Taking charge of bacteria in food

It is easy for food to become contaminated with bacteria, but far more difficult to recognise the fact. The standard test for counting bacteria takes up to two days, which can seem a long time if someone is seriously ill and requiring medical attention. A new capillary zone electrophoretic test can detect bacteria in food within seven hours, then identify and count them.

Bacterial contamination of food is common but very few food-borne bacteria are harmful. The Texas A&M Horticultural Program reminds us that only about 20 of the many thousands of bacteria cause food poisoning, while 90% of the cases in the US are attributable to just 8 common species, including Salmonella and E. coli. Although raw foods are not sterile, they do not contain sufficient microorganisms to be harmful, so good food care will prevent human illness. Most bacteria induce vomiting, abdominal pain and diarrhoea, ranging from minor to severe, but a few can go further. Listeria monocytogenes and Clostridium botulinum can cause spontaneous abortion or death.

The Bad Bug Book collates all the information on food-borne pathogenic microorganisms and natural toxins from several agencies: the FDA, CDC, NIH and the USDA Food Inspection Service. A new database to help monitor food pathogens was also recently announced. Called Combase (Common Database for Predictive Microbiology), it is an internet-based, publicly and freely available database of food microbiology data, set up jointly by the US Agricultural Research Service and the UK Institute of Food Research.

Bacteria are difficult to detect on food because, at least in the first few days of infection, they do not change the appearance or taste and do not emit any telling odours. Suspect foods must be tested. The classic method is plate counting, which involves growing a sample in the lab and counting the number of visible colonies that develop after a certain time. When bacteria grow on a nutrient, they form distinct colonies. In general, a colony is derived from a single bacteria cell, so a count can be obtained.

Plate counting is well established, but has one main drawback. It takes 24-48 hours for the colonies to grow. This weakness has been addressed by researchers from the Departments of Food Science & Technology and Analytical Chemistry at the University of Cordoba in Spain, who have developed a capillary electrophoresis method for analysing bacteria, with an overall analysis time of less than 8 hours.

Separation under CE relies on differences in electrophoretic mobility, which are influenced by several factors, including the charge on the analyte. In the case of bacteria, they exist in solution as colloidal particles that have an overall surface negative charge. One bacterium will have a different charge to another, so they can be separated during CE. With a test mixture of 5 bacteria, Miguel Valcarcel and colleagues examined the conditions for good separation within a reasonable time scale.

The optimised conditions are described in Anal. Chem. 2004, 76. They include a phosphate running buffer at pH 7.0 to which were added calcium chloride and myoinositol hexaphosphate, which had the synergistic effect of improving the separation between the bacteria and sharpening the peak shapes during UV detection. Separation and detection took a mere 30 minutes, the remainder of the aforementioned 8 hours being taken up by sample preparation.

Although bacterial colonies did not have to be grown, unlike plate counting, the food samples did require enrichment, to concentrate the bacteria for analysis. This was accomplished by mixing the sample with phosphate buffer and dehydrated tryptosa soya broth, a standard bacterial lab medium. After 7 hours, the solution was centrifuged and the bacteria retrieved by filtering them out of the liquid. UV detection requires about 10⁸ cfu/ml of bacterium in solution (cfu = colony forming units) and this CE method gave linear calibration over the range 10⁸-10¹⁰ cfu/ml.

In tests with corn flakes, juice, baby food and frankfurters, each contaminated with more than one bacterium, all bacteria were clearly detected. They were also counted and compared with the amount measured on the same samples by plate counting. The results were remarkably similar, confirming the validity of the new CE method.

Apart from the advantage of speed, taking 3-6-fold less time than plate counting, the CE method measures the amount of bacteria present. It also identifies the individual bacterial species from the electrophoretic migration order and time, which will help investigators to trace the source of the contamination.

Colourised low-temperature electron micrograph of a cluster of E. coli bacteria. Individual bacteria are oblong and coloured brown.(Image: ARS)

Microbiologists use computer-assisted laboratory methods to record growth of Listeria monocytogenes bacteria on ready-to-eat meat products. (Image: ARS)