1.Introduction Sample Collection and Preservation Methods Wet mount Preparation Normal saline 0.85%...

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 Feces

8. 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


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.


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


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.


Parasites

Protozoa

CystTrophozoite

Oocyst

Helminths

Platyhelminths

Cestoda

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).


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.



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.


• 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”



• 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



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.


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.


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.


• 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.)


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.


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


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.


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


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 uppermost

3. 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.


• 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.)


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



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.


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.)


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.


Notice

o 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.


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.


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.)


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.




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.


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


Wet mount procedures

Examine the slide on microscope:o 10X o 40X


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


1.Introduction Sample Collection and Preservation Methods Wet mount Preparation Normal saline 0.85%...

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