Microbiological testing of foods: what, why, how

Complexity in food testing arises from the food (matrix), the need to detect low numbers of target microorganisms in the presence of potentially similar background microflora, the potential use of testing to demonstrate compliance and the high cost (not just financial) of getting it wrong. Microbiological criteria for food specify the method of analysis¹ because “test results are dependent on the analytical method used”². Several bodies are involved in the development of standardised methods, and laboratories may have to run several methods for the same target to meet client needs. The current review of Standard 1.6.1 of the Food Standards Code³ and the July 2012 collaboration agreement struck between the International Organization for Standardization (ISO) and the Association of Analytical Chemists (AOAC International)⁴ should hopefully reduce the workload for food laboratories.

Microbial contamination is not uniform throughout a food⁵ and test results may not paint the right picture if an unrepresentative sample is examined. Mitigation of the risk of reporting on an unrepresentative sample includes the use of at least 10 g sample for testing and, when the results are used for assessing the quality of a batch of product, the use of a sampling plan which requires the removal of a number of samples for testing.

From a food safety perspective, there is a desire to detect low levels of pathogens in a food because of the potential for multiplication in the time between production and consumption. A minimum of 25 g is routinely taken for the examination for pathogens such as Salmonella and Listeria monocytogenes.

Food microbiologists must also consider the need to optimise the recovery of their target. Processing of foods may cause sub lethal injury and although the microorganisms are viable (and pose a potential health risk), they may not be culturable. The use of a resuscitation step in pathogen testing is designed to overcome, at least in part, this problem. Some food ingredients may inhibit the growth and subsequent isolation of the target organism. For example, the antibacterial compounds in cocoa containing products, such as chocolate, must be neutralised by skim milk when testing for Salmonella⁶.

Tests for indicator organisms and enumeration of bacteria are very much a feature of food (and water) microbiology. Indicator organisms are used to assess the potential for the presence of pathogenic bacteria. Tests for indicator organism/s seek out those microorganisms that are universally present in human faeces in greater numbers than faecally transmitted pathogens. Such tests must be reasonably easy and cheap. Non-specific bacterial counts, such as Standard Plate Counts provide a measure of the amount of bacterial load in a food, and are used to provide some measure of its microbiological quality.

The context for testing dictates what tests are appropriate. When testing is part of quality assurance programs, the aim is to verify that products have been suitably processed and that hygienic production conditions have prevailed. Tests for indicator organisms and bacterial count are common.

Testing is also used to meet product specification/s in trade agreements and to verify compliance with microbiological criteria set by regulators. A combination of tests for indicator organisms, Standard Plate Counts and pathogens is normally used. Such results can have financial and legal ramifications and it is important that they stand up to independent scrutiny. That is, results must be valid (do they provide a true picture of the item examined?) and reproducible.

It was recognised back in the 1890s, that results generated by different laboratories could not be compared “because of the substantial lab to lab variation in methods”⁷ and that there was a need for standardised methods.

Standard methods are consensus methods and aim to be the best practicable. That is, they must have acceptable test performance characteristics, be able to generate a timely result (results must be available before foods are consumed or past their shelf-life), do not need specialised equipment or special training of the analyst, and the cost of analysis must not be so prohibitive as to prevent its widespread use^1,8. The first manual of standard methods (for water analysis) was published in 1905^7,8.

Today, a large number of organisations, such as the International Organization for Standardization (ISO), the European Committee for Standardization (CEN), the North American based Association of Analytical Chemists (AOAC International) and Standards Australia are charged with developing standard methods. The decision on which standard method to follow is largely driven by trade agreements and regulatory compliance. ISO works closely with CEN and their joint ISO/EN standard methods are referenced in European Commission regulation². Thus, ISO/EN methods are widely used in Europe and AOAC methods, in North America; and their trading partners follow suit.

Standards Australia is recognised by the Australian Government as the peak non-government standard organisation. In the early days of method standardisation, Standard methods were developed along industry lines. Thus, there were the AS 1142 series for eggs and egg products; AS 1095 series for the dairy industry and AS/NZS 1766 series for food. Methods were developed by technical committees in response to requests from stakeholders, mostly regulatory agencies. This was obviously duplicitous and, commencing in 1987, the egg and dairy methods were transferred to the AS 1766 series.

Around 2001, the Standards Australia Food Microbiology Committee proposed to adopt international standards whenever possible, in line with Australia’s obligation under the World Trade Organization Treaty on (the reduction of) Technical Barriers to Trade (WTO TBT)⁹. A restructure of Standards Australia Food Technology committees was then undertaken so that the local committee structure aligned with those in ISO/TC 34 (Food Products) and a new technical committee, FT-024-01 (renamed FT-035 in 2011) was constituted to mirror ISO/TC 34/SC 9, Microbiology and ISO/TC 34/SC 5, Milk and Milk Products (dairy microbiological test methods)⁸. The AS 5013 series commenced with this change. Most of the standards in this series are ISO clones, some with Australian Annexes which either clarify requirements in the standard or document variations that apply in Australia.

Microbiological criteria (referred to, in Australia, as Food Standards) are set to protect public health. Standard 1.6.1 of the Australian New Zealand Food Standards Code “lists the maximum permissible levels of foodborne microorganisms that pose a risk to human health in nominated foods, or classes of foods”¹⁰. This Standard prescribes the methods of analysis – AS/NZS 1766 for food and AS 4276 for packaged water in line with Codex Alimentarius recommendations¹. Alternative methods may be used, but they must be demonstrated (using AS/NZS 4659) to be equivalent to that prescribed.

Methods are prescribed in legislation^1,2,10 as that ensures that the same measures are used to assess compliance. However, at times the legislative tool lags behind changes in the standardisation community. This has been the case in Australia where the current methods referred to in the Food Standards are no longer available: since 2004, the AS/NZS 1766 methods have been gradually migrated across to the AS 5013 series. This is expected to be rectified soon as Standard 1.6.1 is currently under review³.

Many of us have realised that the ISO methods did not meet our needs and we hope to influence the development of ISO standards by attendance at the annual plenary meeting and more importantly, through participation in method development working groups. Australia is currently represented in 6 working groups: for meat and meat products, method validation, Cryptosporidium and Giardia in foods, General requirements and guidance for microbiological examinations and psychrotrophic microorganisms.

At the international level, ISO is collaborating on an AOAC project [International Stakeholder Panel on Alternative Methods (ISPAM)] on the harmonisation of the microbiological criteria for alternative methods (i.e. validation and verification requirements) and in June 2012, ISO and AOAC International signed a cooperation agreement that will allow them to jointly develop and approve common standards. This will hopefully eliminate the need for laboratories with a wide client base to run several methods for the one target.