3.3: Lab Procedures- Operating a Microscope

Brightfield Light Microscope General Operating Procedure:

The following step-by-step operating procedure should be carefully observed when first using the Nikon Alphaphot-2. Go through these steps now and again when carrying out the procedures for parts B, C, and D. This instrument is designed for fast-simplified use and correct operating technique should soon become automatic.

1.  Plug in and turn on the in-base illuminator.

2. Raise the nosepiece using the coarse adjustment knob. This provides greater access to the stage when the slide is in position.

3. Rotate the nosepiece so that the 10X objective is in operating position. You should feel a definite position stop for the objective. Turn the nosepiece clockwise and counter-clockwise until you are familiar with this position stop.

4. Open the iris diaphragm approximately half way.

5. Place the slide in the stage slide holder securely. Be sure the slide has the specimen side up. Roughly center the specimen over the light coming from the condenser.

6. Move the microscope condenser by means of the condenser rack and pinion knob until the top of the condenser is approximately the thickness of a piece of paper beneath the slide.

7.  Lower the objective using the coarse control knob until it reaches a stop. Do not force the knob. The stop should be obvious if you are moving the controls gently. Again, become familiar with the feel of the position stop.

8. View through the eyepieces and without disturbing the coarse adjustment setting, slowly rotate the fine adjustment knob in the appropriate direction until the specimen detail is in sharpest possible focus.

9. Adjust the interpupillary distance between the two eyepieces by sliding the eyepiece plates in or out.

10. If your eyes are very different the left eyepiece tube is adjustable to compensate for refraction differences of the eyes. The correct procedure is to bring the specimen into sharpest possible focus with a fine adjustment knob using the right eyepiece only, while closing your left eye. To focus for the left eye, view the specimen with the left eye only and turn the knurled collar until the specimen is in sharp focus. Do not adjust the fine adjustment knob during this procedure.

11. Once the specimen detail is in sharp focus using the 10X objective, it is then possible to rotate the nosepiece to other objectives without changing the position of the coarse adjustment knob. Very little refocusing with the fine adjustment is required since the Nikon Alphaphot-2 microscope objectives are parfocal. The term parfocal means that if an object is in focus with one objective lens, it will be in focus with all of the lenses.

Make certain that the portion of the specimen you wish to view is exactly centered in the field of the low-power objective. This is necessary because the microscopic field of view is smaller under high magnification than under low magnification.

Remember that the iris diaphragm setting must be changed whenever a different objective is used. As magnification increases the condenser iris diaphragm is opened as required. With these microscopes you are effectively increasing the light reaching the higher power objective. Adjustment of the condenser iris diaphragm results in proper contrast for viewing specimens under varying magnifications.

12. Try the highest magnification, the l00X-oil immersion objective. To focus with the oil immersion lens proceed in the following manner:

a)     Revolve the nosepiece so that there is no objective directly over the specimen. This allows enough space so that a drop of immersion oil may be placed on the specimen.

b)    Place one drop of immersion oil in the center of the circle of light formed on the specimen slide.

c)  Turn the nosepiece until the 100X objective snaps into place. The objective should be in the oil but must not touch the slide.

d)    Rotate the fine adjustment knob to obtain a sharp focus of the specimen. Remember to make the adjustments noted in step 10.

 

Problems:

Certain mechanical difficulties, real or apparent, may be encountered while operating your microscope.

A common problem is the failure of the fine adjustment to turn in the direction required for sharp focusing. This indicates that it has been screwed to the limits of its threads, either upward or downward, as the case may be. Screw it back to about one-half the thread distance, use the coarse adjustment to raise or lower the objective sufficiently to bring the specimen into view, and then refocus with the fine adjustment.

If the coarse adjustment fails to lower the objective sufficiently to bring the specimen into view, the fine adjustment has been screwed up too far and should be screwed down to about half its thread distance.

Any other problems? Burned-out light bulb? Focus knobs difficult to turn, grinding as they move, or moving by themselves? Specimen going in and out of focus as the stage is moved? Did you write the word "up" on your microscope slide? Is it still facing up? Do not attempt to fix these yourself. Get your instructor.

 

Proper Care of the Microscope:

The compound microscope used in microbiology is a precision instrument. Its mechanical parts, such as the calibrated mechanical stage and the adjustment knobs, are easily damaged and all lenses, particularly the oil immersion objective, are delicate and expensive. Thus, the instrument must be handled with care. The following rules, cautions, and maintenance should be observed:

1.  Use both hands when carrying the microscope; one firmly grasping the arm of the microscope, the other beneath the base. Avoid sudden jars.

2.   To keep the microscope and lens systems clean:

•   Never touch the lenses. If the lenses become dirty, wipe them gently with lens paper.

•   Never leave a slide on the microscope when it is not in use.

•   Always use lens paper to remove oil from the oil-immersion objective after its use. Do not wipe the lower power objectives with the same piece of lens paper used to clean the oil-immersion objective. If by accident oil should get on either of the lower power objectives, wipe it off immediately with clean lens paper.

•   Keep the stage of the microscope clean and dry.

3.  To avoid breaking the microscope:

•  Never force the adjustments. All adjustments should work freely and easily.

•  Never allow an objective lens to jam into or even to touch the slide or cover slip.

•  Never focus downward with the coarse adjustment while you are looking through the microscope. Always incline your head to the side with eyes parallel to the slide, so that downward movement can be arrested before the objective touches the slide.

•  Never exchange the objectives or eyepieces of different microscopes and never under any circumstance remove the front lenses from the objectives.

•  Never attempt to carry two microscopes at one time.

4.         Storage of the microscope:

• Make sure the immersion oil has been removed from the lens.
• Put the low power (10X) objective into position and turn the stage all the way down.
• Be sure the slide holder does not extend beyond the left edge of the microscope.
• Wrap the electrical cord around the cord hanger.
• Your TA will place the microscope in the cabinet.