Introduction and Basic Technique

INTRODUCTION AND BASIC TECHNIQUE

Proper use of the microscope is integral to the practice of microbiology. Bacteria, which can be as small as less than a micron in size, are at the limits of resolution in most light microscopes. Hence it is essential that the microscope is correctly set up to produce maximum resolution and ease of use. The microscopes in the Hetzel laboratory are modern compound binocular microscopes which require little adjustment for optimal use but do need some attention to basic technique.

PARTS OF THE MICROSCOPE

BASE

  • On/off switch
  • Light Intensity Adjustment
  • Light window

BACK

  • Fine and coarse focus adjustment (these move the stage up or down)
  • Condenser movement knob
  • Stage stop lever

STAGE

Slide holder and adjustment knobs

CONDENSER

  • Iris diaphragm lever
  • Filter and filter holder

NOSE PIECE

Objective lenses

  • 4x
  • 10x-low power
  • 40x-high power
  • 100x (oil immersion) – black ring


PRISM HOUSING

Oculars

  • one fixed focus, one adjustable focus
  • Adjustable eye width scale
  • Revolving head lock

NOTE: the filter and oculars are not fixed in the microscope and may fall out if the microscope is tilted.


SETTING UP

  1. Place the microscope on the bench in a position where you can comfortably sit and look down the oculars.
  2. Turn the nosepiece such that the 10x lens is directly over the stage.
  3. Wind the stage up (coarse focus knob) until it is about 1 cm below the bottom of the 10x lens.
  4. Wind the condenser right up until it is just under the stage.
  5. Open the iris diaphragm about one quarter.
  6. Turn on the power.
  7. Adjust light level to about 8.
  8. Place a suitable slide on the stage and position the stained area over the light window.
  9. Focus with coarse adjustment – from the starting position, you should only need to wind the coarse focus down, away from the 10x lens. If the slide does not come into focus, look at the stage, wind it back up again and start again.
  10. When in focus using the coarse adjustment, adjust the fine focus. This should only require a half turn either way. If the slide needs more adjustment than this, redo the coarse focus first.
  11. If 100x oil immersion is required, swing the objectives out of the way, add a drop of immersion oil to the slide and swing the 100x lens into the oil.
  12. Adjust the fine focus only (if the slide is already focused, only a slight adjustment should be necessary).
  13. Adjust the oculars to your eye width and focus.
  • Pull the oculars apart to their widest separation.
  • Place your eyes just above but not resting on the lenses.
  • Slowly push the oculars together until a single image is observed.
  • Note the reading on the scale.
  • Place a tissue over the ocular with the adjustable focus. Look down the other lens and adjust the fine focus until sharp. Swap the tissue to the other lens. Adjust the focus on the adjustable ocular until a sharp image is seen.
  1. Observe your specimen!
  2. Pack the microscope away at the end of the session:
  • Ensure the slide has been removed and placed into the sharps bin.
  • Move objective lens to the 10X magnification.
  • Wipe oil off all objectives (using 95% ethanol if necessary) and the stage.
  • Turn light source to 1 and switch off power.
  • Wrap cord around microscope ensuring the cord is not bent backwards.
  • Replace the cover.

TROUBLE SHOOTING

Problem Cause Remedy
Cannot fine focus, coarse focus OK Slide upside down Turn slide over
Image refractile Iris diaphragm closed Open iris half way
Image blurry – cannot adjust coarse focus correctly

Slide dirty or Objective lens dirty (oil)

 Clean slide or lens with tissue. If problem persists use lens cleaning solution.
No image or partial image (light on) Objective lens not in place Click lens into position

 

Last modified: Monday, 27 November 2017, 12:36 PM