inherent in the material, others in the technique. Errors of +/- 90% in counts o f the order of 10^-10^ mL‘^ are not unusual even with the best possible technique (Collines et a l, 1989). It is therefore, necessary to combine the maximum o f care in technique with a statistical interpretation of results. They can be interpreted only if the product is regularly tested and the normal range is known.
The basis for these techniques is; material containing the bacteria is serially diluted and a portion of each dilution is placed in or on suitable culture media. Each colony developing is assumed to have grown from one viable unit, which as indicated earlier, may be one organism or a group of many.
Standard Plate Count
This is the industry standard method for the enumeration of viable cells. It has been shown to be an effective tool for isolating organisms, obtaining them in pure cultures, classifying and identifying them (Hattori, 1988). The Standard Plate Count (SPC) is often known as the Viable Cell Count (VCC) or simply as plate counting.
The plate count technique is relatively straightforward. A cell sample is serially diluted until single colonies can be detected on solid culture medium. Sterile, sealed universal bottles or test tubes containing 9 mL of sterile diluent as well as a number of agar culture plates and sterile pipettes are pre-prepared. 1 mL of the cell sample is aseptically pipetted into 9 mL of sterile diluent. 1 mL of this first dilution is then transferred to the next sample of diluent until the initial sample has been diluted 10 times. The number of dilutions obviously depends on the concentration o f the cell sample which can be estimated by spectrophotometry. Usually the last six diluted samples are tested. 0.1 mL of a dilution is evenly spread over the entire surface of the plate. This is accomplished by the used of a spreader that has been sterilised by immersing in methanol and flaming. The process is repeated for the other dilutions. In practice usually the last few dilutions need be plated out.
Plates are incubated for a set period of time (depending on the growth rate o f the organism). The number of colonies on each plate is identified by dividing the underside of the dish with gridlines and marking colonies in a particular grid with an indelible pen and tallying with a hand-held counter. To be statistically accurate a plate needs to contain between 30 and 300 visible colonies (Cappuccino and Sherman, 1987). The cell concentration of the original sample is calculated by
multiplying the dilution factor by the number of colonies on any plate. Colonies are counted.
For large work loads automated systems are essential. Semi-automatic systems replace the pen that marks the glass above the colonies with an electronic counter which displays the numbers counted on a small screen. Fully automated systems use a television camera or laser beam to scan the plate and results are displayed on a screen.
Plate counts are the most commonly used method for determining viability. Advantages of this method include:
Only viable / reproducing cells are counted Allows isolation of discrete colonies
Easy to master, only requires knowledge of basic aseptic technique Useful test for contamination of samples
Indole plates provide a rapid test for loss of enzyme activity. In the case of biotransformation ability analysis of P. putida ML2
There are however a number of disadvantages of plate counting:
Accuracy
Labour intensive Suitability of organism Incubation time
State of stress of organism Culture conditions
Accuracy, as has been mentioned, can be very low. This is often due to the inherent problems with the test itself. Aggregation of cells gives rise to single colonies. Bacteria are seldomly evenly distributed throughout a sample. Operator errors are also responsible for dilution inaccuracies and the inability to discriminate between colonies too small for analysis.
The equipment preparation, serial dilution and the subsequent plating of samples are all extremely labour intensive.
Chapter 3 - Analytical developm ent Introduction
Suitability of the organism for the test. This method cannot be applied to cells forming inseparable chains or cells which attach themselves to certain solid material. When testing natural cell populations, many of the bacteria present in a sample may not grow on the medium used, at the pH or incubation temperatures employed or within the time allowed (Folt et a l, 1988). This is not a problem for known bacteria. Dehority and Grubb (1980) showed how aggregation of cells can lead to problems. Cold storage of anaerobic bacteria from bovine rumens for short periods of time gave rise to increased plate count values when compared to unstored samples. This is claimed to be due to the fact that the cells form aggregates, which equate to one colony forming unit, but the action of cold storage somehow separates the aggregates, increasing the number of CPU's.
Long incubation times are not only inconvenient but can lead to inaccuracies. The state of stress of the organism can affect the time taken for visible colonies to appear. This is due to the phenomenon of growth delay, which will be discussed in more detail later. This is the time taken before the organism's lag phase in which it repairs any stress-induced damage. In the case of severely stressed cells this growth delay could be as long as the incubation time allotted for the SPC analysis. In which case a reduced number or even no visible colonies would have appeared by the counting time, although a lengthened incubation could change this. Although this factor has an adverse effect on results, growth delay analysis can be used as a quantitative measure of the stress placed on a cell population (Takano and Tsuchido, 1982).
Culture conditions, the recovery of stressed cells depends on the ambient temperature and the composition of the diluent as well as the culture conditions. Certain diluents may cause problems eg saline or distilled water as they may be lethal for some organisms. Diluents, when used direct from the fridge can cause cold shock which may prevent organisms from reproducing.
Other methods of determining viable bacterial numbers based on a serial dilution followed by culture on suitable medium include:
Pour Plate Method Roll-Tube Method Drop Count Method Droplette Method Surface Count Method Plate Loop Count
• Membrane filter counts
• Most Probable Number Estimates • Rapid Automated Methods
Many of these techniques are only of use for organisms that are thermally robust enough to retain viability after immersion in molten agar. P. putida ML2 is unlikely to survive this sort of treatment, however other organisms, such as E. coli, is unaffected by temperatures upto 45°C. All of the techniques retain the same advantages and disadvantages of the basic plate count technique. More information has been included as Appendix 3.1 to provide reference for future work using different organisms.
Biophotometrv
A semiautomatic method for quantifying viable bacteria is described by Vosbeck et al. (1984). The bacterial effect of the antibiotic rifampicin was tested on the organism Staphylococcus aureus. Viable cell numbers were quantified by the analysis of growth curve data. Growth curves were constructed using absorbance measurements made by a multichannel biophotometer. Use of this particular instrument allowed the possible simultaneous monitoring of upto 176 samples. Vosbeck claims a very close correlation with SPC results.
As with the SPC the biophotometric method is based on the organism's growth kinetics. It therefore shares many of the advantages and disadvantages associated with plate counts. These potentially include long incubation periods and dilution errors at high concentrations.
A more specific disadvantage would be the suitability to P. putida ML2. After agitated and aerated growth the change to static culture in a cuvette could lead to metabolic changes which would be reflected in altered growth kinetics.
One problem that this technique does not share with plate counts is cost. The purchase and maintenance cost of a multichannel biophotometer is prohibitively high for use in this study. However there are a number of advantages of this technique:
• Semi-automation of the process allowing the analysis of upto 176 small samples simultaneously.