University of Kentucky College of Agriculture

The Vaillancourt Laboratory

Department of Plant Pathology

University of Kentucky


DAPI staining of conidia

(method of Dr. Mirabito)

Preparation of conidia

1. Collect spores from 2-4 week old plates of C. graminicola. Wash the spores three times in sterile distilled water.

2. Count the spores with the hemocytometer. Dilute to 1 X 104 spores per milliliter in sterile distilled water.

3. Do not leave the spores sitting in water for longer than an hour or two. Place 100 microliter drops of the spore suspension onto plastic cover slips, place in a moist chamber in the 30 degree incubator to allow spores to adhere and germinate for the desired time (most spores will be fully germinated within 4 hours).

Fixation Procedure

1. Do the fixation steps in the fume hood. Place cover slips with adhered spores in a glass Petri dish. Remove the water drop with a pipette. Pipette 400 microliters of para-formaldehyde fixative onto each coverslip to cover the adhered spores (see the recipe at the end of this protocol).

2. Leave the spores in the fixative for 30 minutes. Then remove the fixative solution with a pipette.

3. Wash the coverslips with the adhered spores by agitating them gently in 20 mls of TBS buffer in a glass Petri dish (recipe at the end of this protocol).

Staining Procedure

1. Stain the fixed spores by agitating the coverslips gently in 20 mls of DAPI staining solution in a glass Petri dish for 5 minutes (recipe at the end of the protocol).

2. Wash the coverslips by agitating them gently in 20 mls of TBS buffer. Repeat wash three times.

3. Wash the coverslips in 20 mls of sterile distilled water. Place the coverslips on glass slides for microscopy.

Microscopy

1. Turn on the fluorescence light source. Don't turn it off until it has been running at least 20 minutes. Record in the log book each time the light is switched on and off.

2. Remove the fixed analyzer slider D (see page 16 in the Axioskop operating manual, part # 6.12)

3. Slide the reflector slider 3 Fl into the first position (all the way to the left) to bring the filter into place (see page 18 of the Axioskop operating manual, part #7.5).

4. When microscopy session is complete, please return the microscope to the proper orientations for light microscopy.

 


Recipes

Para-formaldehyde Fixative Solution

6% para-formaldehyde in 100 mM PIPES buffer, pH 6.5

TBS Buffer

10 mM Tris, pH 7.5

200 mM NaCl

0.02% azide

DAPI Staining Solution

0.7 micrograms of DAPI per ml TBS buffer


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