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Micro Testing in a Recall

It is important that food businesses understand the meaning of microbiological test results and have a broad

understanding of laboratory test methods. No time is this more important than during a contamination

incident or recall situation, where high level decisions have to be made based upon microbiological test

results and confidence relies upon choosing the right laboratories and test methods. In this article, we

explain the meaning of some commonly misunderstood microbiological terms and compare the use of

traditional and rapid methods.

Colony Forming Unit (cfu)A cfu is a compact mass of bacterial cells growing on an agar plate which is visible to the naked eye. The number of colonies on an agar plate is expressed as colony forming units (cfu) per unit volume or weight or area of the sample, e.g. cfu/g, cfu/ml, cfu/swab area.

Each colony originates from a single bacterial cell, or a clump or chain of cells. Bacterial cells are rarely evenly distributed throughout a food sample, so there will be variations in the cfu count between samples of the same batch.

Viable

In food microbiology, the term viable means that bacterial cells are alive and able to grow. The formation of colonies on an agar plate indicates that at least one cell in the food sample was alive and able to replicate.

Some rapid methods detect the presence of genetic material from the target micro-organism, and not necessarily whether any live cells are present.

Enumeration tests or plate count method

Laboratories use an enumeration (or plate count method) to determine the numbers of bacteria in a food sample. Since the number of bacteria in a food sample is unknown, and could be very high, the laboratory carries out a serial dilution of the original sample (1:10, 1:100, 1:1000 etc.). A proportion of each diluted sample is then inoculated on to an agar plate. The plates are incubated at warm temperatures for 1 to 3 days to encourage the growth of colonies. From the set of plates, the Microbiologist chooses the best plate to count; a plate with between 30 and 300 colonies is ideal. This number of colonies is then multiplied by the dilution factor to give an estimation of the cfu/g or cfu/ml of micro-organisms in the original food sample.

Typical enumeration tests are Total Viable Count (TVC) on plate count agar, or E.coli on MacConkey agar. Whether the result is acceptable or not depends on the type of micro-organism present and the category of food; is the product ready-to-eat or raw? In very general terms, counts of 10-100 cfu/g are low and counts above >1000 cfu/g are high.

Key terms you need to understand

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Test results given as < cfu/g or > cfu/g

Quite often, laboratories report results as less than or greater than. The table below provides guidance on how to interpret these results.

Result reported by laboratory Interpretation

<1 cfu/ml This indicates that a direct plate of a liquid sample (such as milk or juice) was carried out and no colonies grew. This means that 1 ml of a neat liquid sample was inoculated directly on to an agar plate without any dilutions. Since no colonies grew on the direct plate, there are less than one cfu’s per ml of the sample.

This does not mean there are no micro-organisms in the original sample; it means that using the given test method, no colonies grew, so the levels are below the limit of the test.

<10 cfu/g There were no colonies growing on the agar plate derived from the first dilution.

With solid food samples, it is not possible to carry out a direct plate as the food has to be mashed up into a liquid form so that the sample can be pipetted onto an agar plate. Testing of solid food will require one or more dilutions.

<20 cfu/g The examples given above describe the results when a 1ml sample of the diluent is inoculated on to an agar plate. Sometimes it is difficult for that much liquid to be absorbed onto a plate, so the lab may pipette 0.5ml onto the plate instead. The limit of detection where 0.5mls of a 10-1 dilution is plated out is <20 cfu/g.

Greater than results, e.g. >1000 cfu/g

When results are expressed as greater than it means that there were too many colonies to count.

Greater than results mean that there are a high number of micro-organisms in the original sample, or that the laboratory has not performed enough dilutions to obtain a count. It is important that laboratories are made aware if samples are likely to be highly contaminated so they can carry out the appropriate number of dilutions.

Detected/Not detected test (Also known as presence/absence)

For pathogen testing, laboratories use qualitative methods to detect the presence of a particular pathogen in a food sample. These methods typically involve a number of steps to recover and grow viable cells of the target organism, followed by the use of a variety of confirmatory tests to confirm the result/species.

Since the first stage of these methods usually begins by taking a 25g or 25ml sample of the food, the results are expressed as detected or not-detected in 25g or 25ml. A negative (or not-detected) result means that the target organism was not detected in the 25g/25ml portion of the food sample that was tested using the given method; it does not mean that there are no pathogens present in a whole batch of food.

Presumptive resultA presumptive, or ‘suspect’ result is reported when the method has detected growth of micro-organisms which appear to be typical of the target micro-organism. The result cannot be confirmed until further tests are performed.

When presumptive results are not confirmedMicrobiological testing methods have been designed to encourage the growth of the target micro-organism and filter out unwanted micro-organisms, however occasionally a ‘rogue’ micro-organism gets through the initial steps of the method and can appear like the target organism. For example, some Bacillus species can grow on Listeria agar plates. Also, sometimes the target micro-organism may not behave in a normal way or the colonies may not look typical if the target micro-organism in the food sample has been stressed or damaged by the food manufacturing process.

A presumptive result may also be reported when using rapid methods; for example, if a sample is tested positive with PCR, it means that the method has detected the presence of DNA from the target organism, but it does not necessarily mean that there are viable bacterial cells present in the food sample.

It is for these reasons that the laboratory will report the result as presumptive and then go on to carry out confirmatory tests. Food businesses can view the reporting of presumptive results as a warning that a pathogen maybe present in their food. It is wise not to withdraw or recall food based upon a presumptive result alone. Nevertheless, it is good practice for the business to thoroughly investigate and prepare for the eventuality of a confirmed pathogen by retrieving production records for the suspect batch of food.

Confirmation testsOnce a presumptive result has been reported, confirmatory tests need to be performed to confirm if the suspect organism present is the target organism. Confirmatory tests could be a combination of biochemical, serology or DNA based checks, or analysis by mass spectrometry.

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AccreditationAlways ensure that your laboratory is accredited by a recognised national or international body to carry out the specific test they are undertaking for you. They must also be accredited to do that test in the type of food that is being tested. If they don’t have that accreditation, there may be some doubt about the reliability of the results.

Labs aren’t perfectIf you receive an unusual result, always check that there have been no errors or cross contamination in the lab. That happens. Before you make very important decisions, challenge the lab to check they are confident in their results.

Positive releaseIn some circumstances it may be necessary to screen raw materials or finished product prior to release/dispatch. Usually positive release focuses upon food safety criteria, for instance checking for absence of Listeria prior to despatch of a high-risk food.

Rapid vs traditional culture methodsTraditionally, testing food samples for pathogens has meant using the established ISO methods, which involve several steps to recover and grow the target organism on selective medium (laboratory media designed specifically for the target organism). The ISO methods are considered by some to be the “gold standard” of microbiological method and are given as the reference methods in EC 2073/2005 (Microbiological Criteria for Foodstuffs). Official control samples (tests carried out by the authorities) are tested using ISO methods. However, these methods are slow. It requires 4-5 days to get to the presumptive stage and a further 2 days for confirmatory tests, by which time the food product may have already been sold and consumed. These lengthy ISO methods have highlighted the need for rapid test methods, particularly for short shelf-life products or where positive release is required.

Rapid tests shorten the time to presumptive result by 1-2 days; some can provide presumptive results in <10 hours. This is great if the food samples have tested negative, however a positive presumptive result will still need to be confirmed, which usually takes a further 1-2 days.

The table below illustrates the timescales for the detection of Listeria monocytogenes using ISO compared to a popular rapid method, ELISA.

Result stage ISO Method ELISA

Presumptive 4 – 5 days 2 days

Confirmation 2 days 1-2 days

Total time for confirmed positive result:

6 – 7 days 4 days

The most common rapid tests for pathogens used by contract testing laboratories are PCR (Polymerase chain reaction) and ELISA (Enzyme-linked Immunosorbent Assay). PCR methods detect the presence of a proportion of DNA which is unique to the target micro-organism. ELISA uses the principles of antibody-antigen reactions to detect presence of pathogens. Recently there has been some interesting developments in rapid methods, including the use of phage technology, which utilises viruses that infect pathogens to detect presence of a pathogen. Alternatives to traditional confirmatory tests are also becoming more widely available, for example with the use of mass spectrometry and DNA sequencing to confirm species.

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Variability of resultsWhy do I get different micro results from multiple samples of the

same batch?

Micro-organisms are rarely evenly mixed throughout a food product. With multiple component products such as ready meals and sandwiches, there will be even greater variation as each component will have a different microbial loading and the micro-organisms will behave differently in each component, depending on the levels of moisture, acidity, salt etc. There will also be an element of variability in the test method.

What is an acceptable variation?

Results within 0.2-0.5 log unit are considered to be within normal variation. For example, with a target of 1000 cfu/g (log 3), acceptable results can range from 300 to 3000 cfu/g and be considered microbiologically equivalent. Since micro results are expressed as log counts, a result of 20,000 cfu/g is not twice as bad as a sample with 10,000 cfu/g.

Hygiene indicatorsEnterobacteriaceae, coliforms and E.coli tests are hygiene indicator tests. These are very useful tests since they are easily performed in the laboratory with results available in 24 hours, and they give information about the hygienic conditions of food manufacturing and adequate heat processing. Enterobacteriaceae is the most widely used of the hygiene indicator tests since it covers a wide range of micro-organisms, including environmental contaminants and enteric pathogens.

Need help?

For more information on how RQA can help get in touch at:

contact@rqa-group.com or call us on +44 (0)118 935 7242

RQA can help with laboratory consultancy and training, including specific training on microbiology for food businesses which includes lab result interpretation. We can also help with recall planning, training and simulation exercises.