Real-time microbiology, just more jargon or does it really offer benefits to the microbiologist? Dr Peter Silley reports.
As its name suggests, real-time microbiology indicates that data is available in real-time rather than having to wait 24 hours or longer for the result and as such it provides benefits not only to the microbiologist but also, when used in a manufacturing context, to the whole production team.
While real-time microbiology can embrace a range of technologies, the Don Whitley Scientific approach has been to focus on the well-established impedance technique using rapid automated bacterial impedance technique (RABIT).
Impedance microbiology
So just what is impedance, it can be defined simply as the resistance to flow of an alternating current as it passes through a conducting material.
What is important is to understand that impedance is a multi-component parameter embracing resistance and capacitance signals, the magnitude of each being in part dependent on the frequency of the ac current. This is important when comparing instruments and can often lead to some misunderstanding with respect to the size and nature of the electrical signal. Microbial metabolism usually results in changes in both conductance and capacitance. The importance of temperature control in any impedance system is critical, as a temperature increase of 1oC will result in an average increase of 0.9 per cent in capacitance and 1.8 per cent in conductance (Eden and Eden 1984).
It can be readily appreciated that changes in impedance of the growth medium result directly from the changes taking place in the bulk electrolyte. Substrates in microbiological growth media are generally uncharged or weakly charged but are transformed into highly charged end products as organisms follow normal metabolic pathways. This results in an increase in the conductivity of the test medium. It is important to stress, however, that the principles of medium design, fundamental to traditional microbiology, are equally if not more important in impedance microbiology. In the first instance, a medium must be chosen which will support and select for growth of the test organism. Secondly, that medium needs to be optimised for an electrical signal. The growth of some organisms, particularly yeasts and moulds, does not result in large changes in impedance. This is considered to be due in part to the fact that they do not produce strongly ionised metabolites, but non-ionised end-products such as ethanol.
An impedance system can therefore be considered simply as measuring net changes in impedance in the culture medium at regular intervals. When a test is initially set up the user defines the detection criteria and when the rate of change of impedance exceeds this pre-determined value the system will detect growth. The time required to reach the point of detection is referred to as the time to detection (TTD) and is a function of the size of the initial microbial population, the growth kinetics of the test organism and properties of the test medium. For a given test protocol, the TTD is inversely proportional to the initial microbial loading of the sample (Fig. 1).
Indirect impedance
High salt concentrations are routinely used in many selective media. The resultant high impedance readings of these media are outside the normal working range of the direct impedance technique. However, using the indirect technique these problems can be overcome by monitoring microbial metabolism via the production of CO2 (Owens et al 1989). In this instance potassium hydroxide is added to the impedance tube across the electrodes. The inoculated culture medium is in a separate chamber and is not in contact with the electrodes or potassium hydroxide. The unit is tightly sealed such that any CO2 produced as a result of normal metabolism is absorbed by the potassium hydroxide causing a resultant decrease in impedance.
The work of Bolton (1990) has shown the indirect technique to be a powerful tool for working with strains of Staphylococcus aureus. Listeria monocytogenes, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophilia and Salmonella spp. Furthermore, it now means the medium does not necessarily need to be optimised for electrical response, allowing media previously considered unsuitable to be used for impedance applications. Applications of indirect impedance technology are of considerable potential for anyone with a requirement for a rapid, easily manageable, highly sensitive system for monitoring and quantifying CO2 production whether in a whole cell or isolated enzyme studies.
The technology has been with us for more than 20 years and there is considerable bibliography available concerning its use for determining microbiological loading of raw materials and finished products and for its use in preservative efficacy testing (Silley and Forsythe 1996).
Real-time microbiology simply reflects a continuous apicture' of the interaction between the bacteria under test and the test system itself. As a standard impedance output is generated continually, data is available in real-time and therefore there is no need to wait for a pre-determined period before results are available.
Applications in process control
The concept of impedance microbiology has recently been taken further and fitted into production systems within the food industry where it dovetails with the increasing use of hazard analysis critical control point (HACCP) systems.
In a manufacturing situation productivity and microbiological quality and safety of products depend very much on the controls that can be exercised during production. In this respect quality control at the end of the production chain has long since been proven to be inadequate. Processes out of control invariably lead to economic loss that in many cases is the result of microbiological contamination. Social evolutions and technological developments are forcing industry to continuously adjust its production processes and to introduce new microbiological techniques in response to continual challenges. Microbiologists are no longer able to only operate at the end of the production chain, they must detect microbiological problems, in a faster and more accurate way and must fit the microbiology into the industrial process.
Microbiological test methods are critically evaluated. Fast, sensitive and specific tests are urgently needed and new combinations of modern analysis techniques focusing on the process are being developed. Together with colleagues from other disciplines, the microbiologist and process technologist have become assessors of microbiological risks as well as participants in process improvement projects and the TQM process.
It is only to the extent that combinations of modern microbiological analysis techniques will be coupled in a creative way to the quality management methods, that one can truly speak of process-integrated microbiology. This is an approach in which the responsibility of the microbiological process control is as much as possible handed over to the process operators. It includes not only obtaining a fast testing result, but also the use of SPC. As such the continuous process improvement can be launched including all possible implications for the complete organisation. Only then, microbiology will be able to fully play its in-process role and integrate effectively and efficiently in the TQM system of a company.
SPC software is available as an extension to the RABIT for Windows instrument and has been developed with an industrial partner in a commercial setting. It allows RABIT to be used for an on-line continuous monitoring (Fig. 2) of production processes and presents the data collected over time as easy to interpret charts. The information is updated automatically with new results allowing the process operator to access the data and bring about process changes in the plant.
In addition to increasing confidence in product quality this approach will also help identify cost savings in process control, a factor which will certainly appeal to all those attempting to drive costs out of the process. Impedance clearly has a future.
Enquiry No 51
Dr Peter Silley is with Don Whitley Scientific Ltd, Shipley, West Yorkshire, UK.
