Contact: Kristin Searcy

  1. Use PBS to make a desired dilution of the bacterial sample.
  2. Add 4’,6-diamidino-2-phenylindole (DAPI) to 5 -10mL of the diluted bacterial sample for a final DAPI concentration of 2.5 μg/mL.
  3. Allow DAPI stain to incubate for 15 minutes in the dark.
  4. Pre-moisten a 0.45-μm pore size, 25-mm diameter nitrocellulose backing filter and a 0.2-μm pore size, 25-mm diameter pre-stained black membrane filter with MQ water.
  5. Overlay the black filter over the backing filter on the filtering device, attach tower with clamp, and pour the stained sample into the tower.
  6. Filter the sample at 16 in Hg vacuum pressure until complete.
  7. Remove the clamp and tower from the filtering apparatus, and with tweezers, remove the black filter.
  8. 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.
  9. View slide under 630x magnification.
  10. Using 0.0009 mm2 field (one square grid at 630x), count the number of bacteria per field. (I aim to count ~10 bacteria/field).
  11. Count a total of ~200 bacteria.
  12. Complete another count on the same filter.
  13. 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.)
  14. 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)

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