Adjusting the Microscope
Written by Frank Prince-Iles
Scheme for setting up a simple
- make sure the 10x eyepiece is in place at the top of the draw tube
- raise the body tube a few inches above the stage - by looking from
the side and turning the course focus knob
- rotate the nosepiece and click the lowest power objective into place
above the stage (usually a 10x)
- adjust the illumination if using a mirror, turning the flat side of
the mirror towards the light source so that light is reflected up
towards the condenser
- rack the condenser up to within 2mm below the stage and adjust the
iris diaphragm until it is half open
- place the specimen on the stage making sure that the cover glass is
uppermost and secure it with either the stage clips or the mechanical
- adjust the angle of the mirror so that a spot of light appears on
the slide directly below the objective lens
- looking from the side and using the course control knob, lower the
objective until it is just above the slide
- look through the eyepiece. Adjust the mirror to give an even amount
- use the course control knob to slowly rack the objective upwards and
look through the eyepiece until the specimen is in focus. (Tip) it is
sometimes easier to focus on the edge of the cover slip to start with
as this gives a nice clean edge when in focus - whereas mucus can
sometimes be difficult "to find"
- use the fine focus to obtain the sharpest possible image
- if the light is too bright either use a bulb with a lower wattage
(if using a table lamp to illuminate the mirror) or adjust the iris
diaphragm to reduce glare
- focus the light source onto the slide by slowly racking down the
condenser - watch that this does not affect the mirror angle. Adjust
the condenser and iris diaphragm to give optimum illumination.
Ideally, once the condenser is set in the optimum position, there
shouldn't be any need to keep altering it.
While this long list may seem daunting, it is because I have tried to
cover every step. You will also note that much of it revolves around
optimizing the light source if it is mirror based. With a fixed light
source many of these steps can be ignored. After you have set up the
microscope a few times it should become second nature.
For general microscope work, a magnification between 40X and 400X is
usually sufficient and will allow for relatively easy identification of
most common ectoparasites.
When talking about higher magnification it is important to understand
the difference between simply magnifying the size and increasing the
amount of visible detail. With high-power microscopy the most important
consideration is the image resolution, which means being able to
distinguish two objects that are close together as being separate,
For technical reasons there is a limit to the amount of resolution that
can be obtained with an ordinary light microscope. Usually, beyond 1000X
magnification there is no significant increase in resolution, and any
extra magnification merely gives an increase in size without any extra
As you might guess, increasing resolution comes at a price - and means
investing in more expensive components such as oil-immersion objectives
and condensers. For example, a fairly good oil-immersion objective lens
can cost over £300! It is possible to increase resolution beyond 1000x,
but the type of equipment required is prohibitively expensive.
The reasons for using oil-immersion objectives are technical - but
basically the oil reduces light being reflected back from the slide as
well as bending the light rays inwards to focus on the specimen - giving
more light, less interference and therefore better detail
Using an oil-immersion objective
Focus on the specimen using a dry objective lens (40x), making sure
that the specimen is central in the field of view.
Move the dry objective to one side and place a drop of oil on top of
the specimen. Swing the oil-immersion lens into place, ensuring that it's
front lens makes contact with the oil drop. If it doesn't, slowly lower
the objective while watching from the side until it just touches the drop
If you are using modern parfocal lens, which means that all objectives
are approximately in focus at the same setting, the specimen should
already be roughly in position. Look down the microscope and make any
necessary fine adjustments to the focus, iris diaphragm and condenser to
obtain the best image. Be very careful not to lower the objective onto the
slide as the working distance with high magnification lens is very narrow.
After use it is important to clean the oil from the lens using a soft
tissue. It should be cleaned using a wiping movement, rather that a
circular, scrubbing movement as this might scratch the lens. Any dried on
oil can be removed using xylene, but be careful as this could dissolve the
cement securing the lens!
So what can you see at 1000x mag?
You will see the internal structures of algae and parasites - if you
can get them to stay still! However, at this magnification we would
normally be looking at bacteria and other micro-organisms. In honesty, you
may be initially disappointed with what you actually see. Most
micro-organisms are transparent and even at this magnification are
difficult to see with an ordinary bright-field microscope. With special
stains they are visible, but still fairly small and insignificant (see
Gram stained bacteria
as viewed with a light microscope
The same bacteria
viewed using a scanning microscope
However, as understanding develops it is possible to determine
significant information and interest from high-power microscopy. Special
staining techniques such as Gram and acid-fast staining enable us to
determine the basic group of bacteria - for example most bacterial fish
pathogens are Gram-negative and stain red with Gram staining.
It is also possible to determine approximate size and thereby
differentiate between long Flexibacter. sp and the smaller ulcer-related
bacteria such as Aeromonas sp. The shape of the bacterium can also be seen
as either straight rods, bent rods or round (cocci).
While conclusive identification can only be determined by culturing the
bugs and subjecting them to biochemical testing, high powered microscopy
enables us to go part way to discovery this normally hidden, fascinating
The most common cause of disappointing results is poor setting up of
the microscope before use. The performance of almost all microscopes can
be improved if a little time is spent focusing before use. No matter what
quality microscope you buy, it makes sense to get the best possible image
it is capable of.
An often neglected part of the microscope is the substage condenser,
found on all but the most basic of instruments. The condenser focuses and
concentrates the light uniformly onto the specimen. Most importantly,
because it controls the size of the cone of light illuminating the stage.
It also controls resolution - i.e. how sharp or fuzzy the image is.
Ideal illumination is obtained by critical focusing which ensures that
the specimen and light source are properly centred and focused, with just
the right amount of light to give a clear, uniformly bright image.
Setting up for critical focusing
Put a prepared slide on the stage and bring it into focus with the 10x
or 20x objective
Next focus the condenser. How this is done depends on whether light
source has a field diaphragm such as found with Köhler-type illumination.
Köhler lighting systems have an iris or field diaphragm which controls
the aperture of light going into the condenser. Although this form of
illumination is gaining it popularity, it is would not normally be found
on the average hobbyist microscope.
Focusing the condenser on a microscope without an iris diaphragm is
carried out by removing the slide from the stage and placing a piece of
thin card half-way across the light source aperture. Adjust the condenser
- not the stage or objective - by racking it up or down until the card is
in sharp focus when viewed through the eyepiece lens. This usually occurs
just as the light interference halo turns from blue to red.
At this point the condenser is properly focused and should not need to
be adjusted again. With Köhler lighting systems focusing is carried out
in exactly the same way - only focusing on the leaves of the diaphragm
rather than a piece of card.
Centering the condenser
Next, it is important that the condenser is centred or it
will focus the light at some point to the side of the specimen. To centre
the condenser close down the iris diaphragm and remove the eyepiece.
Looking down the viewing tube you will see a small point of light (see
diagrams). Make sure that the light appears in the centre of the tube by
adjusting the condenser positioning screws. This takes a bit of fiddling
about - but is simple once you are used to it.
|with the eyepiece removed, look down
the viewing tube when you will see something like the diagram on
the left - an off-centre light spot. Adjust the condenser
centering screws until the light is in the middle
The iris diaphragm
The final step is to adjust the substage or iris diaphragm. This is
done with a lever or screw found on the condenser which works in a similar
fashion to that of the iris of a camera. As the iris is opened it allows
more light into the condenser. The iris diaphragm is often used
incorrectly to control light intensity. While this might seem logical, its
proper use is to control the size of the cone of light entering the
objective lens. The correct iris diaphragm setting varies with each
objective. Consequently it needs to be re-adjusted every time you change
To set up the iris diaphragm look down the viewing tube without the
eyepiece in place and slowly open up the iris diaphragm until the circle
of light just about fills the viewing tube. Finally replace the eyepiece,
re-focus and adjust the light intensity if your microscope has rheostat
|Once the light spot is central, open
up the condenser iris until the light just fills the field of
view. Replace the eyepiece
Provided that your lens and slide are clean you should now
get the best possible image.
Read further in Part 3