Using Your Microscope
buying a microscope
Written by Frank Prince-Iles
FishDoc
Part 1

An essential piece of equipment

Without a microscope it is simply impossible to tell the difference between a water quality and a parasite problem. The microscope should be considered the most basic of tools in fish disease diagnosis - indeed, an accurate, full diagnosis of the disease and its cause just isn't possible without a microscope!!

A modern monocular microscope

Why is it so important?

Unusual behavior such as heavy breathing, rubbing, flashing, lethargy is often taken as a sign of parasite disease - yet the same behavour can be due to water quality problems, internal organ disease or many other causes. Without a microscopic examination of both the skin and gills it is simply impossible to tell the difference and any treatment undertaken is based on nothing more than guesswork! While simply taking a guess as to the cause and treatment required may work it is just as likely to make matters worse. So microscopy is a vital and basic step in diagnosing and treating fish disease.

Buying a microscope

They come in a wide range of types and prices, costing less than a hundred to several thousand pounds - although fish keepers generally do not require an expensive model. There is also a market for good second-hand 'scopes.

It really depends on what you want to use it for. Most of the common parasites such as flukes, white-spot and Trichodina can be easily identified with the cheaper models, but a better quality model is required for critical examinations of cell structures and some small parasites. Generally speaking as prices increase you are paying for better engineering, illumination and optics

Models for fish keepers

There are two basic styles of microscope available. The cheaper monocular model has a single viewing tube (as above) - which is fine for occasional use. The binocular models enable you to view with both eyes, giving a better field of view. If you want to take photos or video - then you will really need a trinocular model with a dedicated phototube.

Two other considerations which can make a considerable difference are illumination and the stage.

Illumination

The better the lighting - the clearer the image. Most of the cheap models have an understage mirror which reflects light to illuminate the slide. This can pose problems in dull conditions or lead to contortions with table lamps to try and improve the illumination of the specimen being studied. By far the best option is a fixed or plug-in understage light system, which gives a consistent amount of light. A basic plug-in system can be purchased for under £50 and, in my opinion, is money well spent. More expensive microscopes have built-in halogen lamps with brightness control

Stage

The stage is the part of the microscope where the slides are placed for viewing. As you will appreciate, only a small part of the slide can be seen at any time and the slide needs to be moved to see other parts. Incidentally, the view that can be seen at any time is called the field of view, which reduces at higher magnifications.

On the cheaper models the slide is held by clips and has to be moved manually - but this cannot be done smoothly and it is virtually impossible to return to a given position on the slide.

All but the most basic of models have a mechanical stage fitted with vernier screws that allows you to move the slide smoothly and examine it in a methodical pattern. Again, this usually adds less than £50 to the basic cost and is invaluable when scanning the slide for parasites.

There are different qualities of mechanical stage- and some of the more robust stages can be quite expensive.

Specimen needs to be as thin as possible

Before any specimen can be examined under the microscope, a slide has to be prepared. No matter how good your microscope is, the final image can only be as good as the slide you are viewing, so proper preparation is important.

A normal compound microscope works by passing light up through the viewed specimen, so it is important that the sample or specimen is a thin as possible. This means working with relatively small amounts of algae, mucus, sediment or whatever. This is particularly important when viewing mucus sample as some of the smaller, transparent parasites might not be seen if the preparation is too thick.

Slide and cover glass

Ideally a new slide and cover glass should be used for each preparation, but in practice slides and slips will be re-used many times. In which case it is important that they are clean and free of smears ( cleaning with alcohol will help remove smears - propan-2-ol from a chemist or drug store).

1. The specimen is placed, together with one drop of pond water onto the centre of a clean slide. Do not use tap water or distilled water as these may kill any parasites present. If the specimen is thick, use a seeker needle to gently spread it as thin as possible.
2. A glass or plastic cover slip is then lowered gently on top making sure that no air bubbles are trapped. The best way to place a cover slip is to hold two opposite edges between first finger and thumb. Holding the slip at a 45o angle, place the bottom edge on the slide just to one side of the specimen and then slowly lower the cover slip until it is flat. You can use a seeker needle to help lower the slide by placing it under the cover glass and slowly lowering it into position
3. Once the cover slip is in position apply a small amount of pressure with a seeker needle to spread the sample under the slip and squeeze out any air. Don't apply too much pressure; just enough to spread the sample. Do not use your finger as a finger print may contaminate the cover glass.
4. Practice makes perfect - so try making slides of all sorts of things; blanket weed, algae, mulm etc.

A sequence of steps to take

There is a clear sequence of steps to take to achieve perfect viewing. These are detailed in the box below. A word of warning: Take care when adjusting the focus knobs that you do not advance the objective lens onto the slide! It is very easy to break the slide and possibly damage the objective.

When setting up the focus it is best to view from the side and lower the objective so that it is nearly, but not, touching the slide. Now adjustments can be made while viewing through the eye-piece and slowly winding the objective UP and AWAY from the slide - this way you avoid any potential damage to either the slide or microscope.

Getting things in focus

Once the specimen is in focus, fine adjustments in illumination and iris aperture can be made to improve viewing.

The slide should be scanned systematically, usually by finding the top corner of the cover glass and then moving the slide slowly across the stage to the adjacent corner. When the opposite side is reached the slide is moved up until a new field of view is visible and then moved slowly across to the other side. This is repeated until the bottom of the slide is reached.

Higher magnification are obtained by rotating the nosepiece turret and selecting another objective and then re-focusing.

Parasites are often transparent

Since many parasites are transparent to light it is often necessary to use various techniques to highlight them. The two most popular methods are phase contrast and darkfield. Both of these methods are outside the scope of these pages, but essentially they manipulate the light so that transparent objects are more readily visible. These specialist methods usually mean adding special condensers of objectives to your microscope. While these methods are useful they are not essential for fish disease diagnosis.

If there is a problem with viewing any specimens with an ordinary brightfield microscope it is possible to increase the contrast by racking down the condenser or closing up the iris aperture, although it does reduce resolution.

Read further in Part 2