In this article we will discuss about the Microscopic Examination of Stool for Parasitic Infestation of Guts.
A. Sample:
(i) Freshly voided stool in a stool collection container is preferable, especially for trophozoites of E. histolytica and G. lamblia and helminthic larva.
(ii) NIH swab for ova of E. vermicularis.
(iii) Sigmoidoscopic specimen of large gut ulcer areas — for trophozoites of E. histolytica.
B. Transport:
Stool should be examined as quickly as possible. If there is delay, it is preserved with Polyvinyl Alcohol (PVA). Stool: PVA ratio should be 1:10. Otherwise one part stool sample in 3 parts 10% formaldehyde in normal saline solution may be used.
C. Direct saline (0.85%) and iodine (1:1 mixture of 0.5% Lugol’s iodine and 50% Acetic acid solutions) mount preparation:
i. Label a clean dry glass slide with the specimen number.
ii. Wear protective latex gloves.
iii. Place one large drop each of normal saline and iodine on the centre of either end of the slide.
iv. Using a wooden or broom-stick applicator, lift as small portion of stool from several part of the sample. Take care not to miss the mucous and visible bloody areas.
v. Make an uniform emulsion of the stool on the slide with the normal saline drop. The emulsion should not be too thick or too thin.
vi. Using the same applicator, make a similar emulsion with lugol’s iodine.
vii. Discard the applicator in freshly prepared 1% sodium hypochlorite solution.
viii. Place cover slips on emulsions on slide, taking care not to form air bubble.
Microscopic examination:
Using low power objective (10 x) scan the mounts thoroughly for helminthic ova and larva. Next scan the mounts with high power objective (40 x) for protozoa.
Characteristic movement of the protozoa, often is of diagnostic importance, is demonstrated in saline mount. Intracellular details (e.g.. nucleus, nuclear chromatin, chromatoid bar, glycogen vacuole etc.) are better visualised in iodine mount.
D. Concentration methods:
Indication:
Scanty parasite in stool and difficulty in demonstration by direct wet mount.
Methods:
1. Floating method:
(a) Saturated sodium chloride solution floatation.
(b) Zinc sulphate centrifugal floatation.
2. Sedimentation method:
(c) Formol ether sedimentation technique.
(a) Saturated NaCl solution floatation method:
i. In a small container place about 10 g. of stool.
ii. Using an applicator and with gradual addition of saturated sodium chloride solution (sp. gravity 1.2) make an emulsion. Continue the procedure till the container is full almost to the brim.
iii. Coarse materials on top are then removed.
iv. Add little more saline to form a convex meniscus at top.
v. Place carefully a glass slide (size large enough to cover the open end of the container) in contact with convex meniscus of saline.
vi. Wait for 15 minutes.
vii. Carefully lift the slide and invert. Place a cover slip over the preparation side.
viii. Examine under microscope, first directly, and then, if required, with addition of lugol’s iodine.
The method is useful for demonstration of fertilised eggs of Ascaris lumbricoides, Enterobius vermicularis, hookworm, Trichuris trichiura. Unfertilized egg of Ascaris, ova of Cestodes and Trematodes, protozoal cysts cannot be demonstrated by this technique, as these are sedimented.
(b) Zinc sulphate centrifugal floatation:
i. In a 15-20 ml capacity centrifuge tube mix 10 gm. of stool with distilled water to form a suspension.
ii. Filter the faecal suspension through two layers of gauze in a funnel into another centrifuge tube.
iii. Spin at 1800 r.p.m. for 5 minutes. Discard the supernatant, and re-suspend the deposit in freshly added distilled water. Repeat centrifugation continue the procedure of washing repeatedly till the supernatant is clear.
iv. Then add Zn SO4 solution (sp. gr. 1.180) to re-suspend the deposit. Centrifuge at 1800 r.p.m. for 3 minutes.
v. Place few loopful of surface fluid from meniscus on a glass slide. To one add one drop of lugol’s iodine. Place cover slips and examine microscopically.
The method is useful to detect egg of hookworm, fertilized egg of Ascaris, Trichuris, tapeworm and protozoan cysts. It fails to demonstrate un-fertilised Ascaris eggs, nematode larvae, eggs of most trematodes and large tapeworms (e.g.. Taenia).
(c) Formol ether sedimentation technique:
i. Mix ½ t.s.f. stool in normal saline thoroughly. The suspension is filtered through 2 layers of gauze in a funnel, into a 15 ml centrifuged tube (screw capped).
ii. Discard the supernatant after centrifugation at 1800 r.p.m. for 5 minutes.
iii. Re-suspend the deposit in 10 ml of 10% formalin and allow to stand for 5 minutes.
iv. Then add 3 ml ether and close the screw cap. Shake the content vigorously.
v. Next centrifuge at 1500 r.p.m. for 10 minutes.
vi. Sediment is to be used for preparation of wet mounts and smears for staining by modified Z.N. method or Trichrome stain.
The method is useful for demonstration of all varieties of parasitic elements in faeces. This is a must for detection of oocysts of coccidian protozoa (e.g.. Cryptosporidium, Cyclospora, Isospora and Microspora) in diarrhoeic stool of AIDs cases.
Microscopic Morphology of Helminthic Eggs in Stool in Wet Mount:
Ascaris lumbricoides:
Three type of morphology is found in stool:
(a) Fertilised egg,
(b) Unfertilized egg,
(c) Degenerated egg (in older sample without preservative).
Fertilised Egg (Fig. 8.1):
Shape:
Round or oval.
Size:
Length 60-75 μm x breadth 40-50 μm.
Colour:
Bile stained (brown or yellowish brown).
Saturated salt solution:
Egg floats.
Features:
1. Large un-segmented ovum at innermost part.
2. Coarse yolk granules obscures the nucleus.
3. Inner vitelline membrane surrounds the ovum. Often difficult to see.
4. Ovum with vitelline membrane is surrounded by thick translucent shell.
5. Two crescentic space in between ovum and vitelline membrane at two poles.
6. Outer coat — a bile stained thick albuminous coat showing rugosity or mammillations. Sometimes, this coat is found missing, when it is called decorticated egg.
Un-fertilised Egg (Fig. 8.2):
Shape:
Elongated, narrow and more elliptical.
Size:
Length-about 80 μm x breadth about 55 μm.
Colour:
Bile stained.
Salt floatation:
Heaviest of all helminthic eggs, does not float.
Features:
1. Irregular outer albuminous coat
2. Thinner egg shell with vitelline membrane
3. Ovum atrophied
4. Soap bubble like mass of granules
Degenerated Egg:
Similar to fertilised egg, but mammillation is absent (decorticated), ovum not visible, yolk is coarser.
It is to be noted that both fertilised and unfertilized ora may be found in the same sample.
Trichuris trichiura (Fig. 8.3):
Shape:
Barrel like with protuberant mucous plug at each pole.
Size:
Length-about 50 nm x breadth about 25 μm.
Colour:
Dark brown to yellow brown due to bile staining.
Floatation:
Floats in saturated solution of common salt.
Features:
1. Un-segmented bile stained granular mass is the ovum, encased in egg shell.
2. Smooth egg shell is bilayered. Outer one is bile stained and inner one is translucent.
[Differential diagnosis: Capillaria philippinensis egg looks similar to Trichuris trichiura egg. Former is distinguished by smaller size (45 x 21 μm), peanut shape, bipolar flattened mucous plugs and pitted egg shell].
Enterobius vermicularis (Fig. 8.4):
Shape:
Asymmetric. Ventral side flattened and dorsal side convex — giving planoconvex appearance.
Size:
Length 50-60 pm x breadth about 30 pm.
Colour:
Colourless, transparent, non-bile stained.
Floatation:
Floats in saturated common salt solution.
Features:
1. Coiled larva inside the egg shell.
1. Egg shell is transparent and double layered.
(Instead of stool, the egg is better demonstrated in morning perianal NIH swab).
Hookworm (Fig. 8.5):
Shape:
Oval or elliptical in shape.
Size:
Length 65 μm x breadth 40 μm.
Colour:
Colourless. All layers are non-bile stained.
Floatation:
Floats in saturated solution of common salt.
Features:
1. Ovum is segmented in 4 blastomeres.
2. Ovum is immediately surrounded by hyaline shell membrane.
3. Egg shell is transparent.
4. Clear space between egg shell and ovum.
Hymenolepis nana (Fig. 8.6):
Shape:
Spherical or oval.
Size:
Diameter 35-45 μm.
Colour:
Colourless. No element is bile stained.
Floatation:
Floats in saturated common salt solution.
Features:
1. Two distinct membranes encloses an oncosphere.
Outer membrane is thin and colourless.
Inner membrane, embryophore, encloses the oncosphere and shows two polar knobs, from which polar filaments (6-8) originates.
2. Oncosphere is colourless and shows 3 pair of hook lets.
3. Space between outer membrane and embryophore is filled with yolk granules and polar filaments.
[Differentiating points from H. diminuta : H. diminuta egg is larger, outer shell is yellowish and is devoid of polar filaments].
Taenia Spp. (Fig. 8.7):
Shape:
Spherical.
Size:
Diameter 31-43 μm.
Colour:
Brown (bile stained).
Floatation:
Do not float in saturated common salt solution.
Features:
1. Outer shell:
Transparent. Often missing in voided stool. Represent the remnant of yolk mass.
2. Inner membrane, embryophore:
Brown (bile stained), thick walled, radially striated. Encloses the oncosphere.
3. Oncosphere:
14-20 μm in diameter, shows 3 pairs of hooklets.
[By morphology T. solium and T. saginata eggs cannot be differentiated; both looks same. For species identification examination of proglottides (segments) or scolex (head) is necessary].
Microscopic Morphology of Protozoa Cysts in Wet Mount Examination of Stool:
Entamoeba histolytica:
A. Trophozoite (Fig. 8.8B):
For demonstration of E. histolytica trophozoite, saline mount of mucous part of freshly voided stool is to be examined. It is desirable to use a warmer stage of microscope when temperature is below 30°C.
Features:
1. Slow gliding movements.
2. Pseudopodia is thrown from one end only, with a jerky movement resembling ejection under high pressure. This is followed by flowing of the entire endoplasm.
3. Shape:
No fixed shape.
4. Size:
Range 18-40 nm. Average 20-30 μm.
5. Cytoplasm:
Outer ectoplasm— Clear translucent.
Inner endoplasm — Granular. RBC is found here.
(Cytoplasm looks very clean, compared to dirty look of Entamoeba coli cytoplasm, due to phagocytosis of bacteria).
6. Nucleus:
Spherical. 4-6 μm in diameter. Eccentric position. Only faint outline is visible in saline mount.
[In stained preparation —
i. Karyosome — Dot-like, central position, surrounded by a clear halo.
ii. Nuclear membrane — delicate, lined by regularly arranged fine chromatin granule.
iii. Linin network — of fine thread, arranged radially].
B. Pre-cystic stage (Fig. 8.8A):
Size:
Smaller —10-20 μm.
Shape:
Round or ovoid with a small blunt pseudopodium.
Nucleus:
Same as trophozoite.
Endoplasm:
No RBC.
C. Cyst (Figs. 8.9, 8.10, 8.11):
Size:
‘Small race’ — 6-9 μm; ‘Large race’ 12-15 μm.
Shape:
Rounded.
Cyst wall:
Refractile membrane.
Nucleus:
According to stages of development, it might be uninucleate, bi-nucleate or quadrinucleate mature cyst. Nucleus is stained by iodine and bears similar characters as the trophozoite or precyst.
Cytoplasm:
(a) Early Stage:
1. Chromatoid or chromatoidial bars — 1 to 4 in number.
In saline mount — Refractile oblong bar with ends rounded.
In iodine mount — No stain.
In iron-haematoxylin stain — Black stained.
2. Glycogen Mass:
In saline mount — Space in cytoplasm in iodine mount — Stains brown.
[Maturity of the cyst is associated with —
i. Nucleus number changes from one to four,
ii. Chromatoid bars and glycogen vacuole disappears].
Giardia intestinalis:
A. Trophozoite (Fig. 8.12):
Found and better demonstrated in saline mounts of freshly voided diarrhoeic stool or after purgative.
Size:
Length 14 μm x Breadth 7 μm.
Shape:
Flat view — Tennis or badminton racket like.
Side on view:
Longitudinally split pear like.
Dorsal surface:
Convex.
Ventral surface:
Concave.
Anterior end:
Broader and rounded.
Posterior end:
Tapered.
Symmetry:
Bilaterally symmetrical. All the organs are paired.
Features:
1. Axostyle — Two central supporting rod, running longitudinally.
2. Sucking disc — Found on ventral surface at anterior end.
3. Nuclei — Two.
4. Flagella — Four pair.
Movement:
Looks like a falling leaf in saline mount of freshly voided diarrhoeic stool.
B. Cyst (Fig. 8.13):
Better demonstrated in iodine mount. Iodine stains the internal structures.
Size:
Length 12 μm x Breadth 7 μm.
Shape:
Oval or elliptical.
Cyst wall:
Refractile.
Nucleus:
Four in number, forming a cluster at one end.
Axostyle:
Set diagonally.
Rudimentary flagella — arranged in pair across the axostyle.
Space between cell wall and cell membrane:
It is pronounced at the posterior end.