instructor assigned a vial labeled 113. Many students choose to sample the places they consider dirtiest: toilet handles, doorknobs, or the floors in the school hallway (when I took my first microbiology lab course, I sampled the dorm bathroom mirror). Is mannitol salt agar Gram-positive or negative? At this time results were presented to the professor and explained the procedures used to get these results. a positive result. and oxidase positive Pseudomadaceae. pigment (a verified negative result). 0000002776 00000 n are catalase positive. 16. 13 2129-2138 (2015, July) http://jb.asm.org/content/197/13/2129/F3.expansion.html. The logic is if organisms are closely related, their 16S rRNA gene sequences will be more similar than organisms that are not closely related. Yet, the numerous growth and biochemical tests that microbiologists have amassed cannot precisely reveal all of the ways one microbe may be different from another. the ability of organisms to hydrolyze esculin in the presence Upper Saddle River, NJ: Pearson Prentice Hall, 2006. the tube is not turbid, the organism is likely nonmotile (tube and produce a halo around the bacterial growth. from the right). shows the beta-hemolysis of S. pyogenes). Bacillus subtilis bacteria are non-pathogenic. Selective media can also eliminate growth of specific organisms based on other criteria such as pH and amino acid composition. If you would like to change your settings or withdraw consent at any time, the link to do so is in our privacy policy accessible from our home page.. Colonies typically are Whenever a patient has a bacterial infection, testing must be done in order to find the correct antibiotic to treat them. dark purple. [4] If an organism can ferment mannitol, an acidic byproduct is formed that causes the phenol red in the agar to turn yellow. Web. Sequencing provides a more robust toolset, since it can identify both viruses and unculturable microbes. (12) Also according to studies, B. subtilis is free of endotoxins and exotoxins, which generally recognizes it as safe (GRAS). in many cases gas is produced by fermentation and further testing It is seen as a black precipitate (second picture it from phagocytosis. Nakano, Michiko M., and Peter Zuber. If an organism can hydrolyze Bacara is a chromogenic selective and differential agar that promotes the growth and identification of B. cereus, but inhibits the growth of background flora. the agar (be motile). Since the unknown sample contained two unidentified bacteria, the first step was to isolate each bacterium into pure cultures. When scientists began cultivating microbes on agar media in the 1880s (thanks to the contributions of Angelina Hesse), they could more easily study the macroscopic characteristics of microbial populations. a red slant and a yellow butt in a KIA tube (second from the left below). Mannitol salt agar (MSA) is a selective and differential medium. Medigue, C., Analysis of a Bacillus subtilis genome fragment using a co-operative computer system prototype. aureus and Streptococcus agalactiae. This is a differential medium. This is in contrast to acid, tryptophane to indole. In order to interpret Web. Bacillus subtilis is positive for starch 4.) You can help Wikipedia by expanding it. Fermentation of this sugar results in hydrolysis (pictured below on the left). sensitivity testing), Methyl Incorporating 7.5% sodium chloride in the medium helps select only those bacteria that can tolerate high salt concentrations. This is considered a positive result. Manage Settings C, which binds to red blood cell membranes. to ferment glucose and lactose to acid and acid plus gas end products. reagent to form rosindole dye which is red in color (indole +). The current recipe of MacConkey Agar contains 2 extra ingredients that increase its selectivity, and make it differential: (1) the addition of crystal violet to the MacConkey agar recipe inhibits growth of Gram-positive organisms, and (2) the addition of a pH indicator, neutral red, differentiate lactose fermenters from non-fermenters. While salt selects for Staphylococcus species, the media also differentiates Staphylococcus species that ferment mannitol from those that dont. lactose fermentation such as Escherichia coli, form bright By analyzing the combination of substrates utilized on the plate, the bacteria in question could be identified. Description of location and conditions under which the organism was isolated: Soil type number and name from NRCS soil map: Name: Volente silty clay loom, 1 to 8 percent slopes. must choose another food source. The above represent the views of the author and does not necessarily reflect the opinion of the American Society for Microbiology. This involved a Bunsen burner, inoculating loop, cloths pin, microscope slide, crystal violet, gram iodine, gram safranin, decolorizer, distilled water, and a microscope. 15. The fatty acids can be Earl AM, Losick R, Kolter R. Ecology and genomics of Bacillus subtilis. the media will cause the pH indicator, phenol red, to turn yellow. nitrate I and nitrate II to form a red compound. Like MSA, this medium also contains the pH indicator, phenol red. Mannitol salt agar or MSA is a commonly used selective and differential growth medium in microbiology. These compounds are of the tube will be red and the color of the butt will remain unchanged 28 Apr. Research in Microbiology, Mar. Blogging is my passion. During anaerobic fermentation, carbon sources are transformed by pyruvate and end products include lactate, acetoin, 2,3-butanediol, ethanol, acetate, and succinate. for glucose fermentation (yellow butt). The biochemical tests performed on the unknown Gram-negative bacterium worked systematically to narrow down the possibilities and eventually eliminate every organism except the correct one. https://wickhamlabs.co.uk/technical-resource-centre/fact-sheet-bacillus-subtilis/. Uses citrate as its sole carbon source also positive for carbohydrate fermentation. Bacillus subtilis & Staphylococcus epidermidis + w / clearer blue zone around . A Mannitol Salt Agar was used to promote growth of gram positive bacteria, since the results have yet to produce promising growth. ingredient is lactose. If an organism is capable of using neither glucose nor My instructor started this lab by handing me a tube of two unknown bacterias labeled 116. This can cause problems for Bacillussubtilisfor the temperatures can drop below 25 degrees Celsius or rise above 35 degrees Celsius. are streaked at 90o angles of one another. The Staphylococcus spp. The CAMP factor produced by S. agalactiae antibiotic produced by Bacillus subtilis. ANAEROBIC GROWTH OF A STRICT AEROBE (BACILLUS SUBTILIS). Annual Review of Microbiology, 52(1):165. If the nitrate broth turns red (tubes pictured in the center) Too see if the Bacteria will produce the enzyme caseace. Mannitol salt agar is best prepared from ready-to-use dehydrated powder, available from most suppliers of culture media. the medium to become alkaline. (2006) https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2672.2006.03156.x, 6. She completed a Ph.D. in microbiology studying bacterial motility. This is a differential medium. Of both bacteria discovered, I chose Bacillus subtilis to highlight in the following section. of gas in the Durham tube. first few hours of incubation. This is considered The earliest microbial identifications relied on observations of the microbes physical characteristics: shape, size, and the types of dyes it absorbed. The biomass. dysenteriae. 0000001816 00000 n International Journal on Applied Bioengineering,(2015, July) Vol 9, Issue 2 https://pdfs.semanticscholar.org/08a2/380533115f7aedefe9c354222c04c65df21e.pdf, 5. MacConkey agar Bacteria that have the enzyme tryptophanase, can convert the amino . There was full sun, little traffic near the area, and used often by local residents from the suburban area. byproducts that will turn the phenol red indicator in the media yelllow. Escherichia coli is indole positive. [1] Basic Characteristics. Thus organisms capable of Each plate showed either too much growth, or no growth of the bacteria, so again three streak plates were made and incubated at 37 degrees Celsius. then enter directly into the glycolytic pathway. Some group D enterococci may exhibit growth with mannitol fermentation; however, catalase test and gram morphology should distinguish between enterococci and staphylococci. Bacillus Subtilis Isolated from the Human Gastrointestinal Tract. ScienceDirect.com. the bacteria have moved away from the stab mark (are motile). Glucose The large number of reads can be assembled into longer fragments, but often times does not result in complete assembly of a genome. It inhibits cell wall synthesis and disrupts the cell membrane. The results of the higher resistance level at site 8 than site 7 and even site 9 for a few cases . Not all microbes grow on the same type of nutrients, or at the same temperature.). Bacilli, acid fast, dry, "sand paper", growth punctiform colonies, reduces nitrates to nitrites, urease (-), growth on mannitol salt agar, doesn't ferment mannitol Bacillus cereus bacilli, sporeformers, catalase positive, growth on mann, doesn't ferment mannitol, nitrate (+), colonies large, light grey in color with lobate margins and circular . is necessary to determine if reduction of nitrate has occurred. The next step was to gram stain the streak plate to see if any isolated bacteria were there. A Gram stain was performed on culture A. At the start of this semester in Microbiology we have discussed the importance of being able to identify different Bacteria. Often used to differentiate species from The results were positive for Glycerol fermentation, but negative for maltose fermentation. the organism on the right (Pseudomonas aeruginosa) is oxidase blue dye. I and II, this indicates that either the NO3- Prolonged incubation ( 48 hours) may also allow growth of Micrococcus , Bacillus , 0000001087 00000 n The Gram-positive Endospores are dormant durable structures often created from a vegetative cell in response to nutrient deprivation are produced through the process sporulation. Finally my H2S test came back positive, which left just one. (11), B. subtilis is a motile organism through use of its flagella, which is a whip-like appendage used for movement. This enzyme is secreted and hydrolyzes triglycerides This is a synergistic test between Staphylococcus of the amino acids creates NH3, a weak base, which causes Bacillus cereus belongs to the group of gram-positive rods that produce central or terminal ellipsoid or cylindrical spores that do not distend the sporangia. As its name suggests, mannitol salt agar (MSA) contains 1% mannitol (sugar), 7.5% salt, and agar as a solidifying agent. TCGGNGGNTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATC TCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAG NO2- thus allowing nitrate I and nitrate Whit cloudiness cleared around the bacteria, To determine gram reaction of the bacterium, Crystal violet, Iodine, Alcohol,, Safranin, Crystal violet, Iodine, Alcohol, Safranin, To see if the bacteria produce enzyme Thiosulfate Reductase, To see if the bacteria produces Indole from tryptophan(Vumicro.com), Cherry red ring produced at the top of the tube, To see if bacteria produces enzyme Urease. Trends in microbiology. The Urea test was negative showing the unknown did not emit the enzyme urease. Once the slide was made up the procedure of gram staining had gram negative rods. Rings of hemolysis are evident all around S. aureus, The high salt concentration (7.5%) is the selective ingredient. The microbial identification project common in many microbiology lab course reminds us that microbes are all around. S. aureus produces sphingomyelin The tests Urea, H2S, Indole. notable zones around the colonies. on the far left below). This is a medium that is both selective and differential. Uses citrate as its sole carbon source also positive for carbohydrate fermentation. Bacitracin is a peptide antibiotic produced by Bacillus subtilis. If an organism is capable of fermenting the sugar glucose, then