Microscopy

A simple microscope the first device used to examine microbes.  This simple, single lens microscope was invented around 1600 by Zacharias Janssen, but was not immediately used to examine microorganisms.  Antoni van Leeuwenhoek was one who discovered this vast diverse world  in 1676 as he examined his “animalcules”  for the first time. He is most noted for these studies because he submitted his findings among the first recorded descriptions, thus paving the way for true scientific study of the tiny life forms.

The most commonly used microscope used today is the compound light microscope.  It uses two lenses and a light source to illuminate the specimen.  The ocular and an objective lens are used together to create a greater total magnification.

The resolving power of a microscope is just as important as its magnification abilities.  Resolution is the minimum distance between two points allowing them to be observed separately. The maximum resolving power of a light microscope is 0.2 micrometers.  Beyond this, the image is no longer clear as detail merges and becomes indistinguishable to the viewer’s eye.  Effective magnification for a light microscope cannot be much higher than 1000x.   This is the magnification produced with the use of the oil immersion lens of the microscope used in this lab.

The resolving power is limited by the wavelength of the light used to illuminate the specimen.  Some microscopes use wavelengths shorter than visible light resulting in the ability to see much greater detail than the common light microscope.  These include the electron microscope.  The cost of this equipment is great, and the preparation of the specimen can be complex.  This is not equipment commonly seen in undergraduate microbiology courses.

One disadvantage of the light microscope, which allows light to pass directly through the specimen is the limited ability to view living organisms.  The stains used to clarify transparent structures of a specimen kill it preventing the observation of the characteristics of the living organism.   The dark-field condenser directs light to the specimen laterally.  The beam is reflected to the viewer’s eye.  The object appears bright against a dark background.   The phase-contrast microscope uses a lens and condenser system which takes advantage of the differing refractive indices of cell parts.  ( The different speed in which light passes through or is reflected by the different cell parts.) These will be used to view protozoa early in the  lab course.

The last type of scope that will be employed in this course is the dissecting scope.  This equipment is useful to examine objects that can be seen with the naked eye, but it allows the viewer to see it in greater detail.  We will use this to examine some of the properties of the fungus, mold, and colonies of bacteria.
 

INTRODUCTION TO YOUR MICROSCOPE

  1.  ocular lens
  2.  dial for ocular focus adjustment
  3.  body
  4.  nosepiece
  5.  objective lenses
 scanning objective
 low power objective
 high power objective
 oil immersion lens
  6.  stage platform
  7.  stage clips/lever
  8.  condenser
  9.  iris diaphragm lever
10.  mechanical stage adjustment knobs
11.  light source
12.  light switch
13.  base
14.  coarse adjustment knob
15.  fine adjustment knob  (fine focus)

Scope Use and Care

1. Plug the microscope into the outlet on the opposite side of your work bench.  Make sure the cord is not in your work area but remains behind the scope.  Also,  check that the cord is not in an area that the scope is at risk of being pulled from the lab bench. 
   
2. Pick up the scope and gently put it down in front of you.  Never scoot it because the vibration caused by the nonskid bottom of the scope on the lab bench can damage the scope. 
   
3. Place a prepared slide on the stage platform.  The stage platform has a mechanically operated slide holder.  A slide must be place flush with the right angle made by the holder on the right, then held in place by releasing the stage clip.  The slide is held in place by tension.  If you force the slide under the clip, it will spring the mechanism, and it will not longer hold the slide in place. Let your instructor help you with this on your first try.
   
4. Check the light switch.  Make sure it is set around number 7.   It should rarely be set to a greater number than 7 because this sends too much power to the bulb. 
   
5. The stage platform has an opening for the transmission of light.  The size of this opening can be adjusted by a lever to the iris diaphragm as needed to increase or decrease light passing through the specimen on the slide centered over the opening.  Make sure the iris diaphragm is open allowing the light to pass to your slide. 
   
6. Below the stage is a condenser which further controls the light.  The best position for the condenser is usually just barely below the top surface of the stage platform.  The best way to check this is to raise it gently all the way up, then bring it back down about 1-2mm.  This is one of the most frequent problems students have when they are unable to focus their scopes.  Make sure the condenser is in the appropriate place, then there is no reason to move it during the lab exercises.
   
7. The nosepiece bears four objective lenses.  The shortest is the scanning objective, and it has the lowest magnification power, magnifying 4X.  The ocular lens magnifies 10 times.  Total magnification is the product of the ocular and objective lens.  This means an object in magnified 40X when examined using the scanning objective.  Turn the scanning objective in place over the light beam.  You will always begin with this objective. 
   
8. Center the slide using the mechanical stage adjustment knobs. 
   
9. Bring the scanning objective as close to the slide as possible by turning the coarse adjustment knob.  (Do not force it, and watch carefully from the side, not through the oculars!) 
   
10.  Now, while observing through the ocular for the first time, slightly turn the coarse-adjustment knob.  You are moving the objective away from the slide.  This pattern is essential to avoid breaking the slide and/or the objective.  Stop when the object becomes clear in the field of view.  If you see two circles of view, you will want to stop here and adjust the ocular lenses according to the directions in the oculars section. 
    
11. If you cannot see the object, try moving the slide slightly with the stage adjustment knobs.  Start back at step 9. 
    
12. The image is focused by using the coarse-adjustment knob first, and then the adjacent fine adjustment knob(fine focus).  You should not have to turn the fine focus more than one full turn to bring the object into sharp focus 
    
13. If you want to magnify the object further, turn the objective lenses so that the low-power objective is directly over the specimen.  The object observed using the scanning objective lens should still be in the center of the field of view.  This property is known as parcentral or parcentral.  Also, you should only use the fine focus in order to see the object.  At this point, do not use the coarse adjustment knob.  It should not take much more than a full turn to refocus the object at the greater magnification.  This property of your microscope is called parfocal. 
    
14. This process should be repeated for the high power objective lens.   Refer to instructions on the use of the immersion oil to use the longest objective lens (oil immersion lens)

The ocular lenses magnify 10X.  They can be moved in relationship to each other so that a student may adjust them to his individual face width.  The width (distance apart) should be adjusted so that one lighted circle (field of view) is seen simultaneously with both left and right eyes. When looking through the oculars, the eyes should not brush the lenses; rather, there should be ½ to 1 inch between the eyes and the lenses.  (See board demonstration by instructor.)
 
If you wear glasses, it is possible to adjust the ocular lenses to accommodate the difference of vision between the right and left eyes.  This will allow viewing without the use of eyeglasses.

The left ocular is adjustable in focus with respect to the right ocular.  Therefore, when focusing on a specimen for the first time each lab session.

    1.  Use only the right ocular while focusing with the coarse-adjustment, then use the fine focus to
         make the object clear.
    2.  Using the left ocular alone, turn the numbered dial at the base of the left ocular until the image           is clear.
    3.  Look through both oculars.

Use of Immersion Oil

The oil immersion lens is the objective lens of greatest magnifying power, 100X, giving a total magnification of 1000 X.  To improve resolution and use of light, an oil of the same refractive index as the glass slide and the objective lens is placed between the two.  If the light beam passes through glass-air-glass, some rays are bent (refracted) and lost.  Passing through glass-oil-glass, none are lost.

Follow steps listed in microscope use to focus clearly using the 40x (high-powered) objective.  This is also known as the high-dry objective.

Turn the nosepiece so that there is room between the high-dry and the oil-immersion lens to place a drop of oil directly on the slide over the light beam.

After the drop of oil is in place, carefully watch to see if the objective will clear the slide safely as you drag the oil-immersion lens through the drop of oil and click it into place.

Increase the light using the iris diaphragm.  Only increase the light using the light source adjustment after opening the iris diaphragm.

Look through and adjust the fine-focus and light slightly.

If the image does not appear, do NOT go back to the high-dry lens.  You may swing the nosepiece around to use the scanning objective (4X) and then 10X.  You cannot use the high-dry when oil is on the slide.  Instead swing the nosepiece back past the scanning objective to return to the oil.  This bypasses the high-dry.  Ask the instructor to demonstrate this technique if you are struggling with use of the oil-immersion lens

If observing several slides with oil in sequence, the oil-immersion lens does not need to be cleaned between each slide.  However, thorough cleaning of the lens with lens paper is necessary before returning the microscope to the cabinet for storage since oil tends to dry and harden over time.  Also, through away the lens paper immediately after use.  You do not want to accidentally use it to clean other lenses.