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The determination of the antimicrobial activity of disinfectants and antiseptics is a complex procedure (1, 2). Published reports have shown that in suspension test methods for bactericidal activity of disinfectants, significant differences in microbicidal effects occurred both within and between laboratories (4). One of the reasons for the differences could be the relative complexity of the procedure. The determination of the virucidal activity of disinfectants and antiseptics is even more complex (3, 6). Therefore, virucidal tests must use the simplest methods possible, since methodological complexity will add extra variation to the results even when using well-standardized methods. In most tests, to stop the inactivating action of the chemicals, it is necessary to dilute the mixture to prevent damaging the cell culture on which the viruses are counted after inactivation. This approach requires the use of high virus concentrations in the mixture or the testing of great volumes of mixture, or both. Using high virus concentrations has two drawbacks. First, high virus concentrations are very unlikely to reflect the real-world situation. Second, they can cause virus aggregation, which has been a cause of concern among different authors studying the virucidal activity of disinfectants and antiseptics (8, 12, 13). Furthermore, testing large volumes of mixture is expensive in terms of both materials and labor.

We have recently described a method that we have called VIRADEN (for virus adsorption enumeration) (9, 10), which is based on the direct enumeration of viruses adsorbed into nitrate-acetate cellulose membranes. The characteristics and performance of the method make it a priori most suitable for testing the virucidal activity of disinfectants and antiseptics.

To assess it, we chose different chemicals used either as disinfectants (glutaraldehyde and chlorine) or as antiseptics (iodine and chlorhexidine digluconate). Glutaraldehyde, chlorine, and iodine are known to have virucidal activity (11), whereas chlorhexidine digluconate (Hibitane) (11) is inactive for naked viruses. The inactivation was determined in parallel with the standard methods used for this purpose and with the method modified by counting the viruses with the VIRADEN method. In order to compare the methods all the inactivation experiments were performed as indicated by the present standard methods also.

Viruses, cells, and media. All experiments were performed with the attenuated poliovirus type 1 strain Lsc-2ab. Viruses were propagated on the buffalo green monkey kidney continuous cell line (BGM). The cells were grown in Eagle's minimum essential medium (MEM autopow; ICN Biomedicals Inc., Aurora. Ohio) containing 5% fetal bovine serum, 2 mM L-glutamine, 26.8 mM NaHCO₃, 100 U of penicillin ml¹, and 100 µg of streptomycin ml¹.

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Enumeration of viruses. (i) VIRADEN. Viruses were enumerated by the VIRADEN method as previously described (10). Briefly, the virus suspension was amended by adding MgCl₂ · 6H₂O to a final concentration of 0.05 M MgCl₂. The viral suspension was then filtered through a 47-mm-diameter 3-µm-pore-size cellulose nitrate filter as indicated, at a flow rate never exceeding 200 ml per min. When the entire sample had been filtered, the membrane filter was washed by passing 100 ml of sterile 0.05 M MgCl₂ through it.

(ii) Standard plaque assay. Virus suspensions were titrated by the plaque assay method on confluent monolayers of BGM cells as described elsewhere (5).

Assessment of the virucidal activity of chemicals. The disinfectants were first diluted to twice the test concentration. Then 10 ml of this solution was added to 9 ml of sterile distilled water. The pH of the solution was measured and adjusted to between 6 and 7. The reaction tube was placed in a water bath set at 22 ± 2^oC (mean ± standard deviation) and left for about 3 to 5 min before 1 ml of the virus suspension containing approximately 10⁶ PFU per ml was added. After the addition of the virus suspension, the contents of the tubes were gently mixed. The contact time was measured from this point. At the desired time interval, 1 ml of the reaction suspension was removed with a sterile pipette and immediately transferred to either 10 or 100 ml of sterile distilled water or phosphate-buffered saline (PBS), with or without a neutralizing agent depending on the disinfectant or antiseptic tested. The 10-ml tubes and the 100-ml bottles were stored at 4°C until tested.

View larger version (7K): [in this window] [in a new window]   FIG. 1.   Kinetics of inactivation of poliovirus type 1 measured by counting viruses by the standard plaque assay (; solid line) and VIRADEN (; broken line). Shown are graphs for dilutions of 1/5,000 (A) and 1/1,000 (B) of a 100-ml solution containing 2 g of iodine, 2.5 g of potassium iodide, and 50% ethanol.

View larger version (7K): [in this window] [in a new window]   FIG. 2.   Kinetics of inactivation of poliovirus type 1 measured by counting viruses by the standard plaque assay (; solid line) and VIRADEN (; broken line) in solutions of 1 mg (A) and 2 mg (B) of chlorine per liter prepared from a 3% commercial chlorine solution (bleach).

View larger version (8K): [in this window] [in a new window]   FIG. 3.   Kinetics of inactivation of poliovirus type 1 measured by counting viruses by the standard plaque assay (; solid line) and VIRADEN (; broken line) in 0.1% (A) and 0.05% (B) glutaraldehyde prepared from a standard 25% glutaraldehyde solution.