Use of a microscope in laboratory

The microscope is an essential instrument for the diagnosis of disease. It is a preci- sion instrument and requires careful maintenance to prevent damage to the me- chanical and ocular parts and also to stop fungi from obscuring the lenses. 

1. Components of a microscope 

The various components of the microscope can be classified into four systems: 

— the support system 

— the magnification system 

— the illumination system 

— the adjustment system. 

Support system (Fig. 3.1) 

This consists of: 

— the foot (1) 

— the limb (2) 

— the revolving nosepiece (objective changer) (3) 

— the stage (4) 

— the mechanical stage (5), which gives a slow con- trolled movement to the object slide. 

Magnification system (Fig. 3.2) 

This consists of a system of lenses. The lenses of the microscope are mounted in two groups, one at each end of the long tube — the body tube. 

● The first group of lenses is at the bottom of the tube, just above the preparation under examination (the object), and is called the objective. 

● The second group of lenses is at the top of the tube and is called the eyepiece. 

Objectives

Magnification

The magnifying power of each objective is shown by a figure engraved on the sleeve of the lens (Fig. 3.3):

— the x 10 objective magnifies 10 times;

— the x 40 objective magnifies 40 times;

— the x 100 objective magnifies 100 times.

Fig. 3.1 Components of the support system of a microscope
1: foot; 2: limb; 3: revolving nosepiece; 4: stage;
5: mechanical stage.

Fig. 3.2 Components of the magnification system of a microscope

Fig. 3.3 Objective lenses

Fig. 3.4 Numerical aperture

(The x 100 objective is usually marked with a red ring to show that it must be used with immersion oil.) 

Some microscopes are fitted with a x 3 or x 5 objective instead of a x 10 objective. 

Numerical aperture 

The numerical aperture is also engraved on the sleeve, next to the magnification (Fig. 3.4), for example:

— 0.30 on the x 10 objective 

— 0.65 on the x 40 objective 

— 1.30 on the x 100 objective. 

The greater the numerical aperture, the greater the resolving power (see below). Moreover, the greater the numerical aperture, the smaller the front lens mounted at the base of the objective. The front lens of the x 100 objective is the size of a pinhead, so handle it with care. 

Other figures that may be marked on the sleeve 

The sleeve may also display: 

— the recommended length in millimetres of the tube (between the objective and the eyepiece) — usually 160 mm; 

— the recommended thickness in millimetres of the coverslip used to cover the object slide — e.g. 0.16 mm. 

The screw threads of all objectives are standard, so the objectives in the revolving nosepiece are interchangeable. 

Working distance 

The working distance of an objective is the distance between the front lens of the objective and the object slide when the image is in focus. The greater the magnify- ing power of the objective, the smaller the working distance (Fig. 3.5): 

— x 10 objective: the working distance is 5–6 mm 

— x 40 objective: the working distance is 0.5–1.5 mm 

— x 100 objective: the working distance is 0.15–0.20 mm.

Fig. 3.5 Working distance of an objective

Resolving power 

The resolving power of an objective is its ability to reveal closely adjacent details as separate and distinct. The greater the resolving power of the objective, the clearer the image. 

The maximum resolving power of a good medical laboratory microscope is about 0.25 mm (the resolving power of the normal human eye is about 0.25 mm). 

Immersion oil increases the resolving power by conserving many light rays that would be lost by refraction if a dry objective were used.

Fig. 3.6 An eyepiece

Eyepiece

Magnification 

The magnifying power of the eyepiece is marked on it (Fig. 3.6): 

— a x 5 eyepiece magnifies the image produced by the objective five times; 

— a x 10 eyepiece magnifies the image 10 times. 

If the object is magnified 40 times by the x 40 objective, then by five times by the x 5 eyepiece, the total magnification is: 5 x 40 = 200. To calculate the total magnification of the object observed, multiply the magnifying power of the objective by that of the eyepiece. Microscopes used in medical laboratories have a magnifying power of between x 50 and x 1000. Certain eyepieces have a calibrated graticule. These eyepieces are used to measure the size of an object under the microscope (e.g. protozoan cysts).

Binocular microscopes 

Binocular microscopes (two eyepieces but using only one objective at a time) are generally recommended. They are less tiring for the eyes than monocular micro- scopes when long examinations have to be made. Electric illumination is, however, essential for using the x 100 objective. 

Illumination system 

Light source 

An electric light source is preferable, since it is easy to adjust. It is provided either by a lamp built into the microscope beneath the stage, or by an external lamp placed in front of the microscope. 

Mirror 

The mirror reflects rays from the light source onto the object. One side has a plane surface, the other a concave surface (Fig. 3.7). The concave side forms a low-power condenser and is not intended to be used if the microscope already has a con- denser.

Fig. 3.7 A microscope mirror

Condenser 

The condenser (Fig. 3.8) brings the rays of light to a common focus on the object to be examined. It is situated between the mirror and the stage. The condenser can be raised (maximum illumination) and lowered (minimum illu- mination). It must be centred and adjusted correctly.

Diaphragm 

The diaphragm (Fig. 3.9), which is inside the condenser, is used to reduce or increase the angle and therefore also the amount of light that passes into the condenser. The wider the diaphragm the greater the numerical aperture and the smaller the detail seen. But the contrast is correspondingly diminished.

Fig. 3.8 A condenser

Fig. 3.9 A diaphragm

Filters

In some microscopes coloured filters (particularly blue filters) are fitted below the condenser. These can be left in place or removed according to the type of prepara- tion being examined.

Adjustment system (Figs. 3.10 and 3.11) 

This consists of:
— a coarse adjustment screw
— a fine adjustment screw
— a condenser adjustment screw
— condenser centring screws
— an iris diaphragm lever
— mechanical stage controls.

Coarse adjustment screw
This is the largest screw. It is used first to achieve an approximate focus.


Fine adjustment screw
This moves the objective more slowly. It is used to bring the object into perfect focus.

Fig. 3.10 Microscope adjustment system

1: coarse adjustment screw;

2: fine adjustment screw;

3: condenser adjustment screw;

4: condenser centring screws;

5: iris diaphragm lever.

Condenser adjustment screw 

This is used to raise the condenser for greater illumination or to lower it to reduce the illumination. 

Condenser centring screws 

There may be three screws placed around the condenser: one in front, one on the left and one on the right. They are used to centre the condenser exactly in relation to the objective. 

Iris diaphragm lever 

This is a small lever fixed to the condenser. It can be moved to close or open the diaphragm, thus reducing or increasing both the angle and the intensity of the light. 

Mechanical stage controls 

These are used to move the object slide on the stage: one screw moves it backwards and forwards and the other screw moves it to the left or right (see Fig. 3.11). 

2. Setting up the microscope 

When a new microscope is received in the laboratory, it is important to know how to set it up correctly. 

Positioning the microscope 

Place it on a firm level bench (check with a spirit level) of adequate size but not too high. The microscope must be placed in the shade away from the window. Place a square felt pad under the microscope. If no felt is available, use a piece of heavy cloth. 

Setting up a lamp for the microscope 

If the microscope has a mirror, you can make a lamp to provide illumination. A porcelain holder for a light bulb is fixed on a wooden base and the whole is encased in a wooden or tin box with an opening for the light (Fig. 3.12). Cut slits in the top of the box to enable the bulb to cool. 

Alternatively, a flap can be fitted above the opening to serve as a shutter (Fig. 3.13). Use a 100 W opaque electric bulb of the “daylight” type (blue–white). 

Fitting the accessories 

Screw the objectives into the revolving nosepiece, following this order in a clock- wise direction: 

— x 3, x 5 or x 10 objective; 

— x 40 objective; 

— x 100 oil-immersion objective. 

The screw threads are standard. After you have screwed in the objectives: 

● Put the eyepiece(s) in place. 

● Fix the condenser under the stage. 

● Fix the mirror on the foot.

Fig. 3.12 Setting up a lamp for the microscope

Fig. 3.13 Alternative light source for the microscope

Positioning the lamp

If electric illumination is to be used, place the lamp 20 cm in front of the micro- scope facing the mirror. Adjust the position of the lamp so that it shines on the centre of the mirror (Fig. 3.14)

Fig. 3.14 Positioning the light source

If the lamp is fitted with a lens, the filaments of the bulb are projected on to a piece of paper covering the mirror. This makes it possible to centre the beam more pre- cisely. In some models the bulb is turned until a clear image of the filament is obtained. 

Preliminary adjustment of the mirror 

Use the plane side of the mirror. Remove any coloured filters. Open the iris dia- phragm to the maximum. Raise the condenser. Place a piece of thin white paper over the lens at the top of the condenser (Fig. 3.15). 

The piece of paper should show an image of the electric bulb, surrounded by a circle of light. Adjust the mirror so that the image of the bulb is in the exact centre of the circle of light (Fig. 3.16). If daylight is being used, adjust the mirror so as to maximize the amount of light passing through the condenser. 

Fig. 3.15 Adjusting the mirror

Fig. 3.16 Image of the light source, as seen through the condenser

Fig. 3.17 To centre the condenser, first close the diaphragm

Fig. 3.18 Raise the condenser until the edges of the circle of light are in focus

Centring the condenser (if centring is provided for) 

It is very important to centre the condenser correctly. This is often overlooked. 

1. Place a slide preparation without a coverslip on the stage. Lower the condenser. Open the iris diaphragm. Examine with the lowest-power objective (x 3, x 5 or x 10). Look through the eyepiece and bring the slide into focus. 

2. Close the diaphragm. A blurred circle of light surrounded by a dark ring appears in the field (Fig. 3.17). 

3. Raise the condenser slowly until the edges of the circle of light are in sharp focus (Fig. 3.18). 

4. Adjust the position of the mirror (if necessary) so that the circle of light is in the exact centre of, or superimposed upon, the bright area surrounded by the dark zone (Fig. 3.19). 5. Adjust the centring screws of the condenser so that the circle of light is in the exact centre of the field (Fig. 3.20). Then check with the other objectives.

Fig. 3.19 Adjust the position of the mirror to centre the light source

Fig. 3.20 Use the centring screws of the condenser to centre the light source

Adjusting the diaphragm 

Open the diaphragm completely. Remove the eyepiece and look down the tube: the upper lens of the objective will be seen to be filled with a circle of light. Close the diaphragm slowly until the circle of light takes up only two-thirds of the surface (Fig. 3.21). Do this for each objective as it is used. 

Adjusting the eyepieces 

Selecting the eyepiece 

The x 5 and x 10 eyepieces give good results in the medical laboratory. The high- power eyepiece increases magnification but there may be no great increase in de- tail. The eyepiece to use is a matter of individual choice. 

Binocular adjustment 

When a binocular microscope is used, the interpupillary distance (the distance be- tween the pupils of the eyes) can be adjusted to suit the operator. 

Focusing the eyepieces 

One of the eyepiece holders (usually the left) has a focusing collar (Fig. 3.22). If the collar is on the left eyepiece holder, close your left eye and, using the x 40 objective, bring the image into focus for your right eye with the right eyepiece. 

Then close your right eye and look through the left eyepiece. If the image is in focus, no adjustment is needed. If the image is not clear, turn the focusing collar until it is in focus. The microscope is now adjusted to suit your own binocular vision. 

3. Focusing the objective 

Low-power objective (x 10) Rack the condenser down to the bottom. Lower the objective until it is just above the slide preparation. Raise the objective, using the coarse adjustment screw, until a clear image is seen in the eyepiece.

Fig. 3.21 Adjusting the diaphragm

Fig. 3.22 Focusing the eyepieces

Occasionally a clear image cannot be obtained although the objective has been lowered as far as possible.This is because the fine adjustment screw has been turned right to the end.Turn it back as far as it will go in the other direction and then focus by raising the objective. Rack the condenser up slightly if there is insufficient illumination. 

High-power objective (x 40) 

Rack the condenser half-way down. Lower the objective until it is just above the slide preparation (the working distance is very short — about 0.5 mm). Using the coarse adjustment screw, raise the objective very slowly until a blurred image ap- pears in the field. Bring into focus using the fine adjustment screw. Raise the con- denser to obtain sufficient illumination. If the microscope has no condenser, use the concave side of the mirror. 

Oil-immersion objective (x 100) 

Perfectly dry, stained preparations must be used. Place a tiny drop of immersion oil on the part to be examined (use synthetic oils, which do not dry, in preference to cedarwood oil, which dries quickly). Rack the condenser up as far as it will go, and open the iris diaphragm fully. Lower the x 100 objective until it is in contact with the oil. Bring it as close as possible to the slide, but avoid pressing on the prepara- tion (modern objectives are fitted with a damper). Look through the eyepiece and turn the fine adjustment screw very slowly upwards until the image is in focus. If the illumination is inadequate, use the concave side of the mirror as recommended for the x 40 objective. 

Important: In most modern microscopes, it is not the objective holder but the stage which is moved up and down by the coarse and fine adjustment screws to bring the image into focus.

Depth of the microscope field

The image is seen in depth when a low-power objective is used. When the high- power objectives (x 40, x 100) are used, the depth of focus is small and the fine adjustment screw must be used to examine every detail from the top to the bottom levels of focus of the object observed (e.g. the different nuclei in a spherical amoeba cyst).

Images seen under the microscope

Remember that the circle of light seen in the eyepiece is called “the microscopic field”.

How to establish the position of images seen

Images observed in the microscopic field can be placed in relation to the hands of a clock. For example, a schistosome egg is placed at “2 o’clock” in Fig. 3.23.

Fig. 3.23 Establishing the position of images seen under the microscope

Inversion of images 

The image seen is inverted by the lenses: 

● Objects seen at the bottom of the microscopic field are actually at the top. ● Objects seen on the left side of the microscopic field are actually on the right.

Moving the object 

If you move the slide in one direction, the object examined moves in the opposite direction (Fig. 3.24).

Fig. 3.24 Moving the object

Changing the objective 

Modern microscopes are made so that the object remains more or less in focus when you change from a low-power objective to a more powerful one. If this is not the case for your microscope, raise the nosepiece before changing to the more powerful objective and refocus. Before changing objec- tives, make sure that the object examined is in the middle of the field, so that it is not lost after changing the objective. 

4. Use of an ocular micrometer 

The size of microorganisms or substructures of organisms can be measured by microscopy using an ocular with a cali- brated micrometer disc. The micrometer disc has a scale that is usually divided into 0.1-mm and 0.01-mm subdivisions (Fig. 3.25).

A stage micrometer is used to calibrate the ocular micrometer.

Fig. 3.25 An ocular micrometer disc

Materials 

● Binocular microscope 

● Ocular with a x 10 magnification 

● Ocular micrometer disc 

● Stage micrometer 

● Lens paper 

● Immersion oil. 

Method 

1. Unscrew the eye lens of the ocular. 

2. Place the micrometer with the engraved scale face-down in the ocular. Use lens paper to handle the disc. 

3. Replace the lens carefully. 

4. Place the ocular with the micrometer in the ocular tube of the microscope. 

5. Put the calibrated stage micrometer on the stage of the microscope and focus on the scale. You should be able to clearly distinguish the 0.1-mm and 0.01- mm subdivisions. 

6. Adjust the stage micrometer so that the 0-mm line coincides with the 0-mm line of the ocular micrometer. 

7. Look for another set of lines where the scale of the stage micrometer coincides with that of the ocular micrometer. This set of lines should be as far away from the 0-mm line as possible (Fig. 3.26). The distance between the two coinciding sets of lines varies, depending on the magnification of the objective of the microscope. 

8. Count the number of 0.1-mm subdivisions of the stage micrometer scale be- tween the 0-line and the second set of coinciding lines. 

9. Count the number of subdivisions of the ocular micrometer scale between the 0-line and the second set of coinciding lines. 

10. Calculate the propor tion of a millimetre that is measured by a single ocular unit using the following formula:

Fig. 3.26 Calibration of an ocular micrometer with a stage micrometer

Example 

For a microscope with a high-power objective (x 40), the calculation is as follows:

Important: Corresponding objectives should not be exchanged for a calibrated ob- jective, but must be separately calibrated. The ocular containing the micrometer disc should be stored until required. Each microscope that is to be used for meas- uring the size of organisms must be individually calibrated.

5. Dark-field microscopy

To obtain a dark field a special condenser with a blacked-out centre is used. If this is not available it is possible to obtain a dark field under the x 10 and x 40 objectives by inserting a disc or stop in the filter holder below the condenser.

The stops must be made of a material through which light cannot pass and must be the correct size for the objective in use. If the stop is too small, too much light will pass into the objective and a dark field will not be obtained.

If the stop is too large, insufficient light will be available to illuminate the specimen.

6. Routine maintenance

Microscopes must be installed in a clean environment, away from chemicals. Workplaces should be well ventilated or permanently air-conditioned (intermittent use of air conditioners produces condensed water). The microscope needs daily attention to keep it in good working order and thus to ensure reliable laboratory results. Optical instruments should not be kept for long periods in closed compartments since these conditions also favour fungal growth which can corrode optical surfaces. Special care is required in hot and humid climates.

Cleaning the microscope

Microscopes are used to investigate biological tissues and fluids and must therefore be decontaminated at regular intervals.

Materials

● Clean pieces of old cloth and a fine linen handkerchief

● Special lens tissue paper or, if unavailable, white absorbent paper or medical- grade cotton wool

● A piece of chamois leather, if possible (otherwise a non-fluffy rag)

● A small bottle of cleaning solution (see below)

● A plastic cover

● A small rubber bulb and, if possible, a soft camel-hair brush (or a fine paint- brush or blower for cleaning lenses)

● A desiccator 15–20 cm in diameter containing not less than 250 g of dry blue silica gel (which indicates humidity by turning pink).

Method

Cleaning the optical surfaces

The optical surfaces (condenser, objectives, eyepieces) must be kept free of dust with a fine paintbrush, a soft camel-hair brush (Fig. 3.27) or a blower. If dust is found inside the eyepiece, unscrew the upper lens and clean the inside.

Fig. 3.27 Cleaning the objective lenses using a soft camel-hair brush

Oil residues on the lenses should be removed with special lens tissue paper, absorbent paper or medical-grade cotton wool.The optical surfaces may be finally cleaned with a special solution, consisting of the following: 

— 80% petroleum ether (boiling point 60–80°C) 

— 20% 2-propanol. 

Note: Do not use 95% ethanol, xylene or toluene for cleaning the lenses, since these substances dissolve the cement.They can, however, be used for cleaning mirrors. 

Cleaning the instrument 

Heavy contamination can be removed with mild soapy solutions. Grease and oil can be removed with the special cleaning solu- tion described above. The instrument should then be cleaned with a 50 : 50 mixture of distilled water and 95% ethanol. This mixture is not suitable for cleaning the optical surfaces. The mechanical parts (coarse adjustment screw, fine adjustment screw, condenser focusing and mechanical stage) should be periodically cleaned and lubricated with machine oil to make them run freely.

Maintaining the microscope 

When you carry out repair and maintenance procedures, take care not to confuse the condenser centring screws with the condenser clamp screws. To maintain the microscope proceed as follows: 

● Check the mechanical stage. 

● Check the focusing mechanism. 

● Remove any fungal growth. 

● Check the diaphragm. 

● Clean all mechanical parts. 

● Lubricate the microscope according to the manufacturer’s instructions. 

● Check the spring load on the specimen clamp. Too high a tension may result in breakage of slides and damage to the clamp. 

● Check the optical alignment. A dim appearance of the specimen is often due to misalignment of the optical parts rather than to insufficient light. 

Precautions 

● Never dip the objectives in xylene or ethanol, as this may cause the lenses to become detached. 

● Never use ordinary paper to clean the lenses. 

● Never touch the lenses with your fingers. 

● Never clean the support or the stage with xylene or acetone. 

● Never clean the inside lenses of the eyepieces and objectives with cloth or paper (this might remove the anti-reflective coating); use a soft camel-hair brush, a fine paintbrush or a blower instead. 

● Never leave the microscope without the eyepieces unless the openings are plugged. 

● Never keep the microscope in a closed wooden box in hot humid countries. ● Never press the objective on to the slide, since both the slide and the objective may break. Take care when focusing the microscope.

● Keep the mechanical stage clean. 

● Do not dismantle the optical components, as this may cause misalignment. The optical surfaces should be cleaned with lens cleaning tissue or soft tissue paper. 

● Never put the microscope away with immersion oil on the objective. Remove any oil daily. Mild soap solution is suitable for most cleaning. 

● Use organic solvents only in accordance with the manufacturer’s recom- mendations. 

● Never carry the microscope by the limb with one hand; use both hands, one under the foot, the other holding the limb. 

● When changing a bulb, avoid touching the glass with your fingers, as finger- prints reduce the intensity of illumination. 

● To maximize the lifespan of bulbs, adjust the voltage with a dimmer switch to give the lowest required light intensity. 

● If the mains voltage fluctuates excessively, use a voltage stabilizer. 

Additional precautions to be taken in hot climates 

Dry climates 

In hot, dry climates the main problem is dust. Fine particles work their way into the threads of the screws and under the lenses. This can be avoided as follows: 

● Always keep the microscope under an airtight plastic cover when not in use. 

● At the end of the day’s work, clean the microscope thoroughly by blowing air over it with a rubber bulb. 

● Finish cleaning the lenses with a soft camel-hair brush, a fine paintbrush or a blower. If dust particles remain on the surface of the objective, clean it with special lens tissue paper. 

Humid climates 

In hot, humid climates and during the wet season in hot, dry climates, fungi may grow on the microscope, particularly on the surface of the lenses, in the grooves of the screws and under the paint, and the instrument will soon be useless. This can be prevented as described below. 

Always keep the microscope under an airtight plastic cover when not in use, to- gether with a dish filled with blue silica to dry the air under the cover. (The silica will turn red when it has lost its capacity to absorb moisture from the air. It can be simply regenerated by heating in a hot-air oven or over a fire.) The microscope must be cleaned daily to get rid of dust. 

These procedures must be carried out regularly, and are essential in conjunction with repair and maintenance procedures.