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Suspension tests are widely used under laboratory conditions in the development, verification, and registration of biocidal products. The simplest test is the determination of the MIC—the lowest concentration of a biocide that inhibits the growth of a test organism—which is widely used in the evaluation of antibiotic activity against bacteria. A series of biocide dilutions (usually in growth media specific for the test organism) are inoculated with a known concentration of the test organism and then incubated to determine the MIC. This method is useful for evaluating the efficacies of biocides against a wide range of vegetative organisms, such as bacteria and fungi, and in the development of product preservation. MICs are limited, as many biocides react with organic and inorganic constituents of the growth media and are therefore not available for activity against the test organism; examples of this include oxidizing agents, halogens, and aldehydes. As biocide MICs are generally at relatively low levels, they have limited use in demonstrating the various formulation effects that can enhance the efficacy of an antiseptic or disinfectant. Similar to MIC determination, the minimum microbicidal concentration (e.g., the minimum bactericidal concentration) can be determined by exposing the test organism to biocide or biocidal-product dilutions for a fixed time and then determining at what concentration no growth is observed. These tests are not widely used to evaluate biocidal activity.

Time-kill, or D-value, determinations are used to study the effects of a biocidal product over time. In their simplest form, the test organism at a known concentration is added to the product, and samples are removed over time to determine the concentration of test organisms remaining (Fig. 1.18). An important consideration in these tests (as for any microbicidal test) is the need to stop the activity of the biocide at a required exposure time, which is referred to as neutralization. With chemical biocides, neutralization can be by physical removal (the most obvious method being filtration), dilution, or chemical sequestration or inactivation. Filtration is achieved by passing the sample through a 0.1- to 0.4-μm-pore-size filter, trapping the organisms (usually bacteria and fungi), and allowing the biocide to pass through into the filtrate. Chemical neutralizers include sodium thiosulfate (for some oxidizing agents and halogens), Tween and lecithin (for quaternary ammonium compounds [QACs] and chlorhexidine), and sodium sulfite or glycine (for glutaraldehyde). It is important that, other than the neutralization of the biocide, no inhibitory substances (including the neutralizer itself) be present or formed that could inhibit the growth of the test organism on incubation; for example, chlorhexidine has affinity for certain filter materials, which can subsequently inhibit the growth of the test organism on incubation of the filter on growth media. It is therefore important that positive, negative, and neutralization growth controls be included to ensure the correct interpretation of results.

FIGURE 1.18 Typical time kill, or D-value, determination. A known concentration of the test culture is exposed to the biocide, samples are withdrawn at various times and neutralized, and the population of survivors is determined by incubation on growth medium. The actual exposure can be conducted at various temperatures, in the presence or absence of test soils, or under other test conditions.

Following exposure and neutralization of the biocide, the survivor population can be determined qualitatively or quantitatively. Quantitative methods determine the number of survivors by direct enumeration, e.g., by plating onto growth agar for bacteria and fungi (as shown in Fig. 1.18). The data from this analysis can be plotted as a time-log reduction relationship (Fig. 1.19).

From this plot, the decimal reduction time, or D value, can be calculated; it is defined as the time (e.g., in minutes or seconds) at a given temperature required to kill 1 log unit (or 90%) of a given microbial population under stated test conditions (Fig. 1.19). It is usual for the average D value to be determined as the negative reciprocal of the slope (m) of the plotted relationship (−1/slope):

where Δlog N is the change in log₁₀ microbial population, ΔT is the change in time, and m is the slope of the survivor curve.

FIGURE 1.19 Determination of the D value on microbial exposure to a biocide.

FIGURE 1.20 Typical survivor curves on biocide exposure. Curve 1 is concave downward, curve 2 is sigmoidal, and curve 3 is concave upward.

Graphical presentation of the microbial response to a biocide over time can also be useful for analysis of the biocide–microbial-population interaction. Although the data presented in Fig. 1.19 demonstrate a linear relationship, typical biocide survivor curves are often found to be nonlinear (examples are shown in Fig. 1.20). Practically, these data can have many interpretations. For example, curve 1 suggests an initial lag phase in biocidal activity that could be due to the presence of interfering soils or microbial clumping, which limits biocide access to the test microorganism. Curve 3 may be indicative of insufficient biocide concentration or the presence of a microbial sub-population (or mixed population) with greater resistance to the biocide.

Qualitative methods simply determine the presence or absence of growth in a sample, although the data from the analysis can be used to estimate the actual population present by serial dilution if it is accepted that the microbial population is randomly distributed (i.e., not clumped) (Fig. 1.21).

FIGURE 1.21 Qualitative and semiquantitative population determinations.

By serial dilution of the initial sample (in this case, using 10-fold dilutions) in growth media and monitoring of growth, the highest dilution demonstrating growth must have contained ≤0 viable cells and can be used to estimate the most probable number in the initial sample (Fig. 1.22). This analysis is also referred to as a “fraction-negative” determination. In certain situations, these methods can be used to estimate D values (a representation of a test is shown in Fig. 1.22).

In this case, the fraction of observed growth or no growth can be used to estimate the number of survivors in the sample at a specific exposure time. Different mathematical equations are used, for example, the Halvorson-Ziegler equation (to estimate the surviving population):

where Nt is the population at time t, n is the number tested, and r is the number sterile.

Therefore, if we take the example from Fig. 1.22, we can generate the following table:

Time	n	r	n/r	Nt
1	4	1	4	1.4
2	4	2	2	0.7
3	4	3	1.3	0.3

From this analysis, the D value can be estimated by plotting (as described above) or using mathematical equations, like the Stumbo-Murphy-Cochran equation:

FIGURE 1.22 D-value estimation using most probable number estimations.

where t is the exposure time, N₀ is the initial population, and Nt is the population at time t.

Clearly fraction-negative methods are relatively restrictive, but they are often used in combination with direct-enumeration methods to estimate the number of survivors in the quantal (10² to 10⁻² CFU/ml) range of the survivor curve (see section 1.4.3).

The effects of various types of physical and chemical variables can be tested by using suspension methods. Physical effects include the product temperature and pH, while chemical effects include the biocide concentration or dilution, formulation type, and interfering substances, such as organic (e.g., serum and blood) and inorganic (e.g., heavy metals and hard water) soils. These effects are often important considerations in the practical use of the biocidal product.

Examples of some standardized suspension tests are given in Table 1.18.