Microbial analysis of food
- Food is a good culture medium for growth of many microorganisms.
- The presence of microorganisms in food causes decomposition and spoilage of food.
- Therefore, microbial analysis of food is important in quality assurance of food.
- Microbial analysis of food gives idea about:
- The quality of food:
- By determining type and number of microorganisms in food and by comparing this standard value, we can evaluate quality of food
- The sanitary condition of food:
- Microbial analysis helps to predict possible contamination of food.
- The storage condition:
- By microbial analysis we can detect whether food is stored in proper condition or not.
- The types of microorganisms in food:
- By microbial analysis, we can detect pathogen or their toxic product if present in food.
- The quality of raw material:
- It gives quality of raw materials used in food.
- Evaluation of effectiveness:
- Microbial analysis also evaluates the effectiveness of preservative methods employed.
Steps in microbial analysis of food:
1. Sampling:
- Sampling is an important step in microbial analysis.
- Sample of food can be obtained from processing line from storage house or from market.
- Sample:
- The sample should be representative of whole lot of food.
- Sample should be collected randomly from the lot.
- The principle is to avoid bias and to draw sufficient number of sample units.
- Sampling device:
- Sterile spoon pipette, stirrer, swab etc. are used for taking samples.
- Sterile scissors, knife, can opener etc. are used for opening food packages.
- Liquid and small particles are sampled with sampling tube, spoon, droppers etc.
- Large food materials (e.g. Cheese) are cut by knife.
- For surface samples, knife, swabbing or sticky cello phone tape is used.
- Sticky cello phone tape is pressed on the surface of solid agar to transfer microorganisms.
- Number of samples:
- If food is examined for Salmonella, usually co-samples are collected 5 samples are needed for other organisms.
2. Techniques for sample collection:
- When the product is in big container: Steps in sample collection are:
- Outer surface of container is cleaned by washing or by 70% ethanol.
- Container is opened with the help of sterile cutting instrument.
- If the food is in bulk, the sample are collected from various places within container.
- In case of liquid sample, it is first agitated to form homogenous mixture and sample is taken.
- When the food is in small container:
- When the sample is in small container, they should be taken directly to the lab for analysis.
3. Preparation of sample:
- For solid food sample: Solid food is mixed with sterile diluent in mechanical blender to obtain homogenous suspension. For example, when 10gm food is added to 100ml diluent, 10-1 dilution is obtained.
- For liquid food sample: Liquid food is serially diluted to obtain 10-1, 10-2, 10-3 dilution.
4. Transportation of sample:
- Multiplication and death of sample (microorganisms) should be prevented during transport of sample.
- The sample should be delivered to lab immediately.
- Perishable and unfrozen sample should be cooled to 0-5oC and transported in insulated container.
- Frozen product must be maintained in frozen condition until analysis.
- Sampling reports should be prepared and sent with sample.
5. Sampling report:
- The report should provide the following information:
- Name and address of person collecting sample.
- Date, time, place and purpose of sample collection.
- Nature of food
- Name of manufacturer, importer, seller, batch number, manufacture data, expiry date etc.
- Method of sampling, size and no. of samples, temperature of product at the time of sampling.
- Suggested test can also be given.
6. Sampling plan:
- Choice of sampling plan depends upon the organism present in food, the nature of food and whether the food is raw or processed or eaten directly, type of consumer i.e. normal, infant, old person or ill person.
- Sampling plan is basically a statement of criteria of acceptance based upon examination of required number of sample units by specific method.
- Sampling plan are of two types:
- Two class plan:
- It consists of three specification ‘n’, ‘c’, and ‘m’.
- This plan is simplest and is used to ‘accept’ or ‘reject’ food product.
- E.g. Two class of certain food for coliforms is:
- N=5, c=2 and m=102
- It means 5 sample must be tested.
- In order for that to be acceptable not more than two samples must contain greater than 100 coliforms per grams.
- The food will be rejected, if 8 or more sample units exceed 100 organism per grams.
- e.g. n=5, c=0, m=0 (Salmonella). It means food will be rejected if any one sample out of five contained organisms.
- Where, n= number of sample unit to be tested.
- m= the maximum number of organisms per gram. Value above this is non-acceptable.
- c= the maximum no. of allowable samples that may extend the microbiological criteria ‘m’.
- when this number is exceeded, the lot is rejected.
- Three class plan:
- It consists of four specifications i.e. n, c, m, and M.
- M- the quantity is used to separate marginally accepted quality from unacceptable quality.
- Number or value above M in any sample are unacceptable.
- e.g. n=5, c=2, m=100, M=150 e.g. food A. coliform
- It means 5 samples must be tested. If not more than two sample.
- 2 sample exceed equal or more than can organism, it is acceptable if any one sample.
- Sample contain equal or greater than M, than sample is non-acceptable.
I. Sample procedure for dairy product:
- Milk and milk product:
- No. of samples:
- Fluid milk= 5 field samples
- Dried milk and ice cream = 10 field samples
- Cheese = 5 field samples
- Butter = 5 field sample
- Methods of analysis:
- Enumeration of mesophilic aerobic bacteria
- Enumeration of coliforms
- Enumeration of Staphylococcus aureus (cheese)
- Detection of Salmonella
- For butter, enumeration of yeast and mold as well as coliforms.
i. Microbial analysis of Milk:
- In general market milk is sampled in the container in which it is sold.
- Sterile plunger is used for sampling and 10ml sample is taken. The collecting sample is put in sterile screw capped tube and immediately send to lab.
- Before performing test, the sample is mixed by inverting up and down for more than 10 times.
- For milk bottle, the top is flamed, cap removed with flame forceps.
- For plastic packets, the top and corner of packet is swabbed with spirit.
- After allowing to air dry, the swabbed area is cut with sterile scissors.
- The collected milk is tested by methylene blue test resazurin test, MPN test.
- Three routine tests prescribed for milk are:
- Plate count at 32oC: serial dilution is made and pour plating is done from tubes of 10-2– 10-8 dilution.
- For raw milk, DMC is usually done.
- Coliform test: It is done by MPN
- Mesophilic aerobic bacteria in Milk:
- Raw milk should not exceed total plate count = 2x 105/ml
- Pasteurized milk should not exceed total plate count = 2x 104/ml
- At 37oC, coliform = 0 (best)
- At 42oC, fecal coliform = 0 (best)
- For dried milk,
- Mesophilic aerophilic bacteria = n=5, c=2, m= 5X 104, M = 5×105
- Coliform= n=5, c=2, m=2, M=102
- Staphylococcus aureus = n=5, c=1, m=10, M=102
- Salmonella = n=10, c=0, m=0
ii. Microbial analysis of Cheese:
- Cone sample is taken with the help of borer.
- The collected sample is made into finer pieces with a sterile grater. A grater is a device with a rough surface for grating food.
- The holes remaining after borings are sealed with paraffin wax.
- 10gm of grated sample is mixed with 90ml of warm diluent and homogenized in a blender.
- Serial dilution is performed.
- Plate count is performed on yeast extract milk agar. Similarly, coliforms can also be counted.
- For direct microscopic examination, thin film of slices of cheese are made. They are defatted with xylol. Xylol is washed and smear is stained and examined.
- For cheese,
- Staphylococcus aureus, n=5, c=1, m= 103, M=104.
iii. Microbial analysis of Ice cream:
- If the ice cream is in packet of small size, it should be taken to lab in frozen condition (dry ice can be used).
- Melting ice cream should not be taken from the field.
- The wrapper is removed aseptically and content are put in sterile screw called tubes.
- The collected sample is allowed to melt in the lab below 20o C.
- 10ml sample is weighed in a screw capped jar and serial dilution is performed in 0.1% peptone water.
- Plate count is performed from each dilution.
- Mesophilic aerobic bacteria, n=5, c=2, m=2.5X105.
- Coliforms: n=5, c=2, m= 102, M=103
- S. aureus: n=5, c=1, m=10, M=102
- Salmonella: n=10, c=0, m=0
iv. Microbial analysis of Butter:
- The sampling of butter is done by using sterile knife.
- 10g of butter is emulsified in 90ml of warm water (40-45oC) diluent and plate count is performed.
- Cultures are made on agar free nutrient medium.
v. Microbial analysis of Yoghurt:
- Yoghurt is sampled from the container in which it is fermented using spoon or spatula.
- Double dilution of yoghurt is prepared to 0.1% peptone water.
- 5ml of dilution is mixed with 5ml of melted LS differential medium.
- It is poured in plates, cooled, to solidify and incubated around 43oC for 48hrs.
II. Sampling procedure for meat and meat products:
- No. of field samples=5
- Method of analysis:
- Enumeration of mesophilic aerobic bacteria
- Detection of Salmonella
i. Microbial analysis of Carcass sampling (raw meat):
- Sterile large swab is taken and they are moistened in 0.1% peptone water.
- The carcass is wiped with the swab.
- The carcass is again cleaned with dry pad.
- The pads are taken and 250ml of diluent is added in the pad to disperse the microorganisms from the trap.
- Serial dilution is performed.
- Plate count is made ta 20oC.
- For detection of Salmonella, plating is done in selective medium.
ii. Microbial analysis of Chopped meat (raw meat):
- It may be fresh, minced or chopped meat.
- Several random samples are taken so as to detect universally distributed microorganisms.
- The sample is mixed with diluent and mixed well.
- Sample is serially diluted.
- Plate count is performed from dilutions 10-4 to 10-8.
iii. Microbial analysis of Poultry:
- Swab with lengthy rod is taken and it is sterilized.
- For each chicken, 2 swabs are taken. One for swabbing outside the body and other for swabbing body cavity of chicken.
- The swabs are rinsed in 100ml peptone water.
- Sample is serially diluted.
- Plate count is performed for total count and coliform count using selective media.
- To test for Salmonella, the dilution should be enriched in selective or tetrathionate broth and sample is further processed.
iv. Microbial analysis of Fresh fish:
- The whole surface of fish is either swabbed or washed with sterile 0.1% peptone water +1% NaCl in sterile plastic bag.
- Appropriate dilutions are prepared and total count is performed in marine agar which is incubated at 20oC for 5 days.
- Cultures can also be done in glucose tryptone media, macconkey agar is or media containing 5-10% NaCl for halophilic bacteria.
v. Microbial analysis of Eggs:
- Outer surface of egg shell is swabbed with sterile swab. Sterile swab must be moistened in peptone water prior to swabbing.
- Then appropriate dilution is made from the swab.
- Inner portion of egg can be taken as sample and diluted.
- No. of field samples:
- Salmonella = 10 field samples
- Mesophilic bacteria = 5 field samples
- Coliforms = 5 field samples
- Methods of analysis:
- Enumeration of mesophilic aerobic bacteria (TPC)
- Enumeration of coliforms
- Detection of salmonella
- For TPC= n=5, c=2, m=5x 104, M=106
- For coliforms = n=5, c=2, m=10, M=103
- For Salmonella = n=10, c=0, n=0