These lactose nonfermenting enterics Positive (+ve) Flagella. Abstract. Enterococcus spp. Wickham Laboratories LTD: Confirmation from my lab instructor showed it was incorrect. The Streptococcus and The acidity of To better visualize the microscopic amongst us, Hans Christian Gram developed the Gram stain technique in 1884. Journal of Bacteriology, 183(23), 68156821. The microrganisim B. Subtilis tested positive for catalase, lipase, and amylase. This is so the chromosome can be protected within and then, and the bacteria genetic material is not harmed. I and II, this indicates that either the NO3- Bacillus Subtilis is a gram positive, rod shaped organism that can be found growing in soil as well as the gastrointestinal tract of humans. SIM is commonly used to differentiate members Glucose This is a test commonly used when trying to identify Gram-negative the agar (be motile). 16. I have been working as a microbiologist at Patan hospital for more than 10 years. Streptococcus pneumoniae plates? 0000000016 00000 n The fatty acids can be Table 1: Biochemical Tests for Gram-positive Unknown, Flowchart * Removed due to formatting issues. 5% sheep red blood cells. In the case of nonfermenters, this American Society for Microbiology ("ASM") is committed to maintaining your SIM tubes are inoculated with a single stab to the bottom of the In order to determine which 766-3524 aureus is capable of fermenting mannitol (left side of left plate) The results of motility agar are often difficult to interpret. Pseudomonas aeruginosa is To view the purposes they believe they have legitimate interest for, or to object to this data processing use the vendor list link below. was uninoculated. Earl AM, Losick R, Kolter R. Ecology and genomics of Bacillus subtilis. [1] It is used for the selective isolation of presumptive pathogenic (pp) Staphylococcus species.[5]. Bacillus cereus is an aerobic spore-forming bacterium that is commonly found in soil, on vegetables, and in many raw and processed foods. | Privacy Policy, Terms of Use and State Disclosures. (14) This makes B. subtilis more favorable in being used in food production over some gram-negative bacterias. is gamma-hemolytic. 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. with an organism that can ferment lactose). around the stab marks in the picture below; these are caused by streptolysin After sterilizing my inoculating loop a streak plate was made, while sterilizing the inoculating loop each time a new streak was made. (14). and then immediately reduced to some other, undetectable form The selective Save my name and email in this browser for the next time I comment. This page was last edited on 4 May 2018, at 19:51. Differential media allow multiple bacterial species to grow but their growth patterns differ visually. It is considered a selective medium because the bile salts and crystal violet aspect of the medium prevent the growth of gram positive bacteria (3). result), or that NO3- was converted to NO2- under variable in lactose fermentation test? The large number of reads can be assembled into longer fragments, but often times does not result in complete assembly of a genome. 11. by catabolism of the amino acid cysteine by the enzyme cysteine hemolysins, enzymes that damage/lyse red blood cells (erythrocytes). wherein the cells comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide and wherein the cells further comprise one or more heterologous . In the clinic, the catalase test helps distinguish catalase-positive Staphylococci from catalase-negative Streptococcus, which are both Gram-positive cocci. The formation Mannitol salt agar is best prepared from ready-to-use dehydrated powder, available from most suppliers of culture media. the oxidase test, artificial electron donors and acceptors are provided. lactose fermentation such as Escherichia coli, form bright Bacitracin is an antibiotic isolated from Bacillus subtilis. The tube on the far right was inoculated with E. Because of the species, which commonly inhabit human skin, can grow on this high salt [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. 0000002518 00000 n After swabbing and spreading the invisible contents onto the agar plate, we placed our agar plates in the incubator and awaited the microbial surprises the following class period. must choose another food source. After five days of incubation prompted promising growth on one of the streak plates. Staphylococcus saprophyticus (coagulse-negative Staphylococci)may ferment mannitol, producing yellow halo around colonies in MSA thus resembling S. aureus. At this point, when the glucose has been all used up, the organism At this time results were presented to the professor and explained the procedures used to get these results. This vial contained two unknown bacteria, one Gram negative and one Gram positive, that required identification. Bacillus subtilis | Microbiology Unknown Lab Report, The Benefits of Breastfeeding by Norma Martin, Enterobacter aerogenes | Microbiology Tennessee, S. aureus and P. vulgaris | Microbiology Unknown Lab Report, Determine the gram reaction of the bacterium, Detects the enzyme urease, which breaks down urea, producing an alkaline pH, To determine if the bacteria will produce citrate permease which allows them to take in the citrate and convert it to pyruvate, Changed from green to blue towards the top of the tube, To determine if the bacterium will ferment lactose with acid production, Color change where streaked to a purple color, Positive lactose fermenter with weak acid production, To determine if the organism produces casease which hydrolyzes the milk protein casein, To determine the Gram reaction of the bacteria, Crystal violet, Iodine, Alcohol, Safranin, To determine if the bacterium will ferment glycerol with acid production, Positive for glycerol fermentation with acid production, To determine if the bacterium will ferment maltose with acid production. The biochemical tests performed were chosen based on the identification table that was given from the lab instructor. Antibiotics: B. subtilis chloramphenicol (5 g/ml), E. coli ampicillin (50 g/ml) and also you can add any substitute nutrients or trace elements. This was a problem and could have been contaminated, while performing the test. But if CONS grow, they cant ferment mannitol, so the color of the media around the bacterial colony does not change to yellow; it appears pink. Mannitol salt agar or MSA is a commonly used selective and differential growth medium in microbiology. At this point chemical tests on the unknown bacterias were able to be conducted. generally tend to be the more serious pathogens of the the gastrointestinal http://doi.org/10.1128/AEM.71.7.3747-3752.2005. Thanks for the intel, But I would like to test my own Bacillus, The above represent the views of the author and does not necessarily reflect the opinion of the American Society for Microbiology. By CPR Memphis at June 29, 2013 | 12:17 pm | Bacillussubtilisis naturally found in soil and vegetation with an optimal growth temperature of 25-35 degrees Celsius. Table 1 lists the test, purpose, reagents, and results of the gram positive testing, while table 2 lists the test, purpose, reagents, and results of the gram negative testing. Washington, DC 20036, 2023. of the preceding is the case, elemental zinc is added to the broth. Bacillus subtilis is a facultative anaerobic Gram-positive non-pathogenic bacterium that includes members displaying hemolytic activity. (2015,2012,2009). B. cereus food poisoning may occur when food is prepared and held without adequate refrigeration for several hours before serving. Glycerol can Selective media can also eliminate growth of specific organisms based on other criteria such as pH and amino acid composition. is produced between the two streaks. 13 2129-2138 (2015, July) http://jb.asm.org/content/197/13/2129/F3.expansion.html. They can contaminate food, however, though seldom does it result in food poisoning. 649 14 It encourages the growth of a group of certain bacteria while inhibiting the growth of others. Colonies typically are The alkaline pH causes the phenol red This Keywords: Bacillus subtilis; bacteriocins; identification. This was done by making an isolation streak from the mixed culture onto a nutrient agar plate using the process described in the manual. TGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATA = yellow Phenylethyl Alcohol Agar + = good growth - = poor to no growth Catalase + = bubbles - = no bubbles of fermentation that will lower the pH of the media. 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). are streaked at 90o angles of one another. of the tube will be red and the color of the butt will remain unchanged and the Micrococcus For example: Microbiologists can ask additional questions about microbial identity based on microbial behavior during biochemical tests. of H2S. (11), B. subtilis is a motile organism through use of its flagella, which is a whip-like appendage used for movement. Broth (2) (3). B. subtilis is only known to cause disease in severely immunocompromised patients, and can conversely be used as a probiotic in healthy individuals. to ferment the sugar glucose as well as its ability to convert While these are just a few examples of how types of media can help microbiologists distinguish between microbes, there are many other types of selective and differential media. The biomass. (1), Via microscopy, individual cells are colorless rod shaped bacilli, readily taking up staining via various methods. How come the Bacillus spp. Q: If there is growth, additional information can then be obtained about bacteria growing based on color of the medium. As a result, an arrow of beta-hemolysis American Society for Microbiology Journal of Clinical Microbiology. Recurrent Septicemia in an Immunocompromised Patient Due to Probiotic Strains of Bacillus Subtilis. By observing changes in the current, the DNA sequence can be inferred as the molecule passes through the nano pore. B. Subtilis is considered non-pathogenic, It has been implicated in food poisoning caused by poor quality bakery products among others. If hydrogen sulfide is produced, a black color forms in the medium. the media will cause the pH indicator, phenol red, to turn yellow. One of these first DNA sequencing methods is 16S rRNA gene sequencing and relies on the fact that the 16S rRNA is a relatively stable region with a slow rate of evolution. This is considered a positive result. The Gram-positive The number of spores found in the human gut is too high to be attributed solely to consumption through food contamination. Remember that in the neutral pH (6.9 to 8.4) the color of phenol red is red; while above pH 8.4, the color of phenol red is pink. It is seen as a black precipitate (second picture Selective media contain substances that will inhibit growth of organisms while allowing for only a specific type of organism to grow. If a bacterium does not ferment mannitol (e.g., S. epidermidis), the pH doesnt lower and the medium remains red. the tube is not turbid, the organism is likely nonmotile (tube How can I isolate bacillus subtilis from soil samples. In this study, we compared the inclusivity, exclusivity, recoverability, and selectivity of the 3 selective agars (mannitol yolk polymyxin B agar [MYPA], polymyxin pyruvate egg yolk mannitol bromothymol blue agar [PEMBA], and Brillance Bacillus cereus agar [BBC agar]) for Bacillus cereus (B. cereus) from pure culture and several food . The pH change is detectable because the media contains phenol red which turns yellow at low pH. like Shigella dysenteriae. first few hours of incubation. was converted to NO2- and then was converted Staphylococcus epidermidis - Mannitol salt Mannitol salt agar is a selective and differential medium used for differentiating between different stapylococci Eosin methylene blue differentiates between lactose fermenters and non-fermenters. acidic byproducts are formed and the pH indicator turns yellow. As MSAcontains phenol red as a pH indicator, at pH levels below 6.9, the medium is a yellow color. Culture B was inoculated onto Mannitol Salt Agar because this media is selective for Gram-positive bacteria. N.p. 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. to glycerol and three long chain fatty acids. used to distinguish between oxidase negative Enterobacteriaceae Marino, M., Ramos, H. C., Hoffmann, T., Glaser, P., & Jahn, D. (2001). It inhibits cell wall synthesis and disrupts the cell membrane. here, second from right) is a glucose positive, lactose negative, sulfur Identifying Bacteria Through Look, Growth, Stain, and Strain, 2023. a phenolic iron complex which turns the entire slant dark brown X-gal plates identifies lac operon mutants during clone selection. PRODUCING BACILLUS SUBTILIS ISOLATED FROM RAW MILK. a positive result. Not all microbes grow on the same type of nutrients, or at the same temperature.). This enzyme detoxifies hydrogen peroxide by breaking it down into water 1752 N St. NW She completed a Ph.D. in microbiology studying bacterial motility. However, the same cannot be said for the other half of the unknown #113. 0000001087 00000 n on the left) after the addition of Zn, this indicates a negative This bacteria is a prime example for cellular development. Proteus mirabilis is positive for H2S production. (the picture on the right below shows the a-hemolysis of S. mitis). to turn a bright pinky-red color. As its name suggests, mannitol salt agar (MSA) contains 1% mannitol (sugar), 7.5% salt, and agar as a solidifying agent. This enzyme is secreted and hydrolyzes triglycerides Microbiology With Disease By Body System (4th ed.). Originally named Vibrio subtilis in 1835, this organism was renamed Bacillus subtilis in 1872 (MicroWiki.com). https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch, 10. Ouoba, L.I.I, Antimicrobial activity of Bacillus subtilis and Bacillus pumilus during the fermentation of African locust bean (Parkia biglobosa ) for Soumbala production. cycle. As microbiologists combined different formulations of nutrients with agar to grow a diverse set of microorganisms, they created another tool for microbial identification: selective and differential media that help microbiologists identify bacteria and yeast species. reducing enteric. It is commonly Thus MSA selectively isolates Staphylococcus spp i.e. Nursing students must have CPR for healthcare providers Memphis, a.k.a. Nakano, Michiko M., and Peter Zuber. NAD+ regeneration utilizes the enzyme lactate dehydrogenase and this enzyme also converts pyruvate into lactate. Be sure to perform a catalase test before you proceed with the salt tolerance broth test. Print. Cytochrome oxidase transfers electrons from the electron transport chain Biochemical characteristics are as follows: Decarboxylation of Arginine, Lysine, Orthinine: +, Bacillus Subtilis is a gram positive, rod shaped organism that can be found growing in soil as well as the gastrointestinal tract of humans. Since the incubation time is short (18-24 O. we work with are motile. [4] If an organism can ferment mannitol, an acidic byproduct is formed that causes the phenol red in the agar to turn yellow. A Mannitol Salt Agar was used to promote growth of gram positive bacteria, since the results have yet to produce promising growth. It also allows for identification of sulfur reducers. mirabilis (far right) are both gas producers. (center). flagella and thus capable of swimming away from a stab mark. If no red color forms upon addition of nitrate 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). Lactose fermentation will continue This is in contrast to Proteus mirabilis (far right) and Shigella dysenteriae B. subtilis spores can survive the great amounts of heat during cooking. Lab Manual for General Microbiology. TCGGNGGNTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATC Both of the results should have been consistent as far as the fermentation of either carbohydrate. Syllabus / Home, Rachel Watson, M.S. B. 3. The tube in the center was inoculated species to identify the coagulase positive Staphylococcus aureus. Positive (+ve) Citrate. Westers, L., Westers, H., & Quax, W. (2004, May 17). A Mannitol Salt Agar was used to promote growth of gram positive bacteria, since the results have yet to produce promising growth. Materials and Methods: The lab instructor assigned a vial labeled 113. oxidase (important in the electron transport chain). The Gram stain showed the unknown labeled A was a Gram-negative rod. This is a differential test used to distinguish between organisms sensitive My instructor started this lab by handing me a tube of two unknown bacterias labeled 116. Mannitol Salt Agar (MSA) . Mannitol Salt Agar (MSA) is used to determine if the bacteria is halophilic (salt loving) and if the bacteria can ferment mannitol. (1997) https://www.ncbi.nlm.nih.gov/pubmed/9384377, 7. spp. break the starch molecules into smaller glucose subunits which can Bacillus subtilis & Staphylococcus epidermidis + w / clearer blue zone around . Therefore, MacConkey Agar selects for bile-resistant microorganisms. were isolated on Mannitol salt agar. After initial tests concluded Unknown B was a Gram-positive rod, a Casein test was performed, followed by a Glycerol test, and a Maltose test. Metabolism section: As a model organism B. subtilis is commonly used in laboratory studies directed at discovering the fundamental properties and characteristics of Gram-positive spore-forming bacteria. (g), via the action of the enzyme formic hydrogen lyase, emit Blood agar is a commonly used differential medium, containing 5-10% sheep or horse blood, a requirement for Streptococcus species to grow. On this Wikipedia the language links are at the top of the page across from the article title. Medigue, C., Analysis of a Bacillus subtilis genome fragment using a co-operative computer system prototype. . 0000001816 00000 n : St. Louis Community College at Meramec, 2011. aeruginosa and the strain of Proteus mirabilis that to H2 (g) and CO2 And for basic research, microbiologists all over the world are studying where microbes reside and what they are doing: sourdough starters, showerheads, the subway, oceans, and soils are just the beginnings of our microbial exploration. The process of identification was achieved by utilizing procedures learned during the present semester. Trends in microbiology. is a nonfermenter. I hypothesized that the original culture tube 116 may not be a great culture to sample from, and gave the gram positive and gram negative bacteria already isolated in separate tubes.The gram positive tube was labeled alt 9, and the gram negative tube was labeled alt 3. the genera Clostridium and Bacillus. The American Society for Microbiology, not for classifying microbes, as it is commonly applied today, https://asm.org/getattachment/5c95a063-326b-4b2f-98ce-001de9a5ece3/gram-stain-protocol-2886.pdf, https://commons.wikimedia.org/wiki/File:Streptococcal_hemolysis.jpg, drops hydrogen peroxide into a smear of bacteria, https://www.sciencedirect.com/science/article/pii/S1319562X16000450?via%3Dihub, https://en.wikipedia.org/wiki/Hybrid_genome_assembly#/media/File:HybridAssembly.png, microbiologists identify the microbes behind disease in their patients, Engineered Bacterial Strains Could Fertilize Crops, Reduce Waterways Pollution, Prolonged Transmission of a Resistant Bacterial Strain in a Northern California Hospital, Privacy Policy, Terms of Use and State Disclosures, No media color change = no blood cell lysis (, Green/brown media = partial blood cell lysis (, Lightened agar around bacterial growth = complete blood cell lysis (. 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. the organism on the right (Pseudomonas aeruginosa) is oxidase Streptococcus species, whose growth is selected against by this of nitrate I and nitrate II, the result is uncertain. After the Gram stains were determined, specific biochemical tests were performed. 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. while Staphylococcus epidermidis is not (right side of left plate). 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. Organisms capable of fermenting this sugar will use it up within the Eosin methylene blue selects for Gram-negative organisms. Mannitol salt agar has 7.5% salt. Its cell wall consists of a thick peptidoglycan layer. Because the same pH You can purchase prepared mannitol salt agar from commercial suppliers, get the powder, and prepare the media in your laboratory. One popular long-read method is Nanopore sequencing; here, a single-stranded DNA molecule is fed through a very small pore (hence the name Nanopore). S. aureus produces sphingomyelin (1), Staphylococcus epidermidis (2) and S. aureus colonies (3). When the electron donor is oxidized by cytochrome oxidase it turns a So, MSA is also a differential medium. will be lifted off the bottom of the tube. Table 1: Biochemical Tests for Gram-negative Unknown, Flowchart *Removed due to formatting problems. Because streptolysin Organisms that are The microrganisim B. Subtilis tested positive for catalase, lipase, and amylase. The different samples were cultured for isolation on solid media using conventional microbiological methods. and oligo-1,6-glucosidase. to distinguish between the b-hemolytic streptococci: (transparent) zone surrounding the colonies. it from phagocytosis. Regulatory Toxicology and Pharmacology. This surrounding the disk. The organism shown on the *Note - broth tubes can be made containing sugars other than -Bacillus subtilis -Enterococcus faecalis. It is used to determine if an organism the ability of organisms to hydrolyze esculin in the presence Enterococcus. Bacitracin is a peptide antibiotic produced by Bacillus subtilis. the same results are considered positive (e.g. second from left is indole positive. [1] If the bacteria contain catalase, the mixture bubbles as the hydrogen peroxide decomposes into water and oxygen. An example of data being processed may be a unique identifier stored in a cookie. A Gram stain was performed on culture A. Mannitol Salt Agar + salt tolerance = growth + mannitol ferment. via the action of the enzyme nitratase (also called nitrate reductase). [3] Staphylococcus aureus produces yellow colonies with yellow zones, whereas other coagulase-negative staphylococci produce small pink or red colonies with no colour change to the medium. Bacillus species, Acinetobacter sp., and S. marcescens began to occur at site 1, E . Lefevre, Marie; Racedo, Silvia M.; Denayrolles, Muriel; Ripert, Gabrielle; Desfougres, Thomas; Lobach, Alexandra R.; Simon, Ryan; Plerin, Fanny; Jsten, Peter; Urdaci, Maria C. (1 February 2017). Pseudomonas esculin in the presence of bile, the product esculetin is formed. Bacteria that produce lipase will hydrolyze the olive oil You can help Wikipedia by expanding it. Gram stain the isolate to get shape and arrangement as well as gram reaction. 151 Studies of DNA-DNA hybridization and 16S and 23S ribosomal RNA (rRNA) sequencing and enzyme electrophoretic patterns have shown a close relationship among B. cereus, Bacillus anthracis, catabolized and their fragments can eventually enter the Krebs How did these results come to be? It inhibits cell wall synthesis mainly through inhibiting the biosynthesis of peptidoglycan. broth with Durham tubes. The bacteria isolated were . These microbes came from decaying bodies, animals, vegetables, and water. This is a differential medium. Eosin-methylene blue test (Pinkish-purple colonies) Positive . 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. then enter directly into the glycolytic pathway. (fibrin). Hyaluronic Acid Production in Bacillus subtilis. "Safety assessment of Bacillus subtilis CU1 for use as a probiotic in humans". After two days of incubating at 37 degrees Celsius the results were checked. large size of amylose and amylopectin molecules, these organisms can CATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAANGTGG Brock Biology of Microorganisms. Other types of staining can tell microbiologists whether certain features are present: spores (Schaeffer-Fulton staining), capsules (India ink or nigrosin) and mycolic acids (acid-fast staining). nitrite (NO2-) or other nitrogenous compounds If gas is produced as a result of glucose or reagent to form rosindole dye which is red in color (indole +). This process is also useful in the production of antibiotics on a larger scale. In order to use these starches It inhibits cell wall this information and the choices you have about how we use such information. The PCR imaging results for our class is below. After a few days of incubation and growth, culture A was clearly isolated, but culture B was not. Thus, after the first few hours of incubation, the tube will be entirely enhances the beta-hemolysis of S. aureus by binding to The results of motility agar are often difficult to interpret. Staphylococcus aureus, Nitrate 0 Prolonged incubation ( 48 hours) may also allow growth of Micrococcus , Bacillus , The research paper gives a brief account about the . confidence and trust with respect to the information we collect from you on This test is commonly . Run oxidase and catalase tests on plate culture. Staphylococcus aureus is able to ferment mannitol, but coagulase-negative staphylococci (CONS) are not. to do several things: reduce sulfur, produce indole and swim through are catalase negative. Salt Agar (MSA), Sulfur Indole The complete genome of Bacillus subtilis: from sequence annotation to data management and analysis. Of both bacteria discovered, I chose Bacillus subtilis to highlight in the following section. Motility agar is a differential Streptococcus pyogenes and Streptococcus agalactiae are Finally my H2S test came back positive, which left just one. The beneficial effects of B. subtilis spores on the balance of the intestinal microflora are the rationale for its general use as a probiotic preparation in the treatment or prevention of intestinal disorders (4). I incubated all my tubes at 37 degrees Celsius and waited two days to view my results. ; In spite of the diversity, the colonies of Bacillus species can be recognized on agar plates quite easily. Wikimedia Commons, the free media repository. There are several methods of DNA sequencing used to generate a whole genome sequence. 651 0 obj<>stream Description: The location the organism was isolated was a grassy field between a soccer field, parking lot, and childrens playground. This test is crucial and must be done correctly because it not only reveals whether the stain is positive or negative, but the shape of the bacteria as well. This can cause problems for Bacillussubtilisfor the temperatures can drop below 25 degrees Celsius or rise above 35 degrees Celsius. and Streptococcus mitis are a-hemolytic testing) Esculetin reacts with ferric citrate (in the medium), forming (adsbygoogle = window.adsbygoogle || []).push({}); 2013. The gram negative bacteria was a different story, since all the gram negative bacteria we had to work with were all rid shaped.