Microbiology Student Lab Number 25

Major Unknown Date: Before Due Date

Unknown gram positive: Bacillus subtilis Unknown gram negative: Enterobacter aerogenes

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Received a broth media containing two unknown organisms. Spore staining indicated that the gram positive bacterium was a spore former which eliminated the possibility that the bacterium was of the following genera: Mycobacterium. The FTM test indicated the unknown bacteria to be an obligate aerobe. megaterium. The media was incubated at 37˚F after inoculation with a loop onto the Simmon Citrate slant. 4. Lactobacillus. Isolated cultures of gram positive bacteria had to be extracted from the nutrient agar whose colonies were visually distinct from that of the gram negative colonies. Results of Gram staining indicated the presence of a gram positive streptobacillus of average size and gram negative staphylobacillus of average size. 2. Staphylococcus. This eliminates the possibility that the bacterium is of the following genera: Streptococcus. The FTM was incubated at 37˚F for twenty-four hours. LAB PROCEDURES Day 1 July 23. A test for catalase production was done to confirm Bacillus species. Rhodococcus. the broth mixture was streaked onto a nutrient agar plate and a MacConkey Agar plate. 2009 1. This eliminated the possibility that the bacterium could be of the genera Clostridium. Day 2 July 27. 2009 1. The motility test was incubated at 37˚F for twenty-four hours. The motility test indicated that the unknown bacteria to be moderately motile. Gram stained sample from colony to confirm it is gram positive. 2. 2 .I. Both plates were incubated at 37˚F over a period of four days. subtilis. sphaericus and B. Day 3 July 28. To isolate. 4. 3. 5. Corynebacterium. It further eliminated the possibility that the bacterium could be Bacillus cereus or Bacillus thuringiensis. B. The slant cultures seem to grow better at 37˚F but was also capable of growing at 25˚F. Conducted spore-staining. 5. Sporosarcina urea. it confirmed results that concluded the bacterium to be of Bacillus species. and Kurthia zopfil. 6. B. One is known to be gram negative and the other gram positive. The MacConkey agar inhibited gram positive growth. Samples were also taken to conduct a motility test. The gram positive colony had a rhizoid configuration with thread-like margins and umbonated elevations. Though it did not eliminate any bacterium. samples were taken from the same colony to inoculate an FTM to detect respiration requirements. and Micrococcus 3. Upon confirmation. 2009 1. Gram stain the sample to ensure the presence of both unknown species in the unknown broth mixture. 2. The slants were also used to determine the optimum temperature as one slant was placed at 37˚F and the other at 25˚F and incubated for twenty-four hours. Gram Positive Unknown Organism: Bacillus subtilis A. A Simmon Citrate test was conducted to determine citrase production and to differentiate between B. Second gram stain confirmed streptobacillus confirmation. Further samples were taken to produce a working slant culture and a reserve slant culture. stearothermophilus.

This test eliminated the possibility the unknown could be B. megaterium. 2. 2009 1. Day 5 July 30. This coincides with the capabilities of Bacillus subtilis. Voges-Proskauer reagent A and reagent B were added to the mixture. the mixture turned red which indicated a positive reading for acetoin.6. Day 4 July 29. B. after a period of approximately ten minutes. Though the mixture appeared green at first. A Voges-Proskauer test was conducted by inoculating the media with the unknown and incubated 37˚F for twenty-four hours. negative for gas 3 . A glucose test was done to confirm B. The Simmon Citrate test indicated the bacterium was capable of producing citrase. sphaericus and B. 2009 1. megaterium or B. sphaericus. B. stearothermophilus. MEDIA TESTS AND TEST RESULTS Table 1: Gram Positive Media Tests and Results Test/Media Gram stain Spore stain FTM Motility Sample with H2O2 Simmon Citrate Voges-Proskauer Durham glucose tube Biochemical/physiological characteristic Determining cell wall structure Determining spore formation Determine oxygen requirements Determine motility Determine catalase production Determine citrase utilization Acetoin production Glucose fermentation to acid with gas Result Positive. After the Voges-Proskauer media was incubated with the microbe at 37˚F for twenty-four hours. streptobacillus Positive Obligate aerobe Moderately motile Positive Positive Positive Positive for acid production. subtilis. stearothermophilus. B. The test was to detect the production of acetoin and to differentiate between B. subtilis. 3. The test result showed that the bacteria could ferment glucose and produce acid. Citrase production indicates that the unknown could not be B. which only left B. The glucose test was incubated over night for 37˚F. subtilis. Results for the glucose were read. but not gas.

FLOW CHART TO DETERMINE UNKNOWN GRAM POSITIVE BACTERIA Gram Positive Bacillus Cocci Non-Spore forming Spore forming Anaerobe Aerobe Glucose fermenter Glucose non-fermenter Citrase Positive Citrase Negative VP Positive VP Negative Figure 1 : Testing Route to Determine Unknown Gram Positive Bacteria 4 .C.

subtilis is considered to have low virulence to humans (Bacillus subtilis Final Risk Assessment [updated 2007]). subtilis is capable of movement by peritrichous flagella (Bacillus subtilis Final Risk Assessment [updated 2007]). The ability of B. B. acidity and salinity. peptide antibiotics. therefore might only be transient inhabitants of the body. as well as away from toxins from competing microorganisms (Sonenshein 2002). HABITAT Bacillus subtilis is an obligate aerobe and a ubiquitous organism commonly found in water. Research from literature confirms that B.D. subtilis must have mechanisms to not only protect itself and find nutrients. subtilis is also capable of producing a variety of enzymes to both cycle nutrients and ward of offensive organisms as mentioned above. B. humidity and nutrient concentrations. SPECIAL CHARACTERISITCS Bacillus subtilis was the first Gram positive bacteria to be sequenced. During these times. However. When it is active. the organism is not biologically active (Bacillus subtilis Final Risk Assessment [updated 2007]). soil. It is one of the most widely used bacteria for the production of enzymes. however. though some studies have shown that they are capable of producing toxins that may be responsible for food poisoning. however lacks mechanism for attachments. CLINICAL SIGNFICANCE Bacillus subtilis is not known to be pathogenic to humans. The endospore can withstand extreme conditions of high heat. subtilis utilizes chemotaxis and uses its flagella to move towards nutrients. air and decomposing plant residue (Bacillus subtilis Final Risk Assessment [updated 2007]). In the soil or water. but also fend off competitors. subtilis to produce antifungals have also lend to the production of fungicides and treatment for agricultural crop protection. because their natural habitat is in areas where competing bacteria. subtilis is capable of producing endospores. E. To overcome its adversities. F. protease. The endospore staining test showed that B. fungus and other microorganisms exist. ribosides and amino acids. B. the organism is capable of growing in a wide range of temperatures. The result of the motility test showed that the organism was motile. inosine. Because of its’ ubiquity. they are capable of producing insect toxins. It may also be capable of growing on the human body. 5 . it must compete with other microbes and is also exposed to variations in temperature. B. a topical antibiotic used to prevent infections (Stein 2005). B. which would allow it to survive harsh times with limited nutrients and aversive environmental conditions. subtilis is capable of producing over two dozen antibiotics including bacitracin. It also has industrial applications which allow for the production of amylase. and antifungals (Bacteria Genomes – Bacillus subtilis [updated 2009]).

II. it could not be Flavobacterium. 5. Both plates were incubated at 37˚F over a period of four days. The gram negative colonies had a round configuration with smooth margins and a raised elevation and it was a white or cream in color on the nutrient agar. The motility test indicates the unknown bacteria to be highly motile. To isolate. The FTM test indicated the unknown bacteria to be facultative anaerobe. The second gram stain confirms gram negative bacterium in staphylobacillus configuration. Further samples were taken to produce working slant cultures and a reserved slant culture. LAB PROCEDURES Day 1 July 23. the broth mixture was streaked onto a nutrient agar plate and a MacConkey Agar plate. 5. Escherichia coli. Proteus and Pseudomonas. The lactose test was incubated at 37˚F for twenty-four hours. Samples were also taken to inoculate a motility media. 6. 2009 1. Day 3 July 28. Shigella . 2. The slant cultures indicated that the organism grew better at 37˚F than 25˚F. This eliminates the possibilities that the unknown bacterium could be Branhamella. Escherichia coli and Enterobacter. and incubated for twenty-four hours at 37˚F. 6 . Vibrio angiullarum. 2009 1. Moraxella catarrhalis. and Rhodospirillum rubrum. Spirillum serpens. Gram stain the sample to ensure the presence of both unknown species in the unknown broth mixture. 4. Day 2 July 27. The MacConkey agar inhibited gram positive growth. The color of the colony on the nutrient agar indicates that the organism cannot be Chromobacterium because Chromobacterium grows on nutrient agar in low convex colonies with a dark metallic sheen. The media was also incubated at 37˚F for twenty-four hours after inserting the organism into the media. Received a broth media containing two unknown organisms. A glucose test was conducted to differentiate between Proteus. Results of Gram staining indicated the presence of a gram positive streptobacillus of average size and gram negative staphylobacillus of average size. 3. The slants were also used to determine the optimum temperature as one slant was placed at 37˚F and the other at 25˚F and incubated for twenty-four hours. 2. 4. the test was incubated at 37˚F for twenty-four hours. or Klebsiella. After inoculation. 2. Salmonella and Pseudomonas. Because the organism is motile. The FTM was incubated at 37˚F for twenty-four hours. Isolated cultures of gram positive bacteria had to be extracted from the nutrient agar whose colonies were visually distinct from that of the gram negative colonies. Gram Negative Unknown Organism: Enterobacter aerogenes A. Neisseria. Gram staining of sample from colony to confirm cell wall configuration. 3. Enterobacter. 2009 1. A lactose test was done to test for lactose fermentation and to differentiate between Citrobacter. A SIM test was done to test for indole and H2S production to differentiate between Citrobacter. Samples were taken to inoculate an FTM to detect oxygen requirements. One is known to be gram negative and the other gram positive.

The results of the lysine test were vague. the test results were not clear because the solution looked only opaque rather than a definitive purple for a positive result or a yellow for a negative result. This test left only the possibility that the organism was of the genus Enterobacter. The ability to ferment lactose indicates that the organism could not be Proteus. 3. The test was incubated at 37˚F. Day 6 August 3.Day 4 July 29. The glucose test did not further eliminate any species that had not already been eliminated by the lactose test and the motility test. 2. The Glucose test indicated the organisms were capable of fermenting glucose and producing acid and gas. 2009 1. which tests for the decarboxylation of lysine into an alkaline end product. Salmonella. The lactose test indicated the organism was capable of fermenting lactose to produce acid and gas. The media was allowed to incubate for a total of four days. The SIM test indicated that the organism did not produce indole. A lysine test was done to differentiate between Enterobacter cloacae and Enterobacter aerogenes. 2009 1. The SIM test also tested for hydrogen sulfide production. which would indicate that the unknown organism was Enterobacter aerogenes. Day 5 July 30. 4. 5. Morganella or Serratia marcescens. MEDIA TESTS AND TEST RESULTS Table 1: Gram Negative Media Tests and Results Test/Media Gram stain FTM Motility Durham glucose tube Durham lactose tube SIM Lysine Biochemical/physiological characteristic Determining cell wall structure Determine oxygen requirements Determine motility Glucose fermentation to acid with gas Lactose fermentation to acid with gas Test for H2S production and indole production Tests for lysine decarboxylase Result Negative Staphylobacillus Facultative anaerobe Highly motile Positive for acid production and gas production Positive for acid production and gas production Positive for indole production Positive for H2S production Positive for lysine 7 . The negative result eliminated the possibility that the organism could be Citrobacter freundii. B. which also had negative results. The test eliminated the possibility that the organism could be Escherichia coli and Citrobacter diversus. Acaligenes faecalis. The only test differentiate between Enterobacter cloacae and Enterobacter aerogenes is a lysine test. Pseudomonas. After twenty-four hours. however with the assistance of the lab professor. it was interpreted to have a slight purple hue indicating that the test was positive. 2009 1.

C. FLOW CHART TO DETERMINE UNKNOWN GRAM NEGATIVE BACTERIA Gram Negative White colonial growth Non white colonial growth Cocci Bacillus Motile Non-motile Lactose non-fermenting Lactose fermenting Indole positive Indole negative H2S positive H2S negative Lysine positive Lysine negative Figure 2 Testing Route to Determine Unknown Gram Negative Bacteria 8 .

septic arthritis. Treatment for Enterobacter aerogenes infections are usually with use of antimicrobial therapies. osteomyelitis. Early studies have shown that E. endocarditis. they can also be opportunistic pathogens. sewage. and can be found in feces. water. Most exogenous infections occur as a result of antibiotic treatments. It is part of the normal flora of the human and animal intestines. The response to third generation cephalosporins and extended spectrum penicillin varies. F. urinary tract infections. aerogenes. lipid A. aerogenes. skin and soft-tissue infections. mediators for systemic inflammation (Enterobacter Infections 2008). The Lipid A acts as a stimulus for release of cytokines. Infection is rare amongst healthy individuals. CLINICAL SIGNIFICANCE Though Enterobacter aerogenes is part of the normal flora of humans. The microorganism is becoming increasingly more significant because of the rise in resistance of Enterobacter aerogenes infections to antibiotics and its association with nosocomial infections. lower respiratory tract infection. HABITAT Enterobacter aerogenes is a facultative anaerobe with an optimum temperature of around 37˚F. E. aerogenes is the most common form of infection. and ophthalmic infections (Enterobacter Infections 2008). E. aerogenes is more closely related to Klebsiella than to E. E. (Janda 2005). and there are current debates about reclassifying E. especially common as a nosocomial pathogen. there is a rising concern with E. They are also resistant to first and second generation cephalosporins. Nosocomial infections of E. aerogenes can cause several infections including bacteremia. aerogenes developing drug resistance. some dairy products and hospitals (Microbial Glossary). Fourth generation cephalosporins and carbapenems are fairly effective against E. cloacae. Enterobacter aerogenes virulence is due to the lipopolysaccharides. It has also been isolated from soil. which acts as an endotoxin. venous catheter insertions or surgical procedures. It can be differentiated from Enterobacter cloacae by lysine deaminase test. intra-abdominal infections. resistance has been reported to those drugs as well. SPECIAL CHARACTERISTICS Enterobacter aerogenes is motile through peritrichous flagella. 9 . aerogenes are generally resistant to narrow-spectrum penicillin drugs. Sources of infection can be endogenous from colonization of the microbe on the skin and in the gastrointestinal tract or exogenous as a result of the ubiquitous nature of the organism (Enterobacter Infections 2008). however. However.D.

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