Preparation of Permanent Mounts | Zoology

In this article we will discuss about the preparation of permanent mounts of various organisms.

1. Euglena:

Living Euglena, for study, may either be collected from a patch of stagnant water having decaying green leaves or may be obtained from cultures in laboratory. For purpose of study in the living condition take a few euglena in a dropper or a micro-pipette either from the culture, which is maintained in laboratory or from the bottom of a small patch of stagnant water where leaves of plants etc. are decaying.

Now pick a clean slide and put a drop of Mayer’s albumen on top of it. Rub this albumen with index finger on whole of the slide top (surface) in order to make a smooth, thin and homogenous film.

Now, put a drop of the water, containing material, collected from pond, in the centre of slide on the same side the film is spread. Now, observe the movements & irregular shape of the animal under microscope in low magnification, Draw various stages of locomotion at an interval of half minute.

For detailed anatomical study a permanent stained mount may be prepared by following procedure:

Take the same slide after completing the study in living condition. Now take a small piece of filter paper or blotting paper and put its one edge in contact with the fluid having Eugiena on the slide. As a result of this the excess of fluid shall be absorbed by paper and the Eugena present in it shall stick to the surface of slide through Mayer’s albumen film.

Now, kill and fix these Eugiena either by putting the slide in inverted position over the mouth of a smic acid bottle in such a way that the Eugiena could be exposed to fumes of osmic acid, or put a drop of 100% alcohol or 1% acetic acid or Shaudin’s fluid over the sticking Eugiena.

Now, wash the slide in tap water if osmic acid or acetic acid is used, otherwise bring the slide through descending grades of alcohol (90%, 70%, 50%, 30%) to finally tap water, if 100% alcohol or Shaudin’s fluid is used. After washing thoroughly with water stain with Borax carmine dehydrate in 50, 70, 90 and Absolute acohyl, clear in xylol and mount in Canada balsam. Observe under microscope.

2. Paramaecia & Vorticella:

Methods are just the same as described for Eugiena. The only precaution to be taken is that before putting on slide, the drop of culture fluid having animals, put a drop of weak gelatin solution or 1% agar solution or a few fibres of cotton wool in order to check the fast movement of these animals. Picro-indigo carmine or Picro-carmine or Borax-carmine stain should be used for colouring.

3. Monocystis:

It is an endoparasitic protozoan found in the reproductive organs, especially seminal vesicles, of male earthworms. Before the commencement of actual study seminal vesicles should be exposed in living earthworm, because they live inside the cells of seminal vesicles.

The extraction of animals is a tortuous and complicated process. To begin with, take a living, large size & mature earthworm and put it in a disecting tray. Make a longitudinal cut in the mid-dorsal line of the animal in the region of clitellum and expose the seminal vesicles.

Now, with the help of a pair of forceps take out ail the four seminal vesicles from both sides. Put a few drops of physiological saline (7% NaCI) or tap water in a watch glass and put in it the four seminal vesicles. Take the arrow head from your dissecting box and start crushing the seminar vesicles with it till they turn into a thin white paste in the watch glass.

Now, pick a clean slide and rub on one of its surface thoroughly with a small drop of Mayer’s albumen with your index finger. Take a few drops of seminal vesicle paste and spread it in the middle over the same surface of slide on which you have applied albumen.

Observe under microscope, first in low power and then in high magnification. Locate different stages of the parasite. If no stage is visible repeat the same process afresh with another slide till the slide shows desired stage. Now, leave the slide to dry up and put a few drops of 100% alcohol over the entire area on which the paste is spread. Leave the alcohol to evaporate.

Now, bring the slide to tap water and after thorough washing it use Haematoxyline and Eosin (double staining) in their usual way. Dehydrate, clear in Xylene and mount in canada balsam. Observe under low magnification and locate the trophozoit, gametocyte and sporocyst stages, then study and sketch under high magnification Fig. 46.

4. Rectal Ciliates:

The term rectal ciliates is used for all those protozoans which belong to class ciliophora and which live permanently as entoparasite in the rectum and large intestine of frog (amphibians). These are usually three in, number i.e., Opalina, Balantidium and Nyctotherus.

It is not necessary that all three could be available from every frog. It is just possible that none of them might be available or only one of them may be available in a particular frog. For purpose of extraction a living frog is anesthetized by chloroform or through pithing or asphyxiation.

The rectum and lower part of intestine are taken out from the frog and are cut open in a watch glass. Their contents are thoroughly washed with phisiological saline (0-7% NaCI) and are collected in the watch glass. Now take a clean slide and apply thin film of Mayer’s albumen on top surface of it.

Put a drop of the contents, from the watch glass in middle of the top surface of slide and observe under low magnification. Find out whether ciliates are present or not. If ciliates are present absorb the excess water with a piece of filter paper or blotting paper and fix the animals by using 100% alcohol or Osmic acid bottle in the same way as in case of Eugiena.

Now, bring the slide to water and use double staining method (Halematoxylene and Eosin). Dehydrate, clear in Xylene and mount in Canada balsam. Study detailed structure under high magnification and make a sketch.

5. Preparation of Slide from Culture of Pond Water:

Collect some water from a stagnant pool or pond from along the bottom with some living plants and some decaying leaves etc. Leave this water in a beaker in a dark place at room temperature (37°C) for two to three days. Now, keep one or two drops of this water on a slide and observe under low power of microscope. You may find Paramaecia, Eugiena, Vorticella and even Hydra.

Fix these organisms by adding two drops of fornaline or absolute alcohol in pond water over the slide. Stain with borax carmine and mount through a process discussed in the procedure of staining. For detailed structure of organisms see diagrammes in this book and draw them.

6. Paramecium Feeding Experiment with Congo Red:

From the culture medium place one or two living Paramaecia in a cavity slide along with two to three drops of culture water. Now, put a few particles of Congo red dye at one edge of the cavity and observe under the micro­scope. The Paramaecia ingest the particles through cytostome.

The food vacuole formed, at the end of cytopharynx, travels, along a definite route in an almost clockwise cyclic fashion till the undigested particles are removed to out side through cytopyge.

Draw the diagramme as shown below in Fig. 50 :

7. Spicules of Sponge:

For purpose of obtaining spicules of sponges take a small piece of body wall of Sycon. Put the piece in a test tube in a little KOH solution (10% aqueous solution of KOH) and boil it thoroughly. Take care that the tube should be kept at an accurate angle with its mouth away from the person holding it.

Boiling should continue till the cells are dissolved and a mass of differently shaped shiny and spiny spicules settle down at the bottom of the tube. Decant the KOH solution and fill the tube up with tap water. Shake the tube vigorously closing its mouth with your thumb. Let the spicules settle to bottom of the tube and decant the water. Repeat the washing.

Now, take a few spicules of each type through a dropper or a brush and put them in the middle of slide. Dehydrate up to 70% alcohol, stain with Picro-indigo-carmine, or Borax-carmine dehydrate up to 100% alcohol, clear in Xylol and mount in canada balsam. Observe under low magnification of microscope and make a sketch Fig. 51.

8. Gemmule of Sponge:

The gemmules which are provided in laboratory for purpose of study are already in 70% alcohol. Take one or two gemmule and pass them through 50% and 30% alcohol and bring them to water. Now, put these gemmules in test tube in 1% KOH solution and boil for a few minutes (not more than 5 minutes). Decant the KOH and wash the gemmules thoroughly with tap water through vigorously shaking the tube.

Now, transfer these gemmules to a staining tube and dehydrate in 50% and 70% alcohol. Now, stain in Picro-indigo-carmine, or Borax-carmine, dehydrate up to 100% alcohol, clear in xylol and mount in canada balsam. Study and sketch under low power of microscope Fig. 52.

9. Spongin Fibres:

Spongin fibres constitute the skeleton of common bath sponges belonging to genus Euspongia. For obtaining spongin fibres take a small piece of dried bath sponge Boil in 1% KOH solution for 5 mts. or till the cells are dissolved and spongin fibres are settled at bottom.

Dehydrate up to 70% alcohol, stain in Picro-indigo-carmine, or Borax-carmine dehydrate in 100% alcohol, clear in Xylol and mount in Canada balsam. Observe under low magnification of microscope. Fig. 53.

10. Hydra:

The study of entire hydra in living condition may conveniently be made by picking a hydra, through dropper, from either culture in laboratory or from aquatic plants in any pond or ditch having stagnent water and decaying material. As the animal is capable to swim or move rapidly they are conveniently studied on a cavity slide.

However, a fixed, narcotized or stained animal may easily be studied on an ordinary slide. After completing studies on living animals, they may be killed and fixed either by putting a drop of Bouins fluid over them or putting a Menthol crystal in water in which they are kept and the after removing the menthol-water, by putting a few drops of formalin.

After fixing the living animals, they are thoroughly washed with water, dehydrated, stained by using Borax carmine or picro-indigo- carmine, dehydrated in 100% alcohol, cleared in xylol and mounted in canada balsam. They may be observed, now, under low magnification of microscope Fig. 54.

 11. Obelia Colony, Medusa and other Coelenterates:

The coelenterates like Obelia colony, Bougainvillia, Tubularia, Sertularia, Campanuiaria, Pennaria, Plumularia, the material, stain it with borax-carmine or picro-indigo-carmine, dehydrate in 90% alcohol pass through 100% alcohol clear in Xylol and mount in canada balsam. Observe under microscope in low or high magnifications as required Fig. 55- Fig. 56.

12. Planarians:

Planarians are freshwater helminthes belonging to order Turbellaria. They may be collected from near the stones in a patch of stagnent water. – For purpose of microscopic studies a planaria e.g., Dugesia is put in the middle on top of a slide.

A little grease is applied on surface of slide around the planarian and another slide is put on top of the planarian. The two slides are tied with a strong thread and sufficient pressure is applied on the slides in order to make the planarians flattened and to let them assume their natural form because otherwise they will curl and study will be difficult.

Now, put these planerians, as such, in Acetic- formal-alcohol (AFA) for 6-10 hrs. depending on the size of specimen. Now, take out the animal, along with slides, rinse and remove the slides slowly and gently. Wash the specimen thoroughly in 70% alcohol.

Stain in Gower’s carmine and differentiate in 70% alcohol. Dehydrate in 90% and absolute alcohol, clear in xylol and mount in canada balsam. For mounting such thick specimens special care and special methods are adopted. Put small triangular pieces of coverslip in a circular fashion around the specimen. The pieces should be equal to the thickness of the specimen.

Now, flood the centre of the slide with Canada balsam after putting the animal in centre in proper orientation. Put the coverslip above the specimen in such a way that it should cover the glass pieces completely. Leave it to dry, preferabely in an oven Fig. 57.

13. Fasciola Hepatica:

Liver flukes may easily be collected from any butchery where sheep’s and pigs are slaughtered. In laboratory, they may easily be collected from bile duct of sheep’s. For this purpose bring a complete infected liver of sheep from the nearest butchery.

Cut open the bile duct and take out the flukes in 0.78% saline at a temperature ranging between 37°c and 40°C. Wash the animals thoroughly in physiological saline (0-78% Nacl) and put them between two slides on the inner surfaces of which enough grease has been applied.

Tie the free ends and middle of the slides with a strong thread and put them in acetic-formol- alcohol (A F A) for 10 to 20 hrs. Now, take out the animal, along with slides, rinse and remove the slides slowly and gently. Wash the specimen thoroughly in 70% alcohol.

Stain in Gower’s carmine and differentiate in 70% alcohol. Dehydrate in 90% and absolute alcohol, clear in xylol and mount in Canada balsam. For mounting such thick specimen’s special care and special methods are adopted. Put small triangular pieces of coverslip in a circular fashion around the specimen. The pieces should be equal to the thickness of the specimen.

Now, flood the centre of the slide with Canada balsam after putting the animal in centre in proper orientation. Put the coverslip above the specimen in such a way that it should cover the glass pieces completely. Leave it to dry, preferabely in an oven Fig. 58.

14. Septal Nephridium of Earthworm:

Take a preserved earthworm, stretch and pin it in a dissecting tray. Now, cut it along mid dorsal line behind the clitellum. Take a small piece of septum, which extends from alimentary canal up to body wall Wash it thoroughly with-water, dehydrate up to 70% alcohol, stain in Borax carmine and again dehydrate through 90% and absolute alcohol, clear in xylol and mount in canada balsam.

For isolating only one nephridium use two sharp needles and operate on the slide under binocular (fig.59).

15. Pharyngeal Nephridia from Earthworm:

Pharyngeal nephridia are present in the region of pharynx in 4th to 6th segments. They appear as a bunch of tubules in the inter segmental space above the pharynx. Cut open the earthworm in the aforesaid region and pick a small part of the bunch.

Ascertain under microscope. When satisfied, wash with water dehydrate thoroughly in 30%, 50%, 70% alcohol, stain in Borax carmine .dehydrate through 90% and absolute alcohols, clear in Xylol and mount in Canada balsam Fig. 60.

16. Blood Glands from Earthworm:

They are found mixed with pharyngeal nephridia. They are dark brown, small rounded structures. Proceed as for pharyngeal nephridia above. (Fig. 61.)

17. Ovary of Earthworm:

Take either a preserved or a fresh and earthworm. Stretch and pin it in a dissecting trey. Now, make a cut in the mid dorsal line starting just behind the clitellum towards anterior end. The two flaps, so cut open, should be pinned on sides. Cut the alimentary canal transversely after 15th segment.

Now pull the alimentary canal upwards and forwards holding its cut hind end. Pulling of the alimentary canal should be slow and gradual. As soon as you reach the anterior edge of clitellum, near septum between 12th and 13th segments, hold the alimentary canal vertically.

Near the septum along the mid ventral line of alimentary canal look for a pair of dark blackish brown hearts. Now, search for two tiny white dots, which may be found either attached to hearts or attached to integument just close to hearts. With the help of fine forceps take out both these ovaries and put them on slides on which a thin film of Mayer’s albumen has been applied.

The albumen will hold the ovaries in place and will not let them washed away with reagent. When slightly dry, wash in tap-water stain with borax carmine after dehydrating up to 70% alcohols and pass through 90% and absolute alcohol, clear in xylol and mount in canada balsam Fig. 62.

18. Parapodium from Nereis:

For the preparation of slides of parapodia instead of complete animal, only one or two segments shall be provided to each student. The parapodia are lateral extensions of segmental skin. With the help of scissors cut the parabodium near its base, wash thoroughly with water, dehydrate up to 70% alcohol, stain with Borax carmine, completely dehydrate in 90% and absolute alcohol, clear in Xylol and mount in Canada balsam Fig. 63.

19. Salivary Glands of Leech:

Take a fresh narcotised leech. Put it in a path dish and press the animal from behind forward. This process will remove the blood from the alimentary canal. Repeat this process two to three times or till the blood stops coming out of its mouth.

Now pin up the animal in dissecting tray after stretching the animal with ease as far as possible. Now, with the help of scissors or with the help of a blade make a cut in the anterior one inch of the animal along the mid dorsal line up to the position of pharynx.

With the help of a scalpel remove the muscular and botryoidal tissue from the body-wall and pin down the two flaps of the body-wall in the dissecting tray. Locate the buccal cavity and try to search the salivary glands among the radial t muscles which extend from the buccal cavity and pharynx to the body wall. Pick up a bunch of few of these glands and observe under low power of microscope.

If you are satisfied, that you have picked up the salivary glands, proceed to dehydrate. Stain in Borax carmine after 70% alcohol and wash them in 90% alcohol. Rinse them in absolute alcohol, clear in Xylene and mount them in D.P.X. Observe under microscope and draw the diagram as shown below in Fig. 64.

20. Nephridium of Leech:

After squeezing the leech to remove the blood from its alimentary canal and pinning it down in dissecting tray make a cut throughout the length of the animal along its mid-dorsal line. Remove the alimentary canal and botryoidal tissue. Locate the dark lateral hazomocoelomic channel and white vas deference running along either lateral sides.

On the outer side of vas deferens, in each p segment, lies a white coiled nephridium. Removing all attached botryoidal tissue & muscles detach the nephridium from the body wall. Wash it with water in a stainng tube and proceed to stain it with Borax carmine. Mount in D.P.X and observe under microscope. Draw the diagram as shown in Fig. 65.

  21. Gill Lamella of Unio:

Take a small piece of unio’s gill, preferably from inner lamina. With the help of ‘ two needles separate the two lamellae of this lamina. Now select the inner lamella and wash it thoroughly with water. Use Borax carmine for staining. Dehydrate, clear in xylol and mount in candada balsam Fig. 66.

22. T. S. Ospharidium of Pila:

Take a preserved pila and break off the shell. Look for an aperture on the left side of the body of pila, the Inhalent siphon. Now, see through the inhalent siphon. Attached to the inner dorsal surface of mantle, you will find a small protuberance – the Ospharidium.

Take out the entire ospharidium along with a little piece of mantle. Now, hold the piece of mantle between first finger and thumb of left hand. Take your razor or blade and cut a few longitudinal sections. Put them in water, select the best section and stain it with Borax carmine. Dehydrate, clear and mount in canada balsam. Fig 67.

23. Radula of Pila:

After exposing the pila by removing its shell and mantle locate the buccal mass, which is present just behind the tentacles as a large soft muscular nodule. Remove the integument from above the buccal mass. Now take it out by cutting the muscles from around with the help of scalpel and after cutting the tentacles.

Now place the buccal mass in a watch glass in water Make a shallow incision with the scissors and locate a hard chitinous brownish ribbon-like structure in the middle of buccal mass – The radula. Take out the radula with the help of forceps and treat it with 5% cold KOH for 10 minutes.

Press it between two slides and tie the slides with rubber bands and leave for 10 minutes. Now you can prepare the slide by simply dehydrating it in 90% and absolute alcohol for 5 minutes each and after washing with Xylol for two minutes. Else, you can display in watch glass if asked by the teacher Fig. 68.

24. Statocyst of Prawn:

Take an antennule either from the animal or else you will be provided one in the laboratory. Look along the dorsal surface of its precoxa and locate the depression. Now, make incisions, one each, along its lateral edges. Holding the posterior end of upper flap of precoxa pull it upwards and forwards.

Locate a round greyish structure attached to inner surface of precoxa. Hold it between thumb and first finger of the left hand and cut few thin sections. Select one section, wash, stain with Borax carmine, dehydrate, clear in Xylol and mount in Canada balsam Fig. 69.

25. Hastate Plate of Prawn:

For the study of hastate plate take the cardiac stomach of prawn. Cut open the cardiac stomach along mid dorsal line. The hastate plate is present along its floor. It is a triangular structure of chitinous nature. With the help of scissors cut along its edges. Wash thoroughly in water and stain in picro-indigo-carmine or Borax carmine Fig. 70.

  26. Salivary Glands of Cockroach:

Take a fresh anesthetized cockroach. Pin it in a dissecting tray. Cut open the animal making one incision on either lateral sides. Remove the tergal plates through the help of forceps. Look for the alimentary canal. Stretch and display the alimentary canal.

Pour water in tray so that the animal is completely submerged. Look for a pair of whitish, flattened and glandular structures floating in water, attached to the anterior end of alimentary canal in-front the crop. Cut the alimentary canal from behind the crop.

Cut the alimentary canal from behind the crop. Lift the crop and pull it forwards. Now, with the help of needles separate the salivary ducts from the crop. Trace the two ducts up to neck region. Now, trace the common efferent salivary duct inside the neck up to hypo pharynx.

Take out both the glands along with hypo pharynx and put them in watch glass. For mounting, spread the glands on a slide and fix them with 5% formalin in 70% alcohol. For purpose of staining they may be treated with Borax carmine. Fig. 71.

27. Pecten from Scorpion:

Take an anesthetized scorpion. Turn it upside down. Locate a pair of yellowish comb like structures in the second mesosomatic segment along the ventral surface. Remove one of them through scissors and boil with KOH (10%) for about 10 mts. Wash thoroughly with tap water, dehydrate up to 70% alcohol, stain with Picro indigo carmine, put through remaining grades of alcohol, clear in Xylol and mount in Canada balsam Fig. 72.

28. Book-Lung from Scorpion:

Take an anesthetized scorpion and cut open from dorsal side. Remove the carapace and all the viscera. Look for whitish, rounded structures in the mesosomatic segments, from 3rd to 6th, situated obliquely on sides along the inner surface. Take out one of them, treat with KOH (5%) and wash thoroughly with water. Use Borax carmine as stain, dehydrate, clear and mount Fig. 73.

29. Culture of Drosophila Fly:

Take a specimen jar and place some apple pulp or banana pulp along the bottom of the jar. Also, add 2 gms of moldex dissolved in 40 ml of boiling water to the fruit pulp. Keep this jar in open for a day so that the Drosophila flies may visit the food. If they enter the jar trap them inside. If no fly visits the food insert a pair of Drosophila fly into the jar and cover it with a muslin cloth.

Keep the jar in open for about a week. At the end of one week or 10 days you would observe the larve crawling above the pulp. You may use the larvae for chromosomal studies or meiotic preparation. If you want to study and prepare the life cycle leave the larvae as such and observe them to pass through various moultings till they develop into adult flies.