256
Exercise 19
information about the heterotrophic potential of the microbial community but yields
little insight into their in situ rate processes. All plating media are highly selective,
and none will give "total viable counts" [cf. review of Caldwell (1977) and Austin
(1988)]. Direct counting of aquatic bacteria has been greatly facilitated by staining
the bacteria with fluorescent dyes, filtration onto polycarbonate (Nucleopore)
filters stained black with irgalan dye, and examination with epi-illuminated
fluorescence microscopy (Francisco et aI., 1973; Hobbie et aI., 1977; Bowden,
1977; Biihrer, 1977).
Direct counting of bacteria and other natural microflora is considered the most
reliable method for the evaluation of community dynamics [e.g., Daley (1979) and
Sorokin and Kadota (1972)]. Estimates of biomass from cell numbers and average
cell dimensions improve further the estimates of microbial dynamics, particularly
when coupled to direct measures of community bacterial productivity. The ensuing
exercise demonstrates one approach to bacterial community analyses by
enumeration. Automated image analysis systems now are being developed that
will permit these tedious analyses to be accomplished rapidly and with greater
accuracy than can be done manually. However, these systems are expensive and
may not be routinely available for many years.
Detection, enumeration, and size evaluations of small bacteria and cyanophytes
are difficult. The binding of organic dyes to the cells, however, can produce
fluorescence when excited with light of appropriate wavelengths. As a results.
cells can be differentiated from other particles at the lower limits of detection fOf
light microscopy.
Numerous fluorescing dyes are available. Acridine orange binds with DNA and
RNA of living cells. When excited with light (436 or 490 nm), the DNA complex
fluoresces green and the RNA complex red. Unfortunately, nonliving particles
may also become stained and fluoresce. In waters containing much suspended
seston, other fluorescing stains can be used for epifluorescent enumerations. For
example, DAPI [4'6-diamidino-2-phenylindole ] is specific for DNA of cells (Porter
and Feig, 1980). When excited with light at 365 nm, the DNA-DAPI complex
fluoresces blue at or above 390nm, while DAPI associated with non-DNA material
may fluoresce a weak yellow.
Procedures
1. Obtain water samples with a clean Van Dorn or similar nonmetallic water sampler
from several strata within a lake or stream ecosystem. Flush the sample bottles
well. Preserve to achieve a 5% glutaraldehyde solution. Record the initial and final
volumes to determine the dilution factor. Keep the samples on ice and in the dark.
2. Set up the filtration unit with a large-pore Millipore filter on the fritted glass base.
Mount the black Nuclepore (see "Apparatus and Supplies," p. 268) ofO.2-.um pore
size on the other filter and assemble the funnel unit. All filters should be moistened
with distilled water.
3. Filter an appropriate water sample (e.g., 0.3 to 3 ml from productive waters; 5 ml
from an oligotrophic water) with a low ( < 0.3 atm) vacuum. Release the vacuum
immediately as filtration is completed. Rinse the funnel walls briefly with sterile,
filtered (0.2-.um) distilled water.
4. Add 2.0 ml 0.01 % acridine orange stain (2 ml stock plus 18 ml filtered water) to
the funnel and expose for 2 min. (DAPI at a concentration of 50.ug/ml would be
exposed similarly at 2 ml for 2 or more min.) After staining, draw the stain through
Exercise 19
information about the heterotrophic potential of the microbial community but yields
little insight into their in situ rate processes. All plating media are highly selective,
and none will give "total viable counts" [cf. review of Caldwell (1977) and Austin
(1988)]. Direct counting of aquatic bacteria has been greatly facilitated by staining
the bacteria with fluorescent dyes, filtration onto polycarbonate (Nucleopore)
filters stained black with irgalan dye, and examination with epi-illuminated
fluorescence microscopy (Francisco et aI., 1973; Hobbie et aI., 1977; Bowden,
1977; Biihrer, 1977).
Direct counting of bacteria and other natural microflora is considered the most
reliable method for the evaluation of community dynamics [e.g., Daley (1979) and
Sorokin and Kadota (1972)]. Estimates of biomass from cell numbers and average
cell dimensions improve further the estimates of microbial dynamics, particularly
when coupled to direct measures of community bacterial productivity. The ensuing
exercise demonstrates one approach to bacterial community analyses by
enumeration. Automated image analysis systems now are being developed that
will permit these tedious analyses to be accomplished rapidly and with greater
accuracy than can be done manually. However, these systems are expensive and
may not be routinely available for many years.
Detection, enumeration, and size evaluations of small bacteria and cyanophytes
are difficult. The binding of organic dyes to the cells, however, can produce
fluorescence when excited with light of appropriate wavelengths. As a results.
cells can be differentiated from other particles at the lower limits of detection fOf
light microscopy.
Numerous fluorescing dyes are available. Acridine orange binds with DNA and
RNA of living cells. When excited with light (436 or 490 nm), the DNA complex
fluoresces green and the RNA complex red. Unfortunately, nonliving particles
may also become stained and fluoresce. In waters containing much suspended
seston, other fluorescing stains can be used for epifluorescent enumerations. For
example, DAPI [4'6-diamidino-2-phenylindole ] is specific for DNA of cells (Porter
and Feig, 1980). When excited with light at 365 nm, the DNA-DAPI complex
fluoresces blue at or above 390nm, while DAPI associated with non-DNA material
may fluoresce a weak yellow.
Procedures
1. Obtain water samples with a clean Van Dorn or similar nonmetallic water sampler
from several strata within a lake or stream ecosystem. Flush the sample bottles
well. Preserve to achieve a 5% glutaraldehyde solution. Record the initial and final
volumes to determine the dilution factor. Keep the samples on ice and in the dark.
2. Set up the filtration unit with a large-pore Millipore filter on the fritted glass base.
Mount the black Nuclepore (see "Apparatus and Supplies," p. 268) ofO.2-.um pore
size on the other filter and assemble the funnel unit. All filters should be moistened
with distilled water.
3. Filter an appropriate water sample (e.g., 0.3 to 3 ml from productive waters; 5 ml
from an oligotrophic water) with a low ( < 0.3 atm) vacuum. Release the vacuum
immediately as filtration is completed. Rinse the funnel walls briefly with sterile,
filtered (0.2-.um) distilled water.
4. Add 2.0 ml 0.01 % acridine orange stain (2 ml stock plus 18 ml filtered water) to
the funnel and expose for 2 min. (DAPI at a concentration of 50.ug/ml would be
exposed similarly at 2 ml for 2 or more min.) After staining, draw the stain through