Protocol name: CTC staining
Prepared by: Monica Ponder / Date Created/Revised: 5/27/03
Purpose: To qualitatively determine if microbes are metabolically active under different conditions. As indicated by the reduction of tetrazolium dye by the electro transport system which then fluoresces under a certain wavelength. / Reference(s):

Comments:

Items you will need:

5- Cyano 2,3-ditoyl tetrazolium chloride- Polysciences Inc. catalog #19292

DAPI- Sigma D9542

0.45um Hemiacetate filters- Millipore HAWP02500

25mm Glass fiber prefilters- Millipore AP1502500

Black polycarbonate filters- Millipore GTBP02500

37% formaldehyde- Sigma

75 x 38 mm Corning glass slides- VWR 48300-160

35 x 60 mm coverglasses- VWR 48404-137

Should use 4 replicates at least for these experiments and always use a positive control and a negative control (formaldehyde fixed).

If all you want to know is whether or not it is active which is what I usually do then you need only a small volume.

Sterile 1.7 milliter cryovials with 1 ml of media and inoculated with 20 ul of culture.

To eliminate questions of residual metabolic activity the CTC should be added (2mM final concentration) a few hours after inoculation.

After addition of CTC the vials must be kept in the dark because visible light spectra inactivated the dye.

Next incubate the cells for several hours to 2 weeks and then fix with 10% total volume of filter sterilized 37% formaldehyde.

Visualizing the cells

  1. On filter apparatus place(use tweezers) a Millipore pre-filter, followed by a hemi-cellulose filter then wetted with MilliQ so that no bubbles are present. On top of these place the GTBP filter from Millipore. This filter should be wetted from the underlying moisture and no bubbles should be visible.

  1. Add 2.5 mls of MilliQ, 10 ul of DAPI (final concentration of 10 mg/ml) and 1 ml of the CTC/cell mixture and mix in the dark. Incubate for 5 minutes to give time for the DAPI to bind. Add another 1.5 ml of MilliQ and mix with pipette.
  2. Turn on vacuum ( water faucet through a flask adapter) so that the solution drips through slowly. Make sure the apparatus is covered with foil or dark conditions are maintained throughout the process.
  3. After the solution has filtered place the GTBP filter in the drying oven briefly until dry (surface should appear dull).
  4. Clean a large microscope slide and coverslip with ethanol and allow to airdry to remove any dust that would refract.
  5. Place 2-3 drops (roughly the size of the filter) of type FF immersion oil on the slide and place the filter on top. Make sure to put the side with the cells facing up.
  6. Add half a drop of immersion oil to the coverslip and place on top of the filter. Seal the slide with nail polish and place in covered box at 4°C until visualized.