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Bacillus Cereus Testing

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Process of identifying and testing for bacillus cereus - including test results and expected finding
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  Lab Write-up - The bacillus hidden in the food INTRODUCTION:  A dangerous case of food poisoning is reported. A dozen people are hospitalized. The patients have severe vomiting but no diarrhea. Patient O is identified and a sample specimen is isolated and taken. It is noted that the bacteria is a very fast grower. This specimen was maintained on NA slants throughout the semester after it had been isolated through the streak plate method. Colony and cellular morphology were determined by microscopic observation and various staining techniques. Metabolic and environmental growth was also tested for.  After taking note of the specimen’s various characteristics and looking up the different microorganisms listed in the identification extract of Bergey’s Manual of   Systematic Bacteriology, it is obvious that this specimen is bacillus cereus . “ Bacillus cereus, an infectious cause of foodborne illness, accounted for 2% of outbreaks with confirmed etiology that were reported to CDC during 1973-1987 .”   B. cereus  is a bacterium that produces toxins. These toxins have two different effects  –  one causes diarrhea while the other, called emetic toxin, causes nausea and vomiting. These bacteria can multiply quickly at room temperature. It is known as the cause of Fried Rice Syndrome”, due to the fact that the bacteria is classically contracted from fried rice dishes that have been sitting at room temperature for hours. METHODS: Instructions from the Microbiology lab workbook and handouts from Dr. Havner were applied in the multitude of tests performed on the specimen. Cultural characteristics and growth were observed/ tested through subculture on Nutrient Agar broth plate and slants. Cellular growth characteristics were found after the specimen was stained by negative and simple stain techniques. Gram stain characteristics were determined by standard Gram staining techniques which confirmed form of growth and determined the potential of the bacteria being a spore former. Endospore formation was then tested for using the endospore staining procedure outlined in the reference.  An Acid Fast stain was performed to test if bacterium was an intestinal based bacterium or if it was of the family mycobacteriums which are also rod shaped.  Aerobicity was analyzed by an NA deep stab and was confirmed during motility testing. Oxygen is a very reactive/destructive element and test determined if bacterium produced the enzyme superoxide dismutase which would enable it to use oxygen.  Differential media included Ma nnitol Salt, Eosin Methylene Blue, and MacConkey’s Agar plates. Optimal growth temperature was analyzed by incubation of the specimen at 4º C, 24º C, 37º C, and 60º C. Optimum pH for growth was determined by inoculation of Nutrient Broth tubes adjusted to pH = 3, 5, and 9 and subsequent incubation at optimum growth temperature. Optimum osmotic growth and salt tolerance was analyzed after inoculation of NA plates for various levels of NaCl concentration.  A test with blood agar as the media was used to see what type of hemolysis the bacterium was capable of. A Triple sugar Iron slant differential for the use of glucose, lactose and sucrose was also performed. The IMViC series of tests (Indole, Methyl Red, Voges-Proskauer, and Citrate) were performed on this bacterium as outlined by the lab manual. Antibiotic sensitivity testing included incubation in the presence of PML antibiotic disks containing Tetracyclin, Erythromycin, Streptomycin, Ampicillin, Penicillin and Novobiocin. Carbohydrate utilization was tested using carbohydrate fermentation tubes with Durham tube collection as well as inoculation of Triple Sugar Iron (TSI) tubes. Nutrient requirements were determined for this specimen by inoculation of starch, urea, Milk agar, and Gelatin agar plates followed by incubation at the optimal growth temperature. Casein utilization was proven to be negative by the use of Litmus Milk tubes. Sulfide production was tested using both the TSI and Motility Agar tubes. Finally enzymatic presence was tested by standard Catalase and Oxidase tests as described in the reference RESULTS: Cultural characteristics on NA plates and NA slants of the bacterium revealed that colonies of this specimen were large, filamentous and had a flaky texture to them. Growth was abundant and there was no pigmentation. Negative and Simple stains revealed this bacterium was a short, rod shaped bacteria. Form of growth was random. The Gram stain designated this bacterium as Gram Positive. The Acid Fast staining was negative. Endospore staining showed the presence of a high number of spores located at the ends. The NA Deep stab revealed that the bacterium was a facultative anaerobe. This specimen was found to have no growth on MacConkey’s  agar and no growth on EMB. It had very little growth on the Mannitol Salt Agar but did not utilize mannitol or produce any acid. Physical factor analysis indicated that this bacterium is halotolerant (higher salt concentrations did not affect growth) and grows best at a slightly acidic pH level and at  37º C. The specimen’s growth was rapid and spread from the stab entry point throughout the tube. This lead to the classification of the specimen as motile. Of the antibiotics tested, this specimen was sensitive to Tetracyclin, Erythromycin, Streptomycin and Novobiocin (although it was becoming resistant and starting to grow on Novobiocin). It was only resistant to Penicillin and Ampicillin, which both have the same mode of action and work by inhibiting cell wall formation. The bacterium was found to be gamma hemolytic. The bacteria displayed, through test results from the TSI tube, an affinity for glucose. It fermented glucose first and did not produce any significant amounts of sulfides or gas. The carbohydrate fermentation tubes confirmed the specimen’s ability to utilize glucose, lactose, and sucrose as indicated by the TSI tube. However, only glucose was actually fermented. No gas was produced in any of the carbohydrate fermentation tests. Acid was produced only in glucose fermentation. The specimen grew on the both Casein Agar plate and the gelatin agar but did not utilize either of them. The presence of urease was indicated by a positive Urea slant. IMViC series characterized the specimen as Indole negative, MR positive, VP and Citrate negative. The Catalase test was positive while the Oxidase test was negative. CONCLUSION: The specimen is a sample of bacillus cereus . The fact that this was a gram positive bacillus that produced spores narrowed the list of possible microorganisms considerably. Only clostridia and bacillus form spores. Of these, only bacillus can grow in oxygen and since the NA deep stab proved it was a facultative anaerobe, it was definitely a bacillus. Of the 3 bacilli listed in the Identification extract, aerobicity (facultative) and temperature favored (37º C) were the most significant factors in speciation. Bacillus cereus grows rapidly at both room and clinical temperatures; however, its optimum growth temperature is 37º C. The symptoms reported by the patients - severe vomiting but no diarrhea and the environment the bacterium was transmitted (through food, perhaps left out too long) are common for infections of bacillus cereus. References Paul A. Granato, Morello, Josephine A, and Helen Eckel. Mizer. Lab Manual and Workbook in Microbiology  . New York: McGraw-Hill Higher Education, 2010. Print.   Bacillus Cereus. Food Poisoining  . U.S. Department of Health & Human Services, n.d. Web. 18 Nov. 2013. http://www.foodsafety.gov/poisoning/causes/bacteriaviruses/bcereus/ Bacillus Cereus Food Poisoning Associated with Fried Rice. Centers for Disease Control and Prevention . Centers for Disease Control and Prevention, 19 Sept. 1998. Web. 18 Nov. 2013. http://www.cdc.gov/mmwr/preview/mmwrhtml/00025744.htm Bergey’s Manual of Systematic Bacteriology  (extract), 1 st  Edition, Williams and Wilkins, Baltimore, 1986
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