Microscopes and how to use them

Duncan Griffiths

Duncan Griffiths Copyright © January, 2003, D, GRIFFITHS, ALL RIGHTS RESERVED


Across the world koi enthusiasts enjoy the koi hobby with a kind of love-hate relationship, folks adore their living jewels but most if not all live in fear of their pride and joy becoming diseased and ill. Upon realizing that something is amiss with their pets, some will have the resource to do an accurate diagnosis; some will take the sick fish to a dealer for an opinion. It is a fact for a larger percentage of koi hobbyists; they will begin the process of second guessing the problem without any real means of diagnosis, then apply shot gun medications, in an attempt to cure the problem and in many cases only make matters worse. This can be largely driven by fear of the unknown or maybe a perception by an individual that he / she, is not capable of microscopic diagnosis.


The aim of this article is to show that ALL hobbyists are capable of using a microscope and how easy it can be. What all the components do and how to adjust them, further, that microscopes need not cost the earth to buy, last but not least the microscopic disease of fishes is really quite fascinating. Although disease is not desirable and we could all happily live with our koi without it occurring, the disease aspect can be a part of the hobby that is just as enjoyable and interesting as the viewing of a nice koi in pleasant surroundings


Microscope types

The most essential piece of equipment the koi owner will ever own for the diagnosis of fish disease is the compound microscope. Even for those of you who have access to an available friendly expert for your diagnosis, the purchase of a microscope and learning how to use it and recognize disease; will be invaluable to obtaining an early head start on a disease in your koi. The earlier you can diagnose and treat a disease the much better the chance of recovery in a sick koi. Microscopes come in many forms and also many prices. They are as varied in quality and price as how big your budget is.


Prices start from around £20/25 for a child's microscope from a toyshop or child's educational center to highly specialized compound microscopes costing thousands of pounds, to, electron microscopes costing hundreds of thousands of pounds and every price you can think of in between.

I am going to assume no one reading this article has access to an electron microscope so we will not be discussing those< big smile>.
We *will* be discussing the basic light compound microscopes.


As mentioned microscopes are available for children in toy and hobby/craft shops, these are not quite suitable for our purpose as they do not have many of the features of the more professional microscopes that enable finite detailing of parasites, but I will say this, if resources are limited, they will work and you will be able to recognize some parasites all be it in a limited way, it’s a case of any port in storm and one of these is better than no microscope at all. Basic compound Microscopes start from around £60 brand new and prices rise at a steady rate, dependent on how deep your pocket is to around £500. Any microscope with in this price range would be more than capable of doing the job for which we need it. Not forgetting that all microscopes are available as pre-owned and as such substantial savings can be made buying one of these.

The types of microscopes available in this price range start at monocular type {single eyepiece} to binocular types, twin eyepiece {twin binocular type eyepieces}.

Monocular type

Binocular type

Also available within these price ranges are stereo-type microscopes, but these tend to be very low powered. You may just get away with this type for big parasites but you would not detect the smaller types of organisms of which there are many.

Stereo type microscope

We are looking for an instrument that has multiple magnifications from around 40X to 600X for most koi disease diagnosis, these can be obtained within the price range quoted above. Most koi parasites can be seen easily at 100X-150X magnifications

If you are prepared to spend a little more you can improve on the quality of the optics on the scope, like all instruments using lenses they improve and better results are obtained from better optic quality, but all optics these days are pretty good for the purpose they were intended for. We can also move onto microscopes that are Trinocular format These have a binocular eyepiece format with a third tube for attachments such as photographic equipment, a “very nice to have” but not essential.


      Trinocular                  Trinocular with CCTV           with SLR Camera 


Question, All these microscopes including the child's one all have something in common what is it?

"They are all useless if you do not know how they work or how to use them"!
Which leads me nicely to the next section




The Compound Light Microscope





Starting at the very top with the “Eyepiece”, these are optical viewing lenses that vary in magnification most common is 10X, but they are available in anything from around 5X to 20X. The more powerful 20X, unless they are of superb optical quality should be avoided, as loss of image quality will be evident. All eyepieces just merely slip into the tube and are easy to interchange

Eyepiece lenses are commonly available as wide field eyepieces and as the name implies this type of eyepiece would give you a wider field of view of the specimen. 


Continuing down the microscope the next component that is of importance to us, is the all-important objective lenses, usually referred to as just "Objectives" and the revolving turret that holds them stable and in place when viewing.

In normal circumstances four objectives are held in position in the turret, although this can be three or even five. Typical objective format is 4X,X10X, 40X or 60X and the last one is normally 100X combined with a 10X eyepiece will give magnifications of 40X, 100X, 400X or 600X and 1000X,

The way in which we calculate what the total magnification of the image viewed is, Factor the value of the eyepiece with the value of the objective, {by multiplying the two values}.  I.E. 10X Eyepiece value, multiplied by the 10X objective value  = 100X, so the image is being viewed at 100X {times} magnifications.
So to recap, multiply the Eyepiece value by the objective value to get the overall magnification.


Dependent on which objective you think will give you the necessary magnification, needed to view what you are expecting to see under the microscope, you simply rotate the turret and position the appropriate objective over the specimen. This process is helped by the fact the turret has a spring loaded indent that can be felt when the objective is perfectly aligned with the specimen and sub-stage light condenser for viewing. To prevent accidental movement, the turret semi-locks to prevent accidental rotational pressure,


The arrangement of the objectives in the turret is such that as you rotate the turret you will move from the lowest magnification and the smallest objective in size, to the next largest and so on, until the highest magnification and longest objective is reached. Baring in mind that as magnification increases so will the demand for illumination as less light enters the objective, so the light will need to be increased as the magnification increases. It is no accident that the system works this way, if you start viewing at the lowest magnification and therefore the smallest objective after you have first focused the objective, you now have a set distance established from the face of the objective lens to the slide. If you now decide to go to the next magnification as you rotate to the next size, this should swing round with a rough degree of focus without hitting the slide and causing damage.


As far as the 100X objective is concerned, when used in conjunction with the 10X eyepiece this will give a very powerful magnification/image of 1000X, at these extreme magnifications all kinds of image contamination occur from the atmosphere and light diffraction will degrade the image. All 100X objectives are called oil immersion lenses. With this process we add a small drop of thin oil to the surface of the slide and submerge the lens of the objective into the oil, thus eliminating the air from the space between the slide and the lens, in so doing we reduce contamination and light loss, this will result in a greatly improved image.

The oil immersion process is achieved, by focusing the 100X objective then backing the objective away from the slide. We then drop a small sample of oil on the area of the slide to be viewed, then very carefully bring the objective back down, while watching that it does not touch down on the slide, {by viewing from the side if needed} then apply fine focusing to get a sharp image.

An alternative way to do oil immersion is to, focus the sample at 400X/600X, then rotate to 1000X, in this way when the turret and objective is rotated to 1000X it should be in rough focus without striking the slide, but, as you begin to rotate the turret round to the X100 objective stop at the half way mark. Now while the cover slip is exposed drop some oil on the slide, then continue to rotate round till the X100 locks into position and fine focus.
Now was that hard?


Moving on down the microscope, we come to the stage, this is simply a stage to hold the slide and specimen stable for viewing. This can also be a moving stage that is capable of moving mechanically in the X. Y. axis, thus, if your instrument is equipped with a moving sub-stage you can systematically scan the slide leaving no area un-viewed. On entry-level scopes they may not have this facility, so you will have to scan the slide by manually moving the slide around between thumb and forefinger.


Moving down again, we come to the sub-stage condenser and iris and filter holder

The condenser as the name implies condenses some thing. That something is the light for viewing and illuminating the specimen. The condenser concentrates the available light and can be moved up and down so as to focus the light right onto the subject you are viewing, thus giving the best light concentration pin-pointed on the subject for a good well lit image.

The iris is used commonly for controlling the amount of light reaching the subject in the image, but its true purpose is for controlling the contrast of the image, the amount of light needed is determined by the bulb and it's controls, and the iris is cut down or opened to obtain the best contrast for image viewing.


Moving across the microscope, we come to the focus controls both fine and course adjustment, these can be combined in one control, more often than not they are two separate controls, again as the name implies one is for course focus the other for fine. Tip, start with the course close to the slide by viewing from the side as it approaches the slide so as to be sure the objective does not touch down, then course focus by working away from the slide, then fine focus. On thick samples you can rotate the fine focus backwards and forwards as you scan and view, to scan someway through deep samples.


Under the moving stage again we have the one of the most important components the light source.

At entry level, microscopes may have a mirror, which can be tilted to pick up light and deflect it into the condenser as we move up in sophistication; the light source tends to be electric and as you move up again, they will have a variable resistor to turn up or down the light.


Again if you want to get an entry-level microscope of the mirror type, they do work. Just position a halogen desk spotlight in front of the mirror and angle the mirror to deflect the light into the condenser.



Last but not least, the Foot or Platform. This may seem like a very unimportant part but I can assure you nothing could be further from the truth, if you need to view specimens at high magnification and want them stable a heavy and broad foot is needed, I realize that a lot of microscopes come with a horse shoe type arrangement, but by and large the more stable the instrument the better the image and a substantial foot goes along way towards that end.


We have now covered the main components of the microscope now for some of the less common components


Microscope head.

Some of the high end instruments come with inclined viewing tubes for a more relaxed viewing {see schematic picture} and certainly all the binocular/trinocular types have this arrangement, so the head is separate from the tube body. In the microscope head are prisms and/or mirrors to deflect the image from the vertical through to the angle of the viewing tube, in making the head separate this also enables the head to be rotated through 360O with out loss of the image. On entry-level models just above the foot is a joint that can also be used to incline the whole instrument at an angle backwards for the same reason.

Interocular Adjustment {Binocular only} 


Interocular adjustment is needed for binocular Microscopes so the eye pieces suit the interpupillary distance between the eyes, this is done by pushing and pulling the two eye pieces apart until the image looks like one complete round image, with no hint of double vision specimens on view. Once this is achieved, the reading on the optimum distance scale {usually above either the right or left eye piece} must be taken, then this figure is transferred to each eye piece tube {This is a knurled screw ring fitting located below the two eye pieces with a scale drawn on in it white, this achieved by rotating the eyepiece so that the reading around the circumference of each eye piece lines up with a line etched on the outer part of the base of each eye tube I.E. a reading of 60, each eyepiece should be lined up at 60.


               Interocular Adjustment                                 Dioptric Adjustment


Dioptric Adjustment {binocular only}

Further on binocular microscopes; there is also Dioptric adjustment, because with this type of instrument you will be using two eyes instead of one, it maybe the case that you wear spectacles, and your two eyes do not have the same vision capabilities as each other, this feature compensates for that problem Again, this is the knurled screw ring fitting located below the two eye pieces with a scale drawn on in white, this is rotated taking the eye pieces up or down for adjustment.


The way adjustment is achieved in the event one eye is impaired to the other. Shut one eye over one of the eye pieces and focus the image using the normal course and fine adjustment as mentioned above, {looking only through the one chosen eye, and not moving either eye piece from the previously selected interocular setting as described in the previous paragraph}.

When focus is achieved close the first eye and look with the other eye through the other eye piece but now focus using just the Dioptric adjuster ring by rotating the Dioptric ring till the image is in focus with that eye, {note this time do not use the course or fine adjustment for this part of the process} and that’s the job done, on the side of the Dioptric ring is the measurement scale and a marker on the tube. Make a note of all these settings and you can quickly dial in the correct settings for your eyes if somebody else has used the microscope and altered the settings.
If both eyes are the same optically then both numbers will be *roughly* the same on each eye piece, if the scales are different then you have an optical difference from right to left eye

Generally, you will not need to view specimens through your microscope now using spectacles if you wear them, as this finite eye adjustment on the instrument has now compensated for your eyesight.



So we have our microscope and some fish that need inspection for parasites, how do we go about this for the first time?


Whenever you are going to use your microscope it important to get everything prepared first and get your-self comfortable.This would include a suitable platform/table for placing your microscope upon close to a power supply and close to the fish but not to close to water that you risk an electric shock. If the weather is changeable be ready to have the facility to cover the microscope up in the event of rain.

Lay out a number of 3”x1” slides and cover slips, along with a clean cloth, not only to clean and dry your hands but to wipe moisture off the back of the newly prepared slide to prevent sticking of the slide to the sub-stage. Further to this you will of course need a koi and a vat/bath to apply a little anesthetic not to much, we are not looking to work on the fish just subdue the koi to point where we can obtain a sample without having to go and change afterwards, from splashing. {Note: some anesthetics expel parasites off the host notably, “2-phenoxyethenol”} so be mindful of this.


So how do we take and prepare a sample {know as a scrape and scope}

When the koi is subdued, obtain a mucus sample by holding a flat plastic spatula at a 45o angle to the body of the koi, then applying very slight pressure run from the front of the koi; just rearward of the gill plate, along the koi’s flank to the peduncle area {tail muscle) obtaining a small sample of mucus. {If you have never done this before have a friend help to hold the fish}   


Having obtained a mucus sample pick up a 3” glass slide then scrape the sample off the spatula onto the glass slide, by rubbing the spatula face onto the edge of the slide.

Now pick up a cover slip these can either be plastic or glass and are normally 22mm square,While holding the cover slip edge down in contact with the glass slide, using the cover slip, gather the mucus sample into one small tight sample in the center of the slide. Apply a very small drop of pond water to help with this. Then while holding the cover slip over the sample; again edge down, in contact with the glass at an angle of 45deg, allow the cover slip to slowly close down on the sample/slide while holding that first contact edge in place to the slide, in this way you will eliminate as many air bubbles from the slide as possible by pushing them out of the side.

Holding the slide and cover slip between the thumb and index finger apply a little pressure to squeeze the sample out, obtaining a thin sample to view.



Now the part its all been leading to!


Place the sample; cover slip upper most onto the sub-stage and secure into the sub-stage clamps, when satisfied all is secure its time to apply an objective to the sample.A good place to start is with one of the lowest magnification and work up as needed,For me, that would be 100X magnification or the 10X objective.

While observing the microscope from the side bring the objective towards the slide and stop just prior to touch down. On most microscopes there is a stop screw that prevents touch down on the slide, {a form of limiter} it would be a good idea to set this now. Turn on the light source; if a variable light source is used, remember to ensure the bulb is on the lowest setting. {A light bulb turned on at the highest of setting will soon shorten the life of the bulb}


Place your eye to the eye piece and adjust the amount of light for comfortable viewing and at the same time opening and closing the iris, then rotating the course adjuster back the objective away from the slide till an image appears in rough focus. At this point we can further adjust the lighting to suit better viewing, using the light adjuster and / or the iris, finally using the fine adjuster to obtain a sharp image.


Now we can begin to systematically inspect the slide, remembering to start at an edge moving up or down, it makes no difference which way you start out as long as when you reach the end of the slide, you traverse across the slide slightly then travel down or up the slide in the opposite direction. Repeat this until the whole slide has systematically been scanned.

If you discover nothing on the sample you can move to either another slide/sample or move up in magnification to the next natural selection. If you do find a parasite, and it is large enough for you to view and possibly be identified, we now fine tune the focus and play with the light a little more for ultimate viewing.


To start with we will not use only the fine focus tuning to obtain the sharpest image possible. But once this is set we can alter the light to obtain adequate light for the specimen and finally close the iris down to obtain good contrast for viewing the fine intricate detail of the specimen. At this point, the focus can be locked for comfort {only on some models} and if you are not familiar with all fish parasites, a book can be used to compare what you see with what's in the book. Most books these days have parasite photographs taken whilst the camera is looking through the microscope, so the example picture of the parasite is exactly what you are now seeing.

If you have a digital or 35mmSLR camera, with the right adaptors photographs can be taken now if you so wish.

I am hoping to cover photography in a later article.


So we have covered the basics, now you have no excuse to not have a go.I promise you that you will find this a fascinating part of the hobby that I hope you do not get to practice too often.

If you require further information or advice don’t hesitate to contact me further I can recommend a good contact man for microscope products and that ever so important advice
Alan Potter at Brunel microscopes is really helpful in microscope matters www.brunelmicroscopes.co.uk