MICROBIAL LOAD IN MEAT AND MEAT PRODUCTS: How to determine

DETECTION OF MICROBIAL LOAD IN MEAT AND MEAT PRODUCTS

There are several methods that can be used to determine the microbial load in meat and meat products:

1. Total viable count / Total plate count (TVC/TPC)
2. Coliform count
3. Yeast and mold count
4. Indicator food pathogens

1. Total Plate Count – TPC (most common method):

Requirements:

1. Meat sample
2. Pestle and mortar
3. Peptone water (0.1%) 
4. Petri plates  
5. Test tubes (sterilized)
6. Test tube rack
7. Pipettes – 1ml, 10 ml 
8. Spirit lamp 
9. Sterilized molten agar          
10. Incubator

Sampling

Meat sample, a representative sample should be collected in a very sterile atmosphere near a flame so as to avoid air contamination. Generally, a 10 g sample is taken for the making of serial dilutions during the estimation of TPC, Coliform etc. However, there are regulations for certain food poisoning organisms such as Salmonella where we have to collect 25 g sample. Sample may also be taken keeping in view the regulations.

Sample precautions

All scissors, forceps and knives should be presterilised. Sample must be collected in a presterilised petri plate or a beaker in the lab. If the sample has to be collected from some food establishment, the procedure must be aseptic and transportation must be quick and under refrigeration. If longer duration of time from food establishment is expected, method of freezing is required. The time must be very short from the point of collection to its delivery in the laboratory. In case of non-destructive type of sampling, the meat sample is not physically lifted but surfaces are swabbed and the samples are collected aseptically. In case of carcass surfaces of large carcasses or poultry, an area of one square centimeter is swabbed and results expressed on the basis of area and not weight.

Preparation of serial dilutions:

1. Keep the flask containing 90 ml presterilised peptone water (should not be hotter than the normal room temperature to avoid injury to microbial cells) and test tubes each containing 9 ml of diluents ready in the laminar air flow chamber.
2. The meat samples are opened in laminar air flow chamber sterilized by ultra-violet irradiation.
3. About 10 g of sample is aseptically weighed and transferred to a sterile mortar in which 90 ml of sterile 0.1% peptone water is taken from the flask.
4. The sample is homogenized for 2 min using a sterile pestle and mortar to make 10^-1 dilution.
5.  To prepare 10^-2 dilution, 1 ml from 10^-1 dilution is mixed with 9 ml of 0.1% peptone water and for further dilutions the process is repeated in the same manner till the desired dilution level is achieved, depending upon the level of contamination or microbial load expected.
6. Proper mixing in serial dilutions is ensured by shaking the tubes sideways and immediately the cotton plug is replaced to avoid contamination. Preparation of sample and serial dilutions are made near flame in laminar flow apparatus.

Procedure for estimation of total plate count:

1. Required quantity of suitable medium (plate count agar) is suspended in desired volume of distilled water as specified by the manufacturer, followed by boiling to dissolve the medium completely.
2. It is then sterilized by autoclaving at 15 lbs pressure (121^oC) for 15 min.
3. Final pH of the media is adjusted to 7.0±0.2 at 25^oC unless and until it is self achieved as per the manufacturer’s protocol of dilution.
4. The pour plate technique is followed for plating.
5. 1 ml from each appropriate dilution (say 10^-2 and 10^-3 depending upon the expected microbial load) is inoculated in the sterile petri plates already marked with the dilution number.
6. Now pour approx. 16 to 20 ml of molten agar (temperature should be in the range of 40-45^oC at the time of pouring) on the inoculated petri plates.
7. The petri plates are slowly rotated clockwise and anticlockwise so that the inoculum is uniformly mixed with the agar.
8. After the agar has cooled and solidified, the petri plates are inverted and incubated at 37^OC for 24 h.
9. After incubation, colonies are counted in plates with colonies counting between 30 to 300.
10. The number of colonies are multiplied by the reciprocal of the respective dilution and expressed as log₁₀ cfu/g (cfu=colony forming units)

Calculation:

Calculate the number N of microorganisms present in the test sample as a weighed mean using the following equation:                  

                             ∑C        
            N=   -----------------------
                        (n1+0.1n2) D                                   

Where:

• ∑C = the sum of the colonies counted on all the dishes retained from two consecutive dilutions.
• n1= the no. of dishes retained in the first dilution
• n2= the no. of dishes retained in the second dilution
• D= dilution factor corresponding to the first dilution

2. Coliform count

The procedure of sampling, dilution and inoculation is same except the medium used is violet red bile agar (VRBA) and incubation temperature is 35^oC for 24 h. Also, medium is not autoclaved.

3. Yeast and mold/ mould count

The procedure of sampling, dilution and inoculation is same except the medium used is potato dextrose agar (PDA) and incubation temperature is 25-30^oC for 5 days.  

4. Isolation and identification of indicator food pathogens

I. Staphylococcus aureus

S. aureus is isolated from the food by plating after enrichment. Enrichment is done in alternate trypticose sodium broth with 10% NaCl and 1% sodium pyruvate. Selective plating is done on Baired Parker Agar.

Food sample (meat) is inoculated in enrichment broth and incubated at 37^oC for 24 hours. Loop full of culture from enrichment broth is streaked on Baired Parker Agar and incubated at 37^oC for 24 hours. Jet black color colonies are indicative of S. aureus.(should be absent in 50 g of sample).

II. Salmonella spp.

To isolate Salmonella from meat sample, pre-enrichment is done in buffered peptone water. After that, the sample is transferred to enrichment broth of selenite cysteine broth. A loop full of culture is taken from enrichment broth and streaked on Salmonella Shigella Agar (SSA) and Brilliant Green Agar (BGA) and incubated for 24 hours at 37^oC. Pink colonies surrounded by red color on Brilliant Green Agar and colorless and translucent colonies on Salmonella Shigella Agar are indicative of Salmonella spp. (should be absent in 25 g of sample).

III. Escherichia coli

 Enrichment broth of peptone water is used before plating on Eosin-Methylene Blue (EMB) agar on which streaking is done and plate is incubated at 37⁰C for 24 hours. Black centered colonies with greenish metallic sheen are indicative of E. coli.

IV. Listeria spp. 

 5 g of sample is blended with 0.5% peptone water and incubated at 30^oC for 7 days. A loop full of inoculum from the enrichment broth is taken on 7^th day and streaked on PALCAM (Polymixin, Auriflavin, Lithium Chloride, Cephacidine, Aesculine, Mannitol) agar. Plate is incubated at 30^oC for 48 hours. Greyish green color colonies with black hallow centre is indicative of Listeria spp.

V. Vibrio spp.

5 g of sample is mixed in 225 ml of alkaline peptone water with 3% NaCl for enrichment. A loop full of culture is taken and streaked on TCBS (Thiosulphate Citrate Bile Salt Sucrose) agar and incubated at 37^oC for 24 hours. Vibrio cholerae produces large yellow color colonies and bluish green colonies are produced by Vibrio hemolyticus.

VI. Bacillus cereus  

Enrichment of food sample is done in Trypticose Soya Agar and Polymixin broth. A loop full of culture is streaked on MEPA (Mannitol, Egg yolk, Polymixin Agar) and incubated at 37^oC for 24-48 hours. Greyish white and opaque colonies are indicative of B. cereus.

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