284
Assay of Some Common Hydrobiological Techniques
Sorokin et a1. 1996b). Because of the above large size range of planktonic
ciliates, for their adequate quantification in natural waters it is necessary to
use a combination of methods, counting separately their average to large forms
of over 30-40l1m size, accessible for viable counting in a large chamber, and
their smaller fraction less than 30-40l1m cell size also including nanociliates,
which need a special quantification technique. In accordance with this tactic,
the freshly taken samples are discharged as gently as possible into 2-1 glass
jars which may be kept without significant loss for 2-3 h in the shade at a temperature close to that in situ. Treatment of the sample starts from the fixation
of a 40-ml portion poured into the serum bottle, containing 2.5 ml of 10%
glutaraldehyde, after gently mixing it. The fixed, subs ample can be preserved
in a cold place for between 1 and 2h before examination by epifluorescence
microscopy as described above (Sect. 6.1.l.D). After the subsample is thus
fixed, a 100-ml volume of water is poured into a 200-ml beaker, from which
the large ("ciliate") counting chamber 3-4mm deep (Fig. 6.6B) is filled without
air bubbles. The chamber for counting ciliates is separated inside with thin
O.S-mm-thick walls, fixed at 6-7-mm intervals. The water in the chamber is
examined under the binocular stereoscope at x60-x80 magnification, with the
chamber placed in a black background and illuminated by a strong beam of
light from its side. The chamber is examined along the strips made by the above
internal walls, the interval between them being fitted with in the field of the
stereoscope. For sizing of ciliate cells, they are fixed in a separate small
chamber or in a petri dish with 0.1 % glutaraldehyde or a very weak (pale
yellow) Lugol or Utremohl solution. The ciliates which have settled down to
the bottom are picked up with the aid of a fine pipette and examined under
high magnification.
Returning to the fraction of smaller ciliates, their epifluorescence counting requires a certain expertise in making preparations and recognizing their
cells during examination under the epifluorescence microscope. Therefore, the
techniques should be first thoroughly practiced with cultures of naked ciliates
obtained by adding several drops of fish broth to a natural water sample. The
viable ciliates are first counted in the chamber in an appropriately diluted
culture, then they are fixed and counted in the same dilution by the epifluorescence technique. During the examination of ciliates in epifluorescence preparations, it is also possible to record the presence of chloroplasts in their cells,
which is a very common phenomenon (Stroecker et a1. 1989; Sorokin et a1.
1996b). To observe the kind and abundance of food particles in their food
vacuoles, double staining of the preparations can be employed. First, they are
stained with acridine orange, as described above for bacterioplankton (see
Sect. 6.1.2.1), then the subsample thus stained is gently filtered through a black
Nucleopore filter. The filter in the funnel is instantly flooded with primuline
(see Sect. 6.1.1C). If the epifluorescence technique is not available, the fraction of small ciliates may be quantified by a viable count using a small chamber
for counting nanoheterotrophs (Fig. 6.6a). In conclusion, it is necessary to
point out that modern data on the domination of small naked forms in planktonic ciliate populations provide little hope of obtaining reliable estimations
Assay of Some Common Hydrobiological Techniques
Sorokin et a1. 1996b). Because of the above large size range of planktonic
ciliates, for their adequate quantification in natural waters it is necessary to
use a combination of methods, counting separately their average to large forms
of over 30-40l1m size, accessible for viable counting in a large chamber, and
their smaller fraction less than 30-40l1m cell size also including nanociliates,
which need a special quantification technique. In accordance with this tactic,
the freshly taken samples are discharged as gently as possible into 2-1 glass
jars which may be kept without significant loss for 2-3 h in the shade at a temperature close to that in situ. Treatment of the sample starts from the fixation
of a 40-ml portion poured into the serum bottle, containing 2.5 ml of 10%
glutaraldehyde, after gently mixing it. The fixed, subs ample can be preserved
in a cold place for between 1 and 2h before examination by epifluorescence
microscopy as described above (Sect. 6.1.l.D). After the subsample is thus
fixed, a 100-ml volume of water is poured into a 200-ml beaker, from which
the large ("ciliate") counting chamber 3-4mm deep (Fig. 6.6B) is filled without
air bubbles. The chamber for counting ciliates is separated inside with thin
O.S-mm-thick walls, fixed at 6-7-mm intervals. The water in the chamber is
examined under the binocular stereoscope at x60-x80 magnification, with the
chamber placed in a black background and illuminated by a strong beam of
light from its side. The chamber is examined along the strips made by the above
internal walls, the interval between them being fitted with in the field of the
stereoscope. For sizing of ciliate cells, they are fixed in a separate small
chamber or in a petri dish with 0.1 % glutaraldehyde or a very weak (pale
yellow) Lugol or Utremohl solution. The ciliates which have settled down to
the bottom are picked up with the aid of a fine pipette and examined under
high magnification.
Returning to the fraction of smaller ciliates, their epifluorescence counting requires a certain expertise in making preparations and recognizing their
cells during examination under the epifluorescence microscope. Therefore, the
techniques should be first thoroughly practiced with cultures of naked ciliates
obtained by adding several drops of fish broth to a natural water sample. The
viable ciliates are first counted in the chamber in an appropriately diluted
culture, then they are fixed and counted in the same dilution by the epifluorescence technique. During the examination of ciliates in epifluorescence preparations, it is also possible to record the presence of chloroplasts in their cells,
which is a very common phenomenon (Stroecker et a1. 1989; Sorokin et a1.
1996b). To observe the kind and abundance of food particles in their food
vacuoles, double staining of the preparations can be employed. First, they are
stained with acridine orange, as described above for bacterioplankton (see
Sect. 6.1.2.1), then the subsample thus stained is gently filtered through a black
Nucleopore filter. The filter in the funnel is instantly flooded with primuline
(see Sect. 6.1.1C). If the epifluorescence technique is not available, the fraction of small ciliates may be quantified by a viable count using a small chamber
for counting nanoheterotrophs (Fig. 6.6a). In conclusion, it is necessary to
point out that modern data on the domination of small naked forms in planktonic ciliate populations provide little hope of obtaining reliable estimations