230 DISINFECTION
the process has achieved its object. One difficulty with this
approach is that of providing suitable growth conditions for
all of the possible species of organisms which may be pres-
ent. Also, the normal flora is likely to vary with time, and so,
ideally, this method is best applied in the form of a routine
monitoring procedure. Both of these difficulties produce an
increase in the financial cost of this type of approach, but the
reliability of the results obtained is correspondingly high.
The ideal method would involve a combination of these two
approaches: an initial test with known resistant organisms
in order to indicate the upper levels of activity which may
be required; this to be followed by routine monitoring tests
to guard against both unsuspected resistance amongst the
normal flora, and unforeseen breakdowns in the method of
application.
Any testing method which involves the use of surface
films is subject to the problem of physical recovery of the
organisms. In the case of small surfaces, the articles can often
be placed in or on a suitable nutrient medium, and provided
this allows contact between the medium and the organisms
in the film, then growth takes place. Where larger or less
accessible surfaces are concerned, then the simple direct
method will have to be replaced by some kind of sampling
technique. Sampling techniques vary widely and not all are
applicable in all situations. In the case of accessible surfaces,
simple methods such as wiping with sterile swabs may be
used; or alternatively, flooding the surface with a sterile
liquid, some or most of which is then removed by swab or
pipette. An interesting alternative consisting of a sterile, agar
medium, “sausage” was devised by Ten Cate (1965). The
exposed transverse surface of this sausage is pressed against
the surface to be sampled. After sampling a slice is cut from
the sausage and incubated with the exposed side uppermost.
A simple method which is used for sampling skin surfaces
may also be used in other applications. This method employs
adhesive cellophane tape. The adhesive surface is pressed
onto the surface to be sampled, and then removed for trans-
fer to a suitable medium. Where surfaces are inaccessible,
as in pipes and processing machinery, then a rinsing tech-
nique is usually most convenient. The resulting liquid may
be added directly to suitable nutrient media, or, if present
in large bulk, may be filtered through membrane filters to
remove the organisms. The resulting filter membrane is then
transferred to a suitable medium.
One final problem which is of importance in all meth-
ods of testing which involve assessment of viability, is that of
recovery and growth of the test organisms following exposure
to the disinfectant. Organisms which have survived a disin-
fection process often show altered requirements for optimal
growth. Response to physical conditions such as incubation
temperature, as well as to biochemical conditions such as
dependence on certain nutrients, may be completely altered
from that of unexposed cells. While considerable effort has
been made to derive efficient recovery methods (Flett et al. ,
1945; Jacobs and Harris, 1960; Harris, 1963; Russell, 1964)
the problem is so variable and so many combinations of fac-
tors must be considered, that it is far from being solved. There
is general agreement that recovery methods should be selectedwhich will allow maximum recovery of exposed organisms;
but putting this into effect can be extremely difficult.LIQUID DISINFECTANTSSeveral chemical agents have long been employed for destroy-
ing microorganisms, although it is frequently asserted that
such substances are without effect on bacterial spores. With
many agents, however, this is untrue (Sykes, 1970; Russell,
1971, 1982). The most important substances are phenols and
cresols, biguanides, chlorine-releasing compounds and other
halogens, aldehydes, alcohols, quaternary ammonium com-
pounds, mercury compounds, strong acids and alkalis and
hydrogen peroxide. The majority of these are considered in
detail below. Further information is provided by Hugo and
Russell (1982).Phenols and CresolsAlthough Kronig and Paul (1897) and Chick (1908) showed
that phenol was active against spores, the concentrations,
5%, employed, were considerably higher than those needed
to kill vegetative bacteria. More recent studies have indicated
that bacterial spores are not killed even after long exposure to
phenols (Sykes, 1958, 1965; Loosemore and Russell, 1963,
1964; Russell and Loosemore, 1964; Russell, 1965, 1971;
Rubbo and Gardner, 1965; Briggs, 1966). Of the bacterial
spores. Bacillus stearothermophilus is the most resistant to
phenol and B. megaterium the most sensitive (Briggs, 1966).
However, in contrast to its lack of sporicidal activity, phenol
is sporostatic at low concentrations.
Several factors influence the antimicrobial activity of
phenols and cresols (Bennett, 1959; Cook, 1960; Bean,
1967):1) Concentration. These compounds have high con-
centration exponents, h, which, as described above,
indicates that they rapidly lose their antibacterial
activity on dilution. This also means that dilution
procedures can be used to prevent the carryover of
inhibitory concentrations into recovery media when
viable counts or sterility tests are being carried out
(Russell, 1982; Russell et al. , 1979). Studies from
Bean’s laboratory (Bean and Walters, 1955; Bean
and Das, 1966; Bean, 1967) are of interest, for they
show that with dilute solutions of disinfectants
with high intrinsic activity, e.g. benzylchlorphenol,
the high proportion taken up by the cells means that
the concentration remaining is only weakly bacte-
ricidal, so that the surviving cells do not meet lethal
conditions.
2) Temperature. The bactericidal activity of the phe-
nols and cresols increases rapidly with an increase
in temperature. Examples of temperature coeffi-
cient ( u 10 ) against E. coli at 30–40° (Tilley, 1942)
are: phenol 8.4, o -cresol 6.9, p -cresol 5.6.C004_002_r03.indd 230C004_002_r03.indd 230 11/18/2005 10:19:27 AM11/18/2005 10:19:27 AM