Bacillus cereus and Food Poisoning | Microbiology Lab Report

Unknown Microbiology Lab Report

Ken Ly

12/03/13

General Microbiology

Fall 2013

Introduction

There are many different species of bacteria that exist in the world, depending on their pathogenic characteristics.  From determining the cause of a disease to diagnosing a patient, to developing microorganisms for certain antibiotics, it is essential that pathogens are identified.  To this end, a variety of methods were utilized to correctly identify the unknown bacterium.




Materials and Methods

A tube labeled as “unknown # 113” containing two bacteria were given out by the microbiology instructor along with an unknown chart for identification.  Tests and procedures were based on information from laboratory manual 1 ( McDonald et al.2011) were used to identify the unknown #113.  Final results were recorded in Table 1 and Table 2, and flowcharts are also provided.

In an effort to obtain isolated colonies, a pure culture was carefully obtained from the unknown # 113, and inoculated onto the Nutrient agar plate through quadrant technique.  Through this process, two colonies developed, and each was transferred to a separate Nutrient agar plate.  .  Both plates were placed in a 37-degree incubator to obtain a pure culture without contamination and a Gram stain method was used to determine the type and shape of bacterium.  Certain biochemical tests were performed based on the unknown identification chart provided.  The unknown #113 was determined by Gram-positive rod and Gram-negative rod.

Table1. Gram-positive rod bacterium includes tests, purposes, reagents, observations, and results

All of the following tests were performed on this unknown:

1. Methyl red test
2. Casein test
3. Maltose test

Table 2. Gram-negative rod bacterium includes tests, purposes, reagents, observations, and results

All of the following tests were performed on this unknown:

1. Simmon’s citrate test
2. Methyl red test
3. EMB test

 

Results

The unknown #113 cultivated two isolated colonies, which were similar in size. Unknown # 113 had opaque cream-colored heterogeneous colonies. The Gram stain test differentiated between the two by identifying one as a Gram-positive rod and one as a Gram-negative rod.

A Methyl red test was performed on the Gram-positive rod resulting in the yellow broth turning red.  Next, a Maltose test was conducted resulting in the red broth turning yellow indicating it was positive for Maltose.  Finally, the Casein test was performed resulting positive for Casein.

A Simmon’s citrate test was performed on the Gram negative rod resulting in green agar to blue indicating positive for Simmon’s citrate test.  Next, a methyl red test was conducted resulting in the yellow broth not turning red indicating negative for Methyl red test.  Finally, an EMB test was performed indicating positive for lactose.

 

Table 1 & Table2 Results

 

TABLE 1. Gram Positive Rod Methods

TEST	PURPOSE	REAGENTS	OBSERVATIONS	RESULTS
Gram stain	Determining Gram positive or Gram negative bacterium.	Crystal violet dye, Gram iodine, Alcohol, Safranin.	Purple rods.	Gram positive rods.
Methyl red test	Determining the presence of acids from glucose fermentation.	Six drops of methyl red.	Yellow broth turned to red color.	Positive methyl red test.
Maltose test	Determining the presence of acid from glucose fermentation.	None.	Red broth turned to yellow color.	Positive maltose test.
Casein  test	Determining the presence of casease.	None.	Clearing around bacterial growth.	Positive Casein test.

 

TABLE 2. Gram Negative Rod Methods

TEST	PURPOSE	REAGENTS	OBSERVATIONS	RESULTS
Gram stain	Determining Grampositive or Gram negative bacterium.	Crystal violet dye, Gram iodine, Alcohol, Safranin.	Bright pink rods.	Gram negative rods.
Simmon’s citrate agar	Determining the presence of citrate permease.	None.	Green medium turned to blue color.	Positive citrate permease test.
Methyl red	Determining the presence of mixture acid from glucose fermentation.	Six drops of methyl red.	Yellow broth turned to red color.	Negative methyl red test.
EMB agar	Determining the presence of acid from lactose fermentation.	None.	Dark red medium turned to pinkish-purple color.	Positive lactose fermentation test.

 

 




Discussion / Conclusion

After many tests, the unknown #113 was identified to be both Bacillus cereus and Enterobacter aerogenes. This was determined after the unknown #113 was separated into two colonies. The two colonies were then identified by transferred to separate nutrient agar plates to obtain pure colonies.  Gram stain was performed on each colony and one stain showed purple rods which indicates Gram positive rods and one stain showed bright pink rods which determined it to be Gram-negative rods.

Since one colony was determined to be a Gram-positive rod the Methyl red test was performed to determine the presence of acids from glucose fermentation. After the methyl red drops were administered, the yellow broth turned a red color which meant that it was a positive methyl red test. To furthermore determine the identification of the colony, the Maltose test was performed to determine the presence of acid. After incubation, the result was the red broth turning a yellow color which determined that the maltose test was positive. This is true because the red basic broth turned acidic yellow overnight which means the microbes underwent glucose fermentation. Lastly, the Casein test showed clearing around the bacterial growth which indicates a positive Casein test. The clearing around the microbes means that the bacteria produced the enzyme Casease which hydrolyzes the milk protein Casein. Positive methyl red, positive maltose, and positive casein indicate that this Gram-positive rod is Bacillus Cereus.

The unknown #113 also contains Gram-negative rod microorganisms.  Because of this, Simmon’s citrate test was performed and the microorganisms were inoculated onto the surface of Simmon’s citrate agar tube. The green medium turned to a blue color which determines it to be a positive citrate permease test. The agar is used to determine if an organism can use citrate as its sole carbon source. If citrate is used pyruvic acid and carbon dioxide are produced. Carbon dioxide reacts with components of the medium to produce an alkaline compound. The pH levels of the alkaline compound turn the medium blue which indicates the presence of citrate permease. Again, the Methyl red was performed which did not turn red color which indicates a negative test. Finally, the Eosin Methylene Blue (EMB) agar test was done on the microbes. The dark red medium turned into a pinkish-purple color which determined positive for lactose fermentation. The EMB agar contains eosin and methylene blue dyes which is selective because it inhibit the growth of gram-positive. Lactose fermentators metabolize the lactose production acid by-products causing color change in the colony. Weaker fermentation of lactose results in colonies with a pinkish-purple color. If it were a stronger acid, the color would have resulted in a metallic green sheen. Positive citrate permease, negative methyl red, and positive lactose fermentation indicate that the Gram-negative is Enterobacter aerogenes.

Since all of the tests came back clearly the identification of the unknown #113 was correct. The problem that was encountered was from the original tube of colonies given. After the first round of tests, only one of the colonies was identified correctly. The other colony was only able to be obtained by obtaining a second tube. The inability to identify the second colony the first time could have been because of various things; for which exact reason is unsure. It could have been from contamination of the tube prior to being tested, not completely sterilizing the inoculating loop by the flame, or an error in preparing the tube.

Bacillus cereus (B. Cereus) is a Gram-positive aerobic or facultatively anaerobic spore-forming rod bacterium that can be found in food. It is spore-forming and motile and can grow well anaerobically. It can produce toxins that lead to two types of food illnesses.  One type is characterized by diarrhea call enterotoxin, and the other by nausea and vomiting known as emetic toxin. B. cereus is becoming one of the major causes of food poisoning in the industrialized world due to its toxins.   B. cereus is capable of producing beta-lactamase, which could resist beta-lactamase antibiotics; however, it is usually susceptible to treatment with clindamycin, vancomycin, gentamicin, chloramphenicol, and erythromycin². (Granum.2006)

 

 

References

1.  McDonald, Virginia, Mary Thoele, Bill Salsgiver, and Susie Gero(2011). Lab Manual for General Microbiology.
2.  Granum P.E., & Lund T. (2006). Bacillus cereus and its food poisoning toxins. FEMS microbial. Lett. 2, 223-228.