Bacterial Enumeration

An important part of the McMurdo Long Term Ecological Research (LTER) project is monitoring spatial and temporal patterns, and processes that control bacterial production in perennial ice covered lakes. This data set quantifies bacteria concentrations at specific depths in Dry Valley lakes.

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Lake water samples are collected at specific depths with a five-liter Niskin bottle during normal LTER limnological sampling. Sub-samples for bacteria enumeration are decanted into a 1 L amber Nalgene bottle. 18 mL bacteria samples are pipetted from the amber Nalgene bottle into new 20 ml glass scintillation vials for each depth. Bacteria samples are preserved by adding 0.5 ml of buffered to saturation (with sodium borate) formalin (0.2 m filtered) to each sample. Samples are stored at 4 degrees C until preparation for counting. Bacteria samples are counted upon return to Montana State University. A 0.45 micrometer 25 mm diameter membrane filter is placed on a glass fritted filter apparatus base and covered with a 0.2 micrometer 25 mm diameter black polycarbonate filter. An appropriate volume of sample (1.5-6 ml depending on the concentration of cells in each sample) is added to a cleaned filter tower (scrubbed with Alconox, soaked in 10% HCL, rinsed in DIW, rinsed with 95% ETOH). An appropriate volume (0.5 ml SYBR/3 ml sample) of 0.2 µm filtered 25X SYBR Gold solution (10,000X concentrate SYBR Gold Nucleic Acid Gel Stain in 1X TBE buffer) is added to the sample and allowed to incubate in the dark for 15 minutes. The sample is filtered under low vacuum (0.3 atm; 5 in Hg), and the tower rinsed with 1 ml of 0.2 micrometer filtered DI water when a thin layer of sample remains. The filter is placed on a glass microscope slide containing 1 drop of Antifade solution (0.1% p-phenylenediamine in a 1:1 (weight:volume) solution of phosphate-buffered saline and glycerol). 1 drop of Antifade solution is placed on top of the filter before placing a cover slip on the filter. A sample blank is prepared by following the above procedure with .2 micrometer filtered DI water. Cells are counted on a Nikon Optiphot epifluorescent microscope equipped with a DM510 filter cube (Nikon) at a final magnification of 1,000X. The total number of cocci, rods and filaments (defined as bacteria greater than 5 micrometer in length) are counted in at least 10 different fields of view until at least 300 cells are counted yielding a less than 15% counting error. At least 1 digital image is taken at each depth using the Optronics Microfire digital camera system for archiving purposes. cells/mL = ( (S-B) / volume ) * (filter area/field area) where S is the average number of cells per field in the sample, B is the average number of cells per field in the blank, filter area is the area (micrometer square) of the filter containing bacterial cells, field area is the area for each field counted (micrometer square), and vol is ml of sample filtered.