268
Exercise 19
b. A multiple filtration manifold unit, if available, to permit simultaneous vacuum filtration of
up to 12 samples, 25-mm diameter; 12 stoppers (#6).
c. Cellulose acetate membrane filters (e.g., Millipore or Sartorius), 25-mm diameter, 0.22- or
0.45-J1m pore size.
d. Vortex mixer.
e. Trichloroacetic acid (TCA). Caution: TCA is caustic, adheres to skin, and is very corrosil'e.
Measure 30.0 g of TCA into a 200-ml volumetric flask, bring up to mark with pure
water, and mix thoroughly. Carefully transfer to a 250-ml automatic transfer pipet.
Label 15% TCA.
r. Lake or stream water, filtered through 0.45-J1m pore size filters.
g. Polypropylene test tubes, 50 ml.
h. Water bath or oven, 95 to 100°e, with rack for holding tubes during extraction.
i. Ethylacetate and scintillation fluors.
Bacterial Enumeration and Biomass
1. Glutaraldehyde [1,5-Pentanedial], added to field samples to preserve at a 5~~ solution.
Caution: Glutaraldehyde at 50%, the maximum available commercially, is caustic. Use of
25% is recommended as a stock solution. Use only in a well-ventilated area or in a hood.
2. Acridine orange stock solution: To obtain a 0.1 ~;,; solution in 1 % glutaraldehyde, add 4.0 ml
of 25% glutaraldehyde (0.8-J1m pore size filtered) to 100 mg acridine orange in a 100-ml
volumetric flask and bring to volume with water.
3. Polycarbonate Nuclepore filters, 0.2-J1m pore size, 25-mm diameter, stained black by
manufacturer or by soaking 2 to 24 h in a solution of 2 g of irgalan black (Chemical Index,
acid black 107) in 1 liter of2% acetic acid. When stored in the dye, rinse several times in clear
water and use immediately. Alternatively, filters can be dried on absorbent paper and stored
(Francisco et al. 1973; Hobbie et al. 1977).
4. Millipore filters, 1.2-J1m pore size, 25-mm diameter.
5. Filtration unit (25-mm diameter base, funnel, clamp, filtration flask) and vacuum pump.
6. Microscope slides, coverslips, and Cargille Type A nonfluorescent immersion oil.
7. Compound epifluorescent microscope (e.g., Zeiss Standard or Universal or Leitz Ortholux)
with Xenon or other halogen lamp system. Appropriate beam splitters and filters to achieve
excitation at 436 or 490 nm.
References
Austin, B. (ed.). 1988. Methods in Aquatic Bacteriology. Wiley, Chichester. 425 pp.
Autio, R.M. 1990. Bacterioplankton in filtered brackish water cultures: Some physical and
chemical parameters affecting community properties. Arch. Hydrobiol. 117:437-451.
Bell, R.T., G.M. Ahlgren, and I. Ahlgren. 1983. Estimating bacterioplankton production by
the [3H]thymidine incorporation in a eutrophic Swedish lake. Appl. Environ. Microbiol.
45:1709-1721.
Bowden, W.B. 1977. Comparison of two direct-count techniques for enumerating aquatic
bacteria. Appl. Environ. Microbiol. 33:1229-1232.
Bratbak, G. 1985. Bacterial biovolume and biomass estimations. Appl. Environ. Microbiol.
49: 1488-1493.
Bratbak, G. and I. Dundas. 1984. Bacterial dry matter content and biomass estimations. Appl.
Environ. Microbiol. 48:755-757.
Biihrer, H. 1977. Verbesserte Acridinorangemethode zur Direktzahlung von Bakterien aus
Seesediment. Schweiz. Z. Hydrol. 39:99-103.
Caldwell, D.E. 1977. The planktonic microflora of lakes. CRC Critical Rev. Microbiol.
5:305-370.
Coveney, M.F. and R.G. Wetzel. 1988. Experimental evaluation of conversion factors for the
CH)thymidine incorporation assay of bacterial secondary productivity. Appl. Environ.
Microbiol. 54:2018-2026.
Exercise 19
b. A multiple filtration manifold unit, if available, to permit simultaneous vacuum filtration of
up to 12 samples, 25-mm diameter; 12 stoppers (#6).
c. Cellulose acetate membrane filters (e.g., Millipore or Sartorius), 25-mm diameter, 0.22- or
0.45-J1m pore size.
d. Vortex mixer.
e. Trichloroacetic acid (TCA). Caution: TCA is caustic, adheres to skin, and is very corrosil'e.
Measure 30.0 g of TCA into a 200-ml volumetric flask, bring up to mark with pure
water, and mix thoroughly. Carefully transfer to a 250-ml automatic transfer pipet.
Label 15% TCA.
r. Lake or stream water, filtered through 0.45-J1m pore size filters.
g. Polypropylene test tubes, 50 ml.
h. Water bath or oven, 95 to 100°e, with rack for holding tubes during extraction.
i. Ethylacetate and scintillation fluors.
Bacterial Enumeration and Biomass
1. Glutaraldehyde [1,5-Pentanedial], added to field samples to preserve at a 5~~ solution.
Caution: Glutaraldehyde at 50%, the maximum available commercially, is caustic. Use of
25% is recommended as a stock solution. Use only in a well-ventilated area or in a hood.
2. Acridine orange stock solution: To obtain a 0.1 ~;,; solution in 1 % glutaraldehyde, add 4.0 ml
of 25% glutaraldehyde (0.8-J1m pore size filtered) to 100 mg acridine orange in a 100-ml
volumetric flask and bring to volume with water.
3. Polycarbonate Nuclepore filters, 0.2-J1m pore size, 25-mm diameter, stained black by
manufacturer or by soaking 2 to 24 h in a solution of 2 g of irgalan black (Chemical Index,
acid black 107) in 1 liter of2% acetic acid. When stored in the dye, rinse several times in clear
water and use immediately. Alternatively, filters can be dried on absorbent paper and stored
(Francisco et al. 1973; Hobbie et al. 1977).
4. Millipore filters, 1.2-J1m pore size, 25-mm diameter.
5. Filtration unit (25-mm diameter base, funnel, clamp, filtration flask) and vacuum pump.
6. Microscope slides, coverslips, and Cargille Type A nonfluorescent immersion oil.
7. Compound epifluorescent microscope (e.g., Zeiss Standard or Universal or Leitz Ortholux)
with Xenon or other halogen lamp system. Appropriate beam splitters and filters to achieve
excitation at 436 or 490 nm.
References
Austin, B. (ed.). 1988. Methods in Aquatic Bacteriology. Wiley, Chichester. 425 pp.
Autio, R.M. 1990. Bacterioplankton in filtered brackish water cultures: Some physical and
chemical parameters affecting community properties. Arch. Hydrobiol. 117:437-451.
Bell, R.T., G.M. Ahlgren, and I. Ahlgren. 1983. Estimating bacterioplankton production by
the [3H]thymidine incorporation in a eutrophic Swedish lake. Appl. Environ. Microbiol.
45:1709-1721.
Bowden, W.B. 1977. Comparison of two direct-count techniques for enumerating aquatic
bacteria. Appl. Environ. Microbiol. 33:1229-1232.
Bratbak, G. 1985. Bacterial biovolume and biomass estimations. Appl. Environ. Microbiol.
49: 1488-1493.
Bratbak, G. and I. Dundas. 1984. Bacterial dry matter content and biomass estimations. Appl.
Environ. Microbiol. 48:755-757.
Biihrer, H. 1977. Verbesserte Acridinorangemethode zur Direktzahlung von Bakterien aus
Seesediment. Schweiz. Z. Hydrol. 39:99-103.
Caldwell, D.E. 1977. The planktonic microflora of lakes. CRC Critical Rev. Microbiol.
5:305-370.
Coveney, M.F. and R.G. Wetzel. 1988. Experimental evaluation of conversion factors for the
CH)thymidine incorporation assay of bacterial secondary productivity. Appl. Environ.
Microbiol. 54:2018-2026.