Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 11229 and Pseudomonas aeruginosa ATCC 2730 were grown overnight in flasks containing 80 ml Tryptone Soya Broth, TSB (Oxoid), with shaking, at 30 °C. The culture was centrifuged at 4000 rev min ¹ (510  g , Sigma, Harz, Germany; model 3K-1) for 10 min. The resulting cell pellets were pooled and resuspended in 0·1% peptone water. In general, an inoculum level of approximately 1  10⁷ ml ¹ was used, which was made reproducible through the use of a calibration standard produced from an investigation of O.D. readings and plate count numbers.

Each well, except for one of the controls (well 10), in column one received 50 µl of the prepared inoculum. The plate was then incubated at the desired temperature and time, with the analyser recording the O.D. of each well at 600 nm every 10 min.

Using an available spreadsheet, the calculations and time required were minimized with the use of the template which was developed. The template received the raw data from the Bioscreen, and outputted the results in the form of concentrations of preservative with the observed fractional areas.

The values of A, C, B and M are obtained from a non-linear fitting procedure, commonly found on statistical packages such as JMP (SAS Institute, Cary, NC, USA).

In general, plotting the inhibitor concentration on a logarithmic scale gives a characteristic sigmoid-shaped curve. The curve can be split into three principle regions: a region where the presence of the preservative has no effect on the of the organism relative to the control growth (as measured by O.D.), a region where there is increasing inhibition of growth, and a region where there is no measurable growth relative to the control. Terms have been assigned to two specific concentrations, the non-inhibitory concentration, NIC, the concentration above which the inhibitor begins to have a negative effect on growth, and the minimum inhibitory concentration, MIC, which marks the concentration above which no growth is observed relative to the control.

The straight alkyl chain trimethyl ammonium bromide surfactants (CnQAC) were examined. Figure 2 displays the observed and fitted inhibition profiles for a range CnQAC compounds against Staph. aureus. A plot of chain length against the log MIC and log NIC values gave a stepwise drop in susceptibility from n = 8 to 14, Fig. 3. A maximum level of inhibition was obtained at n = 16; thereafter, a decrease in susceptibility was found. In the case of Ps. aeruginosa, a stepwise drop in the log MIC and log NIC values was observed up to n = 14. For chain lengths of n = 16 and 18, complex inhibition profiles were found e.g. Figure 4.

Phenethyl alcohol, PeA, is described as a poorer inhibitor than phenoxyethanol, PoE ( Paulus 1993). An inhibition study was conducted under the same conditions of temperature and inoculum challenge. Under these conditions, it was found that PeA had the lower MIC ( Table 1). The slight difference in molecular weight (122·2 vs 138·2 for PeA and PoE, respectively) cannot account for the 2000 ppm difference between the MIC values.

Dodecylcholine is a quaternary ammonium analogue, but unlike the quaternary ammonium compounds (QACs) used regularly in disinfection, the alkanoylcholines have an expected low toxicity due to an ester link which connects the alkyl chain with the choline moiety. The modified Gompertz analysis of five separate inhibition investigations against Staph. aureus, done on different days by different operators, gave an NIC = 6·16 ± 0·45 ppm and an MIC = 10·09 ± 1·04 ppm. The inoculum challenge for these experiments ranged from 0·9  10⁷ to 1·59  10⁷ organisms ml ¹.

Minimum inhibitory concentration experiments are generally carried out over a specified time, e.g. 24 h. As this technique is based on growth relative to a control, it should allow an estimation of an MIC at any time up to the point of the test termination. In this laboratory, many of the experiments are terminated after 18 h, allowing time for data analysis and the setting up of another experiment(s) based on those results. An analysis of the calculated NIC and MIC values of the inhibition of C14QAC against Staph. aureus, with respect to incubation time, was carried out. The NIC and MIC values were calculated every hour, after an initial 4 h incubation, up to the end of the test. The results are shown in Fig. 5. The figure clearly shows a rise in the MIC as incubation time increases, commensurate with an increase in the NIC. Below about 8 h of incubation, the signal to noise ratio of the control wells is low, resulting in large FA values. The time taken for a substantial increase in the signal to noise ratio will be dependent on the inoculum size and the specific growth rate of the organism.

By examining all the data from the 'basic' MIC dilution technique, it has been possible to place a value on the data normally rejected. Furthermore, it has been found that these data can lead to the placing of an accurate MIC value on the antimicrobial, and also, a value on the non-inhibitory concentration, the NIC, the concentration below which normal growth is observed.

The MIC (and the NIC) are dependent on the conditions under which the experiment was run. These conditions should be specified when reporting the results. The most important conditions that have been observed are the incubation temperature, the organism and the inoculum size used, and a fuller examination of these factors will be reported in the near future.

The NIC is the concentration above which the inhibiting substance begins to have an observable effect on growth. At concentrations below the NIC, growth occurs at a pace equal to the control. The population of microbes is unaffected by the presence of the test substance. In some instances, the FA may exceed one if the test inhibitor leads to better growth conditions than the control. Thus, this methodology could be used for the optimization of growth media as an FA greater than one implies better growth conditions.

Devinsky et al. (1985, 1991) have shown that there is a relationship between the MIC and CMC of quaternary ammonium compounds. Optimum antimicrobial activity of surfactants is strongly related to the ability to form micelles. Essentially, a micelle represents a microbially-inactive form of the surfactant. The lower the CMC, the less free-bulk surfactant available to interact with the microbial membrane. Our hypothesis to explain the inhibition profile shown in Fig. 4 is that premicellization occurs between membrane components of the Gram-negative bacterium and the surfactant biocide. This essentially reduces the effective concentration of the biocide in solution and thus, there is an increase in the FA. As the CMC of the surfactant is approached (416 mg l ¹, Mukerjee and Mysels (1971)), this effect reaches a maximum. This effect may be one of the causes of 'blebbing' ( Jones et al. 1989). At concentrations higher than the CMC, the membrane becomes solubilized by the surfactant and lysis occurs. This phenomenon, in which a lower concentration of inhibitor has a greater inhibitory effect on growth than at a higher concentration, would have been undiscovered if this method of examining the inhibition profile had not been adopted.

At the MIC, no growth relative to the control is recorded. In some cases, the FA appears to increase past the MIC, but this can be related to an increase in opacity caused by the inhibitor itself at the wavelength used. If the MIC is obscured by the inhibitor in this way, then an adaptation of the methodology of Mann and Markham (1998) may be prudent. The MIC is defined by this method mathematically, based on the Gompertz equation. In some cases, the unbridled use of the Gompertz will lead to inaccurate MIC values. For example, the use of the modified Gompertz function with the data shown in Fig. 4 would be unwise. In such cases, other approaches may have to be adopted, such as truncating the data and fitting a regression line to those data. The MIC is defined for a particular incubation time. Figure 5 shows that after about 10 h (dependent on initial inoculum size), this method can provide an accurate NIC and MIC. Changes in both values can occur with greater incubation times, and an examination of the rate of change in the NIC and MIC with incubation time may also be prudent.