NAME: NITHYA LATCHUMY A/P GEEVARETNAM CHETTIAR

MATRIX NUMBER: MLT 11011

LECTURER’S NAME: PROFESSOR MADYA DR. INIT A/P ITHOI

DATE OF SUBMISSION: 5TH APRIL 2013

COLLEGE OF MEDICAL LABORATORY TEHNOLOGY

LEVEL 2/SEMESTER 2

DIAGNOSTIC PARASITOLOGY(DMLT 2822)

ASSIGNMENT: DIAGNOSIS OF INTESTINAL PROTOZOA

INTRODUCTION

Intestinal protozoa can be divided into pathogenic and non-pathogenic protozoans. Laboratory diagnosis is important to differentiate between the harmless and the medically important parasites. This is most often based upon the morphology of respective organisms. There are some steps that need to be followed in order to successfully diagnose intestinal protozoan. The steps include:

Collecting specimens Preparation of specimens Evaluation under microscope

Preparation of stool specimen includes direct smear (wet mount), concentration method, fixative and preservation, staining methods and cultivation.

1

SPECIMENS AND COLLECTION OF SPECIMENSThe specimen is stool as the intestine is the habitat of the protozoa. Duodenal aspirate and material from ulcer or abscess can also be examined. The stool is collected in a dry, clean and leakproof screw-capped container. Loose and watery stool are examined for trophozoites while formed stool is examined for cysts. (Diagram 1.1)

Stool is best examined fresh as motile trophozoites may be seen and increases the chances to discover protozoa.The stool specimen must be free of urine and contaminants.If specimens need to be stored, they must be preserved immediately. They are then sealed well and stored properly.If preservative is unavailable, the specimen is kept in a refrigerator. These specimens are not suitable to examine motile form of protozoa.

2

Diagram 1.1

Screw-capped container to collect and store stool

PREPARATION OF STOOL SPECIMENS

a. DIRECT SMEAR (WET MOUNT)

Direct fecal smears can be used as a quick screening test to check for any intestinal parasite, but the small size of the sample limits its usefulness. Wet mounts are useful for detecting motile organisms. The disadvantages are dirty background and less detection of parasites. Protozoa are often detected via a direct fecal smear, and are best acquired from the surface of fresh feces. The procedure of direct smear is as follow:

First of all, a drop of saline or iodine is placed on a clean glass slide using a dropper.A pea-sized amount of fresh stool is taken using an applicator stick and mixed together with the saline/iodine drop.The smear is mounted with a coverslip and examined under microscope at 40x objective.Trophozoites, cysts, ova can be seen.

3

Feces is mixed with saline using an applicator stick

The fecal material is mixed until it is well dispersed to produce a smear

b. CONCENTRATION METHODS

A concentration procedure is performed mainly to separate the parasites from fecal debris. The concentration procedure not only increases the numbers of parasites in the sediment but it also unmasks them, making them more visible by removing debris.

Formalin ether sedimentation technique

Formalin acts both a fixative and preservative of protozoan eggs, larvae and cysts. The specific gravity of protozoan cysts and helminthes eggs is greater than that of water. Fecal debris is extracted into the ether phase so that the parasitic forms can be separated and then regimented by centrifugation. The procedure is as follow:

A small amount of stool is mixed with 5 ml of 10% formal saline in a centrifuge tube using an applicator stick.

The mixture is then strained into a paper cup containing cotton gauze as a filter. The filtered suspension is poured back into centrifuge tube. 10% formal saline is added until it reaches 8 ml. 2 ml of ether is added. The centrifuge tube is plugged with a rubber stopper and shaked vigorously for few

minutes. It is then centrifuged at 2500 rpm for 2 minutes. The debris plug is loosened with an applicator stick and the supernatant is discarded in a

single hand motion. The sediment is allowed to mix with the remaining fluid and placed on a clean glass

slide. The sediment is mounted with a coverslip and viewed under microscope.

4

BRINE FLOATATION TECHNIQUE

This technique is best used in diagnosis of helminthes but can also be used to recover protozoa which are light weighted. The protozoa which are lighter than the solution will float and can be lifted with glass slide to be examined. The procedure is as follow:

Saturated solution of sodium chloride is prepared (brine solution). A small amount of feces is mixed with 2ml of brine solution in a

bijou bottle. More brine solution is added till the brim of bijou bottle while

stirring. Drops of brine solution are added up to the surface of the bottle

without overspilling. A clean glass slide is placed over the solution surface and left for

30 minutes exactly. The slide is lifted in a single hand motion and examined under

microscope.

5

Bijou bottle used in brine floatation technique.

FIXATIVE AND PRESERVATIONPreservation of specimens is necessary when stool specimens cannot be examined within the prescribed time interval. Various preservatives are available with the two most commonly used being Merthiolate iodine formaldehyde (MIF) and Polyvinyl alcohol (PVA).

FIXATIVE ADVANTAGE DISADVANTAGEPolyvinyl alcohol (PVA) Permanent smears can

be made and stained with trichrome

Zinc is preferred over copper

No mercuric chloride

Staining not consistent Organism morphology

may be poor Copper-morphology of

cysts and trophozoites is poor

Merthiolate iodine formaldehyde (MIF)

Components both fix and stain organisms

Easy to prepare Long shelf life Useful for field surveys Suitable for

concentration procedures

Not suitable for some permanent smears stained with trichrome

Inadequate preservation of morphology of protozoan trophozoites

Iodine interferes with other stains and fluorescence

Iodine may cause distortion of protozoa

10% Formalin All purpose fixative Easy to prepare Long shelf life Good preservation of

morphology of helminth eggs, larvae, protozoan cysts, and coccidia

Suitable for concentration procedures and UV fluorescence microscopy

Suitable for acid-fast, safranin, and chromotrope stains

Compatible with immunoassay kits and

Not suitable for some permanent smears stained with trichrome

Inadequate preservation of morphology of protozoan trophozoites

Can interfere with PCR, especially after extended fixation time

6

UV fluorescence microscopy

Polyvinyl alcohol (PVA)

This is a mixture of fixative and water-soluble resin that is specifically used to fix and preserve trophozoites of intestinal amoebic organisms. These trophozoites are very fragile and will become distorted or disintegrate completely within a few hours after stool passage. This fixative will preserve trophozoites for long periods of time and will make it easier to identify them. PVA is primarily used for preserving fresh specimens to be shipped to central laboratories. Permanently stained films can be prepared from the preserved material. The solution serves as an adhesive as well as a preservative and prevents the loss of organisms during staining. This is advantageous when preparing smears from liquid specimens.

Preparation of reagents : Polyvinyl Alcohol Fixative (PVA

Ethyl alcohol (95 %) 50 ml

Saturated mercuric chloride 100 ml

Glacial acetic acid 10 g

Glycerol 3 ml

Polyvinyl alcohol powder (PVA) 9 g

Procedure:

STEP 1: Mix a portion of feces with three times the amount of formalin.

STEP 2: Allow settling for at least one hour and store in screw-cap bottles.

STEP 3: If shipment or prolonged storing is required, dip the top portion of the tightly fastened container, to include the cap, two or three times into a hot paraffin bath. This will prevent spillage and reduce the rate of evaporation

7

PERMANENT STAININGIt is a routine diagnosis. Two methods are used in permanent staining of intestinal protozoa, trichrome and iron-hematoxylin.

TRICHROME STAINING METHOD

The Trichrome stain is a rapid staining procedure which provides excellent differentiation of internal structures of intestinal parasites as well as facilitating the separation of these organs from background material and artifacts. The Trichrome stain (Wheatley Modification) is to be used for staining of intestinal protozoan cysts and trophozoites in PVA fixed specimens. Sediments from the Formalin-ethyl acetate concentration technique cannot be stained by this method. All liquid stool specimens should receive a trichrome stain as trophozoites will occur in liquid specimens and not formed specimens. Soft stool specimens may be stained if requested or the concentrates are suspicious.

Preparation of staining solution

1) Tincture of iodine: Iodine crystals 7g Potassium iodine 5g 70 % alcohol 100ml

Procedure:

1. The iodine and potassium iodide is grinded in a mortar2. The mixture is then dissolved and rinsed in alcohol3. Tincture is stored in a brown bottle

2) Modified trichrome stain

Chromotrope 2R 0.6g Light green SF 0.3g Phosphotungstic acid 0.7g Acetic acid 1.0ml Distilled water 100 ml

8

Trichrome staining procedure

1. A thin smear is made of fresh stool on a slide2. After the smear is dried, it is placed in PVA fixative3. It is then let to stand overnight4. The smear is then stained as follow :

Solution DurationTincture of iodine 1 minute

70 % alcohol 1 minuteTrichrome stain 8 minute

Acid alcohol 10 secondsAbsolute alcohol 1 minute

xylene 1 minute

5. It is then mounted with deepex or Canada balsam

The outcome of trichrome staining(100x oil immersion)

Cytoplasm stain blue-green Nucleus stain red

9

Protozoa-Entamoeba histolytica cyst

Glycogen vacuoles are clear( dissolved by fixatives Background is usually various shades of green

IRON HEMATOXYLIN STAIN

This staining method gives maximum detail and more reliable outcome but is more time consuming than the trichrome staining. It is often used with fresh stool

Procedure:

1. Direct smear of fresh stool2. Slides are immersed in schaudinn’s solution for overnight3. Dip slides in iodine tincture for 5 min 4. Place slides in 70% ethanol for 5 min.5. Place slides in 50% ethanol for 2 min.6. Wash well with distilled water.7. Place slides in hematoxylin working solution for 10 min.8. Place slides under running tap water (best if tepid) for 10 min.9. Differentiate slides (one by one) in destaining solution or for 30 s.10. Place slides under running tap water for 10 min.11. Place slides in 95% ethanol for 5 min.12. Place slides in 100% ethanol for 5 min.13. Place slides in 100% ethanol for 3 min.14. Place slides in xylene for 5-10 min.15. Mount slides with DPX

10

Entamoeba histolytica

RESULTS (MICROSCOPIC EVALUATION)

11

Entamoeba histolytica cyst- wet mount (high power)

Entamoeba histolytica trophozoite- iron hematoxylin oil immersion

12

Entamoeba coli cyst-iodine stain oil immersion

Entamoeba coli cyst and trophozoite- trichrome stain oil immersion

Iodamoeba buetschlii cyst- iodine stain high power

13

Iodamoeba buetschlii trophozoite and cyst- trichrome stain oil immersion

Giardia lamblia trophozoite- trichrome stain oil immersion

Giardia lamblia cyst- iron hematoxylin stain oil immersion

14

Balantidium coli trophozoite- trichrome stain oil immersion

Balantidium coli cyst in formalin preserved stool- unstained high power

Cryptosporidium parvum oocyst- acid fast stain oil immersion

15