1. Medical Parasitology Lab.
Introduction

2. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Topics
Introduction
Sample Collection and Preservation Methods
Wet mount Preparation
Normal saline 0.85%
Iodine
BMB
Artifacts
Concentration Techniques
Modified Formal- Ether Sedimentation technique
Acid- Ether Sedimentation technique
Flotation Techniques
By using Sheather’s solution
By using Sodium Chloride solution
Staining of parasites
Raed Z. Ahmed, Medical Parasitology Lab.,2012

3. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Topics (cont.)
Detecting of Blood Parasites
Thick and thin Blood smear
Counting of Helminthes Eggs in Feces
Chemical Tests
Fecal PH test
Testing feces for Occult Blood
Fecal fat test
Stool reducing sugar test
Medical Entomology
Raed Z. Ahmed, Medical Parasitology Lab.,2012

4. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Grades
Quizzes: ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ10%
Assignment & Participation: ــــــــــــــــــــــــ10%
Midterm examination:ــــــــــــــــــــــــــــــــــــ 30%
Final examination: ــــــــــــــــــــــــــــــــــــــــــ50%
Practical exam: ـــــــــــــــــــــــــــــــــــ20%
Written exam: ـــــــــــــــــــــــــــــــــــــ30%
Raed Z. Ahmed, Medical Parasitology Lab.,2012

5. General Lab Objectives
To familiarizes the student with the most widely used techniques for detection of parasites.
To be able to identify the parasite stages.
To learn the student, how to deal with risk samples.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

6. What is the stool or feces?
Waste residue of indigestible material (cellulose during the previous 4 days),
Bile pigments and electrolyte,
Intestinal secretions, including mucus,
Leukocytes that migrate from the bloodstream,
Epithelial cells that have been shade,
Bacteria and Inorganic material(10-20%) chiefly calcium and phosphates. Undigested and unabsorbed food
Raed Z. Ahmed, Medical Parasitology Lab.,2012

7. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Fecal Specimen
Fecal specimen are examined for protozoa, helminthes larvae or eggs.
The stages of protozoa found in stool samples are trophozoites and cysts or oocysts.
The stages of helminthes usually found in the stool samples are eggs and larvae, through whole adult worms or segment of worms may also be seen.
Adult worms and segment of tape worms are usually visible to naked eye, but eggs, larvae, cyst, oocyst and trophozoites can be seen only with the microscope.
In order to see these structure, the fecal material must be properly collected and examined.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

8. Parasites stages Parasites Protozoa Cyst Trophozoite Oocyst Helminths
Platyhelminths
Cestoda
Eggs
Adult
Segments
Trematoda
Cercariae
Nematoda
Larva

9. Number of Specimens and Collection Time
No technique is 100% successful in detecting parasites by single stool examination, and at least three serial stools must be examined before a patient can be considered free from infections in which stages of parasites would be expected to be free in the faeces.
Because of the intermittent passage of certain parasites, the possibility of finding organisms is increased by examining multiple specimens.
It is suggested that 3 specimens, collected at 2 to 3 day intervals, should be examined both pretreatment and post treatment (to ensure eradication of documented pathogenic protozoa).
Raed Z. Ahmed, Medical Parasitology Lab.,2012

10. Collection of Fecal Specimen
Because of fragile nature of many intestinal parasites, and the need to maintain their morphology for accurate identification.
Reliable microscopic diagnosis can not made unless the stool is collected properly.
The stool specimen must be enough for satisfactory examination of fresh feces uncontaminated by urine, dirt*, water or other body secretion such as menstrual blood.
If the sample is too small or contaminated with urine, it should not be accepted. Ask the patient to pass another specimen.
* Urine will destroy the amoebic trophozoites and dirt will interfere with examination
Raed Z. Ahmed, Medical Parasitology Lab.,2012

11. Collection of Fecal Specimen (cont.)
Collect the specimen in a clean dry screw-capped top container
Collect the stool with a clean tongue blade or similar object.
The container should be free from antiseptics and disinfectant.
Random specimen: suitable for qualitative testing for blood and microscopic examination.
Timed specimen: for quantitative fecal testing such as fecal fat testing, because of the variability of bowel habit and the transit time required for food to pass through the digestive tract, so the most representative sample is a-3 day collection.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

12. Collection of Fecal Specimen (cont.)
The container with the specimen should be clearly labeled with the following:
Patient’s name or number.
Date and time of collection.
All samples should be accompanied by a requisition form from the physician giving relevant clinical details and recent travel history.
Samples and forms from patient with a confirmed or suspected diagnosis of certain infectious diseases such as AIDS or hepatitis should be clearly labeled with “Biohazard”
Raed Z. Ahmed, Medical Parasitology Lab.,2012

13. Collection of Fecal Specimen (cont.)
Most viable parasites are susceptible to desiccation or temperature variation.
If time lapse between collection and observation is considerable, i.e. more than 4 days, it may be necessary to add some form of preservative to faeces specimen to retain morphology.
Formed samples can be kept in a refrigerator at 4 C° for a short time, but not in incubator.
Any whole worms or segments passed should be placed in a separate container
Raed Z. Ahmed, Medical Parasitology Lab.,2012

14. Preservation methods for fecal specimens
Preservation allows fecal samples to be examined after a delay in delivery or postage or testing.
Many methods for the preservation of stool samples and permanent staining procedures.
The most common fixatives are:
Polyvinyl Alcohol, PVA
Merthiolate Iodine Formalin, MIF
Sodium acetate Acetic acid Formalin, SAF
Formalin.
Bayer’s solution*
The preservatives used have different effect on the various stages of the parasites.
* This technique was found to be very useful, particularly for cyst.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

15. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Formalin
Formalin 4% has been used for many years as an all purpose fixative that is appropriate for helminthes eggs and larvae and for protozoan cyst.
The fixative has a long shelf life.
Concentration methods, like formalin- ether concentration can be performed from the preserved stool samples without loss of concentration abilities.
The major disadvantage of formalin is that permanent staining procedures can't be performed from formalin preserved stool samples.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

16. Raed Z. Ahmed, Medical Parasitology Lab.,2012
PVA
This fixative is recommended for the preservation of the trophozoite and cyst stages of the intestinal protozoa, and also suitable for helminthes eggs and larvae.
The preservation of the two stages of protozoa is excellent.
The PVA is a plastic resin that serves as adhesive for the stool material.
Has a long shelf life ( months to years ).
Concentration methods can’t performed from the specimen preserved by PVA.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

17. Raed Z. Ahmed, Medical Parasitology Lab.,2012
PVA (cont.)
The greatest advantage of this fixative is that a permanent stain can be prepared from stool specimen preserved by PVA, giving excellent result with trichrome staining.
Specimen preserved by PVA can’t be used with immunoassay kits.
Toxic, because contain mercury compound.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

18. Raed Z. Ahmed, Medical Parasitology Lab.,2012
SAF
Good routine fixative for protozoan cyst and trophozoites, helminthes eggs, and larvae.
Has long shelf life. ( months to years).
The preserved stool samples permits concentration techniques, most monoclonal detection kits, and permanent staining.
Unlike the PVA, the SAF fixative has poor adhesive properties when SAF preserved samples are used to prepare permanent stained smears. ( Mayer’s albumin has been recommended as an adhesive.
The combination of SAF preserved material and CB, IHK, and mod. Ziel Neelsen provides excellent staining of protozoan where staining of SAF preserved material with Trichrome gives poor results.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

19. Raed Z. Ahmed, Medical Parasitology Lab.,2012
SAF (cont.)
Specific advantages of the use of SAF are:
SAF preserved material can be used for concentration techniques and permentant stained smears (CB, IHK).
SAF preserved material can be used for some immunoassay methods.
SAF is easy to prepare and has a long shelf life.
Unlike the PVA, the SAF fixative contain no mercury compounds. It is therefore much less toxic than PVA
Raed Z. Ahmed, Medical Parasitology Lab.,2012

20. Raed Z. Ahmed, Medical Parasitology Lab.,2012
MIF
This fixative was originally developed as a screening procedure for intestinal parasites.
MIF combines preservation and staining for most kinds and stages found in faeces.
It’s contains Merthiolate, Iodine, and Formalin.
The preserved material permits concentration techniques.
The major disadvantages are the short shelf life ( duo to iodine) and permanent stained smears can’t be prepared from MIF preserved material.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

21. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Fixative used for the preservation of stool samples: an overview of the advantages and disadvantages:
Formalin 4%
PVA
SAF
MIF
Toxicity
+/-
+++
( duo to Hg )
Shelf life
Long
(months)
(months/years)
Limited
Preparation
Easy
Difficult
Quality of fixation
Egg: ++
Cyst: ++
Cyst: +++
Troph’s: +/-
Troph’s: +++
Troph’s: ++
Formalin ether concentration
Possible
Not possible
Permanent stained smear
Only Trichrome
IHK, CB, mod. Ziel Neelsen
Raed Z. Ahmed, Medical Parasitology Lab.,2012

22. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Preservation of worms
Cestodes
Wash in water to remove the mucus. Large tapeworms such as Taenia can be washed for several hours to relax the musculature, and can then be fixed in 10% formol saline b/w two glass slides to give flatter specimens.
Trematodes
These should be treated in a similar manner to cestodes, and mounted with the ventral sucker uppermost
Nematodes
Adult are washed in saline to remove mucus. Worms up to about 7 cm in length are fixed in hot(60-70˚C) 70% alcohol, which straightens out living worms, except those which have natural curvatures at the head or the tail. Alternatively, they can be fixed in hot 5% formalin.
Large worms such Ascaris lumbricoides can be fixed and preserved in cold 5% formalin
Raed Z. Ahmed, Medical Parasitology Lab.,2012

23. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Stool Analysis
A stool analysis is a series of tests done on a stool (feces) sample to help diagnose certain conditions affecting the digestive tract .
These conditions can include infection (such as from parasites, viruses, or bacteria), poor nutrient absorption, or cancer.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

24. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Stool Analysis (cont.)
Laboratory analysis includes macroscopic, microscopic examination, chemical tests, and microbiologic tests.
The stool will be checked for color, consistency, weight (volume), shape, odor, and the presence of mucus and parasites stages.
The stool may be examined for hidden (occult) blood, fat, meat fibers, bile, white blood cells, and sugars called reducing substances.
The pH of the stool also may be measured.
A stool culture is done to find out if bacteria may be causing an infection.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

25. Fecal Sample Examination
Macroscopic Examination
Color
Consistency
abnormal features
adult worm or segment
Microscopic Examination
WBC/ HPF
RBC/ HPF
Mucus
Yeast
Cyst, trophozoite, or both
Larvae, egg, or both
Chemical Examination
Fecal PH test
Fecal fatty acid testing
Testing feces for Occult Blood
Stool reducing substances testing
Raed Z. Ahmed, Medical Parasitology Lab.,2012

26. Macroscopic Examination
Color:
Brown is normal color, results from the intestinal oxidation of stercobilinogen to urobilin.
Bright red to dark red to black stools occur when iron or bismuth is taken or when there is intestinal hemorrhage.
Pale yellow stools indicate the biliary obstruction, steatorrea, and also associated with diagnostic procedures that use barium sulfate.
White stools occur when there is obstructive jaundice.
Green stool may observed in patient taking oral antibiotic, because of oxidation of bilirubin to biliverdin.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

27. Macroscopic Examination (cont.)
Consistency: degree of moisture, will be a guide as to whether the trophozoite stage or the cyst stage of protozoa is likely to present.
Formed, write “F”
Soft , write “S”
Loose , write “L”
Watery , write “W”
Raed Z. Ahmed, Medical Parasitology Lab.,2012

28. Macroscopic Examination (cont.)
Abnormal features:
If mucus is present writ “M”, and “B” if blood is present.
The presence of mucus coated stool is indicative for intestinal inflammation or irritation.
Adult worm or segments
The feces may have adult helminthes or segments present such as Ascaris lumbricoides, Enterobius vermicularis, or Taenia spp. gravid segment, these can be seen by naked eye.
And frequently motile for several days and may migrate to the top of the container.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

29. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Notice
If several specimens are received at the same time; those containing blood and mucus should be examined first, followed by liquid specimens. These specimens are the most likely to contain amoebic trophozoites ( which die soon after being passed), and must be examined within 1 hour after passage.
Formed specimens may be examined at any time during the first day, but should not be left overnight ( cyst may disintegrate).
Excessive bulky stools may indicate conditions such as giardiasis.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

30. Microscopic Examination of wet mount
Wet mount is the simplest and easiest technique for the examination of feces, and this method should be performed in all laboratories at peripheral level.
A wet mount can be prepared directly from fecal material or from concentrated specimens.
The basic types of wet mount that should be used for each fecal examination are normal saline (0.85% NaCl), iodine, and buffered methylene blue.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

31. Microscopic Examination of wet mount (cont.)
The Saline Wet Mount
Is used for the initial microscopic examination of stool specimens.
It is employed primarily to demonstrate worms eggs, larvae. Protozoan trophozoites, and cysts.
This type of mount can also reveal the presence of red blood cells and white blood cells.
If the presence of amoebic trophozoites is suspected, warm saline (37˚C) should be used.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

32. Microscopic Examination of wet mount (cont.)
The Iodine Wet Mount
Is used mainly to stain glycogen and the nuclei of cysts, if present.
Cysts can usually be specifically identified in this mount.
Trophozoite can not be revealed by this type of wet mount, because iodine kill trophozoite.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

33. Microscopic Examination of wet mount (cont.)
The Buffered Methylene Blue Wet Mount
Should be prepared each time amoebic trophozoites are seen in a saline wet mount, or when their presence is suspected.
BMB stains amoebic trophozoites, but not stain amoebic cysts, flagellate trophozoites or flagellate cysts.
BMB stain is appropriate only for fresh unpreserved specimens.
BMB stain live organism only, it isn’t used on preserved samples in which the organism have been killed
Wait for five minutes to allow the stain to penetrate the trophozoites. It will overstrain the trophozoites in 30 minutes.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

34. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Notice
Formed stool: take the portion of stool from an area to include inside and outside parts of the specimen.
Stool with mucus: if mucus is present, label a second slide with the patient’s name or number. Put a drop of saline on the slide, pick up a small portion of mucus and mix with the saline. Trophozoites, if present, are sometimes more readily found in mucus than in the solid parts of the stool.
Loose watery stool: if mucus is not present, pick up a small portion of the stool (any part) and mix with the saline.
Raed Z. Ahmed, Medical Parasitology Lab.,2012

35. Making Direct smear Microscopy
Materials and reagents:
Microscopic slides.
Cover slips.
Applicator sticks.
Marker or pen for labeling.
Reagents:
Saline solution(isotonic)
Lugols iodine(1% solution)
BMB
Raed Z. Ahmed, Medical Parasitology Lab.,2012

36. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Wet mount procedures
Examine the slide on microscope:
10X
40X
Raed Z. Ahmed, Medical Parasitology Lab.,2012

37. Raed Z. Ahmed, Medical Parasitology Lab.,2012
Result of Examination
If no parasites are found:
“No ova or parasites seen”, and specify whether this result was obtained by direct examination or by a concentration method (name method used).
Never state categorically: “No parasites”
If any parasites are seen, write the scientific name of the parasite with stages
Example: Giardia lambilia cyst
Raed Z. Ahmed, Medical Parasitology Lab.,2012