ZL 4064- Parasitology

Practical 2- Parasitological survey on toad/frog

Introduction

Parasitological surveys are important to understand the potential hosts of certain parasites and to
obtain information on the potential for transmission of these parasites. Amphibians are host to a
wide range of parasites including Protozoa, Trematoda, Cestoda, Acanthocephala, and
Nematoda. They provide a rich parasite fauna for laboratory studies which can be either external
or internal parasites.

Objectives

• To acquire knowledge on the endo and ectoparasites present in Amphibians

• To gain experience on how to conduct a parasitological survey on Amphibians.
• To gain knowledge on the different techniques used in a parasitic survey.
• To be able to identify different endo and ectoparasites present.

Materials

1)Preparation of amphibian saline:

• Distilled water - 100 ml
• NaCl - 0.65 g
• KCl -0 .014 g
• CaCl2-0.012
• NaHCO3 – 0.02g,
• NaH2PO4 – 0. 001 g
• Glucose -0.2 g (Optional)

2)Ectoparasitic survey:
• Paint brush
• Amphibian saline
• Petri dish
• Scalpel
• Slides, Cover slips
• Light microscope

3)Frog dissection and endoparasitic survey:

• Watch glasses, Petri dishes (About 10 from each)
• Dissecting scissor, scalpel, pins
 • Dissecting needle, threads
• Amphibian saline
• Distilled water
• Light microscope
• Dissecting microscope
• Gloves, tissues
• Dissection tray
• Slides, coverslips
• Stoppered tubes about 10
• Pointed Pasture pipette
• Cotton wool
• Absolute methanol

4)Fecal analysis:
• Direct fecal smears
• Toothpicks
• Coverslips, slides
• Saline solution
• Lugol’s Iodin

5)Fecal concentration method

• Centrifuge tube
• Small beakers
• Slides, coverslips
• Pipettes
• 10% Formalin
• Ether

6) NaCl flotation technique:

• Saturated NaCl solution
• Test tubes
• Coverslips, slides
• Pipettes
7) Preservation: Trematodes, Cestodes, Nematodes
• cold 0.85% saline
• Alcohol, Formalin, and Acetic Acid (AFA)
• 70 % Alcohol
• Hot buffered Formalin
• Alcohol and Glycerin

Methods

External examination

First, Examine the toad’s/frog’s body surface well including armpits and webs (both fingers and
toes). If parasites are present, remove carefully using a paint brush. Use the saline solution to
place the brushed materials.
Irrigate both nasal and buccal cavities with the saline solution using a fine pipette and use the
resulted fluid to observe under the light microscope.

Internal Examination

Pin the euthanized toad/frog on the provided dissection tray. Make an incision from the anus to
the buccal cavity through its median line and pin the resulted two skin flaps to either side.
Dissect the exposed internal organs and place them in separate saline solutions. Puncture the
heart with a fine pipette to get the blood and prepare both thin and thick smears. Use each organ
to prepare a tissue smear and observe it under a light microscope. Separate the portions of the
digestive tract such as the esophagus, stomach, duodenum, and rectum using threads. Place the
separated portions in different saline solutions and split them open to observe the constitutes by
preparing smears.

Carry out the fecal analysis for any fecal materials present.

Direct fecal smears-saline and iodine wet mount preparations

Place one saline drop onto the centre of the left half and one iodine drop onto the centre of the
right half of a glass slide (or separate slides). Place similar quantities of faecal matter to each
drop using an applicator stick and mix it well. Cover both using a coverslip and observe under
the light microscope.
NaCl floatation technique

Add 1g of feces into a 2ml of NaCl in a test tube and mix it well. Filter the suspension through a
strainer into a 15 ml test tube. Fill the test tube full using the saturated NaCl to have a convex
solution surface (add last few drops using a dropper). Place a cover slip on top of it and left it for
about 30-45 minutes. Use the cover slip to examine under the light microscope. Count the
number of parasitic stages.

Fecal concentration procedure- formalin-ether

Add a portion (1.0- 1.5 g) of fecal matter into formalin in a centrifuge tube and stir it to obtain a
suspension. Strain the suspension through a 400μm mesh sieve directly into a different centrifuge
tube. Add more than 10 % formalin to the suspension to bring the total volume to 10 ml. Add 3.0
ml of ether to the tube and mix it well (insert a rubber stopper and shake vigorously for 10
seconds). Remove the stopper and centrifuge at 400- 500 g for about 2-3 min to obtain 4 layers;
top layer of ether, a plug of fatty debris that is adherent to the wall of the tube, a layer of
formalin, and sediment. Lose the plug of fatty debris using an applicator stick by a spiral
movement and pour the top 3 layers in a single movement allowing the tube to drain inverted for
at least five seconds. Mix the fluid with the sediment after flowing of the residual fluids on to
sediment. Use this to transfer a drop of the suspension to a slide for examination under a
coverslip and iodine can be used to stain it.

Preservation And Fixations

Trematodes

Fixation/Preservation

Living specimens should be placed in cold 0.85% saline for 30 minutes to several hours
(depending on the size of the worm) before fixation. Place the specimen on a slide in a petri dish
and cover with another slide or a cover glass to flatten the worm (do not apply pressure)

Fixatives

• Alcohol, Formalin and Acetic Acid (AFA)

Should be used hot (60-630C).
• 70% alcohol
Transfer to 70% alcohol for long term storage.
Cestodes

Fixation/ Preservation

• Formalin
Hot (60-63°C) buffered formalin is used. If the whole worms are immediately swirled around in
the container of fixative, they will be rapidly fixed with minimal contraction of the proglottids.

• Alcohol, Formalin and Acetic Acid (AFA)

Fixes tape worm tissue well, however the acid will dissolve the characteristic calcareous
corpuscles of tape worm tissue.

• 70% alcohol
Transfer to 70% alcohol for long term storage.

Nematodes

Fixation/ Preservation
It is recommended that nematode larval stages be killed/ fixed in hot water (60-63°C) and then
be transferred to a fixative. Direct preservation in fixatives may cause the cuticle to become
‘sticky’ and the larvae will become damaged when they adhere to the glass container.

Fixatives
• Alcohol and glycerin
Excellent for most nematodes. Should be used hot (60-63°C).
Can be left in this solution indefinitely (fixed specimens should be checked routinely every six
months for possible fixative replacement)

Check out:
https://www.youtube.com/watch?v=aRZ6CnACUoc
https://parasitology.ou.ac.lk/virtual-diagnostic-techniques-for-parasitology/