Authors: Nina Kammenaya & Jenny Cappuccio
Purpose: To determine the number of cells in a solution
Reference: Kepner & Pratt Microbiological Reviews (1994) 58, 603-615
- DAPI stain (Invitrogen D1306, 10 mg) dissolved in water to 1 mg/mL. Binds to DNA so..likely CARCINOGEN, Store stock at -20 C wrapped in foil. Working solution of 10 ug/mL for daily use in water.
- Glutaraldehyde (25% solution, Sigma G5882) or formaldehyde (40% solution)
Note: both cross-link biomolecules in cells and are TOXIC
- Filtration system: 25 mm dia. VWR# 26316-696
- Filters: Need 1. and either 2. (autofluorescent) or 3. Black (fluorescent i.e. DAPI)
- Bottom support filter 25 mm diameter 1.0-8.0 um porosity (for cell dispersion) (e.g. millipore, Isopore, TTTP02500)
- Anodiscs (best but $$$$) or other 0.2 um, 25 mm dia. for autofluorescent cells (ie cyanobacteria)
- Black Filters for DAPI (millipore, isopore, 0.2 um, 25 mm dia. GTBP02500)
- Use early to mid-log culture with no cell flocks/aggregates visible. If there are some – short gentle sonication should help.
- Prepare 8-10 serial dilutions of the culture. I prefer lower concentration.
- Measure Absorbance in OD, (600 nm for non-pigmented cells, 750 nm for cyanobacteria such as Syn. 6803.)
- Fix cells if counting will be done later (TOXIC). Use glutaraldehyde to a final concentration of 2.5% (vol/vol), or formaldehyde 1.0% (vol/vol). Don’t use formaldehyde for autofluorescent chlorophyll containing cells. Prepare cell solution in dark vial if further DAPI staining incubate on ice 10-30 min
- If the cells possess auto-fluorescence – proceed to 6. If the cells are colorless – they need to be stained. DAPI or SYTO59 or Acridine Orange or other stain can be used. Procedure below is for DAPI
- In a dark vial use DAPI at a final concentration of 1 ug/mL for cultured cells (see ref. for saltwater o soil samples).
- Incubate on ice 7-10 min.
- Set the filtration system up: preferably use a fritted glass filter holder.
- Use 25mm filter of appropriate porosity (use 0.2um white Anodiscs for >2um cyanos). If the cells are rod-shaped – make sure they won’t run through the pores with their narrow side.
To make cell dispersed even more evenly, you can put one filter of higher porosity (1.0-8.0 um) under the trapping filter.
- Add 5-20 ml buffer/media into the filtration funnel. Add known amount of the cell culture (dilution 1) to it (not too much – you will need to count them!). Mix with a tip to homogeneity. Open a vacuum valve and gently! filter the cells. Make sure you have not much of excess liquid on the filter. Wash with equal volume of water to remove background fluorescence for stained samples
- Put one drop of immersion oil on a lead glass slide (ref. says type FF). Fit the filter on it and knock with the glass on a table to get the drop spread under the filter.
- Put one more drop on top of the filter. Cover with the cover slip gently not to move the cells. Place the glass on a wet paper towel and wrap with aluminum foil. Cyanos can be kept at +4C for several weeks, DAPI store at C in the dark for ~1 week.
- Repeat with all the dilutions.
- Determine working area of the filter (ie diameter that is actually in contact with the cells, mine is 16 mm for the VWR set-up)
- Count known area. Or even better take images on fluorescent microscope. Note objective and magnification for pixel to area calculations. Count cells, calculate, and plot vs OD.