Collecting samples for analysis

Whenever possible, samples packaged in individual packages should be collected and sent to the laboratory in their original commercial packaging, sealed and intact. Each packaging unit of an individual package of the product constitutes a sample unit and as many sample units should be collected as required by the sampling plan. If the packaging unit or individual package contains an amount of food insufficient for performing the required analyses and the keeping of counter-samples, a sufficient number of individual packages should be collected as part of one and the same sample unit. At the time of analysis, the contents of these individual packages should be placed together in one single sterile flask, which must be subsequently thoroughly mixed before withdrawing an analytical unit of the mixture. If the product does not allow for such mixing, the analyst should take, from each of the individual packages, portions of approximately equal weight, to compose or complete the analytical unit for that particular sample unit.

In the case of foods contained in vats, tanks or large containers, impossible to transport to the laboratory, representative portions should be transferred from the bulk product to sterile collecting flasks or bags under aseptic conditions.

Selection and preparation of  Containers for the  sampling of  foods contained in non-individual packages

a)  Use flasks or bags with leak-proof caps, made from non-toxic material approved for food contact and, preferably, autoclavable or pre-sterilized. The use of glass flasks or containers is not recommended due to the risk of breakage, contamination of the sampling environment with pieces of broken glass and loss of sample material.

b)  Choose flasks of appropriate size for the amount of food to be collected. To determine the quantity of sample to be collected, consider that each sample unit should contain at least twice the number of analytical units that will be used in the tests and preferably three or four times that amount (to allow for proper separation of counter-samples and prevention of possible spills or losses). Also consider that only three-quarters of the sampling flasks’ capacity should be filled with the sample (to prevent overflow and to allow proper mixing of sample before withdrawing the analytical units).

c)  Non-pre-sterilized flasks and utensils that will

be used to collect food samples (such as spatulas, spoons, scissors, tweezers, openers, corer samplers, etc.) should, preferably, be sterilized individually in an autoclave (121°C/30 minutes) or in a sterilizing oven (170  + 10°C/2h). Some other methods may be used as an alternative, such as flame sterilization, immersion in ethanol and alcohol combustion, and treatment(s) using disinfectant solutions. In the latter case, only disinfectants approved for use on food-contact surfaces should be used, in strict adherence to the manufacturer’s instructions and followed by 12 rinsing cycles with sterile reagent-grade water to remove all residues. Non-sterile flasks or bags showing – after having been subjected to an internal surface washing test – counts of viable microorganisms smaller than 1 CFU/ml of their holding capacity, may be used directly without previous sterilization.

Procedures for the  sampling of  foods contained in non-individual packages

a)  Before starting to collect the sample unit, the whole

mass of the food should be thoroughly mixed, to ensure that the microorganisms will be evenly distributed throughout the food. Next, using appropriate utensils or instruments, withdraw the amount of product necessary to compose or complete the sample unit.

b)  If it is not possible to thoroughly mix the food mass before initiating sampling, portions should be taken from different parts of the content, until obtaining the amount of product appropriate to compose or complete the sample unit. Avoid withdrawing portions of the regions close to the surface or opening of the tank or container.

b.1)   To collect powder samples from different parts of tanks or large packages, corer samplers or vertical double-tube samplers, long enough to reach the center of the food mass, may be used. A different sterile sampler or sampling device

should be used for each sample unit to be collected, or the instrument should be sterilized between one sampling operation and the next.

b.2)   To compose or complete a sample unit with portions taken from different points of foods that consist of one large solid piece, sterile knives, tweezers and forceps should be used to cut the food into smaller pieces.

b.3)   In the case of large blocks of frozen foods, such as frozen fish blocks and frozen seafood blocks, volumes of frozen liquid egg, etc., the most adequate procedure is to use an electric drill (with a previously sterilized drill bit( in combination with a sterile funnel. Insert the drill bit in the funnel (the lower opening inside diameter of which should be only slightly greater than the diameter of the drill bit) and position the bit onto the point of the block from which a sample is to be taken. Turn on the drill and scrapings of the frozen food will move towards the surface and accumulate in the funnel, from where they can be transferred to an adequate collecting flask.

b.4)   when samples are collected using faucets or tubes, the outer part of the outlet should be cleaned with ethanol 70%, sterilized by flame, if the material is fire resistant. The initial amount of product should be discarded before starting collecting the sample material. This will wash out the pipe and remove any accumulated dirt or residue particles.

b.5)   For the sampling of margarine and similar products (“spreads”) ISO 6887-4:2003/Cor.1:2004 recommends removing the external layer (3 to 5 mm) and withdrawing the sample units using a previously sterilized corer sampler. Insert the instrument diagonally, without reaching the bottom, rotate it in a complete circle and pull the sampler out, lifting out a conical portion of the product.

c)  Remember that the external surface of collecting flasks and bags is not sterile. For that reason, do not hold flasks or bags directly above the mass of food, as contaminants may fall or otherwise be introduced into the product. Likewise, never insert a collecting flask directly into the product, but use an appropriate utensil instead to withdraw the sample units.

d)  When withdrawing the collecting instrument filled with collected product, do not hold it above the other pre-sterilized instruments, since spatters of the food may contaminate the instruments that will be utilized later on.

e)  Open the collecting flasks or bags only as far or wide enough to insert the product and close/seal immediately.

f)  Do not touch the internal surface of collecting flasks or bags and their respective caps or closures.

g)  Contaminated foods may contain microorganisms that are harmful to health. These samples should be collected by staff that are well-trained in the handling of microorganisms and who are aware of the care required for protecting their health and safety. In case of doubt, each sample should be treated as if it were contaminated.

 Sampling of  foods involved in  foodborne diseases

Collect and analyze samples of all suspected foods as soon as possible. However, it is of no use to collect samples that have undergone temperature abuse or that are already in a state of partial deterioration. The results of such analyses will be of little or no use to the conclusions of the investigation. If there are no leftovers from suspected meals, one of the following alternatives may be tried: collect samples from similar meals, prepared at a later point in time but under the same conditions, collect samples of the ingredients and raw materials used in the preparation of the suspected meals and collect all containers and cooking utensils used to hold or prepare the suspected meals.

Sampling of   water

These samples must be collected from their original sealed packaging. If there is any desire or need to collect smaller volumes from pack-aging of greater holding capacities, the entire content should be homogenized by inverting the packaging several times in quick succession. Next, the mouth or outlet should be disinfected with ethanol 70% and, under aseptic conditions, the seal broken open with a sterile or flame-sterilized knife or pair of scissors. Do not collect but dispose of the initial volume or run-off and then collect the sample in an adequate sterile flask.

To collect other kinds of water, section 9060A of the   21^st Edition of the  Standard Methods for the Examination of Water and Wastewater  pro-vides the following guidelines:

To collect samples from faucets or pipes, clean the external area of the outlet with a solution of 100 mg/l sodium hypochlorite or ethanol 70%, in addition to flame-sterilizing it if it is made of fire resistant material.

Open the faucet completely and let the water run for approximately 2 to 3 minutes to flush out any debris or impurities and clean out the piping system. Reduce the flow of water to collect a sample without spilling water droplets out of the collecting flask.

To collect water from wells or cisterns with a pump, the water should be pumped out for at least five to 10 minutes to allow the temperature of the water to stabilize before starting the actual sampling. In case there is no pump available, collecting flasks should be prepared by attaching a weight onto the base or bottom and introducing the flask directly into the well. Care should be taken not to contaminate the sample with material and impurities that may have accumulated onto the surface of the water.

To collect water from rivers, lakes or water reservoirs, hold the collecting flask by its base and then lower it into the water until it is totally immersed and covered by the water surface with the mouth of the bottle turned downward. Turn the mouth of the flask into the direction of the water flow with a slightly upward slope, so that the water will be retained. If there is no water flow or current, push the flask forward horizontally, in the direction opposite to that of the hand.

Samples of chlorinated water should have any residual chlorine neutralized immediately after the samples are taken, to eliminate its bactericidal effect against the microbiota  present. To that purpose, 0.1 ml of a 3% Sodium Thiosulfate (Na₂S₂O₃) Solution should be added to the collecting flasks (before sterilization), for each 100 ml of sample to be collected. This amount is sufficient to neutralize 5 mg of residual chlorine per liter of sample. In situations in which the concentration of residual chlorine exceeds 5 mg/l, utilize 0.1 ml of a 10% Sodium Thiosulfate (Na₂S₂O₃) Solution for each 100 ml sample. This quantity is enough to neutralize 15 mg residual chlorine per liter of sample. Sterile plastic bags or flasks, which are commercially available and already contain sodium thiosulfate, may also be used. If the sample is collected and sent to the laboratory by the interested person, without previous neutralization of the chlorine, a sterile sodium thiosulfate solution should be added immediately upon arrival of the sample, under aseptic conditions.

Water samples containing high levels of metals (greater than 1.0 mg/l), including copper and zinc, should be collected in flasks containing EDTA (ethylenediamine-tetraacetic acid), a chelating agent used to reduce the toxicity of metals to microorganisms. This is particularly important if the interval between the time of sampling and the time of analysis is greater than four hours. For that purpose, the collecting flasks should – prior to sterilization – be furnished with 0.3 ml of a 15% EDTA solution for each 100 ml of water to be collected (372 mg/l). Adjust the pH of the solution to 6.5 before use. The EDTA and thiosulfate solutions may be added to the same flask.

References

SILVA, N.D .; TANIWAKI, M.H. ; JUNQUEIRA, V.C.A.;  SILVEIRA, N.F.A. , NASCIMENTO , M.D.D. and GOMES ,R.A.R .(2013) . MICROBIOLOGICAL EXAMINATION METHODS OF FOOD AND WATE A Laboratory Manual . Institute of Food Technology – ITAL, Campinas, SP, Brazil.