2.0 Definition
Bacterial abundance is given in terms of the number of bacterial cells/kg seawater.

3.0 Principle of Analysis
Bacteria are preserved, stained with either acridine orange or DAPI and concentrated onto a membrane filter. This causes the individual bacteria cells to fluoresce green (using acridine orange) or bluish white (using DAPI) under blue or ultraviolet excitation respectively on an epifluorescence microscope. The individual cells are counted in fields of view of known area and the concentration of bacteria in the original sample is
calculated.

4.0 Apparatus
4.1 Any high quality epifluorescence microscope equipped with objectives specifically designed for fluorescence work at <400nm with immersion oil. The numerical aperture of the objective should be high and the focal plane should be constant across the entire field of vision. Total magnification (objective, eye pieces and auxiliary magnifiers) should be at least 1000x.

4.2 A blue filter set (blue excitation 450-490 nm, dichromatic beam splitter 510 nm, barrier filter 520 nm) is used with acridine orange.

4.3 An ultraviolet filter set (ultraviolet excitation 365 nm, dichromatic beam splitter 395 nm, barrier filter 420 nm) is used with DAPI.

5.0 Reagents
5.1 Glutaraldehyde: 25%, Grade II (Sigma)

5.2 Acridine Orange: 80% dye content (Sigma)

5.3 DAPI (4,6-Diamidino-2-phenylindole): (Sigma)

5.4 Manostat Aquet Laboratory Detergent

6.0 Sampling
Samples (95 ml) are measured into a graduated cylinder, then transferred into 125 ml high-density polyethylene bottles. Immediately following collection, the samples are preserved in 5 ml of 0.2 mm prefiltered 25% glutaraldehyde, and stored in the dark at 4° C.The amount of water to be filtered is a function of expected cell number. Following slide preparation, samples should be examined to ensure the proper number of cells (25-100 per field) (Kirchman et al., 1982) and distribution over the field. Samples should be processed, stained and filtered immediately after sampling to avoid loss of bacterial numbers (Turley and Hughs, 1992).

7.0 Procedures
7.1 Acridine Orange: A sample volume necessary to yield approximately 100 cells per field of view (total volume > 2 ml) is combined with 0.05% acridine orange (Sigma, 80% dye content) to a final concentration of 0.005% and filtered at <100 mm Hg onto a 0.2 mm, Irgalan Black stained Nuclepore polycarbonate filter (Hobbie et al., 1977). Uniform cell distribution is obtained by prewetting the ground glass base of the filtration apparatus prior to placement of the wet polycarbonate membrane. After filtration, the Nuclepore filter is immediately mounted while still damp on a slide using Resolve brand immersion oil. The stained bacterial cells can be accurately counted up to one year after preparation if the slides are stored frozen and in the
dark.

7.2 DAPI: A sample volume necessary to yield 25-100 cells per field of view (Kirchman et al., 1982) is filtered onto a 0.2 mm Nuclepore filter prestained with Irgalan Black. After filtration, the filter is covered with approximately 1 ml of the DAPI solution (50 mg/ml), and left to stain in the dark. Some researchers choose to add 0.3-0.4 ml of a 1 mg/ml DAPI solution to the sample when all but 3-4 ml have filtered. After 5-10 minutes, the DAPI is filtered off and the Nuclepore filter is immediately mounted on a slide using Resolve brand immersion oil 518C (n_e =1.518). The stained filters are stored frozen at -20°C in sealed boxes.

7.3 Kirchman et al. (1982) recommend a minimum of 7 fields per filter to be counted per sample. Bacteria are distinguished by distinct morphologies which brightly fluoresce; fluorescing images less than 0.2 mm in diameter are disregarded. An eyepiece of known area should be used during enumeration.

8.0 Calculation and expression of results:
        Bacterial Abundance ( cells / liter) = (C_f * R) / F_s
where:
        C_f = mean number of cells/field
        R = (active area of filter)/(area of field counted)1
        F_s = volume of water filtered (liters)

9.0 Quality control
Accurate measurements of sample filtered and preservative added is important for  accurate estimates.
calibrated automatic pipets should be used for dispensing sample and  preservative.

Accurate, repeatable enumeration of bacterial cells by eye requires experience as well as a  good microscope. New enumerators should train by counting the same samples as an  experienced microscopist until reliable and consistent results are obtained. Periodic  exchange of samples among different microscopists is useful for maintaining data  integrity.

Counts may be calibrated by adding fluorescent microspheres to samples prior to  counting. These are available in a variety of sizes, 0.4-2.0 mm and fluorescence properties  from Duke Scientific Corporation, Box 50005, Palo Alto, CA 94303 USA; tel 800-334-3883.

There is no absolute standard for bacterial counts. Replicate samples drawn from a single  Niskin bottle and prepared and counted in parallel should agree to within +- 15% over the  entire range of abundances encountered if the samples are prepared correctly. The precision of the estimate declines if too few or too many cells are concentrated on the  filter. See Kirchman et al (1982) for a discussion of subsampling and statistical treatments.

10.0 References
Hobbie, J.E., R.J. Daley and S. Jasper. (1977). Use of Nuclepore filters for counting  bacteria by fluorescence microscopy. Appl Environ. Microbiol.33: 1225-1228.
Kirchman, D.L., J. Sigda, R. Kapuscinski, and R. Mitchell. (1982). Statistical analysis of  the direct count method for enumerating bacteria. Appl. Environ. Microbiol. 44:376- 382.
Porter, K.G. and Y.S. Feig. (1980). The use of DAPI for identifying and counting aquatic  microflora. Limnol. Oceanogr. 25:943-948.
Turley, C.M and D.J. Hughes. (1992). Effects of storage on direct estimates of bacterial  numbers in preserved seawater samples. Deep-Sea Res. 39: 375-394.