Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Journal of Applied Microbiology, 2002, Vol. 92, No. 4, pp. 784-789

A rapid method for assessing  the suitability  of quenching agents  for individual biocides  as well as combinations

M.D. Johnston, R.J.W. Lambert, G.W. Hanlon & S.P. Denyer

ABSTRACT

Aims: To develop a novel, rapid method for testing the ability of quenching agents to neutralize disinfectants.

Methods and Results: Tests were performed to determine the suitability of different neutralizers for a range of disinfectants, using a new method based on the Bioscreen optical density analyser. Results showed that during disinfection tests, efficacy could be over-estimated due to poor, or no, neutralization of the disinfectant after a specified time of exposure to the bacteria. The failure to distinguish adequately between bacteriostatic and bactericidal effects can lead to false results during disinfectant testing. Experiments also showed that dilution of the disinfectant, following exposure to the bacteria, was not always sufficient to stop the activity of the disinfectant for chemicals with low dilution coefficients.

Conclusions: The quench test proved to be very quick and easy to perform, with results being available within 18 h. Using the Bioscreen, the test is automated and determines whether dilution into a particular neutralizer is able to inactivate a disinfectant within 30 s.

Significance and Impact of the Study: This new approach allows the efficacy of quenching agents to be determined, prior to undertaking each disinfection study, and can help in the development of more suitable quenching solutions. The test has also been used to find suitable neutralizers for mixtures of disinfectants which might be used during studies on synergistic biocide combinations.

INTRODUCTION

For accurate assessment of surviving organisms following contact with a disinfectant, the activity of the disinfectant must be arrested at the moment of sampling (MacKinnon 1974; Reybrouck 1979; Bloomfield 1991; Sattar et al. 1995; Kemp and Schneider 2000). In early studies on disinfection, efficacy was often over-estimated due to poor, or no, neutralization of the disinfectants after a specified time of exposure to the bacteria (Langsrud and Sundheim 1998). Russell et al. (1979) state that a failure to distinguish adequately between bacteriostatic and bactericidal effects has been responsible for inaccurate conclusions, notably with quaternary ammonium compounds. Quenching activity can be carried out by one of three methods: dilution to a sub-inhibitory level, chemical neutralization or membrane filtration (Russell 1981).

Compounds with high dilution coefficients rapidly lose their activity on dilution (Hugo and Denyer 1987). However, it is not always known whether this is sufficient to remove the disinfectant bound to viable cells. For this reason it is recommended that an appropriate neutralizing agent is incorporated, as well as dilution (MacKinnon 1974; Russell et al. 1979). An inactivating agent must possess certain properties. It must have no inhibitory effect on the microbes themselves and must very rapidly overcome the activity of the antimicrobial compound. If it combines with, and neutralizes the antimicrobial agent, the resultant substance must be non-toxic to the micro-organisms (Russell et al. 1979). The other alternative approach is membrane filtration, which is not a means of inactivation but rather, the removal of antimicrobial from the microbes. Membrane filtration normally requires several washing steps (Cremieux and Fleurette 1991) so it is not an instantaneous method for stopping the action of disinfectant against the test bacteria. It is also a rather cumbersome procedure, which is impractical for carrying out large numbers of disinfection tests.

Before starting any disinfection study, it is essential to establish the most suitable and fastest neutralizing agent. Although many authors mention the importance of the neutralization step and may list suitable chemical inactivators (MacKinnon 1974; Bloomfield 1991; Langsrud and Sundheim 1998), they rarely document the methods used to obtain this information. Russell (1981) is one of the few to do this, but the methods described only assess the quenching ability after 10-20 min in suspension, or involve the use of agar containing inactivators, which may have limited relevance to how the neutralizers would behave in solution.

Langsrud and Sundheim (1998) and Kemp and Schneider (2000) also describe methods used for testing the neutralizing capacity of their neutralizers. They added a portion of the disinfectant test solution to their neutralizer, followed by the bacterial inoculum. After 5 min, the reaction mixture was serially diluted and plated onto agar. A successful quenching agent was one that resulted in survivors (because the neutralizer would have halted the action of disinfectant prior to addition of the bacteria). This test, although effective for assessing the quenching ability of solutions, is time consuming due to the use of solid culture.

The following paper describes a new approach for testing the effect of quenching agents, based on the use of the Bioscreen Microbiological Growth Analyser. The Bioscreen is an optical density reader, which has been described previously (Lambert et al. 1998) and has been used for assessing the quality control of laboratory media (Johnston 1998) as well as for disinfectant testing (Lambert et al. 1998, 1999; Johnston et al. 2000; Lambert and Johnston 2000). This new quench test is rapid, automated by using the Bioscreen, and can determine whether dilution into a specific quenching agent is successful at fully neutralizing (within 30 s) a single compound or a mixture. These experiments also assess whether the effect of dilution alone is sufficient to inactivate a compound or a mixture.

MATERIALS AND METHODS

Preparation of bacterial suspensions for quench testing

Staphylococcus aureus ATCC 6538 was grown overnight in a shaking flask containing 80 ml Tryptone Soya Broth (TSB; Oxoid CM129) at 30°C. The culture was centrifuged (Hettich Rotina model 48R, Tuttlingen, Germany) for 10 min at 1660  g and the resulting cell pellet was resuspended in 10 ml 0·1% peptone water. Cell number was determined by measuring the optical density (O.D.) of cells against known standards and diluted to give 1  10⁸ ml ¹.

Preparation of test disinfectants

The following compounds were prepared in sterile distilled water at appropriate concentrations prior to use: 4-chloro- 3-cresol (CC) and phenol (Fisons); 3-cresol (m-c) and phenylmercuric acetate (PMA; Sigma-Aldrich); 2-phenylethanol (PEA; BDH); silver nitrate (AgNO₃; J.T. Baker).

Preparation of quenching agents

Universal quenching agent (UQA).

UQA (pH 7·0) was prepared from the following ingredients (w/v) dissolved in distilled water and autoclaved: 0·1% peptone, 0·1% sodium thiosulphate, 0·5% Tween 80 and 0·07% lecithin.

Thioglycollate quenching solution.

A 25% (w/v) stock solution of thioglycollic acid was prepared in sterile distilled water (pH adjusted to 7·0 with NaOH). On the day of use, 100 l aliquots of this stock solution were diluted, in 8·9 ml volumes of universal quenching agent, to give a final concentration of 0·25% (w/v) thioglycollate once the disinfectant (1 ml) was added.

Testing the efficacy of quenching agents

A stock solution of each disinfectant or mixture was prepared at a concentration which was anticipated to give greater than 4 log reductions in 60 min. The following were added to each of four universal containers:

(i) 10 ml of the stock disinfectant

(ii) 1 ml of the stock disinfectant into 9 ml UQA

(iii) 1 ml of the stock disinfectant into 9 ml water

(iv) 1 ml of the stock disinfectant into 9 ml thioglycollate-UQA.

Controls used were 10 ml water, 10 ml UQA and 10 ml thioglycollate-UQA (to check cell viability and from which to prepare Bioscreen calibrations). Precisely 30 s after addition of disinfectant to appropriate tubes, all of the above solutions were inoculated with 100 l of the bacterial suspension. This gave an inoculum of 10⁶ ml ¹ in each tube. After a 60 min contact time, solutions were added to TSB in a Bioscreen plate (as described by Lambert et al. 1998). Log reductions for each test sample were calculated from calibration graphs produced in the appropriate control solutions (calculations described by Lambert et al. 1998).

FIGURES

Fig. 1 Calibration graphs which can be used to calculate log reductions from a detection time, obtaine...

Fig. 2 Disinfection kinetics of Staphylococcus aureus with PMA in a suspension test, quenched using UQ...

Fig. 3 Disinfection kinetics of Staphylococcus aureus with PMA in a suspension test, quenched using th...

Table 2 Log reductions obtained from quench tests on biocide mixtures
 

RESULTS

Quench tests on individual compounds

Table 1 shows the log reduction results from quench tests performed on individual disinfectants. If the effect of quenching agents plus dilution was successful (i.e. inactivated the disinfectant within 30 s), there should be survival of bacteria and hence, low log reduction values in the quenched solutions (UQA or thioglycollate-UQA). The quenching process can only be said to have worked if there was inactivation (> 4 log reductions) in the undiluted disinfectant solutions. If the effect of dilution alone was sufficient to inactivate the biocide, survivors would be seen in the disinfectant solutions which were diluted in water prior to addition of bacteria.

Table 1 shows that the combination of UQA and dilution was effective at stopping the action of all biocides tested except PMA. For this agent, > 4 log reductions of the bacteria occurred even after UQA had been in contact with PMA for 30 s, suggesting that the biocide was still active. Significantly, dilution alone was sufficient to halt the action of every agent except AgNO₃ and PMA, which is not surprising since these two compounds have low dilution coefficients (Hugo and Denyer 1987). This means that their activity is not greatly reduced on dilution. Although the activity of AgNO₃ cannot be stopped by dilution alone (> 4 log reductions achieved), it is neutralized when universal quenching agent is used (0·2 log reductions). It was found that PMA could be quenched using thioglycollate-universal quenching agent. The fact that all compounds showed > 4 log reductions when no dilution or quench was employed means that any survivors seen were attributable to the efficacy of the quenching agent or dilution.

Bacterial cells were inoculated into the three controls, 10 ml water, 10 ml universal quenching agent and 10 ml thioglycollate-universal quenching agent (to check cell viability and from which to prepare Bioscreen calibrations). Figure 1 shows that the quenching agents were not themselves toxic to the test organism.

Quench tests on disinfectant mixtures

Table 2 shows that combining UQA with dilution will halt the action of all combinations except those containing PMA. Again, it is evident that mixtures containing either PMA or AgNO₃ cannot be inactivated by dilution alone. Thioglycollate-UQA plus dilution will inactivate all combinations used in this study without being toxic to the micro-organisms. On the basis of these results, it was decided that the combination of UQA and dilution could be used in all cases unless PMA was present in a disinfectant mixture, in which case thioglycollic acid should then be added to the quenching agent.

The effects of using an inappropriate quenching agent to halt a disinfection reaction

Figure 2 shows what can happen when a quenching agent does not inactivate a compound successfully. The figure shows disinfection of Staph. aureus with six concentrations of PMA, which were then inadequately quenched using UQA. It appeared that after 3 min, an increase in contact time had little effect on the log reduction values. From the quench test results (Tables 1 and 2, it is known that there would have been some carry-over of active PMA into the Bioscreen plate when using universal quenching agent, causing growth inhibition in the Bioscreen. This explains why log reductions were observed, even at these low PMA concentrations.

Figure 2 shows a bacteriostatic effect from low concentrations of PMA, while Fig. 3 shows the bactericidal data revealed using an appropriate neutralizer (thioglycollate-UQA). The concentrations of PMA in Fig. 3 were 10 times greater than in Fig. 2, indicating the enhanced efficacy of the thioglycollate-UQA inactivator.

DISCUSSION

The tests performed to assess the quenching ability of different neutralizers on individual compounds showed that the combination of UQA and dilution was effective at halting the action of all biocides tested except PMA. When thioglycollate was incorporated into the quenching agent, the PMA was successfully neutralized as well. Elkhouly and Yousef (1974) also found that thioglycollate was necessary to inactivate PMA.

Langsrud and Sundheim (1998) state that universal neutralization solutions are available for inactivating disinfectants. The label `universal' is given because such an agent will contain many neutralizing compounds, each capable of inactivating a different group of chemicals. It is unlikely that a truly universal quenching agent exists which would be capable of neutralizing all groups of disinfectants while not damaging the microbes. However, a basic formulation can be prepared for inactivating the more commonly-used groups of disinfectants, to which other neutralizing chemicals may be added when required.

Cremieux and Fleurette (1991) state that some inactivators act in a way which is purely chemical neutralization (e.g. for thiosulphate with iodic compounds or thiols with mercurials). Other neutralizers contain phospholipids such as lecithin, or non-ionic surfactants like Tween 80. Non-polar groups on these molecules often lead to the uptake of antiseptics or disinfectants (Cremieux and Fleurette 1991).

In this study, universal quenching agent, UQA, was used as the basic formulation (Lambert et al. 1998), to which other compounds were added when required e.g. thioglycollic acid for mercury compounds (Elkhouly and Yousef 1974). The UQA contains: sodium thiosulphate to neutralize chlorine, hypochlorites, iodine and aldehydes (Gross et al. 1973; Russell 1981; Hugo and Denyer 1987; Bloomfield 1991); Tween 80 for phenols, parabens and quaternary ammonium compounds, QACs (MacKinnon 1974; Hugo and Denyer 1987); lecithin which, in combination with Tween, will neutralize biguanides as well as QACs (MacKinnon 1974; Russell et al. 1979; Hugo and Denyer 1987; Bloomfield 1991). The effect of dilution of the biocide into quenching agent may also be sufficient to halt the action of certain agents, namely phenols, cresols and alcohols (Reybrouck 1979; Russell 1981; Bloomfield 1991). This was found to be the case during this study for all compounds except those with low dilution coefficients. Sattar et al. (1995) state that they and colleagues prefer the use of dilution of the organism-disinfectant mixture at the end of the contact time rather than using a quenching agent. The present study shows that this approach will not always be effective when dealing with compounds that have low dilution coefficients.

When designing these tests, it was decided that a lower level of inoculum would be used than in a normal suspension test to minimize any effects of the microbes self-quenching the biocide (as described by Johnston et al. 2000). It was also important to establish that none of the components of the quenching system were toxic to the bacterial inoculum. For instance, Kayser and Van der Ploeg (1965) and Gross et al. (1973) point out that sodium thiosulphate can be toxic to staphylococci. They recommend that levels of sodium thiosulphate should not exceed 0·1% (w/v). Russell (1981) states that sodium thioglycollate can be used in the inactivation of mercury compounds, but that it too can be toxic to some micro-organisms. This was not found to be the case for the strain of Staph. aureus used in this study when used at a concentration of 0·25%.

The consequences of using an inappropriate quenching agent were clearly demonstrated during this study, when UQA was used unsuccessfully to inactivate PMA. It was clear from the results that there was some carry-over of active PMA into the Bioscreen plate when using this universal quenching agent. This explains why inactivation of Staph. aureus occurred, even at these low PMA concentrations, because the residual disinfectant was acting like a preservative. Denyer (1990) states that antibacterial agents may exert both bacteriostatic and bactericidal effects, often in a concentration-dependent manner. He suggests that bacteriostatic events generally result from a form of metabolic inhibition which is released when the biocide is removed, whereas bactericidal action is caused by irreversible damage to a vital structure or function of the cell (Denyer 1995, 1998). When an effective quenching agent (containing thyoglycollate) was used against PMA, even concentrations of the disinfectant approximately 10 times higher were adequately neutralized. This illustrates the importance of identifying an appropriate neutralizer, before embarking on disinfection tests, in order to avoid false results.

The neutralizer testing approach described in this paper proved to be very quick and easy to perform, with results being available in approximately 18 h (depending on the growth rate of the test bacteria). The test is automated when using the Bioscreen and is able to establish whether dilution into a particular neutralizer can inactivate a disinfectant within 30 s. The fact that the test can be readily performed on combinations of chemicals is extremely important when seeking to screen new biocide formulations which comply with legislation and increasing environmental awareness (Rowbottom 1996). Combinations of chemicals in a synergistic system may offer some viable options (Brown and Winsley 1971; Denyer 1995) by improving efficacy, allowing the use of lower concentrations with less environmental impact, and being cheaper and safer. It is therefore vital that the appropriate neutralizers are used during these studies.

REFERENCES

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2   Brown, M.R.W. and Winsley, B.E. (1971) Synergism between polymyxin and polysorbate 80 against Pseudomonas aeruginosa. Journal of General Microbiology 68 ,367-373.

3   Cremieux, A. and Fleurette (1991) Methods of testing disinfectants. In Disinfection, Sterilisation and Preservation ed. Block, S.S. pp. 1009-1027. London: Lea and Febiger.

4   Denyer, S.P. (1990) Mechanisms of action of biocides. International Biodeterioration 26 ,89-100.

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6   Denyer, S.P. (1998) Mechanisms of action of disinfectants. International Biodeterioration 41 ,261-268.

7   Elkhouly, A.E. and Yousef, R.T. (1974) Antibacterial efficiency of mercurials. Journal of Pharmaceutical Sciences 63 ,681-685.

8   Gross, A., Cofone, L. and Huff, M.B. (1973) Iodine inactivating agent in surgical scrub testing. Archives of Surgery 106 ,175-178.

9   Hugo, W.B. and Denyer, S.P. (1987) The concentration exponent of disinfectants and preservatives (biocides). In Preservatives in the Food, Pharmaceutical and Environmental Industries ed. Board, R.G., Allwood, M.C. and Banks, J.G. pp. 281-291. Oxford: Blackwell Scientific Publications.

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11   Johnston, M.D., Simons, E.-A. and Lambert, R.J.W. (2000) One explanation for the variability of the bacterial suspension test. Journal of Applied Microbiology 88 ,237-242.

12   Kayser, A. and Van der Ploeg, G. (1965) Growth inhibition of staphylococci by sodium thiosulphate. Journal of Applied Bacteriology 28 ,149-157.

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16   Lambert, R.J.W., Johnston, M.D. and Simons, E.-A. (1999) A kinetic study of the effect of hydrogen peroxide and peracetic acid against Staphylococcus aureus and Pseudomonas aeruginosa using the Bioscreen disinfection method. Journal of Applied Microbiology 87 ,782-786.

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21   Russell, A.D. (1981) Neutralisation procedures in the evaluation of bactericidal activity. In Disinfectants, Their Use and Evaluation of Effectiveness, S.A.B. Technical Series 16 ed. Collins, C.H., Allwood, M.C., Bloomfield, S.F. and Fox, A. pp. 45-59. London: Academic Press.

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23   Sattar, S.A., Best, M., Springthorpe, V.S. and Sanani, G. (1995) Disinfectant testing: update. Mycobactericidal testing of disinfectants: an update. Journal of Hospital Infection 30 (Suppl.),372-382.

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