Microbiological Analysis - key considerations

You might have a critical raw ingredient that undergoes minimal, or no, processing where a key food safety, or food spoilage microbe, must be present at less than a certain level. It could be a legal requirement, as part of EU Regulation 2073/2005 on microbiological criteria for foodstuffs. It could be part of a finished product verification criteria, or for the protection of sensitive products and ingredients, or a complaints investigation into a quality or taint defect.

Microbiological results can also be very useful in trend analysis, so that any drift or movement towards an unsafe product, or one where the shelf life may be reduced, can be detected and acted upon, before it is too late.

1. Environmental monitoring

Environmental monitoring can be part of the ongoing suite of tests, or in response to a contamination incident, for example, if Salmonella or Listeria were detected in the final product. This may require a systematic approach to locating the source, by starting with drain sampling, then focusing down on surfaces, or ingredients, once an area is pinpointed.

If routine, then walls, doors or strip curtain divides, working surfaces and key areas of machinery should be included. Tests are usually for background flora (see ‘Spoilage organisms’ below), or indicator (or index) organisms that indicate that pathogens may be present (e.g. Listeria spp. for Listeria monocytogenes; Enterobacteriaceae or faecal coliforms for Salmonella).

Rapid methods for environmental contamination include those using a luminometer to detect Adenosine Triphosphate (ATP). These can be used for surfaces or post clean-in-place (CIP) wash water and give results in 10 to 20 seconds. It is worth noting that at least 10⁴ cfu bacteria are required to trigger a minimal response, with food residues and fruit juice cells, giving a greater response. ATP testing should therefore be seen as a cleaning verification tool, not to determine microbial cleanliness, however, it is a very useful rapid method. Other rapid methods include colorimetric techniques used on liquid processing lines, flow cytometry and DNA/RNA based techniques.

In all cases, baseline levels need to be determined so that upwards trends, or spikes, can be identified and dealt with. It may be that some surfaces or machinery do not give results below the limit of detection, due to their age or cleanability, but this may not contribute to excessive product contamination.

2. Product testing            

1. Spoilage organisms

Before requesting a test suite, it is important to understand the following:

• capability of the test to recover microorganisms
• when the results are required
• sensitivity and the cost of the method.

Dilution of the sample is usually required to be able to enumerate the microflora of a sample. It is helpful to have an approximate idea of the contamination level of a sample, or the laboratory might not be able to enumerate either very low or very high levels of contamination. All methods have a lower limit of detection; a negative result may not mean a microbe is absent which could be significant with respect to pathogens (see below). Dilutions range from 10^-1, for lightly contaminated foods, to as low as 10^-6, for heavily contaminated. When selecting the dilution series, it is important to ensure that pathogens present at low levels, are not missed by using only the more dilute aliquots, for the more common organisms.

The test method chosen will define the microorganisms detected and the count. For bacteria, an Aerobic Colony Count [ACC, also referred to as a Total Viable Count (TVC) or Aerobic Plate Count (APC)] can be conducted using Plate Count Agar, at temperatures ranging from 20 to 37°C, for 48 to 120 hours – see Table 1. Tests to isolate more specialised microorganisms can be carried out at temperatures above or below.

High ACC’s can be expected on unwashed salads and vegetables, and from fermented foods, and therefore testing for this parameter is not useful. This applies to any food or ingredient where high natural levels can be expected.

To determine water quality and safety, a Heterotrophic Plate Count (Total Heterotrophs) is often used, with a two plate two temperature method - see Table 1. The temperature selected is dependent upon the type of sample, its typical temperature and in some cases the country of origin of the method used. A direct comparison of results between methods should therefore not be made and the resultant flora isolated can vary considerably. Table 1 outlines a selection of some of the rationales for ACCs and HPCs.

Table 1: Aerobic colony count protocols for bacteria

Method	Incubation Temperature (°C)	Incubation Time (hours)
American Public Health Association	20 32 or 35	72-120 48
ISO – Total Heterotrophs for water	22 36 or 37	72 48
ISO/BS/EN for food and feed	30	72
Association of Official Analytical Chemists	35	48
Brazilian Ministry of Agriculture	36	48

Whether a food is heat processed, with a kill step during production, or processed without heat, will affect the final flora of the product, and determine acceptable microbial limits, and the types of microorganisms for which to analyse.

Indicator organisms highlight the efficacy of a process, its hygiene status and potential faecal contamination. Index organisms mark the potential presence of a pathogen. Confusingly, the term indicator organism has also been used, by some, for potential presence of a pathogen. Enterobacteriaceae and coliforms are used as indicators of hygiene and process efficacy for pasteurisation, E coli or faecal coliforms as a faecal indicator, and non-pathogenic spores for the efficacy of a high temperature process. Each microbial group has its merits and demerits. Figure 1 shows the relationship between the different groups.

Figure 1: Relationship between Enterobacteriaceae, coliforms, faecal coliforms, Salmonella, E. coli and STEC

Yeast and mould counts can be used as general hygiene indicators, and also in spoilage investigations in key industries, for example soft drinks, confectionery, bakery, and dried foods. Plates are typically incubated at 25°C for five days, however problems can arise with the interpretation of yeast and mould counts in spoilage investigations. There are a number of methods, with different media, temperatures, and each method may isolate a different mould flora, which may not be related to the mould or group of moulds causing spoilage.

Lactic acid bacteria (LAB) are present at high levels in most fermented foods, and therefore their analysis in this type of food is not recommended. They are also environmental bacteria and can be expected in unwashed salads and other vegetables. They can indicate process failure or recontamination after processing, or cleaning failures, in the soft drinks industry, where they can cause considerable spoilage.

Tests for aerobic and anaerobic bacterial spores are often used to indicate process failure. In these cases, it is important to know the thermal process used, because each species’ spores have unique thermal properties. These are subdivided into tests for mesophilic and thermophilic spores. Survival of spores may not indicate process failure, if the product formulation has other control factors, to inhibit surviving spores.

Each test has a minimum turnaround time that is fixed, for example, 5 days for yeasts and moulds; LAB 2 days at 35°C, 3 days at 25°C; aerobic spore counts at 7°C for 10 days, 30 -35°C for 2 days, or 55°C for 2 days. Sometimes these can be extended if the process, or product, includes a sub-lethal thermal process, or other intrinsic control factors which inhibit recovery of viable cells. This is usually outside the scope of the test but can be helpful in investigations.

Coliforms, Enterobacteriaceae, LAB, yeasts and moulds are commonly found on fresh produce, therefore tests for their presence are of limited use. The presence of faecal coliforms or E. coli could indicate faecal contamination of salad vegetables, and the potential presence of Salmonella.

The origin of ingredients can affect the microflora, for spoilage bacteria and moulds, and for pathogen serovars.

b. Pathogens

Isolation methods for pathogens fall into two categories: detection (presence/absence) and enumeration.

i. Detection methods               

These are used where, either the pathogen is present at low numbers in the particular food, is difficult to detect in the food, in a non-homogenous distribution, or poses a severe hazard to the general population, or consumer group, for which the food is intended. There are usually three or four stages, which makes it a lengthy process.

Standard enumeration methods are similar to those for spoilage or environmental organisms, with dilution (often to a lesser extent), because they may not be present at such high levels.  The timescales range from 18 - 24 hours to 4 - 5 days, depending upon the organism and the test method. ISO or national standards body methods tend to be slower but are more likely to work with a greater range of isolates.

It is helpful if the laboratory is able to adapt methods if the samples taken are part of an investigation to determine why a pathogen has been found in a food or in the environment. It is important to remember that a test result is presumptive until confirmed.

Rapid methods can be divided into two types: those that provide a more rapid turnaround for the isolation and detection phase, or a rapid confirmation stage. For the former, a non-selective or selective amplification stage is usually required for a rapid isolation method, or it is likely that the small sample aliquot used for the rapid test may not contain any of the relevant pathogen cells. The end result may or may not require further confirmation, depending upon the type of rapid isolation and detection test used.

ii. Confirmation methods

These are either slow [typically more complex ISO or another standards body (so-called ‘Gold Standard’), which usually work with the widest range of isolates of a particular species], or more rapid techniques. If the latter, it is preferable that they have been accredited against the ‘Gold Standard’, either by the laboratory, or more usually by the national standards laboratory, or similar. A rapid confirmation test should give comparable results to the standard method, otherwise false positives or negatives may result.

Rapid confirmation methods include serological (ELISA or similar), miniaturised multi-well tests (API or similar), or DNA/RNA based tests.

Each method has a minimum sensitivity, i.e. a record of ‘absent’ or ‘not detected’ means to the lowest sensitivity of the method. Similarly, ‘Too Numerous To Count’ (TNTC) means that an incorrect dilution series was used for the test, such that common microbes grew too close together to be able to count as individual colonies.

Microbiological results can be very useful in trend analysis, so that any drift or movement towards an unsafe product, or one where the shelf life may be reduced, can be detected and acted upon, before it is too late.

3. Relevant physical parameters

It can be helpful to request, or to already have information on, key product parameter tests. They can be used to guide the testing suite and include:

• Water activity (a_w) - this can determine which microorganisms are able to grow in the product since each microbe has a very clear a_w below which it cannot grow. The theoretical a_w may not be the same as the actual value. Xerophilic yeasts and moulds are adapted to growth at extremely low a_w and may take some time to grow in the product
• Brix - useful to calculate for juices and concentrates, as like a_w it will determine which microorganisms can grow
• pH - of a product can determine which microorganisms can grow and can also influence their heat resistance at any given value. Some pathogens, and many spoilage microorganisms, are able to grow or survive at low pH. Yeasts and moulds are largely unaffected by low pH over the typical ranges of food and drinks.

3. Shelf life and challenge testing

It is important, when designing a shelf life testing protocol, that appropriate time intervals are considered, particularly if the budget is limited. Are changes in microflora (growth or death) expected at the beginning or the end of the trial period? It may be more efficient to have uneven time intervals, focusing on the expected key period, but still retaining time points until the end of the trial, so that potentially useful data is not lost. In addition, the testing suite could vary, from the start to the end of the trial, for the same reason.

Challenge testing is carried out when an important pathogen is present infrequently, but it’s growth or survival in the product would render it unsafe for consumption. The product is therefore ‘challenged‘, with the microorganism of concern, under typical storage conditions. In this case, the use of strains, their adaptation to the test matrix, the inoculation methods, the experimental matrix (factors x timepoints), the interpretation of results, and the likely background flora are all important factors for the laboratory to consider.

4. Testing specific foods

The IFST Handbook of Microbiological Criteria for Foods, 2^nd edition gives a comprehensive set of criteria for different food types, with a helpful indexed table cross-referencing the individual criteria tables. For example, if biscuits, breakfast cereals, cakes, chocolate, confectionery and potato crisps are selected, then Table N ‘Dried foods - ready to eat’, is referenced. In other cases, more than one table may be referenced. For example, selecting spices points the reader to Table N, as well as Table M ‘Dried foods - to be cooked’ and Table O ‘Dried heat processed foods - ready to eat after rehydration'.