364
Related Techniques
Inoculation 1. Add 0.25 ml virus suspended in culture medium or physiological saline.
2. Incubate the culture at 28°C for 1 h to allow adsorption of the virus.
Tilt the dishes every 15 min during this period to ensure that the cells
do not dry out and that the virus is in contact with the cells.
3. Discard the virus suspension.
Overlay 1. Add 3.0 ml overlay medium (see Comments).
2. Incubate the culture at 28°C for 5-7 days.
3. Discard the overlay medium.
4. Add 3.0 ml of 0.01 % Neutral Red (see Comments).
5. Incubate the culture at 28°C for 2 h.
6. Invert the dishes under a dissecting microscope, illuminate them using an oblique light, and count the number of plaques.
Comments
- Preparation of overlay medium
- Place 0.6 g methylcellulose, 0.1 M 2-(N-morpholino) ethansulfonic
acid and a magnetic stirring bar in a 100-ml screw-capped bottle
and autoclave
- After cooling, aseptically add 100 ml culture medium to the bottle,
constantly stirring with the magnetic stirring bar
- Because methylcellulose dissolves slowly, stir for approximately
30 min. Keep the mixture in the refrigerator at 5°C overnight, and
stir again the next day. Repeat this procedure over several days until the methylcellulose has dissolved completely
- Adjust the pH of the overlay to 6.15 by the addition of sterile 1 M
KOH (this usually requires approximately 5 ml)
Instead of using methylcellulose, some investigators have used 0.75%
agarose (Knudson, 1976) or 1.5% agarose (Brown and Faulkner, 1977;
Wood,1977).
- Preparation of Neutral Red Solution
- Prepare a stock solution by dissolving 100 mg Neutral Red in 100 ml
demineralized water; filter the solution and store it in the dark
- Just before use, dilute the stock solution 1 : 10 with physiological
saline
Troubleshooting
If difficulty is encountered during the assay, check whether the used cells
were at the log phase of growth.
Results
The plaques should have a mean diameter of 670 I-lm, and are pale pink
in color or unstained.
Related Techniques
Inoculation 1. Add 0.25 ml virus suspended in culture medium or physiological saline.
2. Incubate the culture at 28°C for 1 h to allow adsorption of the virus.
Tilt the dishes every 15 min during this period to ensure that the cells
do not dry out and that the virus is in contact with the cells.
3. Discard the virus suspension.
Overlay 1. Add 3.0 ml overlay medium (see Comments).
2. Incubate the culture at 28°C for 5-7 days.
3. Discard the overlay medium.
4. Add 3.0 ml of 0.01 % Neutral Red (see Comments).
5. Incubate the culture at 28°C for 2 h.
6. Invert the dishes under a dissecting microscope, illuminate them using an oblique light, and count the number of plaques.
Comments
- Preparation of overlay medium
- Place 0.6 g methylcellulose, 0.1 M 2-(N-morpholino) ethansulfonic
acid and a magnetic stirring bar in a 100-ml screw-capped bottle
and autoclave
- After cooling, aseptically add 100 ml culture medium to the bottle,
constantly stirring with the magnetic stirring bar
- Because methylcellulose dissolves slowly, stir for approximately
30 min. Keep the mixture in the refrigerator at 5°C overnight, and
stir again the next day. Repeat this procedure over several days until the methylcellulose has dissolved completely
- Adjust the pH of the overlay to 6.15 by the addition of sterile 1 M
KOH (this usually requires approximately 5 ml)
Instead of using methylcellulose, some investigators have used 0.75%
agarose (Knudson, 1976) or 1.5% agarose (Brown and Faulkner, 1977;
Wood,1977).
- Preparation of Neutral Red Solution
- Prepare a stock solution by dissolving 100 mg Neutral Red in 100 ml
demineralized water; filter the solution and store it in the dark
- Just before use, dilute the stock solution 1 : 10 with physiological
saline
Troubleshooting
If difficulty is encountered during the assay, check whether the used cells
were at the log phase of growth.
Results
The plaques should have a mean diameter of 670 I-lm, and are pale pink
in color or unstained.