cleaning agents and disinfectants(Holah et al., 1990; Flint et al., 1997;
Wirtanenet al., 1997).
Thechemical methodsused in the assessment of biofilmformation are
indirect methodsbasedon the utilisation or production of specificcompounds,
e.g. organic carbon, oxygen,polysaccharidesand proteins,or on the biofilm
microbial activity, e.g. livingcellsand ATP(adenosine 50 -triphosphate) content
(Charackliset al., 1982).ATPmeasurement is a luminescence methodbasedon
the luciferine±luciferase reaction. The ATP content of the biofilm is
proportional to the number of living cells in the biofilm and provides
information about theirmetabolic activity. Kinetic data obtainedfor freely
suspendedcells shouldnot be usedto assessimmobilised biomassgrowth, e.g.
biofilm. The ATPmethodis insensitiveand thereforenot suitablefor hygiene
measurementsin equipmentwhereabsolute sterilityis needed,because with
mostof the reagents usedtodaya countof at least 103 bacterialcellsis needed to
obtaina reliableATPvalue(Wirtanen, 1995;Lappalainenet al., 2000).
Important tools in modern biotechnology-related research are based on
microscopicaltechniques. Oneadvantageof microscopicalanalysisis that it
can measuresurface-adheredcells,ratherthan cellsthat havebeendetachedfrom
the surface.Variousmicroscopicaltechniques for studying cell adhesionand
biofilmformationon surface materialsare availableincluding:epifluorescence,
scanningand transmission electronmicroscopy,Fouriertransformationinfrared
spectrometry,quartz-crystalmicrobalanceand infraredspectroscopyas wellas
confocallaserscanningand atomicforcemicroscopying techniques.Fluorescence
is a typeof luminescencein whichlightis emittedfrommoleculesfor a short
periodof timefollowingthe absorptionof light. Fluorescenceoccurswhenan
excitedelectronreturnsto a lower-energyorbitand emitsa photonof light.Many
differentfluorochromeshavebeenusedfor the stainingof microbes in food
samples,biofilmsand environmentalsamples(Wirtanen,1995;Kostya¬l, 1998).
Flowcytometry using fluorescent probes is a direct opticaltechnique for the
measurementof functionaland structuralpropertiesof individualcellsin a cell
population.The cellsare forcedto flowin single file along a rapidlymoving
fluidstreamthrougha powerfullightsource. This technique has beenusedto
determine the viability of protozoa,fungiand bacteria.It measures the viability
of a statistically significant number of organisms (5000±25 000 cells per
sample). The advantages of flowcytometry are accuracy, speed, sensitivityand
reproducibility(Wirtanenet al., 2000b).
In the foodindustry,the firststepis to identifythe biofilmproblemsin a
particular processor site. Subsequently,it is importantto use the best possible
methodsfor isolation and detectionof the biofilmfor furthercharacterisation in
the laboratoryusing molecularbiologyand biochemical methods. Thesemethods
can be utilised in the detectionand identification of microbesin two waysby
performing identification eitherdirectly fromsamplematerial or indirectlyfrom
pureculturesobtained fromthe samples. The two majortechniques appliedin the
molecular detection and identification of bacteriaare the polymerase chain
reactionand the hybridisationtechnique(Maukonenet al., 2003).
Biofilmrisks 49