Sampling and microbiological analysis of foods
For the microbiological analysis of foods, the adequacy and condition of the sample or specimen received for examination are of primary importance. If samples are improperly collected and mishandled or are not representative of the sampled lot, the laboratory results will be meaningless. Because interpretations about a large consignment of food are based on a relatively small sample of the lot, established sampling procedures must be applied uniformly. A representative sample is essential when pathogens or toxins are sparsely distributed within the food or when disposal of a food shipment depends on the demonstrated bacterial content in relation to a legal standard. The number of units that comprise a representative sample from a designated lot of a food product must be statistically significant. The composition and nature of each lot affects the homogeneity and uniformity of the total sample mass. The proper statistical sampling procedure, according to whether the food is solid, semisolid, viscous, or liquid, must be determined by the collector at the time of sampling. Whenever possible, submit samples to the laboratory in the original unopened containers. If products are in bulk or in containers too large for submission to the laboratory, transfer representative portions to sterile containers under aseptic conditions. Sterile sampling equipments and containers should be used for sampling. Use containers that are clean, dry, leak-proof, wide-mouthed, sterile, and have a size suitable for samples of the product. Containers such as plastic jars or metal cans that are leak-proof may be hermetically sealed. For dry materials, use sterile metal boxes, cans, bags, or packets with suitable closures. Sterile plastic bags (for dry, unfrozen materials only) or plastic bottles are useful containers for line samples. Whenever possible, obtain at least 100 g for each sample unit. Submit open and closed controls of sterile containers with the sample.
Deliver samples to the laboratory promptly with the original storage conditions maintained as nearly as possible. When collecting liquid samples, take an additional sample as a temperature control. Check the temperature of the control sample at the time of collection and on receipt at the laboratory. Make a record for all samples of the times and dates of collection and of arrival at the laboratory. Dry or canned foods that are not perishable and are collected at ambient temperatures need not be refrigerated. Transport frozen or refrigerated products in approved insulated containers of rigid construction so that they will arrive at the laboratory unchanged. Collect frozen samples in pre-chilled containers. . Keep frozen samples solidly frozen at all times. Cool refrigerated samples, in ice at 0-4?C and transport them in a sample chest with suitable refrigerant capable of maintaining the sample at 0-4?C until arrival at the laboratory. Do not freeze refrigerated products. Unless otherwise specified, refrigerated samples should not be analyzed more than 36 h after collection.
This sampling plan applies to the collection of finished products under surveillance and/or for determination of compliance for regulatory consideration. It also applies to the collection of factory samples of raw materials in identifiable lots of processed units and/or finished products where regulatory action is possible. It does not apply to the collection of in-line process sample units at various stages of manufacture since those samples do not necessarily represent the entire lot of food under production.
Sample Processing for Microbiological analysis.
Use aseptic technique when handling product. Before handling or analysis of sample, clean immediate and surrounding work areas. In addition, swab immediate work area with commercial germicidal agent. Preferably, do not thaw frozen samples before analysis. If necessary to temper a frozen sample to obtain an analytical portion, thaw it in the original container or in the container in which it was received in the laboratory. Whenever possible, avoid transferring the sample to a second container for thawing. Normally, a sample can be thawed at 2-5?C within 18 h. If rapid thawing is desired, thaw the sample at less than 45?C for not more than 15 min. When thawing a sample at elevated temperatures, agitate the sample continuously in thermostatically controlled water bath.
Various degrees of non-uniform distribution of microorganisms are to be expected in any food sample. To ensure more even distribution, shake liquid samples thoroughly and, if practical, mix dried samples with sterile spoons or other utensils before withdrawing the analytical unit from a sample of 100 g or greater. Use a 50 g analytical unit of liquid or dry food to determine aerobic plate count value and most probable number of coliforms. Other analytical unit sizes (e.g., 25 g for Salmonella) may be recommended, depending on specific analysis to be performed. If contents of package are obviously not homogeneous, macerate entire contents of package and withdraw the analytical unit, or, preferably, analyze each different food portion separately, depending on purpose of test.
Aseptically and accurately weigh unthawed food (if frozen) into jar. If entire sample weighs less than the required amount, weigh portion equivalent to one-half of sample and adjust amount of diluent or broth accordingly. Add 450 ml Butterfield's phosphate-buffered dilution water to blender jar containing 50 g analytical unit and blend 2 min. This results in a dilution of 10-1. Make dilutions of original homogenate promptly, using pipets that deliver required volume accurately. Do not deliver less than 10% of total volume of pipet. For example, do not use pipet with capacity greater than 10 ml to deliver 1 ml volumes; for delivering 0.1 ml volumes, do not use pipet with capacity greater than 1.0 ml. Prepare all decimal dilutions with 90 ml of sterile diluent plus 10 ml of previous dilution, unless otherwise specified. Shake all dilutions vigorously inorder to ensure uniform distribution f the sample through out the diluent. Not more than 15 min should elapse from the time sample is blended until all dilutions are in appropriate media and the time between sample collection and inoculation should not exceed 4 hrs.
The Total Plate Count (TPC)
The total plate count (TPC) method is intended to find out the total number of viable bacteria (total heterotrophic bacteria-THB) in a sample. The no: of total viable bacteria in a given sample can give an account about the quality of the food or the level of microbial pollution in a water body. The determination of THB by total plate count method is done to evaluate general bacteriological quality of the sample being analyzed. To determine the total viable count, a specified volume of the sample is plated and grown on a nutrient medium. After incubation at specific temperatures, the number of colonies developed in each plate is counted assuming that each colony has developed from single bacterial cell. Usually plates with 30-300 colonies are selected for counting. If the expected bacterial load is very high (more than 300), the sample should be serially diluted several times in order to get the colonies within countable range up on incubation.
Procedure:
The sample should be homogenized in a diluent (diluent should be an isotonic solution with neutral pH) before inoculation (the sample to diluent ratio should be 1:9 to form 1/10th or 10-1 dilution of the a sample). One ml from the homogenate should be aseptically transferred into 9 ml sterile dilution blank. This forms the 10-2 (1/100) dilution of the sample. The sample should be serially diluted several times depending on the expected bacterial load. Separate sterile pipettes should be used for each transfer during serial dilution and the dilutions should be mixed well before next transfer. One ml from the respective dilution should be plated (pour plating or spread plating) on a nutrient medium (Nutrient agar/Plate Count agar) in duplicate and plates should be incubated at specific temperatures (depending on the ambient temperature from which the sample is collected). After incubation plates with colonies within countable range (30-300) should be counted and the average number should be expressed in colony forming unit per ml or gm or cm2 of the sample.
Most Probable Number (MPN) of Coliforms
Coliforms are defined as Gram-negative facultative rod shaped bacteria capable of fermenting lactose to produce acid with the liberation of gas. The detection of coliforms in any type of food sample is significant from the public health view point, since they indicates the possible presence of other enteric pathogens. This was based on the premise that coliforms are abundant in human and animal feces and not usually found in other niches. Furthermore, since they can be easily detected by their ability to ferment lactose, it is easier to isolate them than othar gastrointestinal pathogens. Hence, the presence of coliforms in food or water became accepted as indicative of recent fecal contamination and the possible presence of other pathogens. The presence of coliform bacteria in a sample can be quantitatively detected by MPN test using three-tube decimal dilution method. This is mostly done by inoculating the sample into a medium containing lactose and an indicator such as phenol red to detect acid production. Inverted
Procedure:
The sample should be homogenized in a diluent at 1:9 ratio and 10 ml of the homogenate should be inoculated into the first set of three test tubes containing 10 ml double strength media (lactose broth, MacConkey broth, EC broth etc). The next set of test tubes containing 10 ml single strength media should be inoculated with 1 ml sample homogenate and the final set of test tubes containing 10 ml single strength media should be inoculated with 0.1 ml sample homogenate. An inverted Druham?s tube should be placed in each test tube prior to sterilization of the media. All the nine test tubes should be incubated at respective temperatures (44.5oC for fecal coliforms and 37oC for total coliforms) 24-48 hrs. After incubation the no: of tubes showing growth and gas production in each set should be recorded and the MPN index of coliforms per gram or 100 ml sample can be obtained from the standard MPN table.
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