GERBAM — Deuxième Colloque International de Bactériologie marine — CNRS, Brest, 1-5 octobre 1984
IFREMER, Actes de Colloques, 3, 1986, pp. 249-259
28
THE DISPERSION OF ADHERED MARINE BACTERIA BY PYROPHOSPHATE
AND ULTRASOUND PRIOR TO DIRECT COUNTING
M.I VELJI and L.J. ALBRIGHT
Department of Biological Sciences, Simon Fraser University BURNABY, B.C. CANADA
V5A 1S6
ABSTRACT - A technique has been investigated for dispersing adhered marine bacteria from various surfaces
prior to direct counting. The bacterial cells were initially preserved and strengthened by use of 3.7 % formaldehyde (v/v final concentration). Seawater samples were then treated with 0.001 M of tetrasodium pyrophosphate
(final concentration), a sequestering and deflocculating agent. Surface sediment and kelp disc samples were
treated with 0.01 M tetrasodium pyrophosphate (final concentration) after having been suspended in 0.44 M
sodium chloride solution. These samples were incubated for 15-30 min. Subsequently, the samples were treated
with ultrasound ( 100 W) for 30 to 45 s. This technique resulted in an even dispersion of most bacterial cells in the
samples. Bacterial cells dispersed in this manner were then stained with 4’, 6 - diamino - 2 -phenylindole (DAPI)
and enumerated by standard epifluorescent microscopy. Bacterial populations of surface sediment and older
kelp blades which were previously difficult or impossible to enumerate were now found to be easily determined.
A mechanism is proposed to explain the increase in dispersion of the bacterial cells by this technique.
RÉSUMÉ - Une technique pour disperser les bactéries marines des différentes surfaces avant les numérations
directes a été testée. Les cellules bactériennes sont fixées et rendues plus fermes par addition de formaldéhyde à
3.7% (concentration finale v/v). Les échantillons d’eau de mer sont ensuite traités par une solution de
tétrasodium pyrophosphate 0.001 M (concentration finale), utilisée comme agent défloculant et séquestrant.
Les échantillons de la surface du sédiment et des grandes algues sont traités par une solution 0.01 M de
tétrasodium pyrophosphate (concentration finale) dans une solution de chlorure de sodium 0.44 M. Ils sont
alors incubés 15 à 30 mn avant d’être traités aux ultrasons (100 W) pendant 30 à 45 s. Cette technique permet la
dispersion de la majorité des bactéries dans l’échantillon. Les cellules bactériennes, dispersées par cette
technique, sont ensuite colorées par du 4’,6 diamino -2-phénylindole (DAPI) et dénombrées en microscopie à
épifluorescence. Les populations bactériennes du sédiment de surface et des lames âgées d’algues, qu’il était
jusque-là difficile ou même impossible de dénombrer, sont maintenant aisément quantifiées. Un mécanisme est
proposé pour expliquer l’augmentation de la dispersion des cellules par cette méthode.
INTRODUCTION
Bacteria are ubiquitous within the aquatic environment. They occur in and on sediments
(Dale, 1974; Weise and Rheinheimer, 1978), on plants, including seaweeds (Cundell et al.,
1977 ; Roland, 1980), in and on animals (Sieburth, 1979), attached to rocks (Geesey et al.,
1977) and as a major component of detritus (Fenchel and Jorgensen, 1977). A relatively
large proportion of bacterial population within the aquatic environment is adhered ; most
of these cells are attached to sediment particles, plants, animals and detritus.
The direct microscopic count method (Porter and Feig, 1980; Francisco et ai, 1973 ;
Hobbie et al., 1977 ; Daley, 1979) is a very useful way of determining bacterial numbers
within an aquatic sample. This approach works well with unattached bacteria but
bacteria which are attached to surfaces and embedded in colloidal matrices are difficult
and often impossible to observe. This is due to (1) fluochrome, e.g. acridine orange,
249
IFREMER, Actes de Colloques, 3, 1986, pp. 249-259
28
THE DISPERSION OF ADHERED MARINE BACTERIA BY PYROPHOSPHATE
AND ULTRASOUND PRIOR TO DIRECT COUNTING
M.I VELJI and L.J. ALBRIGHT
Department of Biological Sciences, Simon Fraser University BURNABY, B.C. CANADA
V5A 1S6
ABSTRACT - A technique has been investigated for dispersing adhered marine bacteria from various surfaces
prior to direct counting. The bacterial cells were initially preserved and strengthened by use of 3.7 % formaldehyde (v/v final concentration). Seawater samples were then treated with 0.001 M of tetrasodium pyrophosphate
(final concentration), a sequestering and deflocculating agent. Surface sediment and kelp disc samples were
treated with 0.01 M tetrasodium pyrophosphate (final concentration) after having been suspended in 0.44 M
sodium chloride solution. These samples were incubated for 15-30 min. Subsequently, the samples were treated
with ultrasound ( 100 W) for 30 to 45 s. This technique resulted in an even dispersion of most bacterial cells in the
samples. Bacterial cells dispersed in this manner were then stained with 4’, 6 - diamino - 2 -phenylindole (DAPI)
and enumerated by standard epifluorescent microscopy. Bacterial populations of surface sediment and older
kelp blades which were previously difficult or impossible to enumerate were now found to be easily determined.
A mechanism is proposed to explain the increase in dispersion of the bacterial cells by this technique.
RÉSUMÉ - Une technique pour disperser les bactéries marines des différentes surfaces avant les numérations
directes a été testée. Les cellules bactériennes sont fixées et rendues plus fermes par addition de formaldéhyde à
3.7% (concentration finale v/v). Les échantillons d’eau de mer sont ensuite traités par une solution de
tétrasodium pyrophosphate 0.001 M (concentration finale), utilisée comme agent défloculant et séquestrant.
Les échantillons de la surface du sédiment et des grandes algues sont traités par une solution 0.01 M de
tétrasodium pyrophosphate (concentration finale) dans une solution de chlorure de sodium 0.44 M. Ils sont
alors incubés 15 à 30 mn avant d’être traités aux ultrasons (100 W) pendant 30 à 45 s. Cette technique permet la
dispersion de la majorité des bactéries dans l’échantillon. Les cellules bactériennes, dispersées par cette
technique, sont ensuite colorées par du 4’,6 diamino -2-phénylindole (DAPI) et dénombrées en microscopie à
épifluorescence. Les populations bactériennes du sédiment de surface et des lames âgées d’algues, qu’il était
jusque-là difficile ou même impossible de dénombrer, sont maintenant aisément quantifiées. Un mécanisme est
proposé pour expliquer l’augmentation de la dispersion des cellules par cette méthode.
INTRODUCTION
Bacteria are ubiquitous within the aquatic environment. They occur in and on sediments
(Dale, 1974; Weise and Rheinheimer, 1978), on plants, including seaweeds (Cundell et al.,
1977 ; Roland, 1980), in and on animals (Sieburth, 1979), attached to rocks (Geesey et al.,
1977) and as a major component of detritus (Fenchel and Jorgensen, 1977). A relatively
large proportion of bacterial population within the aquatic environment is adhered ; most
of these cells are attached to sediment particles, plants, animals and detritus.
The direct microscopic count method (Porter and Feig, 1980; Francisco et ai, 1973 ;
Hobbie et al., 1977 ; Daley, 1979) is a very useful way of determining bacterial numbers
within an aquatic sample. This approach works well with unattached bacteria but
bacteria which are attached to surfaces and embedded in colloidal matrices are difficult
and often impossible to observe. This is due to (1) fluochrome, e.g. acridine orange,
249