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. cereus 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
Design a method(s) to enumerate the:
Total bacterial count
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. cereus 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)
Glass or disposable “hockey stick” spreader
Pastettes / Pippettes
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. 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...
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