Experimental Procedure and Attachment

Other variables also have to be controlled during the attachment

experiments. During the purification and maintenance of bacterial

strains, subculturing Is often employed. The results from the subculture

experiments show this procedure can greatly affect the attachment

ability of the bacterial species. These changes In bacterial attachment

could be due to variations in the bacterial cell surface characteristics

during the subculturing. Wild type strains of bacteria have been shown

to vary in the quantity of LPS on the surface after subculture (Kikaido,

I960). Changes in the LPS can Influence the hydrophobic or charge

interactions a bacterium can exhibit, and therefore this could influence

their attachment (Xagnusson, 1977). The evidence from these attachment

experiments indicate that the changes observed in attachment ability

became insignificant after five or six subcultures. Although the

bacterial surface after subculture may be different from that in a

natural environment, the use of bacterial cultures which had been

subcultured six times was the only way to control the effects of

subculture on the attachment of the bacterial isolates, although it is and stl1X retain their viability, and therefore the detached bacteria

recognised that subculturing reduced the attachment rate for almost all

the species tested.

The results obtained from the effects of culture age on attachment

are variable, with changes In attachment being observed after long

periods of storage. Reports have suggested that this could be due to

changes In bacterial surface components such as polymers on the surface

of the cell which could change in composition or their effectiveness

with time (Chapter 4). Changes in bacterial attachment with culture age

could also be related to motility with a bacterium becoming less motile

with age (Fletcher 1977). Microscopic inspection of bacteria revealed

that log-phase cultures had greater proportions of motile cells than

stationary phase (Fletcher. 1977). Therefore, the reduction in motile

cells could account for the differences in attachment obtained in these

studies. As explained previously in this chapter, stationary phase

cultures only were used in subsequent experiments to control this

factor. Heeb (1982), has shown that Actinomyces viscosus could be stored

refridgerated for months without significant deterioration in their

ability to attach to beads. This suggests that each bacterium's

attachment must be studied seperately under storage conditions to

determine the changes which occur during storage.

The concentrations of bacteria used in these experiments is

important. Experimental evidence indicates that there are optimum cell

concentrations above which no more attachment occurs (Gordon, 1983).

Carrie (1985) demonstrated that the changing attachment rates of

bacteria was directly related to the bacterial concentration, providing

that the physiological state of the bacteria remained unaltered. The

with an Increasing number of bacteria there would be a greater number

of collisions between the bacteria and the^surface. Therefore, the

opportunities for attachment to take place would increase. Attachment

reaches a maximum value when the surface attachment sites become fully

saturated with bacteria and hence cannot support any more attachment. It

is therefore important to work at bacterial concentrations and for time

periods which prevent or do not allow this to happen. If saturation

occurs within the time period of the experiment, then differences in

attachment rate due to the different environmental factors being studied

would be obscured because under all conditions maximal rates of

bacterial attachment would have been recorded.

In many cases the surface is not fully covered with bacteria, this

could be due to parts of the surface not being available for attachment

(Doyle, 1982>. This could be due to the surface being conditioned in

some adverse way (Fletcher, 1982) i.e. with molecules that inhibit

attachment, or due to bacteria not attaching to the conditioned surface

for some physiochemical reasons. Fletcher (1976), demonstrated that

proteins such as fibrinogen and gelatin adsorb onto a surface inhibited

the attachment of a Pseudomonas species. The mechanism by which these

molecules inhibit attachment vary, depending on the macromolecules

present. Dextrans and LPS, although largely polysaccharide in structure,

are significantly different and so are the mechanisms by which they

inhibit attachment. LPS can inhibit bacterial attachment to a substrata

when LPS is added to the liquid phase with the bacterial cells or when

LPS is adsorbed onto the substrata before attachment. The dextrans,

however, could only inhibit attachment when added to the cells during

aqueous solution, substances are quickly adsorbed onto the surface.

Xacromolecules such as proteins, are generally Irreversibly adsorbed and

tend to mask the original properties of the substratum. The extent to

which these macromolecules obscure the original chemistry of the surface

Is not clear, however, It must be considered during attachment

experiments (Baler, 1981).

The attachment abilities of bacteria can be Influenced by this

conditioning film at the liquid-surface Interface. The substratum charge

can Influence the Ions or charged molecules present on the surface and

this could Influence factors such as pH (Hattori, 1963) and the types of

nutrients present on the surface (Marshall, 1976). If large molecules

are absorbed onto surfaces, they could change their conformation,

becoming more or less accessible to bacteria, and therefore could

influence bacterial attachment to these surfaces.

Problems could also occur due to roughness on the surface (Baker,

1984). This could cause an Increase In attachment due to a larger

surface area being present for attachment or the rough areas could

provide a protected and more favourable area for colonisation. The

roughness of the surface was beyond experimental control and It was

assumed that each surface was affected in the same way. For a single

surface like glass, this could be an acceptable assumption but It Is

accepted that the degree of roughness between surfaces of different

composition would be different. Results therefore have to be Interpreted

with this In mind.

Bright (1983), demonstrated that the ratio between attached and

free-living bacteria depended on the substratum used. In his studies, Inhibited (Pringle, 1966). Whenever a solid surface Is lmmeresed Into an

bacterial activity was more Important In the initial attachment of the

bacterium to hydrophobic surfaces than to hydrophilic ones. Moreover,

different surfaces have been shown to follow a characteristic succession

of microorganisms, so that the final biofllm that forms on one surface

under controlled conditions differed from that formed on another under

the same conditions (Marszalek, 1979: Tamplin, 1990). As the biofllm

that develops on different surfaces varies, only one surface, glass, was

used In subsequent experiments.