Transport and storage of microbiological samples: from the sampling point to the laboratory

The sample keeps changing after it is taken

Hours or days can pass between the moment a sample is taken and the moment the analyst opens it. In that interval, bacteria can multiply if the sample warms up, die if a product that should travel refrigerated is frozen, or remain exposed to a sanitizer that was not neutralized. The final result describes the sample as it arrived at the laboratory, not necessarily as it was in the plant.

The FDA's Bacteriological Analytical Manual (BAM) puts it bluntly: if samples are collected improperly, mishandled or not representative of the lot, the laboratory results are meaningless. This article gathers what the main references say about how to get the sample from the sampling point to the analyst's bench.

Containers and labeling

The BAM recommends sending the product in its original unopened container whenever possible. When that is not possible, portions should be transferred aseptically to clean, dry, sterile, tight-sealing, wide-mouth containers, avoiding glass, which can break. For dry products, sterile bags or cans work; sterile plastic bags should be used only with dry, non-frozen materials. Some specific recommendations:

• Obtain at least 100 g per sample unit.
• Do not overfill bags and prevent closure wires from puncturing them.
• Identify each unit with labeled tape; the BAM advises against writing with a marker on plastic because the ink can pass through it.
• Send as a control an empty sterile container exposed to the same conditions as the sampling.
• Record the date and time of collection and of arrival at the laboratory for each sample.

Temperature by product type

The BAM's general rule is to deliver samples to the laboratory as soon as possible and, as far as possible, maintain their original storage conditions:

• Dry or non-perishable canned products: can travel at room temperature.
• Frozen products: must be kept completely frozen at all times, in pre-chilled containers and rigid insulated shippers.
• Refrigerated products: are cooled on ice to 0-4 °C and transported in a box with coolant capable of maintaining that temperature until arrival. They must not be frozen. Unless otherwise indicated, they should not be analyzed more than 36 hours after collection.
• Mollusks and shellfish: have their own conditions. The BAM indicates examining them ideally within 6 hours and never more than 24 hours after collection.

For liquid samples, the BAM suggests taking an additional unit as a temperature control and measuring it when the sample is taken and when it is received. It is a simple way to document that the cold chain was maintained.

Surface samples: the neutralizer and the clock

With swab and sponge samples there is an additional factor: disinfectant residues. If not neutralized, they can keep inactivating microorganisms during transport and produce a false negative. The 2004 edition of ISO 18593 already pointed out that there is no universal neutralizer and mentioned some common options: lecithin and sorbitan monooleate for quaternary ammonium compounds, sodium thiosulfate for halogen-based products, and catalase or peroxidase for peroxide-based disinfectants. The neutralizer should be chosen according to the sanitizer the plant uses, as discussed in the guide on post-sanitation surface verification.

The current version, ISO 18593:2018, is the reference that Regulation (EC) 2073/2005 sets for sampling processing areas and equipment. It indicates transporting samples refrigerated, between 1 °C and 8 °C, and examining them preferably within 24 hours of collection. For animal carcasses, the same regulation refers to ISO 17604, which describes the sampling methods and the storage and transport rules.

Water samples: residual chlorine

Chlorinated water poses the same problem as sanitized surfaces: the chlorine keeps acting inside the bottle. ISO 19458:2006, the standard for water sampling for microbiological analysis, indicates adding sodium thiosulfate to the bottles before taking the sample. According to the standard, 7.1 mg of sodium thiosulfate pentahydrate are needed to inactivate 1 mg of chlorine, and the usual dose is 0.1 mL of an 18 mg/mL solution per 100 mL of bottle capacity. With higher chlorine concentrations a larger dose is needed. The standard clarifies that thiosulfate does not affect the sample and can also be used in non-chlorinated waters. The interpretation of water indicators is covered in the article on coliforms, E. coli and enterococci in water.

Plan the shipment before sampling

A good share of transport problems can be avoided before the first sample is taken. The BAM recommends notifying the laboratory in advance when perishable samples are being sent, and that coordination makes it possible to confirm three things: that the laboratory will be able to receive and process the samples within the maximum time, what volume or weight it needs for each analysis and what acceptance criteria it will apply at reception. It is also worth defining who is responsible for the box during transit, where the temperature is recorded and how samples of different risk are separated, for example finished product and drain samples, to avoid cross-contamination between them.

Reception at the laboratory

Control does not end at the laboratory door. The BAM describes a reception with clear steps:

• Check the condition of the container: breaks, punctures, loose lids or pierced bags invalidate the sample because of the risk of cross-contamination.
• Verify labeling and records: each unit must arrive identified, with its documentation and, in official samples, sealed.
• Check the sampling plan: that the number of units corresponding to the requested analysis was taken.
• Store if not analyzed immediately: frozen at −20 °C, perishables refrigerated at 0-4 °C for no more than 36 hours and non-perishable or low-moisture products at room temperature.
• Notify whoever took the sample when it does not meet the conditions, to obtain a valid sample and prevent the problem from recurring.

Thawing without altering the sample

The BAM prefers not to thaw before analysis. If necessary, it recommends doing so in the original container, at 2-5 °C, which normally takes up to 18 hours; for rapid thawing, below 45 °C for no more than 15 minutes, with continuous agitation in a thermostatic water bath.

ISO/IEC 17025 includes the handling of test items among its process requirements: the laboratory must record any deviation from the specified conditions when receiving a sample and, if the customer still asks for it to be analyzed, indicate in the report which results may be affected. Reception criteria are one of the questions worth asking any laboratory, as suggested in the article on ISO/IEC 17025 accreditation.

Common mistakes

• Freezing refrigerated products to "preserve them better", which damages the cells and can reduce counts.
• Sending samples on a Friday afternoon to a laboratory that does not receive on weekends.
• Using swabs or sponges without a neutralizer suited to the plant's sanitizer.
• Taking chlorinated water in bottles without thiosulfate.
• Not recording the time of collection, which makes it impossible to show that the maximum time until analysis was respected.
• Putting finished product and drain samples in the same box without separating or labeling them by risk.