ANAEROBIOSIS
Media for SRB are generally best prepared with source water and must have a redox
potential (Eh) of < - 100 mV before SRB growth can occur (Postgate, 1984). In the past,
this low Eh has been achieved by the incorporation of chemical reducing agents such as
thioglycollic acid and/or ascorbic acid into the growth medium (Pankhurst, 1971). The
former is, however, toxic to many strains of SRB (Pfennig, Widdel & Trüper, 1981).
Ascorbate is a poor reducing agent and SRB media containing it - such as that recommended by the American Petroleum Institute (Anon., 1975) - are all too easily oxidized
during transport and handling. In such media, the recovery of SRB may low leading to an
underestimation of the resident population. In samples containing large numbers of
aerobes and facultative anaerobes the recovery with weakly poised media may be increased by the oxygen-scavenging activity of these other organisms.
PRINCIPLE OF METHOD
The phenomenon of oxygen-scavenging has been used in the past for the cultivation of
anaerobic bacteria, such as members of the genus Clostridium by Fortner (1928). More
recently, Tsuneishi and Goetz (1958) used this principle to count SRB by a membrane
filtration technique. The method presented here uses a medium based on sea water which
is reduced by the addition of respiring Pseudomonas putida cells. This medium contains
no toxic reducing agents and is resistant to oxidation through mishandling.
MEDIUM AND METHOD
Ps.putida cells are grown overnight on three plates of Tryptone Soya agar (Oxoid) at
30°C. Growth is removed aseptically and suspended in 3 ml of 0.9% (w/v) saline.
The medium consists of (g/1 sea/source water):
FeSO 4 . 7H 2 O
NH 4 Cl
KH 2 PO 4
Bacto-casamino acids (Difco)
Na ascorbate
Na lactate (70 % w/v)
Resazurin (BDH)
0.3
0.3
0.2
1.0
1.0
3.6 ml
1 tablet
pH 7.5
The medium is distributed as 25 ml lots into universal bottles and autoclaved at 121°C for
15 min. Ten-fold dilutions of the sample are made in the enumeration medium and 10 ml,
1 ml and 0.1 ml aliquots of each dilution inoculated into 3 or 5 replicate bottles. 0.1 ml of
the Ps.putida cell suspension is added to each bottle, which is then completely filled with
sterile medium and screw-capped.
The bottles are incubated at 30°C for 7 days. Although psychrophilic SRB exist in marine
systems, little is known about their distribution and physiology (Postgate, 1984). In N.E.
Atlantic continental shelf and slope sediments no significant differences were found
between SRB counts performed at in situ temperature (4°C) and at 30°C (Battersby,
1983). The only effect of the lower temperature was very slow SRB growth. Similarly, a
Desulfovibrio desulfuricans subsp. aestuarii isolated at 10°C from an East Antarctic pond
exhibited good growth from 20-30°C and only slow growth at 5-10°C (Okasaki and
Ilzuka, 1972). Most marine SRB currently described have optimum growth temperatures
at or around 30°C (Widdel, 1980). SRB growth in the MPN medium is recorded as a
274
Media for SRB are generally best prepared with source water and must have a redox
potential (Eh) of < - 100 mV before SRB growth can occur (Postgate, 1984). In the past,
this low Eh has been achieved by the incorporation of chemical reducing agents such as
thioglycollic acid and/or ascorbic acid into the growth medium (Pankhurst, 1971). The
former is, however, toxic to many strains of SRB (Pfennig, Widdel & Trüper, 1981).
Ascorbate is a poor reducing agent and SRB media containing it - such as that recommended by the American Petroleum Institute (Anon., 1975) - are all too easily oxidized
during transport and handling. In such media, the recovery of SRB may low leading to an
underestimation of the resident population. In samples containing large numbers of
aerobes and facultative anaerobes the recovery with weakly poised media may be increased by the oxygen-scavenging activity of these other organisms.
PRINCIPLE OF METHOD
The phenomenon of oxygen-scavenging has been used in the past for the cultivation of
anaerobic bacteria, such as members of the genus Clostridium by Fortner (1928). More
recently, Tsuneishi and Goetz (1958) used this principle to count SRB by a membrane
filtration technique. The method presented here uses a medium based on sea water which
is reduced by the addition of respiring Pseudomonas putida cells. This medium contains
no toxic reducing agents and is resistant to oxidation through mishandling.
MEDIUM AND METHOD
Ps.putida cells are grown overnight on three plates of Tryptone Soya agar (Oxoid) at
30°C. Growth is removed aseptically and suspended in 3 ml of 0.9% (w/v) saline.
The medium consists of (g/1 sea/source water):
FeSO 4 . 7H 2 O
NH 4 Cl
KH 2 PO 4
Bacto-casamino acids (Difco)
Na ascorbate
Na lactate (70 % w/v)
Resazurin (BDH)
0.3
0.3
0.2
1.0
1.0
3.6 ml
1 tablet
pH 7.5
The medium is distributed as 25 ml lots into universal bottles and autoclaved at 121°C for
15 min. Ten-fold dilutions of the sample are made in the enumeration medium and 10 ml,
1 ml and 0.1 ml aliquots of each dilution inoculated into 3 or 5 replicate bottles. 0.1 ml of
the Ps.putida cell suspension is added to each bottle, which is then completely filled with
sterile medium and screw-capped.
The bottles are incubated at 30°C for 7 days. Although psychrophilic SRB exist in marine
systems, little is known about their distribution and physiology (Postgate, 1984). In N.E.
Atlantic continental shelf and slope sediments no significant differences were found
between SRB counts performed at in situ temperature (4°C) and at 30°C (Battersby,
1983). The only effect of the lower temperature was very slow SRB growth. Similarly, a
Desulfovibrio desulfuricans subsp. aestuarii isolated at 10°C from an East Antarctic pond
exhibited good growth from 20-30°C and only slow growth at 5-10°C (Okasaki and
Ilzuka, 1972). Most marine SRB currently described have optimum growth temperatures
at or around 30°C (Widdel, 1980). SRB growth in the MPN medium is recorded as a
274