product. The inclusion of suchstrains increasesthe cost of the testingand might
ultimatelyselectfor a moretoxicbiocide, although it wouldguaranteethe
efficacyof the biocidein mostcircumstances. The use of more resistant strains
might be recommended for specific usage, for example,mycobactericidal
activity.Veryfewprotocols recommendthe use of a mixedinoculumand
generally these testsare considered to be cumbersomeand proneto variability in
results. However, therehavebeenattempts to developbiocidalefficacytest
using mixed inocula(Bestet al., 1994).
Preparationof inocula
Growthconditions(e.g.temperature, media,chemostator continuouscultures)
affect microbial metabolismand physiology(e.g.outer layer,lipidcontent) and
could altertheirsensitivityto antimicrobials(Russell, 2004).Water qualitycan
also havea profoundeffect,for example on the germination,outgrowthand
sporulationofBacillussubtilisas reported by Knottet al.(1997).
The growthstage of the bacterialpopulationis also essential to control.For
example, exponentialphaseculture ofListeria monocytogenes(Luppenset al.,
2001)andStaphylococcus aureus(Luppenset al., 2002)havebeenshownto be
more sensitive to cationicand oxidising biocides. Nutrient availabilityand
notably limitationcan affectgrowth rate and henceaffectalsobacterialsen-
sitivity to biocides (Russelland Chopra,1996).Thereare considerablediffer-
encesbetween bacteriagrownin continuous culturevs.batchculture,the latter
usually predominantin testingprotocols. The maincriticismof batchculture is
that cellsof differentphysiological agesare present. Pre-treatmentof a microbial
population,for exampleto increase the dispersionof the individualcells, should
be avoided. Pre-treatment with non-ionic (e.g. polysorbate) or cationic
surfactantshavebeenshown to altermicrobial susceptibility,presumablyby
altering the cell membraneproperty. Likewise pre-treatment withpermeabilising
agents suchas ethylenediaminetetraacetic acid (edta), lactoferrin and polycations
will alterthe microorganismsensitivityfollowing an alterationof membrane
permeability. Surprisingly,there is little information on the effect of pH,
temperatureand anaerobiosis duringmicrobial growthand subsequentsensitivity
to antimicrobials(Russell, 2004).It is likelythat pH values will changeduring
microbial growth (Messageret al., 2004a).Alterations of phospholipidcontents
when microorganismsare grownat differentpHs or differenttemperatureshas
beendescribed (Russell,2004).Sincethe alterationsof the microbial cell wall
might be linked to differences in microbial responseto antimicrobials,the strict
control of the growthmediumand conditionsis essentialto ensurethe production
of a reproducibleinoculum.
Detection and countof survivors
Antimicrobialtestingprotocolsusuallyrelyon the detection of survivors as
visible growthand/or the enumerationof bacterial/fungal colonies or virus
plaques. Capacitytests(Section38.4.2)are basedon the detectionof growthbut
do not necessarily quantifythe amountof growthfollowing an antimicrobial
652 Handbookof hygiene controlin the foodindustry