30th , Mar , 2016

Faculty of Veterinary Science

VETERINARY CLINICS

SHAFI’I A. MOHAMED

Microscopic Technique

Rules of using a microscopeAlways carry the microscope in a straight

upright position with one hand around the arm and the other hand under the base. The eyepieces are not attached and will fall out if the microscope is carried at an angle or upside down.

Check out the microscope to make sure all the lenses are clean and the mechanical parts are in working order.

Do not force knobs

Keep the microscope clean. When anything is spilled or otherwise gets on the microscope, clean it up immediately

Clean the lenses with lens paper only. DO NOT CLEAN THE LENSES WITH HANDKERCHIEFS, FACIAL TISSUES, PAPER TOWELS, ETC.--they will scratch the lenses

If you cannot obtain clear focus or good lighting, or if your microscope seems not to be working properly , IMMEDIATELY CALL YOUR INSTRUCTOR.

Never leave any microscope slides on the microscope. Always remove them and return them to their proper place before leaving the room

Always store covered

Types of Microscopes

There are four basic different types of microscopes: light , electron, compound microscope and the dissecting microscope.

• Light microscopes have glass lenses which magnify objects, and light is necessary to illuminate the objects being examined.

Light.M

The Compound Microscope

Compound microscopes are used to examine objects in two dimensions. Very small organisms or cross-sections of organisms are placed on clear glass slides; these objects are viewed as light passes through them.

The Dissecting Microscope

Dissecting microscopes are used to observe material that is either too thick or too large to be viewed with the compound light microscope. While the magnification and depth of field are smaller in the dissecting scope, the field of view is much larger. As its name implies, the dissecting scope is often used to dissect ticks, since it allows for manipulation of material.

Electron Microscopy The Biology Department has an

electron microscope suite, used for teaching students and for research. In these microscopes a beam of electrons (in place of light) and circular magnets (in place of glass lenses) permit the resolution of structures in much finer detail than in an optical microscope

• We have two electron microscopes. The first is a "traditional" transmission electron microscope (TEM) in which an electron beam passes through the specimen. The second is the more recently-developed scanning electron microscope (SEM) in which a beam of electrons scans the surface of an opaque object and produces an image of that surface.

The images are viewed on a cathode tube, or more critically by exposing photographic film. Many of the photographs of cell structure used in your text were taken with an electron microscope.

BLOOD EXAMINATION

Blood examination

The blood is collected from animals through a puncture of jugular vein in horse, camel, cattle, sheep and goat, cephalic vein or recurrent tarsal vein in dog and cat.

Appropriate restraint is very important. An experienced handler is invaluable, but you

must use the owner to restrain the animal It is essential to give him or her clear and specific

instructions. The aim should be to hold the relaxed animal gently but firmly in the optimum position. It is very difficult to bleed a kicking and struggling animal even with forcible restraint, resulting difficult to collect the sample.

General points and technique

It is always easier to hit the vein if the hair is clipped ( electric clippers are much more satisfactory than curved scissors ), how ever hair takes some time to grow and owners often don’t appreciate the bald patch especially on show animals, once you are well practiced in the technique you will find that clipping is only necessary with particularly difficult animals.

There is some controversy over the efficiency of surgical spirit in sterilizing the skin before

venepuncture . If a new sterile needle is used every time, how ever owners often expect visible sterile precautions , and the spirit is very useful to lay the hair and improve visualization of the vein.

It is a good practice to apply pressure over the site of venepuncture with a clean piece of cotton as the needle is withdrawn, and to keep it a half minute or so .

Two blood samples can be collected for laboratory examination.

Whole blood samples: the blood sample is mixed to anticoagulant such as heparin or EDTA or potassium and ammonium oxalate.

It is indicated for hematological examination such as erythrocytic count, leucocytic count, hemaglobin concentration, packed cell volume and blood film.

Serum samples: the blood sample is collected with out addition of anticoagulant, left to clot then centrifuged at 300 rpm for 20 minutes. Only clear serum separates in a clean plastic container for biochemical examination.

Blood film

A drop of fresh blood is placed in one corner end of slide, and spreaded as smear with the help of another slide using its thin edge at an angle 45o . Dry the smear in air, fix in methanol for 4-5 minutes. Wash the slides, dry in air. Stain the smear with Giemsa stain and examine under oil immersion of the microscope for the presence of blood parasites such as Babesia, Theileria, Anaplasma, Trypanasoma and filaria.

1. Fecal examination

The fecal sample is collected directly from the animal. Collection of 5-10 g feces in a clear dry glass container. In delay exam, store the feces

in refrigerator at 4 °C. The feces can be examined by different methods

Not good to do this

A) Direct method: A clean dry glass slide is used. Place a drop of

distilled water in the middle of the slide, add small amount of feces, mix and place a

cover slide. Examine it under microscope for the presence

of parasitic ova. If no parasitic ova is detected it should be examined by qualitative method.

Equipment

B) Qualitative concentration method: Feces is mixed with either of the saturated

sugar, saturated salt solution or 41% magnesium sulfate solution.

The parasitic ova, being lighter float on the top of fluid and can be concentrated for examination

1. Simple flotation method: 1 g of feces mixed with few ml of

distilled water, filtered through a fine sieve. The filtrate is mixed with 4-5 ml of saturated salt solution. It should be then placed in a

tube or cylinder and filled up to the top with solution, cover the tube with glass slide and left it 30-60 minutes at room temperature.

Remove the cover slide and examine under the microscope.

2. Concentration flotation method: 1 g of feces mixed with few ml of distilled water, filtered

through a fine sieve. The filtrate is mixed with saturated sugar solution in a ration of 1:3 in a test tube, mix the contents and centrifuge at 1500 rpm / 5 minutes.

Transfer the small amount of superficial contents of tube on a clean and dry glass slide and examine for the presence of parasitic ova.

The sediment can be examined for eggs of trematodes.

3. Baermen's technique in cattle &horse: Small amount of feces in gauze inside funnel

filled with warm water. After 2 hrs. Examine the first few drop to detect the larva 4. Vida technique in sheep: Pellet of feces mix

with worm water in Petridish for 10 minute then crushed the pellets by forceps examine after 10 minute