A load of new balls

With standardisation becoming necessary in every aspect of life, how do microbiologists keep an eye on their cultures and colonies.

With standardisation becoming necessary in every aspect of life, how do microbiologists keep an eye on their cultures and colonies

Every newspaper, every scientific journal, and all the magazines in the world publish reports that start with the same words. “Tests” they say, “have shown...”

And we all instinctively nod with satisfaction, without pausing to ask the essential questions: Which tests? How shown? How accurately? Who says? And anyway, are the results repeatable? 

These crucial questions may not have been asked often enough in the past, but they are now moving to the head of the list. In an era of compliance, the authorities are starting to ask about our tests. All too often, identical samples sent to different laboratories (and even to the same laboratory, but at various times) have come back with differing results. We all know that, and have long accepted it as a fact of life.

But certification now looms for everyone. People are becoming litigious. Opinions count for less in an era of digital data, and we have to be confident that our results will stand up to repeat examination. The new compliance industry requires repeatability - and standardisation underpins the concept.

The setting of standards goes back to the earliest days of civilisation: six white pepper seeds equally one barley grain; eight barley grains equal one finger’s breadth . . . and official standards for length in the UK date back to the thirteenth century. Metrication was born in 1799 in France, with the metre originally defined as one ten-millionth part of the distance from equator to pole.

Although metrication has invaded science ever since, Imperial measurements still remain in common use in Britain, and to a greater extent in the United States. And although we know that the British and American gallons are different, how many realise that there were two different standards for the inch? The British inch was defined as 2.53998 cm while the US inch was 2.540005 cm. In July 1959, both were officially standardized at 2.54 cm though the US continued to use the earlier value in surveying for decades after that.

Representative results
Microbiologists have been largely left on a limb. We culture organisms, we count colonies, we report. Does anyone stop to consider whether the colony count is real, and whether the result always reflects the number of organisms in the sample? Is the medium giving the microorganisms what they want? Are isolation techniques representative? Can you count? Were there any organisms in the sample to begin with? Most important of all, what controls do you use? In most instances it’s best not to ask.

In the modern world, though, these are the questions people will pose. Every laboratory needs to know exactly how representative their tests might be. In Sydney, Australia, Dr Graham Vesey and his team have developed an ingenious set of standards. The product is in the form of small, freeze-dried rounded pellets, each containing a guaranteed number of viable organisms. The organisms can be released when each pellet is dispersed, and provide a comparable standard against which culture and counting methods can be assessed.

What trade name might you imagine for the product? Pellets, perhaps? Or microspheres? With what you might construe as Antipodean directness, the packaged organisms are marketed as BioBalls (pictured below).

Vesey cut his teeth working on tests for Cryptosporidium at the Thames Water laboratories in North London, and went to Australia to study for his PhD. Mark Gauci qualified in optoelectronics in Sydney and met Vesey while studying for his Masters. They investigated outbreaks of Cryptosporodium and Giardia in the Sydney drinking water and began to experiment with reference samples in 1999. Graham Vesey originally developed advanced methods of staining the protist parasites Giardia and Cryptosporidium at Macquarie University and he and Gauci began to market the system on licence under the trade name EasyStain. The process was licensed in Milwaukee, Wisconsin, back in 1993 and in Sydney (where it had been developed) in 1998.

For a time they worked as Bio Technology Frontiers, but - although the name was snappy - the products weren’t really in the field of biotechnology, so their new Company was given the name BTF. Lucy Cockett, the Company’s Quality Manager who travels the world advising on applications, reveals that one of the main reasons BTF was chosen as the Company name was that, when they came to launch on the world-wide web, BTF was one of the few three-letter domains still available.

With Mark Gauci specialising in laser technology, and Graham Vesey pioneering techniques for studying water-borne pathogens, they were the perfect pair to work on culture methods and cell separation. They began to develop standardised reference samples in 1999 and had soon perfected a guaranteed and repeatable sorting procedure. Their round, pelleted standard samples of parasites were received with enthusiasm, and BioBalls have since been internationally accepted by regulatory authorities.

Once Gauci had perfected the application of laser separation to bacteria as well as protozoa, BTF moved on to produce pellets each containing thirty bacterial cells. The bacteria range from Salmonella and Listeria species to Pseudomonas and Enterococcus. Soon to join the list will be the fungi Candida and Aspergillus, plus Clostridium sporogenes and Streptococcus pyogenes.

Last month BTF received the National Testing and Accreditation Association endorsement for their the BioBall product range under the ISO Guide 34 standard. This makes BioBall the world’s first international quantitative reference standard available for microbiological testing, and it heralds a new era in the standardisation of bacteriological procedures. Now that BioBall products are Guide 34 accredited, ISO accredited laboratories can use these products to simplify their routine quality control procedures.

How good is the product? Routine culture of the microorganisms from each pellet gives an opportunity to assess the success of the sorting procedure. The standard deviation is said to be ±3 plaque-forming units, PFU, and repeated tests show that most pellets truly do contain 30; the lowest count is 28, the highest 33. Three of the species, including E. coli, are now available in pellets containing 1,000 PFU though the claimed accuracy here is within 20%, which seems less impressive though would be viable when distributed through large volumes of water (say, more than a litre).

Where next for the company? Vesey points out that the concept could be adapted to packaging set quantities of many other biological materials, including plant and animal cells, viruses, even gene sequences. Currently BTF are looking at a DNA package for the testing of food products. Within two years, they plan to have a product for PCR standardisation that would allow the food industry quickly to assess the proportion of any foodstuff that is genetically modified. And that, BTF assure me, is only the start.

By Brian J Ford