Medical Parasitology Lab.
Introduction
Topics 1. Introduction • Sample Collection and Preservation Methods • Wet mount Preparation
Normal saline 0.85% Iodine BMB
2. Artifacts 3. Concentration Techniques• Modified Formal- Ether Sedimentation technique• Acid- Ether Sedimentation technique
4. Flotation Techniques• By using Sheather’s solution• By using Sodium Chloride solution
5. Staining of parasitesRaed Z. Ahmed, Medical Parasitology Lab.,2012
6. Detecting of Blood Parasites• Thick and thin Blood smear
7. Counting of Helminthes Eggs in Feces8. Chemical Tests• Fecal PH test• Testing feces for Occult Blood• Fecal fat test• Stool reducing sugar test
9. Medical Entomology
Topics (cont.)
Raed Z. Ahmed, Medical Parasitology Lab.,2012
• Quizzes: 10ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ %• Assignment & Participation: 10ــــــــــــــــــــــــ %• Midterm examination: 30ــــــــــــــــــــــــــــــــــــ %• Final examination: 50ــــــــــــــــــــــــــــــــــــــــــ %– Practical exam: 20ـــــــــــــــــــــــــــــــــــ %– Written exam: 30ـــــــــــــــــــــــــــــــــــــ %
Grades
Raed Z. Ahmed, Medical Parasitology Lab.,2012
General Lab Objectives
1. To familiarizes the student with the most widely used techniques for detection of parasites.
2. To be able to identify the parasite stages.3. To learn the student, how to deal with risk samples.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
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
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
Parasites
Protozoa
CystTrophozoite
Oocyst
Helminths
PlatyhelminthsCestoda
EggsAdultSegments
Trematoda
EggsAdultCercariae
Nematoda
EggsAdultLarva
PARASITES STAGES
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
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.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Raed Z. Ahmed, Medical Parasitology Lab.,2012
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
• The container with the specimen should be clearly labeled with the following:o Patient’s name or number.o 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”
Collection of Fecal Specimen (cont.)
Raed Z. Ahmed, Medical Parasitology Lab.,2012
• 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
Collection of Fecal Specimen (cont.)
Raed Z. Ahmed, Medical Parasitology Lab.,2012
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.
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
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
• 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.
PVA (cont.)
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
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
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
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)
Long(months/years)
Long(months/years)
Limited
Preparation Easy Difficult Easy Easy
Quality of fixation Egg: ++ Egg: ++ Egg: ++ Egg: ++
Cyst: ++ Cyst: +++ Cyst: ++ Cyst: ++Troph’s: +/- Troph’s: +++ Troph’s: +++ Troph’s: ++
Formalin ether concentration
Possible Not possible Possible Possible
Permanent stained smear
Not possible Only Trichrome IHK, CB, mod. Ziel Neelsen
Not possible
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Preservation of worms 1. 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.
2. Trematodes These should be treated in a similar manner to cestodes, and mounted
with the ventral sucker uppermost3. 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
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
• 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.
Stool Analysis (cont.)
Raed Z. Ahmed, Medical Parasitology Lab.,2012
1. Macroscopic Examination Color Consistency
abnormal features adult worm or segment
2. Microscopic Examination WBC/ HPF RBC/ HPF Mucus
Yeast Cyst, trophozoite, or both Larvae, egg, or both
3. Chemical Examination Fecal PH test Fecal fatty acid testing Testing feces for Occult Blood
Fecal Sample Examination
Stool reducing substances testing
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Macroscopic Examination 1. 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
2. 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”
Macroscopic Examination (cont.)
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Macroscopic Examination (cont.)
3. 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. 4. 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
Noticeo 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.
o Formed specimens may be examined at any time during the first day, but should not be left overnight ( cyst may disintegrate).
o Excessive bulky stools may indicate conditions such as giardiasis.
Raed Z. Ahmed, Medical Parasitology Lab.,2012
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
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.
Microscopic Examination of wet mount (cont.)
Raed Z. Ahmed, Medical Parasitology Lab.,2012
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.
Microscopic Examination of wet mount (cont.)
Raed Z. Ahmed, Medical Parasitology Lab.,2012
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.
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
Materials and reagents: Microscopic slides. Cover slips. Applicator sticks. Marker or pen for labeling. Reagents:
Saline solution(isotonic) Lugols iodine(1% solution) BMB
Making Direct smear Microscopy
Raed Z. Ahmed, Medical Parasitology Lab.,2012
Wet mount procedures
Examine the slide on microscope:o 10X o 40X
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
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