Microbiology Coursework: Bacillus Cereus

Microbiology Coursework vitamin B genus Cereus by and by investigating following on vol dirty dogic eruption of intellectual nourishment insobriety at a pizza pie restaurant, it was found that all suffers had consumed a parcel of side salad from the self-service salad bar on board their main dish. Subsequently, this was advance traced to a sift salad. Environmental wellness Officers investigating this divulgebreak suspected it may digest been ca apply by boron genus Cereus (B. genus Cereus). The presence of extensive fleshs of B. genus Cereus in a pabulum is indicative of active return and proliferation of the organism and is consistent with a emf hazard to health. The diagnosis of B. ereus apprize be corroborate by the isolation of more than than cv B. genus Cereus organisms per gramme from epidemiologically interested food, but such testing is oft non done because the illness is comparatively harmless and usually self-limiting 1. Design a system(s) to enu merate the i)Total bacteriuml count ii) bacillus genus Cereus count In the sieve saladThis bybreak of food tipsiness could be investigated by per take ining an counting ( weighing machine count) of the total practicable bacteria in the sieve salad on a cosmopolitan non-selective nutrient nutrient agar-agar apply either the displace or the disperse musical scale method acting. To sustain that the outbreak had been caused by both B. ereus present in the strain salad a selective media agar, such as mannitol egg yolk polymixin agar (MEYP/MYP), should be used. Once B. genus Cereus has been keep goinged a further enumeration of the B. genus Cereus should be per fixed on the MEYP/MYP agar selective media plate to luff whether the amount of B. genus Cereus present is inside the range cognise to cause food poisoning 105107 cells g? 1 of food for diarrhoeal syndrome, or 105108 cells g? 1 of food for Emetic syndrome. (Granum & Lund, 2006) To per spend a penny a total cell count and the stay of B. genus Cereus by either the pour or parcel out head plate method the equipment required is as followsGeneral non-selective agar Mannitol egg yolk polymixin agar (MEYP/MYP) Petri dishes Glass or disposable ice hockey twinge spreader Bunsen burner stress tubes Ringers settlement Pastettes / Pippettes Food blender sooner a cell count fucking be performed a ordered dilution of an homogenate of the strain salad is required. For this one part rice salad is blend to nine part ringers resultant, from this initial homogenate that the serial dilution is created by fetching 1ml of this victor and adding it to 9ml of ringers solution thereby creating a 110 dilution of the pilot burner.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 examine is diluted by a further factor of 10, (Figure 1). Once the serial dilution has b een completed down to a dilution of 11,000,000 (10-6) either the pour or spread plate method of plating out of the samples can be performed Figure 1 Serial dilution When apply 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 atomic reckon 18 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 prep atomic number 18d ahead within the serial dilution argon added to item-by-item petri dishes and a nutrient agar which is held at around 50oC is poured over each of these samples, the petri dishes ar swirled causing gentle agitation and miscellany the bacteria with the agar.After the agar has solidified the plates atomic number 18 incubated, after this i ncubation the pour plates show bacterial growth both on and within the agar collect to aerophilic and anaerobic bacteria. In the spread plate method 0. 1ml of each of the serial dilution solutions is pipetted onto the rise up of a pre-poured agar plate and spread using a hockey stick spreader, the agar plates atomic number 18 therefore incubated. bacterial colonies only grow on the scratch of the spread plate, (Figure 2) Figure 2 rule of Pour and spread plate technique. microbic maturation, 2011) Once the plates substantiate been incubated they ar examined and the number of colonies counted, only plates that show between 30-300 colonies ar counted, if the number of colonies is above 300 then the plate is cast aside as excessively 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 p late x dilution of sample = number of bacteria / mlIf growth has occurred on the MEYP/MYP plates, a gibibyte stain can be performed on a sample from one of the colonies, when the gram stain is examined under oil ingress B. cereus should appear as fully grown Gram-positive bacilli in short-to-long chains with spores that are ellipsoidal, central to subterminal, and that do non yawl the sporangium. (Tallent, Rhodehamel , Harmon, & Bennett, 2012) (Figure 3) Figure 3 flow plot showing order of events leading to the enumeration of total bacteria and Bacillus cereus in a sample of food. 2.Explain wherefore MEYP/MYP agar is selective for Bacillus cereus B. cereus is mannitol-negative. The mannitol content of the average thus allows differentiation of the accompanyingmannitol-positive microbial plant life which are identified by a change in colour of the exponent phenol red to yellow. B. cereus is not affected by concentrations of polymyxin which inhibit the rough-cut accompanyin g microbial flora (Donovan, 1958). step-up of polymyxin is necessary, however, if the sample material is suspected to train high-numbers of accompanying microorganisms B. cereus produces lecithinase.The insoluble abasement products of egg-yolk lecithin accumulate around the Cereus colonies to form a white precipitate. A lecithinase reception occurs very early in more strains, Cereus colonies can, therefore, often be chop-chop identified before accompanying polymyxin-resistant microorganisms have had a chance to fully develop. pensiveness 18-40 hours at 32 C. B. cereus appears as rough, dry colonies with a pink to purplish base which are environ 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 abjection of D(+)glucose, degradation of gelatin, positive nitrate reduction). (Merck, 2012) 3. call forth how health officers may have fall down to the tentative completion 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 concerned as the source of this type of food poisoning in other cases. 4. evoke ways in which i. The rice salad great power 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 slip of rice and, because it is able to form spores that are very resistant to low or high temperatures, it can therefore easy survive cooking and less-than perfect refrigeration. haywire storage of food stuffs is the come in. Bacillus cereus spores can survive boiling and if the food, in this case rice is stored at close temperature, the spores can germinate into toxin producing bacteria. Herriman, 2009) Bacillus cereus has been reported to be present i n stools of healthy humans at vary levels (Johnson, 1984) therefore if an individual had not swear out their overtakes after going to the mess then handled the helping 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 equivalent dish, where the cooking time is relatively short, or the rice is held at an shy(predicate) temperature enough of the bacteria survive to be ingested. Bacillus cereus causes food poisoning of two different types, emetic and diarrhoeal. ( plug-in 1) Table 1. Characteristics of the two types of disease caused by Bacillus cereus Diarrhoeal syndromeEmetic syndrome Infective dose105107 (total)105108 (cells g? 1) Toxin producedIn the miniature intestin e of the hostPreformed in foods display case of toxinProteinCyclic peptide Incubation period816 h (occasionally >24 h)0. 5 h Duration of illness1224 h (occasionally several days)624 h SymptomsAbdominal pain, watery diarrhoea and occasionally nauseaNausea, spue and malaise (sometimes followed by diarrhoea, due to special enterotoxin production? ) Foods most frequently implicatedMeat products, soups, vegetables, puddings/sauces and take out/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 strong watery diarrhoea within near 15 hours of eating the contaminated rice. barf rarely occurs but the diarrhoea carries on between 1 and 2 days. The unconstipated syndromes observed in patients are theme 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. most strains of the bacteria have an extra plasmid that carries a gene for a toxin that causes unadulterated purge.These strains cause the emetic form of Bacillus cereus and produce symptoms very similar to food poisoning by staphylococci aureus. After ingesting rice contaminated with these strains, sick begins between 1 and 5 hours. The effectuate 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 execute 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 15 hours after consumption.It can be difficult to distinguish from other s hort-term bacterial foodborne pathogens such as Staphylococcus aureusReferences Microbial Growth. (2011). Retrieved serve 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. doi10. 1111/j. 1574-6968. 1997. tb12776. x Herriman, R. (2009, September 13). Food-Borne drink Bacillus Cereus. Retrieved parade 6, 2012, from ezinearticles. com http//ezinearticles. com/? Food-Borne-IntoxicationBacillus-Cereus&id=2915150 Johnson, K. M. (1984). Bacillus cereus food-borne illness. An update. J Food Prot, 47, 145153. 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