Each of the isolates in the collections was then incubated in an orbital shaker at 30°C for
10 hours in 2 ml seawater liquid growth medium. These were reinoculated into 50 ml
growth medium and grown at 30°C for a further 12 hours before being used for preparation of experimental agarose plates.
Preparation and Incubation of Plates
The activity of lysozyme-like enzymes was assayed on 0.8 % agarose plates containing
heat-killed target bacteria (McHenery et al., 1979). The bacterial suspensions were
centrifuged at 8 000 x g for 15 minutes and the pellets resuspended in 2 ml sterile seawater.
The suspensions were then adjusted with sterile seawater so that the final optical density
in the agarose medium would be 0.122 at 600 nM, and added to 20 ml agarose medium.
The media were then heated at 65°C for 10-15 minutes to kill the bacteria, and poured
into 8.4 cm diameter petri dishes. Wells of equal diameter (2 mm) were cut into each
agarose plate to receive the enzyme extract.
Eight serial ten-fold dilutions of enzyme extract were prepared and aliquots of 10 p\ of
each of the dilutions were placed individually into the wells and incubated at 20°C for 48
hours. The intact bacteria were stained by the addition of 5 ml of 0.12 % crystal violet to
each plate. After 2 hours, the plates were rinsed with distilled water and the diameter of
the clear zones of lysis was measured. From the number of bacteria per unit surface area
of the plates, the area of the clear zone could be expressed as number of bacteria lysed per
45 hours of incubation at 20°C. The equation for the regression is : Bacterial number lysed
(x 10 5 )y = - 0.92 + 0.11 X. (where X = zone diameter in mm), all calculated values,
(Seiderer et al., 1984).
A ctivity Spectrum of Crystalline Style Extract
An experiment was carried out to determine the activity spectrum of style extract of C.
meridionals, taken from mussels collected under an ambient water temperature of 11 °C.
A total of 43 gut, water column and kelp frond bacterial isolates were cultured as before.
Agarose plates were poured, incorporating cultures of each bacterial isolate separately,
and wells were made within each plate. Into the wells, eight 1:10 serial dilutions of style
extract were pipetted, and the plates were incubated at room temperature for 45 hours.
The zones of lysis were measured, and the enzyme activity was calculated as before. The
plates were divided up into 19 gut isolate plates, 16 water column plates and 8 kelp frond
plates. A cut-off point between susceptible bacteria and resistant bacteria was arbitrarily
assigned to be lxl 0 6 bacteria lysed per µl of style extract. The percent frequencies on
either side of this cut-off point were plotted against the origins of these bacterial isolates.
RESULTS AND DISCUSSION
Lysis of Target Bacteria
Zones of lysis formed the basic assay for lysozyme activity in extracts of the crystalline
style from Choromytilus meridionals. The assay for the activity of this factor is extremely
reproducible and sensitive, due to the use of crystal violet to enhance contrast of low levels
of target bacteria.
The results demonstrated in Figure 1 show the bacterial strains isolated from the gut of C.
meridionals to be 79 % resistant and 21 % susceptible to lysis. A similar spectrum was
found amongst the kelp-frond bacterial isolates, in which 62 % were resistant and 38 %
were susceptible. The bacteria isolated from the water column in the kelp bed are 43%
resistant and 57 % susceptible to lysis. Although the sample size used in this study is too
429
10 hours in 2 ml seawater liquid growth medium. These were reinoculated into 50 ml
growth medium and grown at 30°C for a further 12 hours before being used for preparation of experimental agarose plates.
Preparation and Incubation of Plates
The activity of lysozyme-like enzymes was assayed on 0.8 % agarose plates containing
heat-killed target bacteria (McHenery et al., 1979). The bacterial suspensions were
centrifuged at 8 000 x g for 15 minutes and the pellets resuspended in 2 ml sterile seawater.
The suspensions were then adjusted with sterile seawater so that the final optical density
in the agarose medium would be 0.122 at 600 nM, and added to 20 ml agarose medium.
The media were then heated at 65°C for 10-15 minutes to kill the bacteria, and poured
into 8.4 cm diameter petri dishes. Wells of equal diameter (2 mm) were cut into each
agarose plate to receive the enzyme extract.
Eight serial ten-fold dilutions of enzyme extract were prepared and aliquots of 10 p\ of
each of the dilutions were placed individually into the wells and incubated at 20°C for 48
hours. The intact bacteria were stained by the addition of 5 ml of 0.12 % crystal violet to
each plate. After 2 hours, the plates were rinsed with distilled water and the diameter of
the clear zones of lysis was measured. From the number of bacteria per unit surface area
of the plates, the area of the clear zone could be expressed as number of bacteria lysed per
45 hours of incubation at 20°C. The equation for the regression is : Bacterial number lysed
(x 10 5 )y = - 0.92 + 0.11 X. (where X = zone diameter in mm), all calculated values,
(Seiderer et al., 1984).
A ctivity Spectrum of Crystalline Style Extract
An experiment was carried out to determine the activity spectrum of style extract of C.
meridionals, taken from mussels collected under an ambient water temperature of 11 °C.
A total of 43 gut, water column and kelp frond bacterial isolates were cultured as before.
Agarose plates were poured, incorporating cultures of each bacterial isolate separately,
and wells were made within each plate. Into the wells, eight 1:10 serial dilutions of style
extract were pipetted, and the plates were incubated at room temperature for 45 hours.
The zones of lysis were measured, and the enzyme activity was calculated as before. The
plates were divided up into 19 gut isolate plates, 16 water column plates and 8 kelp frond
plates. A cut-off point between susceptible bacteria and resistant bacteria was arbitrarily
assigned to be lxl 0 6 bacteria lysed per µl of style extract. The percent frequencies on
either side of this cut-off point were plotted against the origins of these bacterial isolates.
RESULTS AND DISCUSSION
Lysis of Target Bacteria
Zones of lysis formed the basic assay for lysozyme activity in extracts of the crystalline
style from Choromytilus meridionals. The assay for the activity of this factor is extremely
reproducible and sensitive, due to the use of crystal violet to enhance contrast of low levels
of target bacteria.
The results demonstrated in Figure 1 show the bacterial strains isolated from the gut of C.
meridionals to be 79 % resistant and 21 % susceptible to lysis. A similar spectrum was
found amongst the kelp-frond bacterial isolates, in which 62 % were resistant and 38 %
were susceptible. The bacteria isolated from the water column in the kelp bed are 43%
resistant and 57 % susceptible to lysis. Although the sample size used in this study is too
429