MICROBIO

PARASITOLOGIC TECHNIQUES — Exam Reviewer Notes

SPC-DRTC Didactics | 1st Rotation: Microbiology

INTRODUCTION

Most parasitic diseases cannot be confirmed by clinical signs and symptoms alone. The parasitology laboratory is essential for:

• Confirming diagnosis

• Ruling out differentials

• Guiding medication choice

• Monitoring treatment response

Key concept: Parasites can only be demonstrated during the patent stage of infection. The specimen and technique chosen depend heavily on the parasite's biology and life cycle.

Common specimens used:

PART 01 — ROUTINE TECHNIQUES

First-line methods. Simple, fast, low-cost. No density-based separation step. Results in minutes. Only a small amount of specimen is examined — light infections can be missed.

1. Direct Fecal Smear (DFS)

Primary target: Motile protozoan trophozoites — also detects cysts, helminth eggs, larvae.

Procedure:

• ~2 mg stool + drop of 0.85% Normal Saline Solution (NSS)

• Apply coverslip and examine

Stains:

• Nair's Brilliant Methylene Blue (BMB) — stains trophozoite nuclei

• Iodine — highlights cyst nuclei and glycogen

Very small sample (~2 mg) → light infections may be missed

2. Kato Thick Smear

Target: Thick-shelled helminth eggs, especially:

• Ascaris lumbricoides

• Trichuris trichiura

Procedure:

• ~50–60 mg of stool placed on a slide

• Covered with pre-soaked cellophane (glycerine–malachite green solution)

• Must be read within 10–20 minutes

Limitations:

• Poor for hookworm eggs (thin-shelled)

• Cannot detect protozoa

• Not suitable for watery stool

3. Kato-Katz Method

Target: Same as Kato Thick Smear + quantifies infection intensity (EPG — eggs per gram)

Procedure:

• Same cellophane method as Kato Thick Smear

• Uses a calibrated template to standardize stool volume

Notes:

• 1% eosin can be added to highlight Schistosoma miracidia

• Requires fresh, formed stool only — not liquid or preserved samples

WHO Infection Intensity Classification (Kato-Katz)

4. Cellulose Tape (Scotch Tape) Method

Target:

• Enterobius vermicularis eggs (pinworm)

• Taenia spp. eggs and proglottids

Procedure:

• Sticky side of clear cellulose tape pressed firmly against perianal skin

• Mounted sticky-side-down on a glass slide

• A drop of toluene or xylene placed under the tape improves visualization

Best collection time: Early morning before bathing, or late at night

Repeat the exam if the first result is negative.

Routine Techniques for Other Clinical Specimens

PART 02 — CONCENTRATION TECHNIQUES

Laboratory methods used to increase recovery of intestinal parasites from stool specimens.

Key points:

• Based on differences in specific gravity between parasites and concentration solutions

• Usually performed using approximately 1 gram of stool

• More sensitive than routine direct fecal smear

Clinical importance:

• Improves detection of protozoan cysts and helminth eggs

• Reduces false-negative results

• Removes fecal debris → cleaner microscopic preparations

• Increases overall diagnostic accuracy

Recommended for:

• Light infections / low parasite burden

• Confirming a negative direct smear

Two Governing Principles

Sedimentation Procedures

Formalin-Ether Concentration Technique (FECT)

• 10% formalin fixes parasites; ether removes fats

• Recovers both helminth eggs & protozoan cysts

• Formalin preserves morphology for long-term storage

• Ethyl acetate is a safer substitute for ether (though removes less fat)

• Gentler, broader recovery

Acid-Ether Concentration Technique (AECT)

• 40% HCl dissolves albumin; ether dissolves fats

• Best for: Trichuris, Capillaria, trematodes, Schistosoma

• Recommended for cat and dog stool

• Limitations: debris plug loss, protozoan cyst destruction

• Stronger recovery but harsher on cysts

Flotation Procedures

Zinc Sulfate Flotation

• 33% ZnSO₄ solution

• Ideal specific gravity: 1.18–1.20

• Best for protozoan cysts

• High specific gravity distorts cysts

Brine (Saturated NaCl) Flotation

• Saturated table-salt solution

• No centrifugation needed — eggs rise on their own

• Cheap

• Shrinks hookworm and Schistosoma eggs

• Not useful for operculated eggs (Clonorchis, Opisthorchis, Heterophyids) — these do not float

Sheather's Sugar Flotation

• Boiled sugar solution, preserved with phenol

• Best for Cryptosporidium, Cyclospora, Cystoisospora

• Phase-contrast microscopy improves visualization

PART 03 — COLLECTION & PRESERVATION OF SPECIMENS

Stool Specimen

Container: Clean, wide-mouthed, waxed cardboard or plastic with tight-fitting lid (retains moisture, prevents spillage)

Required labeling information:

• Patient's name, age, and sex

• Date and time of collection

• Requesting physician

• Requested procedure

• Presumptive diagnosis

• Prior infections

• Travel history

Factors Affecting Stool Quality

Stool Preservatives

Standard ratio: 1 part stool to 3 parts preservative

Collection of Other Specimens

Perianal Swab

• Cellulose (Scotch) tape — sticky side pressed onto perianal skin

• Best collected: early morning before bathing, or late at night

Blood

• Finger-prick must be free-flowing (prevent dilution with tissue fluid)

• Glass slides must be clean and grease-free

• For low parasite density: venous blood drawn and concentrated (e.g., Knott's concentration, membrane filtration)

Sputum

• Best specimen: first morning sputum

• If patient cannot expectorate: induce with 10% NaCl or hydrogen peroxide

• Collect in disposable, impermeable, tightly covered container — send immediately

Urine

• Best specimen: first morning urine (allows overnight concentration)

• Sample is centrifuged and sediment examined

Tissue Aspirates

• Liver — aspirate from the margin/wall of the abscess, not the necrotic center

• Duodenal — via intestinal intubation or the Entero (String) Test

• Cutaneous — sterile needle aspirate taken below the ulcer bed

CSF & Tissue Biopsy

• CSF — collect and examine immediately; centrifuge at 7,000g for 10 min

• Muscle biopsy — small pieces pressed between two glass slides

• Rectal biopsy — most common biopsy type submitted

QUICK SUMMARY COMPARISON TABLE — Routine Techniques

QUICK SUMMARY — Concentration Techniques

Sources: Belizario VY Jr, de Leon WU, eds. Medical Parasitology in the Philippines. 3rd ed. UP Press; 2013. / de Leon WU. Chapter 7: Diagnostic Parasitology.