treatment. In suspension tests where microorganisms are homogeneously
dispersed, samplingfor survivors is relativelyeasy.Thiscontrastswithsurface
carriertests whichmakeuse notably of poroussurfaces. For suchtests,sampling
surviving microorganismsmight be challenging and an additionalstep,suchas
sonication and the use of enzymes or other compoundsfor desorbing the
microorganismsfromthe surface, can beenused,althoughthis mightincrease
the damageto alreadystressed organisms.
The size of the inoculumis particularly importantfor quantitativemethods
that needto demonstrate a reductionin cell number. In this case, the original
inoculummustbe highenoughto demonstratethe required reduction in number,
takinginto considerationdilutionscaused,for example, by a neutralisationstep.
However, a largeinoculummight not necessarily be representativeof microbial
contaminationin practice,and mightcausean inoculum effect' (i.e. similar to soiling) on the activityof a biocide. Nonetheless,most standardprotocols specifythe initialinoculumsize,the samplingsize aftertreatment and the volumeto be platedon recoverymedia. Therecoverymedia mightplayan important role in the survival of damaged microorganisms following an antimicrobialtreatment.Moststandardprotocolsrecommendthe use of complex rich'agar(e.g.tryptone soyaagar)and optimum temperature for microbial
growth. Such growthconditions,althoughoptimum for healthymicroorganisms,
mightbe detrimental for stressed and injured ones(Hurst1977;Doddet al.,
1997).The designof a recovery agar thatfacilitatesthe repair of injured
microorganismshas beeninvestigated,particularlyin relationto foodprocessing
(Czechowiczet al., 1996;Farrellet al., 1998;Kangand Fung,2000; Restainoet
al., 2001).
In addition,protocols that rely on visiblemicrobial growthfollowingtreat-
mentand therefore longincubationperiodsto allowthe formation of colonies
mightnot provide a rapidresponse.The use of rapidcounting techniques,suchas
epifluorescence (Pettipher, 1986;Matsunagaet al., 1995;D'Haeseand Nelis,
2002;Comaet al., 2003; Yamaguchiet al., 2003), flowcytometry (Shapiro,
1990;McSharry, 1994;Endoet al., 1997,2001;Autyet al., 2001;Lehtinenet al.,
2003),bioluminescence(Stewart1990;Stewartet al., 1991; 1996 ; Walkeret al.,
1994;Hillet al., 1994),impedance (Connollyet al., 1993,1994)and micro-
calorimetry (Morgan et al., 2001)havebeenreported. Themeasurementof
`optical density' mightprovide a particularly suitablealternative to plating.
Lambertet al.(1998)investigatedthe use of an automatedcombinedshaker
incubator-opticalreaderto measure the kineticsof inactivationfollowing bio-
cidalexposure. Furtherinvestigations showed the usefulnessof suchan approach
in termsof rapidity,fast screening and adaptabilitywiththe use of different
factors suchas organicload(Lambert and Johnston,2001)and inoculumsize
(Johnstonet al., 2000).Furthermore, comparableresults to a standard test
protocolwereobtained (Lambertet al., 1999;Lambertand Johnston, 2000).
Nevertheless,standardagar-based recoveryprotocolsare easilystandardisedand
are particularlyreproducible and as suchtheyare usedas a referencefor the
development of other alternativecounting techniques (Sheppardet al., 1997).
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