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Microbiology Coursework: Bacillus Cereus

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Microbiology Coursework: Bacillus cereus After investigation following on outbreak of food poisoning at a pizza restaurant, it was found that all suffers had consumed a portion of side salad from the self-service salad bar alongside their main dish. Subsequently, this was further traced to a rice salad. Environmental Health Officers investigating this outbreak suspected it may have been caused by Bacillus cereus (B. cereus). The presence of large numbers of B. cereus in a food is indicative of active growth and proliferation of the organism and is consistent with a potential hazard to health. The diagnosis of B. ereus can be confirmed by the isolation of more than 105 B. cereus organisms per gram from epidemiologically implicated food, but such testing is often not done because the illness is relatively harmless and usually self-limiting 1. Design a method(s) to enumerate the: i)Total bacterial count ii)Bacillus cereus count In the rice salad

This outbreak of food poisoning could be investigated by performing an enumeration (plate count) of the total viable bacteria in the rice salad on a general non-selective agar using either the pour or the spread plate method. To confirm that the outbreak had been caused by any B. ereus present in the rice salad a selective media agar, such as mannitol egg yolk polymixin agar (MEYP/MYP), should be used. Once B. cereus has been confirmed a further enumeration of the B. cereus should be performed on the MEYP/MYP agar selective media plate to show whether the amount of B. cereus present is within the range known to cause food poisoning 105–107 cells g? 1 of food for Diarrhoeal syndrome, or 105–108 cells g? 1 of food for Emetic syndrome. (Granum & Lund, 2006) To perform a total cell count and the confirmation of B. cereus by either the pour or spread plate method the equipment required is as follows:

General non-selective agar Mannitol egg yolk polymixin agar (MEYP/MYP) Petri dishes Glass or disposable “hockey stick” spreader Bunsen burner Test tubes Ringers solution Pastettes / Pippettes Food blender Before a cell count can be performed a serial dilution of an homogenate of the rice salad is required. For this one part rice salad is blended to nine part ringers solution, from this initial homogenate that the serial dilution is created by taking 1ml of this original and adding it to 9ml of ringers solution thereby creating a 1:10 dilution of the original.

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This step is repeated a further 5 times, each time taking 1ml from the dilution created in the previous tube and adding it to 9ml of ringers solution thereby with each step the original sample is diluted by a further factor of 10, (Figure 1). Once the serial dilution has been completed down to a dilution of 1:1,000,000 (10-6) either the pour or spread plate method of plating out of the samples can be performed Figure 1: Serial dilution When using a general non-selective agar both the pour and spread plate methods can be used for enumeration of the total bacteria in the rice salad.

With both methods all plates are performed in triplicate. Along-side the non-selective agar, an agar such as MEYP/MYP selective agar which is selective for B. cereus can be used to confirm that B. cereus is present in the original sample. In the pour plate method 1ml or 0. 1ml of each of the dilutions prepared earlier within the serial dilution are added to individual petri dishes and a nutrient agar which is held at around 50oC is poured over each of these samples, the petri dishes are swirled causing gentle agitation and mixing the bacteria with the agar.

After the agar has solidified the plates are incubated, after this incubation the pour plates show bacterial growth both on and within the agar due to aerobic and anaerobic bacteria. In the spread plate method 0. 1ml of each of the serial dilution solutions is pipetted onto the surface of a pre-poured agar plate and spread using a “hockey stick” spreader, the agar plates are then incubated. Bacterial colonies only grow on the surface of the spread plate, (Figure 2) Figure 2: Method of Pour and spread plate technique. Microbial Growth, 2011) Once the plates have been incubated they are examined and the number of colonies counted, only plates that show between 30-300 colonies are counted, if the number of colonies is above 300 then the plate is discarded as too numerous to count, if below 30 it is discarded as too few to count. After the plates showing between 30-300 colonies have been counted the number of bacteria in the original sample can be worked out using the calculation Number of colonies on plate x dilution of sample = number of bacteria / ml

If growth has occurred on the MEYP/MYP plates, a Gram stain can be performed on a sample from one of the colonies, when the gram stain is examined under oil immersion B. cereus should appear as large Gram-positive bacilli in short-to-long chains; with spores that are ellipsoidal, central to subterminal, and that do not swell the sporangium. (Tallent, Rhodehamel , Harmon, & Bennett, 2012) (Figure 3) Figure 3: flow diagram showing order of events leading to the enumeration of total bacteria and Bacillus cereus in a sample of food. 2.

Explain why MEYP/MYP agar is selective for Bacillus cereus B. cereus is mannitol-negative. The mannitol content of the medium thus allows differentiation of the accompanyingmannitol-positive microbial flora which are identified by a change in colour of the indicator phenol red to yellow. B. cereus is not affected by concentrations of polymyxin which inhibit the common accompanying microbial flora (Donovan, 1958). Addition of polymyxin is necessary, however, if the sample material is suspected to contain high-numbers of accompanying microorganisms B. cereus produces lecithinase.

The insoluble degradation products of egg-yolk lecithin accumulate around the Cereus colonies to form a white precipitate. A lecithinase reaction occurs very early in many strains, Cereus colonies can, therefore, often be rapidly identified before accompanying polymyxin-resistant microorganisms have had a chance to fully develop. Incubation: 18-40 hours at 32 °C. B. cereus appears as rough, dry colonies with a pink to purple base which are surrounded by a ring of dense precipitate. Colonies surrounded by a yellow or a clear zone are not Bacillus cereus.

Further tests should be performed to confirm the identity of Bacillus cereus (anaerobic degradation of D(+)glucose, degradation of gelatin, positive nitrate reduction). (Merck, 2012) 3. Suggest how health officers may have come to the tentative conclusion of B. cereus poisoning. Health officers may have come to this conclusion based on the short incubation time to the sudden onset of illness, and due to rice already being implicated as the source of this type of food poisoning in other cases. 4. Suggest ways in which: i. The rice salad might have been infected by the Bacillus cereus; ii.

The Bacillus cereus could have survived the normal cooking process of the rice; iii. Bacillus cereus causes food poisoning. B. cereus is present in the outer casing of rice and, because it is able to form spores that are very resistant to low or high temperatures, it can therefore easily survive cooking and less-than perfect refrigeration. Improper storage of food stuffs is the issue. Bacillus cereus spores can survive boiling and if the food, in this case rice is stored at ambient temperature, the spores can germinate into toxin producing bacteria. Herriman, 2009) Bacillus cereus has been reported to be present in stools of healthy humans at varying levels (Johnson, 1984) therefore if an individual had not washed their hands after going to the toilet then handled the serving spoon any B. cereus from the hands could be transferred to the serving spoon which in turn could either infect the rice salad or the hand of the person next using the spoon. When rice is boiled and then stored in the fridge without being cooled first, these spores can germinate on the cooked rice and grow well at 4oC.

If the rice is then used in a stir fry or similar dish, where the cooking time is relatively short, or the rice is held at an insufficient temperature enough of the bacteria survive to be ingested. Bacillus cereus causes food poisoning of two different types, emetic and diarrhoeal. (Table 1) Table 1. Characteristics of the two types of disease caused by Bacillus cereus Diarrhoeal syndromeEmetic syndrome Infective dose105–107 (total)105–108 (cells g? 1) Toxin producedIn the small intestine of the hostPreformed in foods Type of toxinProteinCyclic peptide Incubation period8–16 h (occasionally >24 h)0. –5 h Duration of illness12–24 h (occasionally several days)6–24 h SymptomsAbdominal pain, watery diarrhoea and occasionally nauseaNausea, vomiting and malaise (sometimes followed by diarrhoea, due to additional enterotoxin production? ) Foods most frequently implicatedMeat products, soups, vegetables, puddings/sauces and milk/milk productsStarch-rich foods; Fried and cooked rice, pasta, pastry and noodles The form that produces diarrhoea is accompanied by symptoms that are virtually indistinguishable from those caused by the Clostridium perfingens bacteria.

The affected person experiences abdominal cramps and severe watery diarrhoea within about 15 hours of eating the contaminated rice. Vomiting rarely occurs but the diarrhoea carries on between 1 and 2 days. The diarrhetic syndromes observed in patients are thought to stem from the three toxins Hemolysin BL Hbl, Nonhemolytic Enterotoxin Nhe and Cytotoxin K CytK. These enterotoxins are all produced in the small intestine of the host, thus thwarting the issue of digestion by host endogenous enzymes. Some strains of the bacteria have an extra plasmid that carries a gene for a toxin that causes severe vomiting.

These strains cause the emetic form of Bacillus cereus and produce symptoms very similar to food poisoning by Staphylococcus aureus. After ingesting rice contaminated with these strains, vomiting begins between 1 and 5 hours. The effects are fairly short-lived and the digestive system usually returns to normal within about 24 hours. The emetic form is commonly caused by rice that is not cooked for a time and temperature sufficient to kill any spores present, then improperly refrigerated. It can produce a toxin, cereulide, which is not inactivated by later reheating. This form leads to nausea and vomiting 1–5 hours after consumption.

It can be difficult to distinguish from other short-term bacterial foodborne pathogens such as Staphylococcus aureus

References Microbial Growth. (2011). Retrieved March 3, 2012, from The Growth Of Bacterial Cultures: http://classes. midlandstech. com/carterp/Courses/bio225/chap06/Microbial%20Growth%20ss5. htm Donovan, K. O. (1958). A selective medium for Bacillus cereus in milk. J. Appl. Bact. (21), 100-103. Granum, P. , & Lund, T. (2006, January 17). Bacillus cereus and its food poisoning toxins. FEMS Microbiology Letters, 157(2), 223-228. doi:10. 1111/j. 1574-6968. 1997. tb12776. x Herriman, R. (2009, September 13). Food-Borne Intoxication - Bacillus Cereus. Retrieved March 6, 2012, from ezinearticles. com: http://ezinearticles. com/? Food-Borne-Intoxication---Bacillus-Cereus&id=2915150 Johnson, K. M. (1984). Bacillus cereus food-borne illness. An update. J Food Prot, 47, 145–153. Merck. (2012). MYP Agar. Retrieved March 01, 2012, from Merck Microbiology Manual 12th Edition: http://www. mibius. de/out/oxbaseshop/html/0/images/wysiwigpro/MYP_Agar_105267_engl. pdf Tallent, S. M. , Rhodehamel , E. , Harmon, S. M. , & Bennett, R. W. (2012, February 02). BAM: Bacillus cereus. Retrieved March 05, 2012, from FDA U. S. Food and Drug Administration:

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