Food Microbiology - Lab Report Example

Food Microbiology Aim: To perform microbiological assessment of the food items. Introduction: Food Microbiology is the study of the microorganisms which inhabit, produce or contaminate food items. These microorganisms may be deleterious and are responsible for food spoilage. On the other hand, some of these microorganisms are essential for preparing various food items especially the dairy products like cheese, yoghurt and other fermented foods like bread and various beverages like beer and wine (Roberts, D, 2003). Raw and unprocessed food is naturally contaminated with a range of micro-organisms which may include pathogenic forms. The microbiology laboratory is concerned with testing foods to both determine the level of microbial contamination as well as to ascertain the presence of specific pathogen on foods. Results indicate the overall standard in terms of hygiene of food processing and the food chain. The microbial levels permitted for food to be determined safe are regulated by law. The study is carried out in three stages performed as three experiments. In the first experiment, aerobic plate count is done for E.coli count and coliform count on given food item (Roberts, D, 2003, Gilbert et al, 2000). It is evident that coliforms and E.coli are present in human faeces. Their presence in food items can indicate post processing contamination. The aerobic count is used to determine the overall level of microbial contamination of food items and provides an indication for poor processing or post processing techniques especially where the count exceed the legal permitted levels. In the second experiment pre-cooked food is observed for faecal contamination. In the third experiment the quality of milk samples (pasteurized and raw) are checked for the presence of fecal contamination. Experiment 1 Aerobic plate Count at 30'C Materials Stomacher bag Sterile spatula/ forceps/ knife 225 ml of peptone saline diluents 5 X 9 ml of peptone saline diluents Dried plate count agar Sterile spreaders Bagged salad Method: 1. Weigh out 25g of salad in a sterile bag 2. Add 225 ml of sterile peptone saline diluents 3. Homogenizes for 1-2 minutes 4. Place bag in sterile beaker 5. Carry out 1:10 dilutions down to 10-8 6. Mix by rotating in your hand after each dilution 7. Using a sterile pipette, inoculate 0.5ml of the 10-8 dilution into the centre of dried agar plate. As soon as possible, spread the inoculums with a sterile spreader. 8. Repeat for 10-7 to 10-2 dilutions. The same pipette and spreader if started with the most diluted solution. 9. Allow the plates to set for at least 15 mins to ensure the inoculum is absorbed onto the plate. 10. Inoculate at 30'C for 48 hrs. Observation: Observe the Petri dish after 48 hrs for the presence of colonies. S. No. Dilution Factor No. of Colonies 1 10-1 TMTC 2 10-2 TMTC 3 10-3 TMTC 4 10-4 3 5 10-5 167 6 10-6 78 7 10-7 38 8 10-8 3 Calculation: CFU/g = Amount of sample plated X No. of colonies/ Dilution factor CFU/g = 1ml X 78/ 10-6 = 78 X 106 CFU/g CFU/g = 2ml X 78/ 10-6 = 156 X 106 CFU/g = 1.56 X 108 Results The Petri dish with colonies between 15 and 300 were selected to be significant in number whereas Petri plates with TMTC (too many to count) were not considered to be significant. Calculation is performed with the formula mentioned. Plates with dilution factor of 10-8 showed no growth and hence it is reported as < 20 CFU/g of food sample. The amount of sample plated is the volume of dilution pipette on the plate. Average value was chosen to get the exact value. For the dilution factor 10-4 CFU/g is 3 which is not normally expected. Discussion: The serial dilutions, or successive dilution of a specimen e.g. 1:10 dilution equals 1 ml of sample plus 9 ml of diluents, a 1:100 dilution equals 1 ml of a 1:10 dilution plus 9 ml of diluents. This is the process to enhance the probability of finding the most probable microorganism even at higher dilution. If the microorganism is present in the highest dilution then this is depicted when inoculated on the medium solidified on Petri dish. All these process is carried out under the sterilized conditions to avoid any external contamination. Petri plates are incubated for 48 hrs to allow the growth of microbes in the form of colonies. Each colony is derived from a single cell and hence counted as colony forming unit (CFU). The Petri plates are kept in inverted position in order to avoid any kind of disturbance from the water of condensation that is present on the lid of the Petridish (Roberts, D, 2003, Gilbert et al, 2000). For the dilution factor 10-4 CFU/g is 3 which is not normally expected after TMTC in the previous dilution factor plating. The reason could be varied and indicates that while inoculation and pipetting the sample on the medium the tube was not properly shaken and therefore homogeneous distribution of the dilution transferred has not taken place and therefore when the sample was taken out from the tube for plating, negligible microbes have entered. Experiment 2 Objective: To enumeration the number of E. coli in pre-prepared food as an indicator of faecal contamination. The food is considered unsatisfactory at level of more than 10 CFU/g in ready cooked foods. Materials Ready to eat food- cook chill meal Bagged salad Stomacher bags Peptone saline diluents E coli broth NCTC 9001 The standard method for enumeration of E.coli in foods (not dairy, frozen or dried) utilizes coliform medium agar. Method: 1. Prepare a 1:10 dilution of the sample as outline in Experiment 1 dilute in buffered peptone water. 2. Inoculate 0.5 ml of the homogenate onto the centre of a Chromogenic E.coli or coliform medium. 3. Using a sterile spreader spread the inoculum over the agar trying not to touch the sides of the Petri dish. 4. Prepare a positive control plate using E. coli NCTC 9001 plate out for single colony. 5. Incubate at 30' C for 4 hrs +/- 1 hour and then 44.5'C for 18+/- 2 hrs. Observation: Colonies with blue coloration are examined and selected for the presence of '-glusoronidase enzyme activity. On shepherd pie = no growth was recorded On sandwich = TMTC (too many to count) Control = E. coli (growth) Results: Counts of > 100 CFU/G in any food is not satisfactory but in ready to eat or cook-chill foods, >10 CFU/g is unsatisfactory. Discussion: Enzymes in Chromogenic Media glycosides-Enzyme product by E.coli. It clears Chromogenic substance galactoside- Enzymes produced by E.coli and coliform - pink coliform. Presence of E.coli on the inoculated plates is an indication of the fecal contamination in raw or uncooked food items. These contamination is introduced either through the soil infested with the fecal matter or the water which is used for irrigation is infested with the fecal contamination or the containers in which the vegetables/ food items are kept are infested with fecal contamination or the individual handling the food items introduce fecal contamination to the food items. Presence of fecal contamination is totally unsatisfactory and should be taken seriously to eliminate the chances of any kind of infection or disease (Roberts, D, 2003, Gilbert et al, 2000). Experiment 3 Materials Pasteurized milk Raw milk 6 X 12 ml of Violet Red Bile Agar (held at 45'C for no more than 3 hrs) 6 X 5 ml of Violet Red Bile Agar E coli broth culture (NCTC 9001) 3 X 99 ml Saline solution Methods Pasteurized milk 1. Mix the pasteurized sample by inverting 25 times use parafilm to prevent the sample from leaking. 2. Allow the foam to settle. 3. Add 1 ml of milk to the base of a Petri dish. 4. Pour 12 ml of VRBA and mix by rotating clockwise 6 times and then anticlockwise 6 times. 5. Allow the agar to completely set. 6. Pour further 5 ml of VRBA over the set agar. This does not require any mixing. 7. Raw milk: Repeat using the raw sample of milk. However, dilute the sample of raw milk to 10-6 and carry out the count on each of these dilutions on the raw milk. 8. E.coli broth culture: Pipette 0.1 ml of the pre-diluted E.coli broth culture provided into the base of a Petri dish, pour over 12 ml of VRBA, mix as before and allow to set. Overlay with the addition of 5 ml of VRBA. 9. Repeat using the raw milk sample. Observation: S. No. Dilution Factor No. of Colonies 1 10-1 TMTC 2 10-2 TMTC 3 10-3 TMTC 4 10-4 TMTC 5 10-5 9 6 10-6 70 7 10-7 63 Calculation: CFU/g = Amount of sample plated X No. of colonies/ Dilution factor CFU/g = 1ml X 78/ 10-6 = 63 X 107 CFU/g = 6.3 X 108 Results: Examination of the E coli plate- this is the control coliform plate which is used to determine typical coliform colonies. Examination of the milk plates and the number of coliform on each of the plates is calculated. The number of coliform/ ml of milk sample for both the raw and pasteurized samples. The CFU/g for raw milk is 6.3 X 108. Discussion: Milk is the secretion of mammary glands of mammals. It is the "most nearly complete" food for humans and is an excellent culture medium for many microbial species. Freshly drawn milk varies in chemical composition. More important causes of variation in milk quality are the species of mammal, the breed, the age of an individual, the stage of her lactation, her feed, her health and the season of the year. Milk also contains vitamin A, B1 or thiamine and B2 or riboflavin. It also contains small amount of vitamins C and D and smaller quantity of other vitamins. Milk therefore, provides essential nutrients for excellent growth of many bacterial species. Therefore it is must that microbiological examination of milk is performed for checking the quality of milk for various reasons e.g. to judge sanitary quality and conditions of productions, to assess the microorganisms present in the milk they may degrade milk components such as fat, proteins or carbohydrates making the product unpalatable, many milk borne epidemics of human diseases can be caused if milk is contaminated with pathogenic microorganisms. Certain microorganisms produce desirable chemical changes resulting in the production of butter, cheese, yoghurt etc. Various sources of microorganisms have been realized for introducing contamination in milk from the time milk leaves the udder, until it is dispensed into containers, everything which comes in contact with it is a potential source of more microorganisms (Roberts, D, 2003). Milking performed under hygienic conditions with strict attention to sanitary practices will reduce the entry of microorganism into the milk. It is understood that fewer the organisms that can get into the milk, the fewer have a chance to grow. Sanitary care is must to keep the producing animal clean her flanks, udder and teats given special sanitary care just before milking her body can be a source of considerable contamination. The microbial content of air in the milking area is also imperative (Roberts, D, 2003). The CFU/g for raw milk is 6.3 X 108 indicating less significant number of CFU. Proper hygiene and milking conditions always reduce the CFU/g for raw milk. It is therefore evident that these conditions must be emphasized to reduce the microbial contamination. The reductase test is performed which indicates that dye (methylene blue thiocyanate) is blue when oxidized and becomes colorless when reduced. If milk is of excellent quality then decolorization takes more than 8 hrs and if it is of poor quality then decolorization occurs in less than 2 hrs. Heat treatment kills some but not all microbes usually at temperatures below 100'C and this is pasteurization. The pasteurized milk does not show any growth indicating that efficiency of pasteurization process (Roberts, D, 2003). Conclusion: It is very essential to prevent the development of food poisoning and also spread of disease by foods. The microbiological assessment is very imperative tool for the classification of food infections or food intoxications. Food infections are those in which microorganisms present in the food at the time of eating grow in the host and cause disease. Food intoxications are those diseases in which microorganisms grow in the food, producing a substance therein which is toxic to man and animals. Food poisoning is the toxicity introduced into food by microorganisms and their products (Roberts, D, 2003, Gilbert et al, 2000). All the three experiments indicate that some amount of fecal contamination do persist in food items which cannot be ignored. Microorganisms grow at much faster pace and they can deteriorate the food in very short span. Proper hygiene and safety conditions not only reduce their number but also protect the animals and humans from various dreaded infections. Reference: 1. Roberts, D., Greenwood, M., (2003) Practical Food Microbiology, Oxford: Blackwell : Publications 2. Gilbert, R., de Louvois, J., Donovan, T., Little, C., Nye, K., Ribeiro, C., D., Richards, J., Roberts, D. and Bolton, F., J., (2000) Guidelines for the microbial quality of some ready to eat foods at the point of sale. Comm. Dis. Pub. Health. Vol 3. No. 3 pp 163-167. 3. Http://www.hpa-standardmethods.org.uk/pdf/sops.asp Read More