Place the culture on the microscope's stage, turn on the power, select the appropriate lens and focus on the specimen. Adjust the condenser and phase contrast ring to the center if necessary.

Operation method

Program 16.1 Use of the Inverted Microscope

Principle

Place the culture on the microscope's stage, turn on the power, select the appropriate lens and focus on the specimen. Adjust the condenser and phase contrast ring to the center if necessary.

Materials and Instruments

Inverted Microscope Mirror Paper

Move

1. Ensure that the microscope (equipped with a 10×, 20× or 40× objective lens with a condenser and a suitable phase contrast ring) is clean (wipe the stage with 70 % ethanol. If necessary, clean the objective lenses with microscope paper).

2. Turn on the power and adjust the light intensity to the proper intensity starting from the lowest and not directly to the bright light.

3. Check the connection between the light source and the spotter:

(a) Place a stained section, culture bottle, or petri dish on the carrier table;

(b) Close the field-of-view aperture;

(c) Focus on the variable diaphragm;

(d) Adjust the variable diaphragm imaging to the center of the field of view.

4. Adjust the phase contrast ring to the center:

(a) If the microscope is equipped with a phase contrast telescope, insert it into the standard eyepiece position and focus on the phase contrast ring, checking to see that the concentrator ring (white ring on a black background) is coaxial with the objective ring (black ring on a white background);

(b) If a phase contrast telescope is not available, replace the stained specimen with an unstained culture (live or fixed culture), refocus, and adjust the phase contrast ring until optimal contrast is obtained.

5. Observe the cells. 10× phase contrast objective is sufficient for routine observation, 4× phase contrast objective does not provide enough detailed information, and magnification higher than 10× limits the area of observation.

(a) Note the growth phase (e.g., sparse, subconfluent, confluent, dense);

(b) Note the cellular state (e.g., clear, transparent or granular, vacuolated, etc.).

6. examine the culture medium for clarity (e.g., free of debris, floating particles, signs of contamination, etc.), selecting an objective lens with higher magnification as needed.

7. Record the results of the observation.

8. Turn down the rheostat (dim the lights) and turn off the power.

9. Return the culture solution to the incubator or place it on an ultra-clean bench for aseptic operation.

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