recommend time-kill tests of SRB contaminated injection water which again take ca 28
days before a result is obtained.
Principle of the new method:
In an attempt to reduce the delay that is inherent in the API testing methods we have
developed a simple and rapid technique for studying the effect of a biocide on SRB by
measuring the inhibition of :
1) culture absorbance at 580 nm
2) sulphate reduction
- in a logarithmic culture of the test SRB strain.
On average only 4 days are required to complete the test. Although rapid radiorespirometric methods for biocide testing have been described recently (eg. Hardy and Syrett, 1983),
such techniques require relatively sophisticated and expensive equipment and have
attendant safety problems. The method described below is simple, rapid and inexpensive.
MATERIALS AND METHODS
The low iron medium (C) of Postgate ( 1984) is used supplemented with 0,1 g/l ascorbate
and thioglycollate as reducing agents and 0.0002 % w/v resazurin as a redox indicator.
This is dispensed in 9.5 ml amounts into a series of 20 ml capacity test tubes and
autoclaved at 121°C until reduced. The tubes are cooled under a stream of oxygen-free
nitrogen gas, sealed with butyl rubber stoppers and autoclaved with the tops clamped
downfor 15 mn at 121°C. Inoculation of the tubes is by injection of 0.2 ml of the test strain
(30 hours at 30°C in medium C). SRB growth is followed by inserting the tubes into a
colorimeter and measuring the increase in absorbance at 580 nm. In the early logarithmic
phase of growth ( A 580 ca 0.15) dilutions of the biocide are injected into 4 tubes of the same
A 580 and the contents mixed. Control tubes are inoculated with sterile distilled water.
On biocide addition, and after, 3,6, and 9 hours incubation the following parameters are
determined :
1) absorbance at 580 nm
2) residual sulphate in the medium (eg ; BaCl 2 -EDTA titration)
On biocide addition and after 9 hours incubation :
3) the numbers of viable SRB are also determined as described by Postgate (1984).
RESULTS AND DISCUSSION
Figure 1 shows results typical of those obtained by this method. Alkaline glutaraldehyde,
a 5-carbon dialdehyde, was tested against Desulfovibrio desulfuricans NCIB 8400. Biocide increments of 50 ppm were used as this interval is recommended for biocides with
potential off-shore applications (Crouch, 1983). Inhibition of SRB growth occurred at a
concentration of 50 ppm (Fig 1b).The effect of a biocide on a cell is normally dependant
on its concentration and it can be seen from the slight increase and levelling off of A 580 at
50 ppm that glutaraldehyde is bacteriostatic at this concentration. However, at 100 ppm
the decrease in A 580 reflects a bactericidal action with cell lysis. This preliminary observation is confirmed by the counts for viable SRB after 9 hours incubation.
618
days before a result is obtained.
Principle of the new method:
In an attempt to reduce the delay that is inherent in the API testing methods we have
developed a simple and rapid technique for studying the effect of a biocide on SRB by
measuring the inhibition of :
1) culture absorbance at 580 nm
2) sulphate reduction
- in a logarithmic culture of the test SRB strain.
On average only 4 days are required to complete the test. Although rapid radiorespirometric methods for biocide testing have been described recently (eg. Hardy and Syrett, 1983),
such techniques require relatively sophisticated and expensive equipment and have
attendant safety problems. The method described below is simple, rapid and inexpensive.
MATERIALS AND METHODS
The low iron medium (C) of Postgate ( 1984) is used supplemented with 0,1 g/l ascorbate
and thioglycollate as reducing agents and 0.0002 % w/v resazurin as a redox indicator.
This is dispensed in 9.5 ml amounts into a series of 20 ml capacity test tubes and
autoclaved at 121°C until reduced. The tubes are cooled under a stream of oxygen-free
nitrogen gas, sealed with butyl rubber stoppers and autoclaved with the tops clamped
downfor 15 mn at 121°C. Inoculation of the tubes is by injection of 0.2 ml of the test strain
(30 hours at 30°C in medium C). SRB growth is followed by inserting the tubes into a
colorimeter and measuring the increase in absorbance at 580 nm. In the early logarithmic
phase of growth ( A 580 ca 0.15) dilutions of the biocide are injected into 4 tubes of the same
A 580 and the contents mixed. Control tubes are inoculated with sterile distilled water.
On biocide addition, and after, 3,6, and 9 hours incubation the following parameters are
determined :
1) absorbance at 580 nm
2) residual sulphate in the medium (eg ; BaCl 2 -EDTA titration)
On biocide addition and after 9 hours incubation :
3) the numbers of viable SRB are also determined as described by Postgate (1984).
RESULTS AND DISCUSSION
Figure 1 shows results typical of those obtained by this method. Alkaline glutaraldehyde,
a 5-carbon dialdehyde, was tested against Desulfovibrio desulfuricans NCIB 8400. Biocide increments of 50 ppm were used as this interval is recommended for biocides with
potential off-shore applications (Crouch, 1983). Inhibition of SRB growth occurred at a
concentration of 50 ppm (Fig 1b).The effect of a biocide on a cell is normally dependant
on its concentration and it can be seen from the slight increase and levelling off of A 580 at
50 ppm that glutaraldehyde is bacteriostatic at this concentration. However, at 100 ppm
the decrease in A 580 reflects a bactericidal action with cell lysis. This preliminary observation is confirmed by the counts for viable SRB after 9 hours incubation.
618