Microbiology Student Lab Number 25

Major Unknown Date: Before Due Date

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

I.

Gram Positive Unknown Organism: Bacillus subtilis

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

6. A Voges-Proskauer test was conducted by inoculating the media with the unknown and incubated 37F for twenty-four hours. The test was to detect the production of acetoin and to differentiate between B. megaterium, B. stearothermophilus, B. sphaericus and B. subtilis.

Day 4 July 29, 2009 1. The Simmon Citrate test indicated the bacterium was capable of producing citrase. Citrase production indicates that the unknown could not be B. stearothermophilus. 2. After the Voges-Proskauer media was incubated with the microbe at 37F for twenty-four hours, Voges-Proskauer reagent A and reagent B were added to the mixture. Though the mixture appeared green at first, after a period of approximately ten minutes, the mixture turned red which indicated a positive reading for acetoin. This test eliminated the possibility the unknown could be B. megaterium or B. sphaericus, which only left B. subtilis. 3. A glucose test was done to confirm B. subtilis. The glucose test was incubated over night for 37F. Day 5 July 30, 2009 1. Results for the glucose were read. The test result showed that the bacteria could ferment glucose and produce acid, but not gas. This coincides with the capabilities of Bacillus subtilis.

B.

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, streptobacillus Positive Obligate aerobe Moderately motile Positive Positive Positive Positive for acid production; negative for gas

C.

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

D.

HABITAT
Bacillus subtilis is an obligate aerobe and a ubiquitous organism commonly found in water, soil, air and decomposing plant residue (Bacillus subtilis Final Risk Assessment [updated 2007]). Because of its ubiquity, the organism is capable of growing in a wide range of temperatures. It may also be capable of growing on the human body, however lacks mechanism for attachments, therefore might only be transient inhabitants of the body. In the soil or water, it must compete with other microbes and is also exposed to variations in temperature, humidity and nutrient concentrations. To overcome its adversities, B. subtilis must have mechanisms to not only protect itself and find nutrients, but also fend off competitors.

E.

CLINICAL SIGNFICANCE
Bacillus subtilis is not known to be pathogenic to humans, though some studies have shown that they are capable of producing toxins that may be responsible for food poisoning. B. subtilis is considered to have low virulence to humans (Bacillus subtilis Final Risk Assessment [updated 2007]). However, because their natural habitat is in areas where competing bacteria, fungus and other microorganisms exist, they are capable of producing insect toxins, peptide antibiotics, and antifungals (Bacteria Genomes Bacillus subtilis [updated 2009]). B. subtilis is capable of producing over two dozen antibiotics including bacitracin, a topical antibiotic used to prevent infections (Stein 2005). It is one of the most widely used bacteria for the production of enzymes. It also has industrial applications which allow for the production of amylase, protease, inosine, ribosides and amino acids. The ability of B. subtilis to produce antifungals have also lend to the production of fungicides and treatment for agricultural crop protection.

F.

SPECIAL CHARACTERISITCS
Bacillus subtilis was the first Gram positive bacteria to be sequenced. The endospore staining test showed that B. subtilis is capable of producing endospores, which would allow it to survive harsh times with limited nutrients and aversive environmental conditions. The endospore can withstand extreme conditions of high heat, acidity and salinity. During these times, however, the organism is not biologically active (Bacillus subtilis Final Risk Assessment [updated 2007]). The result of the motility test showed that the organism was motile. Research from literature confirms that B. subtilis is capable of movement by peritrichous flagella (Bacillus subtilis Final Risk Assessment [updated 2007]). When it is active, B. subtilis utilizes chemotaxis and uses its flagella to move towards nutrients, as well as away from toxins from competing microorganisms (Sonenshein 2002). B. subtilis is also capable of producing a variety of enzymes to both cycle nutrients and ward of offensive organisms as mentioned above.

II. Gram Negative Unknown Organism: Enterobacter aerogenes

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

Day 4 July 29, 2009 1. The lactose test indicated the organism was capable of fermenting lactose to produce acid and gas. The ability to ferment lactose indicates that the organism could not be Proteus, Pseudomonas, Salmonella, Acaligenes faecalis, Morganella or Serratia marcescens. 2. The Glucose test indicated the organisms were capable of fermenting glucose and producing acid and gas. The glucose test did not further eliminate any species that had not already been eliminated by the lactose test and the motility test. 3. The SIM test indicated that the organism did not produce indole. The test eliminated the possibility that the organism could be Escherichia coli and Citrobacter diversus. 4. The SIM test also tested for hydrogen sulfide production, which also had negative results. The negative result eliminated the possibility that the organism could be Citrobacter freundii. This test left only the possibility that the organism was of the genus Enterobacter. The only test differentiate between Enterobacter cloacae and Enterobacter aerogenes is a lysine test, which tests for the decarboxylation of lysine into an alkaline end product. 5. A lysine test was done to differentiate between Enterobacter cloacae and Enterobacter aerogenes. The test was incubated at 37F. Day 5 July 30, 2009 1. After twenty-four hours, 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. The media was allowed to incubate for a total of four days. Day 6 August 3, 2009 1. The results of the lysine test were vague, however with the assistance of the lab professor, it was interpreted to have a slight purple hue indicating that the test was positive, which would indicate that the unknown organism was Enterobacter aerogenes.

B. 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

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

D. HABITAT
Enterobacter aerogenes is a facultative anaerobe with an optimum temperature of around 37F. It is part of the normal flora of the human and animal intestines, and can be found in feces. It has also been isolated from soil, water, sewage, some dairy products and hospitals (Microbial Glossary).

E. CLINICAL SIGNIFICANCE
Though Enterobacter aerogenes is part of the normal flora of humans, they can also be opportunistic pathogens, especially common as a nosocomial pathogen. E. aerogenes can cause several infections including bacteremia, lower respiratory tract infection, skin and soft-tissue infections, urinary tract infections, endocarditis, intra-abdominal infections, septic arthritis, osteomyelitis, and ophthalmic infections (Enterobacter Infections 2008). Nosocomial infections of E. aerogenes is the most common form of infection. Infection is rare amongst healthy individuals. 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). Most exogenous infections occur as a result of antibiotic treatments, venous catheter insertions or surgical procedures. Enterobacter aerogenes virulence is due to the lipopolysaccharides, lipid A, which acts as an endotoxin. The Lipid A acts as a stimulus for release of cytokines, mediators for systemic inflammation (Enterobacter Infections 2008). Treatment for Enterobacter aerogenes infections are usually with use of antimicrobial therapies. However, there is a rising concern with E. aerogenes developing drug resistance. E. aerogenes are generally resistant to narrow-spectrum penicillin drugs. They are also resistant to first and second generation cephalosporins. The response to third generation cephalosporins and extended spectrum penicillin varies. Fourth generation cephalosporins and carbapenems are fairly effective against E. aerogenes, however, resistance has been reported to those drugs as well.

F. SPECIAL CHARACTERISTICS
Enterobacter aerogenes is motile through peritrichous flagella. It can be differentiated from Enterobacter cloacae by lysine deaminase test. Early studies have shown that E. aerogenes is more closely related to Klebsiella than to E. cloacae, and there are current debates about reclassifying E. aerogenes. (Janda 2005). 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.

WORKS CITED

1. Bacillus subtilis Final Risk Assessment [internet]. [updated 2007 Sept. 24] U.S. Environmental Protection Agency, Biotechnology Program Under Toxic Substances Control Act (TSCA); [cited 2009 August 4]. Available from: http://www.epa.gov/biotech_rule/pubs/fra/fra009.htm 2. Bacteria Genomes Bacillus subtilis [internet]. [updated 2009]. European Bioinformatics Institute (EBI); [cited 2009 August 6]. Available from: http://www.ebi.ac.uk/2can/genomes/bacteria/Bacillus_subtilis.html. 3. Basic Characteristics for Identification of Selected Bacillus Species [internet]. [cited 2009 August 4]. Available from: http://faculty.sdmiramar.edu/dtrubovitz/micro/bacillus.pdf 4. Brown, Alfred. 2009. Bensons Microbiological Application Laboratory Manual in General Microbiology. Short version. 11th ed. McGraw Hill. p. 294. 5. Cappucchino, James G. and Natalie Sherman. 2001. Microbiology A Laboratory Manual. 6th ed. Benjamin Cummings. p. 428-431. 6. Differentiation of Enterobacteriaceae by Biochemical Test [internet]. [cited 2009 August 4]. Available from: http://faculty.sdmiramar.edu/dtrubovitz/micro/Enterobacteriaceae.pdf. 7. Differentiation of Typical Salmonella [internet]. [cited 2009 August 4]. Available from: http://faculty.sdmiramar.edu/dtrubovitz/micro/Salmonella.pdf. 8. Enterobacter Infections [internet]. [updated 2008 August 28]. EMedicine from WebMD; [cited 2009 August 6]. Available from: http://emedicine.medscape.com/article/216845-overview. 9. Janda, Michael J. and Sharon L. Abbot. 2005. The Enterobacteria [internet]. 2nd ed. ASM Press; [cited 2009 August 6]. Available from: http://books.google.com. 10. Microbial Glossary [internet]. Environmental Diagnostic Laboratory (EDL); [cited 2009 August 6]. Available from: http://www.pureaircontrols.com/glossary.html 11. Sonenshein, A.L., James A. Hoch, Richard Losick. 2002. Bacillus subtilis and its Closest Relatives [internet]. Washington DC: ASM Press American Society for Microbiology. [cited 2009 August 4]. Available from: http://books.google.com. 12. Stein, Torsten. 2005. Bacillus subtilis antibiotics: structures, synthesis and specific functions [internet]. Frankfurt, Germany: Institute fur Microbiologie. [cited 2009 August 6]. Available from: http://www3.interscience.wiley.com/journal/118659809/abstract?CRETRY=1&SRETRY=0.

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