- Use PBS to make a desired dilution of the bacterial sample.
- Add 4’,6-diamidino-2-phenylindole (DAPI) to 5 -10mL of the diluted bacterial sample for a final DAPI concentration of 2.5 mg/mL.
- Allow DAPI stain to incubate for 15 minutes in the dark.
- Pre-moisten a 0.45-mm pore size, 25-mm diameter nitrocellulose backing filter and a 0.2-mm pore size, 25-mm diameter pre-stained black membrane filter with MQ water.
- Overlay the black filter over the backing filter on the filtering device, attach tower with clamp, and pour the stained sample into the tower.
- Filter the sample at 16 in Hg vacuum pressure until complete.
- Remove the clamp and tower from the filtering apparatus, and with tweezers, remove the black filter.
- Mount the black filter on a microscope slide using a drop of immersion oil and adhere the cover slip onto the filter with another drop of immersion oil.
- View slide under 630x magnification.
- Using 0.0009 mm2 field (one square grid at 630x), count the number of bacteria per field. (I aim to count ~10 bacteria/field).
- Count a total of ~200 bacteria.
- Complete another count on the same filter.
- Repeat steps 2-13 for the same bacterial sample. (Thus, there will be 2 counts per filter on 2 filters, for a total of 4 counts per sample.)
- Use the following formula to calculate the concentration of bacteria in your sample:
Bacteria(cells/mL) = (N x At) / (d x Vf x F x Ag)
where:
N = number of cells counted Vf = volume of diluted sample filtered (mL)
At = effective area of the filter (mm2) F = number of fields counted
d = dilution factor (Vfinal/Vsample) Ag = area of the field (mm2)