|Basic Malaria Microscopy (part I and II) (WHO - OMS, 1991, 72 p.)|
By the end of this Unit you should:
· be able to demonstrate the use
of the microscope with artificial or natural light
You cannot do your job without a microscope. It is important that you learn how to use it properly, that you understand its limitations and that you know what needs to be done to keep it in good condition.
The microscope that you will use is called a compound microscope. You will need to know the names of some of its component parts:
· so that you can easily carry out instructions during the practical exercises that are part of your training
· so that you can accurately describe parts that may need attention or replacement during the course of your work.
Parts of the compound microscope
All the main parts of a typical compound microscope are illustrated in Fig. 1.
Main tube and body tube
The main tube and body tube are often collectively called the head of the microscope. The head generally slopes towards the user for greater comfort and is then called an inclined head. Polished glass prisms are fitted inside the body tube of an inclined head; these allow the light to bend so that the image reaches the users eye.
The eyepiece, or ocular, is located at the top of the main tube. Most compound microscopes are fitted with binocular heads, which means they have two oculars - one for each eye. Some, however, have only one ocular and are referred to as monocular microscopes.
1. Main tube inclined head
2. Body tube (prism) inclined head
3. Revolving nosepiece
5. Stage (mechanical stage)
6. Substage condenser with iris diaphragm
8. Base (foot)
9. Ocular (eyepiece)
10. Arm (limb)
11. Coarse adjustment
12. Fine adjustment
A number of objective lenses of different magnifications are screwed into the nosepiece of the microscope, which can then be revolved to increase or decrease the magnification of the specimen being examined.
All parts of the microscope are important, but the objective lenses - the lower, magnifying lenses - must be treated with particular care. The lenses are of the best quality and need to be handled very carefully. Sometimes two lenses are glued together, and you must be careful not to use solvents such as strong alcohol solutions or acetone, which could dissolve the glue or cement.
Objectives are referred to by their magnifying power, which is marked on the side. The microscope you will use has the following objectives:
· x 10
· x 40
· x 100 (this objective is often called the oil immersion objective; sometimes it has a black or red ring around it for easy identification).
You will notice in the diagram above that the size of the front lens of the objective decreases as magnifying power increases.
As the magnification differs between objectives, so does the working distance. The working distance is the distance between the front lens of the objective and the specimen on the stage (when the specimen is in focus). The higher the magnifying power of the objective, the shorter is the working distance. Working distances for the standard objectives are likely to be as indicated below (depending on the make of the microscope):
· x 10
· x 40
· x 100
1.81 mm (this is the oil immersion objective).
The mechanical stage
The mechanical stage holds the slide secure and allows the specimen to be moved smoothly backwards, forwards or sideways. Sometimes a scale is fitted to two sides of the stage to show the extent of the movement. This is called the Vernier scale, and it is useful to know how to use it; it can be used to trace a part of the blood film that you need to re-examine or show to your supervisor.
Substage condenser (with iris diaphragm)
The substage condenser is made up of a number of lenses. These centre the light from the mirror, or electric light source, to a central spot on the field. The substage condenser can be raised or lowered to give maximum or minimum illumination.
Inside the condenser is the iris diaphragm. This is used to control the amount of light passing through the condenser. The iris diaphragm consists of a number of interlocking leaves made of a thin metal. It is adjusted by means of a lever.
Filter holder and blue filter
Beneath the iris diaphragm is the filter holder. This is where a blue filter is placed when you use an electric light source for illumination. It has the effect of making the microscope field white rather than yellow.
The mirror is used to direct light from the light source to the microscopic field. The mirror has two sides, one of which is a plane or flat surface and is used with the substage condenser. The other surface is concave and is used without the condenser (the curved surface itself acts as a condenser).
Note: Some microscopes with a built-in illuminator do not use a mirror but have a built-in prism instead, which directs light from the illuminator to the objective/ocular lens system. Others have a removable illuminator, which can be replaced by a mirror when necessary.
The ocular or eyepiece fits into the upper end of the main tube and is what the microscopist looks through when using the microscope. The ocular has its magnifying power marked on it. Magnifying power is the number of times by which it will magnify the image produced by the objective. For instance, with an ocular of x 7 and an oil immersion objective of x 100, the total magnification of the specimen would be 7 x 100 = 700.
Oculars are available in a range of powers. In malaria microscopy, an ocular of x 7 magnifying power is preferred. An ocular of x 6 could also be used, but one of x 10 magnifying power is not recommended.
Oculars fitted to binocular microscopes are called paired oculars and are specially made to suit the microscope in question. On the rim of the oculars you may see the marking x 7P , which denotes a paired set of eyepieces of x 7 magnifying power.
The arm forms a rigid support for the main tube and stage of the microscope. It is strongly made and can be used to carry the microscope around the laboratory. It is recommended, however, that you also support the microscope at the base, with your other hand.
Coarse and fine adjustment
The two adjustment systems - coarse and fine - are used to focus the specimen being examined. The coarse adjustment is for rapid and relatively large movements of the stage (and therefore the specimen); the fine adjustment is for the finer focusing required when the higher powered objectives are used.
It is normal to focus the specimen first with the coarse adjustment and then to use the fine adjustment while the specimen is being examined.
With the oil immersion objective, the coarse adjustment is used in a different way. This will be explained later.
Whatever the shape of the base of the microscope (usually U-shaped or rectangular), it must rest on a firm, flat bench or table. It is essential that the microscope does not wobble while it is being used.
A threaded hole can be seen on the underside of the base. This is to take a screw that secures the microscope inside its storage box during transportation.
Use of the microscope
In the practical sessions you will learn how to use the microscope. You will see how the image (of the specimen) appears larger as you increase the magnification by changing objective lenses.
Initially you will examine specimens given to you by your tutor or facilitator. Some of these will be living organisms in water, others will be everyday objects with which you are familiar (although they will look very different under the microscope).
During these exercises you will learn how to adjust the illumination, and see how to make the best use of the substage condenser and iris diaphragm. You will also be able to use the mechanical stage and the Vernier scale.
You will have time to practise on a monocular microscope and will notice that its illumination is very good. This is the microscope that should be used with the x 100 oil immersion objective when only natural light is available. The binocular microscope, though more restful for long hours of work, needs a reliable electricity supply for illumination; with only natural light available, it is less efficient than the monocular when used with the oil immersion objective.
The light source
A good source of light is needed to examine specimens properly. This may be either daylight or electric light. The electric current may be provided by mains supply or by a battery or generator. Light that is either too bright or too dim will interfere with examination of specimens.
Light from the light source travels the following path, via the substage mirror or the substage lamp:
light source ® mirror (if there is one) ® substage condenser and diaphragm (if the source is artificial) ® specimen ® objective ® ocular
When artificial light is used, a blue filter must be placed between the source and the substage condenser. If a mirror is employed with artificial light, the flat side of the mirror should be used; when daylight is the light source, the concave mirror should be used, without the substage condenser.
Obtaining even illumination
To obtain good, even illumination, the procedure is as follows:
Step 1 Place the specimen slide on the mechanical stage. Using the coarse adjustment, focus on the specimen with the x 10 objective.
Step 2 Making sure that the iris diaphragm is completely open, raise the substage condenser to the point where the field is brightest.
Step 3 Remove the eyepiece and, while looking down the tube, adjust the mirror until the objective lens is fully illuminated.
Step 4 Replace the eyepiece and focus sharply on the specimen using the fine adjustment.
Step 5 Remove the eyepiece again and close the iris diaphragm until the aperture of the objective is two-thirds visible; this will make the specimen appear clearer - it gives maximum resolution.
Step 6 Replace the eyepiece and revolve the nosepiece to select the objective of the required power; you may need to focus slightly at each change of objective. Specific directions concerning the use of the oil immersion objective are given below.
Illumination can be easily adjusted by increasing or decreasing the aperture of the iris diaphragm.
Using the oil immersion objective
When setting up the microscope for use with the oil immersion objective, the following procedure is the best:
Step 1 After arranging the illumination as described in the previous section, observe the rest of the process from the side of the microscope.
Step 2 Using the coarse adjustment, rack up the main tube.
Step 3 Place the slide on the stage of the microscope with the blood film uppermost.
Step 4 When you can see that there will be sufficient space between the stage and the x 100 objective, turn the revolving nosepiece so that the x 100 objective is over the specimen.
Step 5 Place 1 - 2 drops of immersion oil on the blood film in the area which is to be examined.
Step 6 Using the coarse adjustment, carefully lower the objective until the lens is in contact with the immersion oil. Then raise the lens slightly, but allow the lens and oil to remain in contact.
Step 7 Focus the specimen using the fine adjustment, making sure that the lens does not come into contact with the slide. You may alter the illumination if necessary by adjusting the iris diaphragm.
Immersion oil is used between the microscope slide and the objective lens to reduce scattering of the light transmitted by the mirror or illuminator. The oil has to reproduce the optical properties of the glass used for the lenses, and must therefore have a refractive index of 1.515, i.e. approximately 1.5 times the refractive index of water.
When immersion oil is used, the objective lens and the slides must be cleaned at the end of the days work. You can use a soft cotton cloth or lens tissue for the objective lens but remember not to use it to clean other lenses on the microscope. From time to time, dried oil should be removed from the oil immersion objective using xylene (but no other solvent). Oil can be washed off the slides with a small amount of xylene; if no xylene is available, the oil smear can be carefully dabbed with absorbent paper.
Immersion oil can be obtained commercially. In some countries, however, anisole is used for work with the oil immersion objective; this product has the same refractive index as immersion oil. Anisole evaporates from the blood film after some time, so that the film does not need to be cleaned and there is less chance of its being damaged or wiped off. Use of anisole also means that the objective lens does not need to be cleaned.
Care of the microscope
Provided that normal care and common sense are exercised, the laboratory microscope will be useful for many years.
Removing dust and grease
When not in use during the day, the microscope schould be kept covered with a clean cloth or plastic cover to protect the lenses from dust that settles out of the air. Overnight, or if it is to remain unused for long periods, the microscope schould be placed inside its box with the door tightly closed. To protect the objective lenses, the × 10 objective schould be rotated to line up with the ocular.
Oil and grease from eyelashes and fingers are easily deposited on lenses and oculars as the microscope is used; these parts should be cleaned with lens tissue or with very soft cotton cloth.
The oil immersion objective should be cleaned after use. If it is not cleaned, the oil will harden and make the objective useless. A lens tissue or soft cotton cloth is usually sufficient for the purpose. However, the tissue or cloth should never be used to clean other objectives, the oculars or the mirror, otherwise oil will be transferred to these components.
Preventing the growth of fungus
In warm, humid climates it is very easy for fungal growths to become established on lenses and prisms. These growths can cause problems and may even become so bad that the microscope can no longer be used. The lenses may need to be repolished by the manufacturer, which is very expensive and may take several months.
Fungus cannot grow on glass when the atmosphere is dry, and every effort should therefore be made to store the microscope in a dry atmosphere when it is not being used. One of the following methods should be used:
· Keep the microscope in a continuously air-conditioned room.
Note: It is pointless to store the microscope overnight in a room where the air-conditioner operates only during the day.
· Place the microscope in a warm cupboard, i.e. an airtight cupboard in which one or two 25-watt bulbs are constantly alight.
· Keep all lenses and prism heads in an airtight box or desiccator where the air is kept dry by means of active silica gel.
Note: Silica gel is a desiccant - a compound with the ability to absorb water vapour from the air. Self-indicating silica gel is blue when active but becomes pink when it has absorbed all the water it can. It can then be reactivated by heating; it turns blue again as it becomes reactivated. When the silica gel cools it can be returned to the airtight container. Only self-indicating silica gel should be used.
· In locations without electricity but where a kerosene refrigerator is used, place the microscope box on a small shelf sited 20 - 30 cm above the refrigerator chimney. Heat from the chimney will keep the box sufficiently warm and dry to prevent fungus growing on the microscope lenses.
Transporting the microscope
When the microscope is to be transported from one location to another, it is important to ensure that it is properly secure inside its box. The best way to do this is by means of the securing device, which screws into the base of the microscope.