How to Identify Bacillus cereus | Microbiology Unknown Lab

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UNKNOWN LAB REPORT

Unknown Bacterium = B. cereus

Jamie Nichols

Microbiology

INTRODUCTION: 

After becoming knowledgeable of microorganisms and the methods used for indication in the laboratory classroom, this study was done for the identification of an unknown bacterium. There are many reasons for pinpointing the identity of microorganisms.  Reasons vary from identifying a specific organism causing a medical problem as treatments are heavily dependent on the identity of the microorganism, to producing helpful antibiotics such as penicillin produced from microorganisms and food where they take part in preservation.

 




MATERIALS AND METHODS: 

On October 22, 2013 an unknown tube numbered 112 was handed out by our lab instructor. The goal is to determine two separate unknown bacterium, one as Gram positive and one as Gram Negative using various differential tests.  The procedures and sterile techniques learned throughout the course to identify bacteria were applied to these unknown. The laboratory manual procedures by McDonald et. al (1) were followed as instructed unless otherwise noted.

This experiment of finding the unknown bacteria of 112 was first started by inoculating a nutrient agar (NA) plate with the unknown using the isolation streak method. The plate is then inverted and incubated at 37 degrees Celsius for two days. The bacterium grew and was studied based on the physical characteristics. Two distinct different colonies grew. Each colony was isolated to grow purely on its own NA plate. One was labeled “A” and the other “B”.  Gram stains were performed on each microbe to determine the stain color and the shape of the cells. Microbe A was determined to be Gram positive rods and microbe B was Gram negative rods.  Biochemical tests were performed based on the identification table given by the lab instructor. All tests were performed according to the methods stated in the lab manual by McDonald et. al (1).

Table 1 and 2 lists the test, purpose, regent, observations, and results.

Tests performed for Gram positive unknown:

1. Methyl Red- (MR)
2. Glycerol (Gy)
3. Maltose (MI)
4. Casein
5. Oxidase

Test performed for Gram negative unknown:

1. Simmons Citrate
2. Methyl Red (MR)
3. Nitrate
4. Glycerol (Gy)
5. Maltose (MI)
6. Lactose (L)
7. Urea 

RESULTS: 

(This section contains flow charts)

Microbe colony A (Gram positive) had the following morphology on a NA plate: grey, opaque with an irregular surface. After pinpointing it was a Gram positive rod, a Methyl Red-Voges Proskauer (MRVP) test was conducted as well as other testes stated in table 1. These tests and the direction of the lab instructor pointed to Bacillus cereus.

Microbe colony B (Gram negative) had the following morphology on a NA plate: White, irregular, milky colonies. After pinpointing it was a Gram positive rod, a Simmons Citrate Slant test was conducted as well as other tests stated in table 2. These tests pointed to Escherichia coli.

Table 1: Gram positive (A)

Table 2: Gram negative (B)

Flowchart – REMOVED due to formatting issues. 

 




Discussion/Conclusion:

After the completion of the biochemical tests it was concluded that Escherichia coli (E. coli) was the Gram negative of unknown 112. The bacterium was grown and isolated on a nutrient agar plate for further testing. A Gram stain was done and indicated it as a Gram negative rod. The bacterium was then inoculated into a MRVP broth. After incubation the indicator methyl red was added and turned a cherry red color indicating a positive reaction for acid production from glucose metabolism. After a positive Methyl Red (MR) test, the unknown possibilities were then limited to E. coli, Klebsiella pneumonia (K. pneumonia) or Proteus vulgaris (P. vulgaris). Lactose fermentation is a characteristic of both E. coli and K. pneumonia but not of P. vulgaris so a lactose test was then performed. After inoculating a lactose fermentation broth tube and allowing the incubation time the test read positive for lactose fermentation. The evidence of carbohydrate fermentation was seen by the change in color from red to yellow indicating acid production. With P. vulgaris eliminated as it does not produce acid from carbohydrate fermentation only, E. coli and K. pneumonia are left. Because nitrate reduction is a characteristic of K. pneumonia and not of E. coli a nitrate test was conducted. After inoculation and incubation time was complete nitrate I and II were added. There was no color change indicating a negative result of reduction or conversion of NO² or NO³ nitrogenous compounds. Then zinc was added. No change in color indicates a positive result for conversion to some reduced form of nitrogen. This result now eliminates E. coli, leaving K. pneumonia. To confirm K. pneumonia was the unknown a Simmons citrate test was performed to determine if the organism can use citrate as its only carbon source. This is a characteristic of K. pneumonia and not of E. coli. After inoculation and incubation the test results read negative leading to E. coli as an option again. This is a contradicting result as compared to the unknown chart handed out by our lab instructor. A Maltose and Glycerol test were performed however there were no selective differences between K. pneumonia and E. coli. A Urea broth tube was then inoculated and incubated. The hydrolysis of urea produces ammonia which is a weak base. This is a characteristic of K. pneumonia and not of E. coli.  Phenol red was added. The broth did not turn from yellow to red indicating no change in pH. All tests results with the exception of the nitrate test indicate E. coli.  Lab instructor confirmed that the Gram negative rod of unknown 112 was E. coli. The nitrate test described previously, which would have led to an earlier conclusion of E. coli, was likely contaminated during inoculation causing a positive result instead of confirming a negative result.

Testing for the Gram positive bacterium was taking place simultaneously. Originally a round, yellowish colony was grown and isolated on a NA plate. After Gram staining was performed it was determined to be a Gram positive rod. This ruled out all of the cocci bacterium. After many biochemical tests, the results never matched up with a particular bacterium’s characteristics listed on the unknown chart. Two more isolations were attempted and tests were repeated. After consultation with lab instructor it was confirmed that the correct bacterium was not isolated for testing purposes. Contamination of the isolation was the reasoning for the incorrect bacterium grown. At this point a new pure culture of the correct bacterium was received from the lab instructor.  The bacterium was grey and opaque in appearance. A Gram stain was done and indicated it as a Gram positive rod. This again ruled out the cocci shaped bacterium. Bacillus cereus (B. cereus) and Bacillus subtilis (B. subtilis) bacteria were left of the rod shaped bacterium’s to be tested.  A milk agar plate was inoculated with the unknown isolation of bacterium because it differentiates by the production of the enzyme casease that hydrolyzes the milk protein casein. This is a characteristic of B. cereus and not of

B. subtilis. Results after incubation time showed that the enzyme was being produced. The maltose test was then executed to check for acid and gas production by the fermentation of the specific carbohydrate. This test presented a positive result. The red coloring of broth turned yellow revealing acid production, and a bubble present in the durham tube represented the presence of gas.  This test suggests the bacterium’s identity as B. cereus. To further confirm that this was the unknown bacterium a MR test was conducted.  After inoculation and incubation of the bacterium the pH indicator methyl red was added. With no change in color present the test was negative. Acid as a byproduct of glucose fermentation is a characteristic of B. cereus and not of B. subtilis. This is now a contradicting test and suggests B. subtilis bacterium is the unknown. The Glycerol test was then conducted to test for acid production by the fermentation of this specific carbohydrate. The Glycerol broth tube was inoculated, incubated, and then read. Acid production is a characteristic of B. cereus and not of B. subtilis. With no change in color the negative glycerol test suggests the bacterium is B. subtilis. Two tests suggest B. cereus and two suggest B. subtilis. To confirm which bacterium an oxidase test was conducted to identify organisms that produce the enzyme cytochrome oxidase. After inoculating the paper with the unknown and dropping sterile water on top, the test was read within a fifteen second period to avoid false positives. Without the presence of cytochrome oxidase the paper did not turn a purple or blue color deeming it a negative test. This was the confirming test to B. subtilis.

After consulting with the lab instructor, B. subtilis was not the correct bacterium even though test results were suggestive. B. cereus was the correct bacterium inoculated into tube 112.  The MR tests as well as the oxidase test were re-conducted with no change in the results.  These tests should have shown a positive result identifying B. cereus earlier in the testing process as the Gram positive bacterium. The probability for the inaccurate results of the oxidase test could have been that the bacterium was an older culture. In addition the MR test was probably contaminated.

 Bacillus cereus is a Gram-positive aerobic or facultative anaerobic, motile, spore-forming, rod-shaped bacterium that is commonly found in soil and dust or in raw vegetation such as tomatoes or rice (2). It is a bacterium belonging to the Bacillaceae family of the Firmicutes and is recognized as an agent for food poisoning. Most strains of B. cereus are motile but small percentages are non-motile. It is commonly found in soil and is why it can be found in food of plant and animal origins. Some strains are harmful causing foodborne illnesses in humans, while other strains can be useful as probiotics for animals (2). B. cereus is known for producing two types of food-borne illnesses. One type resembles the symptoms and incubation time of Staphylococcus aureus (staph). It is characterized by nausea, vomiting and abdominal cramps and is the emetic (short-incubation) form of the disease (3).

The second type of food-borne illness primarily causes abdominal cramping and diarrhea and is referred to as the long incubation or diarrheal form of the disease. It resembles food poisoning caused by Clostridium perfringens. Both the emetic and diarrheal forms of the illness usually lasts less than a twenty-four hour period after invasion (3).

References

1. McDonald, V., Thoele, M., Salsgiver, B., Gero, S. (2011). Lab Manual for General Microbiology: Bio 203. N.p : Saint Louis Community College at Meramec 

1. Bottone, Ej (2010, Apr. 23). Bacillus cereus, a volatile human pathogen. In PubMed. Retrieved November 29, 2013, from http://www.ncbi.nlm.nih.gov/pubmed/20375358 

1. Todar, K. Bacillus cereus Food Poisoning. In Todar’s Online Text Book of Bacteriology.  Retrieved November 29, 2013 http://textbookofbacteriology.net/B.cereus.html

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