Microbial assessment

MICROBIAL ASSESSMENT OF MEAT ANF MEAT PRODUCTS

  • The bacterial status of the meat is dependent on a number of factors, namely the condition of the animal at slaughter, the spread of contamination during slaughter and the processing and temperature during storage and distribution.
  • The microbiology of meat is normally considered under two criteria,
    • Total bacterial counts, which provide an indication of gross levels of contamination. 
    • Specific counts of species of spoilage/pathogenic bacteria of significance.
  • Indicator organisms provide an indication of the bacteriological quality of meat and meat products.
  • Indicator bacteria are those bacteria that most commonly occur if handling or processing is done unhygienically.
  • Indicator organisms are tested because pathogenic organisms are difficult to test directly.
  • The most common organisms used as indicators are coliforms, feacal coliforms (E. coli), enterococci (faecal streptococci).
  • Staphylococcal counts significantly over l00 /g indicates negligent personal habits of handlers, sanitation or temperature control.
  • The International Commission on Microbiological Specification for Foods (ICMSF) recommended that the general viable count (GVC) at 35°C or at 20°C in case of chilled meat should be less than 107 per gram.

Sampling techniques

  • Destructive sampling is the most efficient means of removing microorganisms from meat for counting and the results (counts) are expressed per unit weight.
  • Destructive sampling is usually performed during investigation of spoilage or public health problem.
  • A non-destructive sampling approach has its limitations in terms of efficiency of removal of microorganisms from the meat surface and the results (counts) are expressed per unit area, usually per Sq. cm or per Sq. inch.
  • It is preferred because of the appearance of the carcass or cut is not affected.
  • A large number of different sampling techniques have been applied to carcass meat and retail meat.
  • The choice of technique is mainly a matter of personal preference and convenience.

Total viable count

  • Total Viable Count (TVC) is otherwise referred to as Standard Plate Count, Aerobic Plate Count, Total Plate Count, Viable Plate Count and Mesophilic Count.

Collection and preparation of sample

  • Destructive sampling involves the collection of composite samples.
  • Care should be taken to take a single block or piece of meat slightly greater than the desired quantity ideally or utmost a second piece to make up the desired quantity (weight).
  • Usually 5g of meat or some times 25g of meat, as in the case of estimation of Salmonella is collected.
  • In non-destructive sampling a, known area should be clearly delineated and marked out using a template and sample collected by swabbing the area by means of a sterile swab (Ready made pre sterilized swabs are available in the market which may be used).
  • Swabbing should be vigorous, complete and should follow a pattern that must be adopted in the collection of each sample to minimize variations due to sampling errors.
  • Moistening the swab prior to sampling in sterile peptone (0.1%) abets the maximum recovery of microbes as this would aid in survival of microbes.

Dilution

  • In case of destructive sampling the sample should be trimmed to attain the desired quantity (weight).
  • Avoid cutting the samples into too many pieces.
  • This sample should be diluted using 45ml of sterile peptone (0.1 %) in the case of 5g samples to entail a 10-1 dilution and macerated for a minute in a stomacher ideally or in a pestle and mortar.
  • If pestle and mortar is used, sterile sand may be used for thorough maceration, to ensure maximum recovery of microorganisms.
  • In case of swab samples the head of the swab is broken into a test tube containing 10 ml of the diluent (0.1 % peptone) to entail a 10-1 dilution.
  • Serial ten fold dilutions are made similarly irrespective of the method of sampling adopted.
  • 1ml of the solution is transferred from the 10-1 dilution to another test tube containing 9ml of the diluent to obtain the 10-2 dilution from which 1 ml is transferred to another test tube containing 9 ml of the diluents to obtain the 10-3 dilution.
  • The process is repeated to obtain 10-4, 10-5 dilutions, so on and so forth.

Plating-pour plate method

  • Place 1ml aliquots of the diluted samples using sterile pipettes in pre identified petri dishes.
  • This should be done immediately after the next higher dilution is made i.e once 1ml of the test sample is transferred to make the next higher dilution.
  • Similarly plating is continued till the required dilution.
  • Use separate sterile pipettes for each dilution.
  • Lift the cover of the petri plate just high enough to insert the pipette while measuring diluted samples of a food into petri plates.
  • Hold the pipette at about 45° angle with the tip touching the inside bottom of the petri plate.
  • Deposit the sample away from the centre of the plate to aid in mixing.
  • Allow 2 to 4 seconds for the sample to drain from the one ml graduation mark to the rest point in the tip of the pipette; then, holding the pipette in a vertical position, touch the tip once against a dry spot on the plate.
  • Do not blowout.
  • Replicate plates may be prepared for each dilution plated.
  • Prepare sterilized molten agar media (plate count agar in the case of TVC) and hold it at 44 - 46°C in a water bath until use.
  • Remove the agar from the water bath; blot it dry with a clean towel to prevent water from contaminating the plates.
  • Pour 12 to 15 ml of liquefied medium at 44°C to 46°C into each plate by lifting the cover of the petri plate just high enough to pour the medium.
  • Avoid spilling the medium on the outside of the container or on the inside of the plate lid when pouring.
  • As each plate is poured, thoroughly mix the medium with the test portions in the petri plate, taking care not to splash the mixture over the edge, by rotating the plate first in one direction and then in the opposite direction, by tilting and rotating the plate, or by using mechanical rotators.
  • Allow agar to solidify (no longer than 10 min) on a level surface.
  • Select the number of samples to be plated in anyone series so that not more than 20 min (preferably 10 min) elapse between diluting the first sample and pouring the last plate in the series.

Incubation

  • After solidification, invert the plates to prevent spreaders, and promptly place them in the incubator.
  • Cultural characteristics can be read after 18 to 24 hours of incubation at 35 to 37oC.

Counting colonies

  • Count colonies with the aid of magnification under uniform and properly controlled artificial illumination, using a tally.
  • Routinely use a colony counter equipped with guide plate ruled in square centimeters.
  • Count all colonies on selected plates containing 25 to 250 colonies promptly after the incubation period.

Computing and reporting

  • Colony counts may be computed by multiplying the total number of colonies (or the average number if replicate plates of the same dilution are used) per plate by the reciprocal of the dilution used.
  • Counts are reported (or estimates thereof) as CFU per g or ml, as applicable.
  • Finally, to interpret the results the value counted is multiplied by the dilution factor, for example, in case a 10-2 plate has 75 colonies, the value, 75 is multiplied by 100 and the result reported as 7.5x 103 CFU/g in case of composite samples.
  • But in case of swab samples this value arrived i.e. 7200 and subsequently the value arrived at is divided by the area sampled say, if 25 Sq.cm, it is divided by 25 and the result reported as 3x 103 CFU/g.

Streptococcal count

  • The collection, preparation of samples, dilution and plating for the determination of streptococcal count is similar to that of the above steps followed in the estimation of Total Viable Count.
  • The plating media used for the determination of fecal streptococcal count in meat is KF Streptococcus agar.
  • Cultural characteristics can be read after 48 to 72 hours of incubation at 35° C.
  • All red or pink colonies are counted as streptococcal colonies.

Staphvlococcal count

  • The collection, preparation of samples, dilution and plating for the determination of staphylococcal count is similar to that of the above steps followed in the estimation of Total Viable Count.
  • The plating media used for the determination of staphylococcal count in meat is Baird-Parker Agar.
  • Cultural characteristics are observed after 24 to 48 hours of incubation at 35°C.
  • Clear zones with grey black colonies in this medium are diagnostic for coagulase positive staphylococci.

Coliform count

  • The collection, preparation of samples, dilution and plating for the determination of Coliform count is similar to that of the above steps followed in the estimation of Total Viable Count.
  • The plating media used for the determination of Coliform count n meat is Violet Red Bile Agar.
  • Cultural Characteristics are read after 18 – 24 hours of incubation at 35oC.
  • Rapid lactose fermenters produce red colonies surrounded by red purple hallow.
  • Slow lactose fermenters and late lactose fermenters produce pale colonies.

Anaerobic count

  • The collection, preparation of samples, dilution and plating for the determination of anaerobic count is similar to that of the above steps followed in the estimation of Total Viable Count.
  • Cultural Characteristics can be read after 48 to 72 hours of incubation at 35o C anaerobically.

Yeast and mould count

  • The collection, preparation of samples, dilution and plating for the determination of yeast and mould count is similar to that to the above steps followed in the estimation of Total Viable Count.
  • Cultural characteristics can be read after 48 to 72 hours of incubation at 30oC.
Last modified: Tuesday, 7 September 2010, 10:36 AM