The dry facts about microbial kill

Prerequisite for achieving hard surface disinfection is a complete wetting of the surface to be cleaned with disinfectant

Do surfaces have to stay wet with disinfectant solution over the whole contact time? This is a frequent question when considering cleaning regimes and validation. Bernhard Meyer, Ecolab’s microbiological support manager for RD&E Health Care EMEA, offers an answer based on recently gathered data
Prerequisite for achieving hard surface disinfection is a complete wetting of the surface to be cleaned with disinfectant. The amount of liquid needed to do this depends on the surface structure. It has been shown that 20–80ml/m² of liquid have to be used to achieve disinfection. But although relatively high amounts of liquid are used, surfaces frequently become visibly dry, before the claimed contact time of the disinfectant has elapsed.
The data presented here indicate that, once started, the microbicidal effect continues after visible dryness of the surface. Hard surfaces, therefore, do not need to be kept wet with the disinfectant over the full contact time, if the claimed concentration and contact time have been determined by methods that are representative of conditions of practice.
The relevance of hard surfaces in the transmission of microbial contamination has been recognised in all areas where cross-infection or the contamination of perishable products have to be avoided. In hospitals,¹ food handling² and in the pharmaceutical and cosmetic industries³ reduction of the microbial burden of surfaces in direct or indirect contact with humans or products is regarded as crucial for the prevention of cross-contamination and cross-infection.
To achieve this reduction, hard surface disinfection is used. In the disinfection of closed systems (clean-in-place – CIP) the amount of disinfectant solution per surface unit is high, and the disinfection process is terminated by a final rinse. In the disinfection of open hard surfaces the amount of disinfectant solution is limited. It is influenced by the mode of application, such as spray, foam or by wiping, and disinfection is not terminated by a final rinse. Instead the disinfectant is left to dry on the surface.

Figure 1: log-reduction of different test organisms in a surface test on glazed tiles using a surface disinfectant with 25g glucoprotamin/100 g as an active ingredient. Reduction was calculated against a control using water. The log viable count is given to indicate the stability of the test organisms to drying.

The question has been raised, as to whether the disinfection process (i.e. the killing of microbes) goes on after the surface is visibly dry. It could be assumed that only the phase during which the surface is visibly wet with disinfectant may be counted as contact time. On the other hand, if the process is not terminated by rinsing the disinfectant away, could the microbial kill process continue even after visible dryness of the surface?
The data presented here will show that disinfection of open hard surfaces is not terminated by visible dryness. Contact times longer than the time needed for drying of the surface can be useful and valid in disinfection, where efficacy under such conditions has been proved using valid methods.
Disinfectants at work
The first step of microbial kill during disinfection is the uptake of the active ingredient in the disinfectant by the cell. Water is needed as a solvent to bring the disinfectant into contact with the cell and to mediate this process. Bansemir⁴ showed that, in addition to the concentration of the disinfectant, the amount of disinfectant solution applied to a surface is also critical. He demonstrated that the desired disinfectant effect cannot be achieved when using less than 20ml/m² of disinfectant on hard surfaces. He also noted that 40-80ml/m² was the limit after which no further improvement could be achieved.
Disinfectants execute their microbicidal effect by chemical or physico-chemical reaction with components of the microbial cell. They interfere with the integrity of the microbial membrane⁵ or react non-specifically with proteins or genetic material inside the cell.⁶ Even after uptake of the disinfectant by the cell, these processes do not progress at unlimited speed, but are time dependent, as with any chemical process. This means that once the disinfectant has been taken up by the cell, the destruction of the microbial cell will continue, even if no further disinfectant is taken up because the disinfectant solution outside the cell has evaporated. The uptake may only be reversed by re-diffusion if the surface is rinsed with clean water.
Testing the kill effect
A hard surface disinfectant based on the active ingredient glucoprotamin was chosen for this test. Because a European standard test for surface disinfection with mechanical action under practical conditions (phase 2 step 2 according to EN 14885) is not yet available, the tests were performed according to the guidelines of the German Society for Hygiene and Microbiology (DGHM).
In brief, test organisms suspended in broth were dried onto standard glazed tiles. The surfaces were then treated with a 0.25% use solution of a disinfectant containing 25g/100g glucoprotamin as the active ingredient. This concentration corresponds to the recommended use dilution of this product for a 4-hour contact time.
In the test, 0.2ml of the use dilution were spread evenly over the entire surface of the tile with an angled glass rod. The test surfaces were left to dry under ambient conditions. After different contact times, the tiles were completely immersed in a recovery liquid that was validated to neutralise the disinfectant immediately and contained quartz sand to re-suspend the test organisms by shaking.

Figure 2: Weight loss of test surfaces after application of 40 and 80 ml/m² of the 0.25% use dilution of the tested disinfectant respectively. The difference to the weight before application is given

The residual count of test organisms was determined by surface plating. Tiles treated with water were used as a control. The log reduction achieved by the disinfectant was calculated as the difference in log colony count of the test tiles and the controls. Log reduction of the test organism and the count of the controls are given in figure 1. It can be seen clearly that under these conditions even the gram-negative test bacteria are rather resistant to drying. Only a minor loss in total count can be seen in the controls. On the other hand, an ongoing kill effect can be demonstrated between 1 hour and 4 hours contact time.
Figure 2 gives the time it takes the same surfaces to dry. After wetting of the surfaces in an analogous fashion to the disinfectant test (i.e. 80ml/m²), in a duplicate test the first surface was visibly dry after 90 minutes. After wetting with half the amount of liquid, the first surface was dry after 60 mins. The gravimetric evaluation in figure 2 shows that in both cases the surfaces were almost dry after 60 mins. None of the surfaces was completely covered with a film of liquid, as determined by visual assessment, after 60 mins. Only small areas of the surfaces were still wet, if at all.
In table 1 drying times of a coated PVC surface under conditions of normal practice are given. Drying times are in the same order of magnitude as those for the test surfaces used before, which indicates that the drying times of these tiles are representative of normal practice.

Table 1: Drying time of different amounts of tap water and 0.25% use dilution of a disinfectant with 25g Glucoprotamin/100g as active ingredient on a coated PVC floor. The amount given was distributed over the surface with a pre-moistened mop
Amount distributed (ml/m2)	Drying time tap water (mins)	Drying time disinfectant (mins)
20	26	14
40	43	28
60	66	92
80	67	101

Continuing effect
The discussed mechanism of action of microbicidal substances and the data presented here show that the microbicidal effect of disinfectants continues, even if surfaces are visibly dry. This can be said at least for those cases, where the active ingredients of disinfectants do not evaporate. Even if after 60 mins the test surfaces were not completely dry, the majority of the surface was no longer visibly wet. Nevertheless an ongoing kill was determined in disinfection trials after 60 mins. Hence, valid contact times for hard surface disinfectants can be determined that are longer than the drying time of the surface after application of the disinfectant.
Prerequisite for this conclusion is that the efficacy of the disinfectant is evaluated by methods that are representative of the drying of the surface under practical conditions. It was also shown that disinfectants tested according to the guideline used here⁷ are effective under practical conditions where the use solution is applied to the surface with a mop or cloth.⁸ This presents evidence that testing according to this guideline is representative for applications in practice.
The effect described here must, however, not be taken as proof for a so-called residual effect of disinfectants. Only after an initial intense contact with disinfectant solution can the kill effect be expected to proceed after visible dryness of the surface. Microbes contaminating the surface after drying do not have this intense contact with the active ingredient of the disinfectant and therefore are unlikely to be killed. Hence, a prophylactic application of disinfectant, to kill a future contamination is not possible.
References
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2. P. Luber, S. Brynestad, D. Topsch, K. Scherer, E. Bartelt, Appl. Environm. Microbiol. 72 (2006) 66-70
3. S. E. Docherty, Pharmaceutical Engineering May/June 1999 36-40
4. K. Bansemir, Swiss Med 7 (1985) Nr. 3 b, 36-39
5. P. Gilbert, L.E: Moore, J. Appl. Microbiol. 99 (2005) 703-715
6. T.E. Cloete, Int. Biodeterior. Biodegread. 51 (2003) 277-282
7. DGHM. Prüfung und Bewertung chemischer Desinfektionsverfahren – Stand 12.7.1991, Hyg.
8. C. Stingl, B. Meyer, Hyg Med 30 (2005) 147-152