The scale of the taskIn any food company, ensuring microbiological

safety and quality is of paramount importance;

consequently much effort is focused on pathogen

detection. The challenge is a global one, as food

producers and retailers increasingly source mate-

rials from around the world. At an international

meeting held last year to set research priorities

on a broad range of foodborne diseases trans-

missible from animals to humans [1], scientists

noted that globalisation and integrated markets

are rapidly changing the way pathogens travel

from country to country. Although cross-border

transmission has been brought to wider atten-

tion with the recent incidence of avian influenza,

in fact diseases caused by organisms such as

Salmonella, Campylobacter and Escherichia coli

have a far greater impact on human health and

the economy. It was pointed out that in Germany

alone, there were 52,000 cases of Salmonella in

2005, and across the EU Campylobacter cases

continue to increase, making it the most common

foodborne disease in Europe.

As well as livestock products, fresh fruits and veg-

etables are often the source of infection, with well-

documented cases of E. coli O157:H7 occurring in

several countries. Prepared salads and vegetables

eaten raw can pose a particular risk.

Some of the economics of foodborne illness

are also worthy of consideration. For exam-

ple, in 2000 the Economic Research Service of

the United States Department of Agriculture

estimated the cost to the economy of five bac-

terial foodborne pathogens (Campylobacter,

Salmonella, E. coli O157 and non O157 STEC/

VTEC, and Listeria monocytogenes) to be

$6.9 billion [2].

There is a significant body of data that highlight

the human and economic impact of foodborne

disease. These data strongly support good

hygiene management at source and during food

processing. Clearly, the microbiology labora-

tory has a central role throughout the produc-

tion chain. In recent years there have been

considerable moves towards the development

of more reliable and more rapid methods for

the isolation, early detection, characterisation

and enumeration of micro-organisms. For the

food microbiologist this is, in practice, largely

about reducing the time taken to produce a total

viable count and the need to arrive quickly at a

presumptive negative or a presumptive positive

result for pathogenic organisms.

Rapid concentrationFor the most part, a food microbiology labora-

tory will be examining raw materials, finished

products or environmental samples that should

be free from pathogens and harbour only very

restricted numbers of potential spoilage organ-

isms. A standard enrichment process is normally

the first part of the protocol for isolating bacteria

from food samples, but for organisms such as E

coli O157:H7 the technique of immunomagnetic

separation (IMS) can give significant improve-

ments to the sensitivity of the method and help

accelerate the process of isolation.

E. coli O157:H7 is frequently found to be

responsible for outbreaks of potentially seri-

ous diarrhoeal illness. Associated with raw and

undercooked meats, contaminated vegetables and

cooked foods, it causes unpleasant bloody diar-

rhoea in most people but it can have more serious

consequences in children, older people and those

Food testing

Food microbiology – a cultured approachby Dr Simon Illingworth and Lisa Baldwin

Figure 1. E.coli 0157:H7 traditional culture method vs IMS culture method.

With food safety issues conti-

nuing to hit the headlines, food

hygiene and the role of the

microbiology laboratory in the

food industry are subject to

considerable scrutiny. The qua-

lity of a food lab’s output is not

simply a matter of ‘a job well

done’ - any failure to quickly

identify a potential pathogen

can have far reaching effects

on human health. We look here

at some recent developments in

approaches to culturing impor-

tant micro-organisms, including

the trend towards more rapid

enrichment procedures, some

of the practical implications of

recently issued ISO guidelines

for the preparation, production

and testing of culture media as

well as the growing use of

chromogenics.

with compromised immune systems, leading to the

development of the haemolytic uraemic syndrome.

Since the presence of even very low numbers of

the organism can produce symptoms, its identifi-

cation in foodstuffs is extremely important. IMS

can improve and speed up this process.

IMS is a sophisticated sorting process that can

be used for the concentration, isolation and/

or purification of micro-organisms from food

samples. It saves considerable amounts of time

by allowing the omission of part of the stand-

ard enrichment process needed when using

conventional culture techniques alone [Figure

1]. When isolating E. coli O157:H7 the use of

traditional culture methods often results in

overgrowth of plates with other sorbitol fer-

menting organisms. This makes it difficult to

identify low numbers of the generally sorbitol

negative O157:H7 colonies. Applying specific

IMS allows concentration of the target cells

from the larger samples (e.g. 1.0 mL) while at

the same time removing non-target cells, thus

greatly improving the chances of isolation.

Using the ISO 16654;2001 O157 method, target

organisms are generally obtained with IMS

after 6 h enrichment, although stressed cells

may require up to 18 h enrichment.

IMS techniques use antibody-coated micro-

scopic paramagnetic particles for the specific

immunomagnetic separation of micro-organ-

isms. The beads used in the Captivate system

(Lab M) for example, have a magnetite core

and a ceramic zirconium oxide coating. When

incubated with a sample, the antibody-coated

beads bind to cell surface antigens forming an

antibody-antigen complex between the beads

and the target molecules. Target cells are thus

“captured”. These are then separated using

a magnetic concentrator from background

organisms and interfering materials. Non-

specifically bound material is removed by

washing, and beads are then plated to selective

media or subjected to other analyses.

The introduction of ISO media

Alongside the desire for more rapid results is

the need to ensure quality and consistency

in the tools used in the lab. An area of grow-

ing importance in food microbiology is

the application of guidelines issued by the

International Standards Organisation (ISO)

for the preparation and production of cul-

ture media (ISO/TS 11133 Microbiology of

food and animal feeding stuffs). Part 1 of the

standard sets out general guidelines on quality

assurance for the preparation of culture media

in the laboratory and Part 2 provides practical

guidelines on their performance testing. The

guidelines take the form of a detailed techni-

cal specification that sets out the minimum

acceptable performance criteria and the meth-

odology and organisms required for quality

control of specific media.

The quality of a culture medium relies on a

number of factors: basic ingredients; correct

formulation; preparation procedures; elimi-

nation of contaminant microbial agents and

appropriate packaging and storage conditions.

ISO/TS 11133-2:2003 sets out the criteria and

methods recommended for performance testing.

It applies to both commercially

prepared ready-to-use and dehydrated media

as well as to culture media prepared from basic

constituents in the user’s laboratory. However,

different, more extensive methodologies apply

to manufacturers compared to end users. By

establishing minimum performance criteria and

stringent protocols for manufacturers, the aim

of the guidelines is for more consistent quality

of commercially made products with a conse-

quent reduction in the extent of quality testing

necessary by the user.

The influence of the International Standards

Organisation in the food industry has increased

significantly in recent years. While the ISO/

TS 11133-2:2003 guidelines currently take the

form of a detailed technical specification, it can

only be a matter of time before they become an

essential standard. Food industry laboratories

are already moving to use ISO-specified media

in the knowledge that these meet strict formula-

tion requirements and have undergone rigorous

testing by the manufacturer. Standardising and

maintaining the quality and consistent perform-

ance of culture media is an important step in

continuing to assure the microbiological safety

of food and food ingredients.

The advent of chromogenicsAlongside the more traditional culture media

now being developed to meet ISO guidelines,

there is a growing trend towards the use of chro-

mogenic media in the isolation of foodborne

pathogens. The first ISO standard specifying the

use of a chromogenic medium for this purpose

was in 2004 and related to the detection and

enumeration of Listeria monocytogenes.

Chromogenic media provide a rapid and accu-

rate means of isolating and enumerating target

micro-organisms based on the detection of

specific enzyme activities. Their development is

arguably one of the most significant advances

in the field of microbiological culture media

in recent years. Not only do they enable faster

detection of specific micro-organisms compared

with classical culture media, they also improve

sensitivity. Depending on the media type and

micro-organism involved, identification can be

accomplished with a much reduced require-

ment for sub-culture or confirmatory tests. The

principle behind the technology of chromogenic

media is straightforward: the activity of bacte-

rial enzymes specific to the micro-organism to

be identified is used to cleave synthetic chro-

mogenic substrates that are incorporated in the

culture media. Importantly, the chromogenic

substrates themselves are colourless; it is only

when they are cleaved by specific enzymes that

they release a characteristic coloured product.

This turns growing colonies containing the rel-

evant enzymes a distinct colour. By choosing an

appropriate selective base medium and adding

a suitable chromogenic substrate, it is possible

to design chromogenic media that allow the dif-

ferentiation and identification of highly specific

groups of micro-organisms.

Chromogenic substrates are formed by coupling

a suitable chromogen with a substrate appro-

priate to the target enzyme. In addition to its

chromogenic properties, the most important

features of a good chromogen are its stability

and insolubility. While the chromogen-substrate

compound itself must be soluble, once cleaved

the chromogen needs to be insoluble to ensure

a build up of colour within the target organism,

rather than diffusing (bleeding) into the sur-

rounding medium [Fig. 2]. In certain media the

interaction of two or more chromogens can be

exploited to create a diagnostic combination or

pallet of colours.

Looking aheadRecent EU hygiene legislation has introduced

the ‘farm to fork’ approach to food safety, and

elsewhere tougher testing regimes are also being

implemented to tackle the issues of foodborne

illness. The contribution of the microbiology

laboratory continues to be significant both in

the development of sampling, testing and man-

agement regimes, and in implementing techno-

logical and regulatory adva nces aimed

at ensuring consistent, reliable detection and

identification of causative organisms.

References1. BfR Federal Institute for Risk Assessment,

Berlin : Priority setting of foodborne and

zoonotic pathogens; 19-21 July 2006:

www.bfr.bund.de

2. USDA Economic Research Service:

Economics of foodborne illness. www.ers.

usda.gov/Briefing/FoodborneDisease/

The authorDr Simon Illingworth, Technical Manager Lisa Baldwin, Product ManagerLab M • Bury, Lancs, UK

Figure 2. Comparison of HAL002 Harlequin Listeria medium incorporating a chromogenic substrate (left) with standard Oxford formula-tion for Listeria isolation (right) following 24 hours incubation.