Feulgen Cytophotometery

E.S.Cole 2005

Slide Preparation

Preparation of reagents

1) Prepare a series of clean Coplin jars. (Use nitric acid to remove pre-existing stain if

necessary). 13 with ordinary lids and 2 with glass lids.

2) Locate or prepare reagents:

Carnoy’s Fixative:

3:1 ETOH: Acetic Acid

add 75 mLs 95% non-denatured ETOH to 25 mLs glacial acetic acid.

ddH2O rinse

5M HCL (glass top Coplin jar)

ddH2O Rinse

Schiff’s Reagent* (See special instructions for preparation.)

ddH2O

“Bleach” (must be made up fresh the day of use!)

18 parts dH2O (180 mLs)

1 part 1 M HCl (10 mLs)

1 part 10% Potassium metabisulfite (Make up fresh!)

(weigh out 1 g, PMBS) place in capped 15 mL centrifuge tube.

Measure out 10 mLs of H2O in a second 15 mL centrifuge tube.

When ready to use, mix water and powder, and then add to H2O and HCl.

2 X ddH2O rinse.

Ethanol series: 30%, 50%, 70%, 95%, 100%, 100%.

Find Euparal vert mounting medium.

3) Set up staining station in Hood (Use gloves and lab jacket).

4) Stain: Fix 5 min in Carnoy’s (unless slides not fixed, then 15 min).

Rinse in dH2O.

Transfer slides to acid for hydrolysis: 20 min. (Time is critical). Rinse.

Transfer to Schiff’s (should be clear) 2hr room temp.

Bleach 10 min. Rinse 2 X.

Dehyderate in ETOH series (2-5 min each). Air dry slides.

Add 3 small drops Euparal vert, and long coverslip. Allow to dry overnight.

Seal edge with mounting medium or nail polish.

Schiff’s Reagent Special Preparation.

1. Dissolve 1 g basic fuchsin in 200 mL boiling distilled water, slowly and carefully.

2. Shake well.

3. Cool to 50 C.

4. Filter and add 30 mL 1 M HCl to the filtrate.

5. Add 3 g potassium metabisulfite in Hood.

6. Bleach overnight in tightly stoppered bottle in dark at room temp.

7. Add 0.5 g activated charcoal.

8. Filter reagent.

9. Store bottle in refrigerator. Lasts in refrigerator up to six months or more.

Use of Cytophotometer.

Operation of Zeiss Microscope & Photometer.

Preparations

1) Adjusting the microscope and aperture alignment.

Turn on scope illumination and MSP21 (toggle switch is back/right).

Turn on “backlight” located at the top of scope (box on purple level, back right-hand

side, rheostat).

Place “silvered” slide onto stage (silver disc is up).

Move silvered region until it half covers the light from the backlight (view through

scope).

Focus on light. (Focus right ocular independently to get clear image of lines). Note:

silvered mirror creates a series of images, depending on focal plane. Proper

image is brightest (true) image not faint (multiply reflected) images.

Use adjustment knobs (purple level, left side) to center back-light circle inside

appropriate aperture. (You select the proper aperture by moving the black slider

top-right of scope/ purple level). E.g. 0.25 mm, 0.63 mm, 1.6 mm.

Be sure the blue filter is pushed in below the stage (back, silver peg on right side of base).

Be sure the condenser lens does not have the flip-in lens engaged and the iris is fully

open.

2) Preparing photometer.

When MSP 21 comes on it will read SELECT MODE. Hold down the SHIFT key

while you press S3. It will now read FILTER AT PLUT 3 =1 TO ___? Press 2.

Taking Measurements (when aperture is aligned and photometer is set).

1) Turn on scope and prepare MSP 21 as described above.

2) Place stained slide on the scope. Use low power (20-40X) to find region of interest (stained specimen). THEN move to 63X Oil immersion for Tetrahymena mics and macs.

3) Position the slide with the selected ocular ring over a blank area of the slide. (No specimens).

4) MSP 21 should be reading: SELECT MODE. Press ADJU.

5) AUTO ADJUST RUNNING appears. Then Adj. V= ___ AD/ ___ ADG1

HV___ V Ref 100.00%.

appears. Hit RET for the background measurement.

6) Disp G1 M1/M2= ___

M1 = ___ % M2 = ___ % appears.

You want the M1 and M2 values to be nearly identical and nearly 100.0%. (best if M1 is slightly larger than M2). These numbers represent the 100% transmittance reference for the two different filters you are using [M1 = 480 nM, M2 = 560 nM]. This method uses a two-wavelength technique to correct for irregularities. To obtain M1=M2=100%, turn up the substage illuminator to a position between 7 and 8 (closer to 7) on the rheostat (lower right). Perform steps 4-6 above. If M1 is greater than M2, turn rheostat down a tiny bit. This produces a warmer light (more red, less blue). Retake values (simply press RET). If M1 is less than M2, turn up the rheostat a bit and retake (more blue, less red). Keep adjusting the rheostat and measuring M1 and M2 until the two values are within 1% of each other e.g. M1=99.8% M2= 99.2 %. If values have drifted far from 100%, repeat steps 4-6 to reset 100% standard. You have now “set” the photometer to read 100% transmittance (no absorption) for both wavelengths over a blank region of the slide. Now you are ready to measure a nucleus.

7) Focus on nucleus of interest, center it in the proper ocular annulus (corresponding to the aperture you slid into place during preparations).

Hit RET for measurements. Disp G0 M1/M2

M1= ____% M2 = ____ % appears.

8) Record M1 and M2 for later calculations. Move to a new nucleus and repeat.

9) For Tetrahymena, we first measure 10-20 micronuclei from 2hr mating pairs as our reference nuclei {G2, 4C standard}. This is done with aperture 0.25 mm. Macronuclei are measured using 0.63 mm aperture. This requires switching form 0.25 to 0.63 mm apertures. After switching apertures, (black slider/ purple level), you must re-center using silvered slide, and again adjust M1/M2 of background light as above.

10) Correcting for aperture size differences. As you select larger apertures (0.25 mm to 0.63 mm or 1.6 mm) you must multiply you r[DNA] by an appropriate size factor. Since the aperture area increases with the square of the diameter, multiply [DNA] by [diameter2 (larger)/ diameter1 (smaller)]². E.g. [0.63/0.25]² = 6.35 X.

E.g. [1.60/0.25]² = 41X .

This can be done after calculating the [DNA] as shown below.

Entering Data.

Find mics and Macs excel spreadsheet blanks. (Search for mic control blank or mac blank.)

Under mic control:

Enter M1 and M2 data under columns A and B respectively in mic control blank. Copy average value “M”.

Under Mac blank: click on first entry under Mac C values to reveal formula. Enter average mic C value you calculated earlier, 2. Value ,2 specifies the number of decimal digits retained. Enter mac M1/M2 data and calculate Mac C values.

Appendix1. The equation for converting absorbance values into DNA content (C values).

The equation:

Mt = B (La)(C)

Where B = aperture size.

Mt = amount of chromophore present in arbitrary units.

La = [1-Ta]

Where Ta = transmission of Low Wavelength light.

Where Q = (1-Tb)/(1-Ta)

And Tb = transmission of High Wavelength light.

ln = natural log.

Note: we need not worry about aperture size (B) if it remains constant. If we change aperture size, we need only concern ourselves with the square of the diameter. (R. Kowles, St. Mary’s College, personal communication). Hence for constant aperture use:

Mt = (La) (C)

Reference: Mendeslohn, M.L. (1958). J. Biophysics and Biochemical Cytology, 4: 407-414.