CLINICAL PARASITOLOGY LABORATORY
SPECIMEN COLLECTION & PROCESSING
TOPIC OUTLINE: ADVANTAGES: DISADVANTAGES:
Collection and Transport Easy to prepare Does not preserve parasite
Fixatives for Preservation morphology adequately for
Processing permanent smears
Stool Screening Method Preserve specimen up to Trophozoites may not be
Other Specimens and Laboratory Techniques several years recovered
Long shelf-life Morphology of cysts and eggs
COLLECTION AND TRANSPORT may fade with time
- Protozoan for (trophozoites and cysts) and Helminth stages Potential health hazard
(egg, larvae, proglottids and adult worms) are found in stool POLYVINYL ALCOHOL (PVA)
samples. - Combined with Schaudinn solution (Zinc Sulfate, Copper
- Typical stool collection protocol: Sulfate or Mercuric Chloride)
3 specimens in 10 days - Trophozoites and cysts of protozoans, most helminth eggs
- Amebiasis can be detected
6 specimens in 14 days - Can be used for preparation in permanent stained smear.
- Medication: Barium, Bismuth & Mineral oil
- Antibiotics and Antimalarial (delayed 2 weeks following the ADVANTAGES: DISADVANTAGES:
therapy) Long shelf-life when stored at Potential health problems
- Samples are collected in a clean, water-tight container with room temperature (Mercury in Schaudinn
tight-fitting lid solution)
- Sample requires: 2 to 5g/ size of a walnut Concentration techniques can
- Urine should not contaminate the stool sample be performed (Two-vial
- Stool should not be retrieved from the toilet bowl system)
- Water should not contaminate the stool sample
- Toilet paper should not be seen in the sample SODIUM ACETATE FORMALIN (SAF)
- Alternative fixative to PVA and Schaudinn fixative
SAMPLE LABELLING
- Used in concentration techniques and permanent stained
Patient’s name
smears.
Identification number
Physician’s name ADVATAGES: DISADVANTAGES:
Date and time of collection Easy to prepare Addition of Albumin is
Age necessary
Gender Long-shelf life Protozoan morphology is not
clear in permanent smears
Trophozoite motility – fresh sample is required Use for preparing smears for Choice of permant stain
Protozoan cysts helminth egg and larvae are not sensitive staining with Modifies acid-fast (Iron Hematoxylin)
Liquid stool samples: Processed within 30mins (presence of stain for Coccidian cysts.
trophozoites)
Semi-formed stool samples: Evaluated within 1 hour MODIFIED POLYVINYL ALCOHOL (PVA)
(presence of trpphozoite and cysts) - Alternative fixative to Mercury-based PVA by using Copper
Formed samples: Can be held within 24 hours Sulfate and Zinc Sulfate
Guideline are not met: the samples must be preserved - Can be used for concentration methods and permanent
smears
FIXATIVE AS PRESERVATION - Do not provide the same quality of preservation of protozoan
- Ideal sample: Freshly collected stool sample morphology
- Substances that preserve the morphology of protozoans and
prevent development of helminth eggs and larvae
ALTERNATIVE SINGLE-VIAL SYSTEM
- Ratio is 3:1 (3 parts fixative and 1 part of stool)
- Single vial systems are free of Formalin and Mercury
TYPES OF FIXATIVE: - Used for concentration techniques and permanent smears
1. Formalin - Used in fecal immunoassays
2. Polyvinyl Alcohol (PVA) - Disadvantage:
3. Sodium Acetate Formalin (SAF) 1. Do not provide the same quality as Mercury-based
4. Modified Polyvinyl Alcohol fixative
5. Alternative Single Vial Systems 2. Organism identification will be more difficult
FORMALIN PROCESSING
- All-purpose fixative for helminths and protozoans MACROSCOPIC
- 2 concentrations: - Consistency (degree of moisture)
5% - protozoan cysts
- Color (normal: brown)
10% - helminth eggs and larvae
- Gross abnormalities (adult worms, proglottids, pus and
Used for routine direct examinations and concentration
techniques but NOT for permanent smears. mucus)
4
, CLINICAL PARASITOLOGY LABORATORY
- Ethyl Acetate is added to a saline-washed formalin-fixed
sample.
- Parasites heavier than the solution settles in the
sediment of the tube.
- Fecal debris are lighter and rises to the upper layer of the
tube.
ADVANTAGES: DISADVANTAGES:
Easy to prepare More fecal debris
Good recovery of parasites Challenging to the
COLOR: INDICATIONS: microscopist.
Brown – color caused by
chemical changes in bilirubin (a Zinc Sulfate Flotation
pigment resulting from the - Principle: Specific gravity
breakdown of red blood cells). - Debris sinks at the bottom of the tube
All shades of brown are - Parasites are lighter thus rises on the tube of the tube
considered normal. - Zinc sulfate (1.18-1.20) is used as the concentration
Green – green vegetable such as solution.
spinach, are common caused of
ADVANTAGES: DISADVANTAGES:
green stool.
More fecal debris is removed Some helminths are dense
Red – can indicate bleeding of
Cleaner preparation, easier
the lower digestive tract or
for microscopic examination
rectum.
Yellow – can be caused by an
infection known as Giardia or PERMANENT STAINS
can indicate bowel - Final procedure in O & P examination
hyperactivity. - Microscope slides that contains a fixed sample that has
Blue – can indicate illness in been allowed to dry and stained
babies or can be the result of - Critical portion of the O & P examination
eating foods that contain blue
dyes Sample of choice is PVA-prepared sample. SAF samples can
White – clean caused by a lack be used but the stain must be Iron Hematoxylin.
of bile (produced in the liver) Slides can be prepared from fresh samples but must not be
and by certain medications. allowed to dry and place immediately into a fixative.
Black – may be caused by Two common stain:
bleeding in the upper digestive Wheatley Trichrome
tract.
Iron Hematoxylin
MICROSCOPIC WHEATLEY IRON SPECIALIZED
1. Direct methods TRICHROME HEMATOXYLIN STAINS
2. Concentration techniques Most widely used Excellent Modifies acid-fast
3. Permanently stained smears permanent stain. morphology of stain
intestinal (Cryptosporidium,
DIRECT WET PREPARATION protozoa. Isospora,
- Direct Wet Mount Cyclospora spp.)
- Slide made by mixing a small portion of unfixed stool with Long shelf life and Nuclear detail are Do not detect
saline or Iodine easy to perform. stained clearer oocysts and spores
- To detect the presence of motile protozoan trophozoites and sharper. (Coccidian/Sporidia)
(fresh samples) Distinct color Modified Iron
- Direct Saline Wet Preparation difference Hematoxylin
between the (incorporates with
- Direct Iodine Wet Preparation
cytoplasmic and Carbolfuschin to
nuclear detect acid-fast
structures. parasites;
combined with SAF-
preserved fecal
samples).
STOOL SCREENING METHODS
- Rapid methods (obtained as kits that contain monoclonal
antibody).
- Commercial antibody is used to detect antigens in patient’s
samples.
- Enzyme Immunoassay, DFA & Membrane Flow Cartridge
CONCENTRATION TECHNIQUES
Technique.
Formalin-Ethyl Acetate Sedimentation
- E. histolytica, G. intestinalis & Cryptosporidum spp.
- Principle: Specific gravity
- Highly sensitive and specific but detects one pathogen at a
time.
4
SPECIMEN COLLECTION & PROCESSING
TOPIC OUTLINE: ADVANTAGES: DISADVANTAGES:
Collection and Transport Easy to prepare Does not preserve parasite
Fixatives for Preservation morphology adequately for
Processing permanent smears
Stool Screening Method Preserve specimen up to Trophozoites may not be
Other Specimens and Laboratory Techniques several years recovered
Long shelf-life Morphology of cysts and eggs
COLLECTION AND TRANSPORT may fade with time
- Protozoan for (trophozoites and cysts) and Helminth stages Potential health hazard
(egg, larvae, proglottids and adult worms) are found in stool POLYVINYL ALCOHOL (PVA)
samples. - Combined with Schaudinn solution (Zinc Sulfate, Copper
- Typical stool collection protocol: Sulfate or Mercuric Chloride)
3 specimens in 10 days - Trophozoites and cysts of protozoans, most helminth eggs
- Amebiasis can be detected
6 specimens in 14 days - Can be used for preparation in permanent stained smear.
- Medication: Barium, Bismuth & Mineral oil
- Antibiotics and Antimalarial (delayed 2 weeks following the ADVANTAGES: DISADVANTAGES:
therapy) Long shelf-life when stored at Potential health problems
- Samples are collected in a clean, water-tight container with room temperature (Mercury in Schaudinn
tight-fitting lid solution)
- Sample requires: 2 to 5g/ size of a walnut Concentration techniques can
- Urine should not contaminate the stool sample be performed (Two-vial
- Stool should not be retrieved from the toilet bowl system)
- Water should not contaminate the stool sample
- Toilet paper should not be seen in the sample SODIUM ACETATE FORMALIN (SAF)
- Alternative fixative to PVA and Schaudinn fixative
SAMPLE LABELLING
- Used in concentration techniques and permanent stained
Patient’s name
smears.
Identification number
Physician’s name ADVATAGES: DISADVANTAGES:
Date and time of collection Easy to prepare Addition of Albumin is
Age necessary
Gender Long-shelf life Protozoan morphology is not
clear in permanent smears
Trophozoite motility – fresh sample is required Use for preparing smears for Choice of permant stain
Protozoan cysts helminth egg and larvae are not sensitive staining with Modifies acid-fast (Iron Hematoxylin)
Liquid stool samples: Processed within 30mins (presence of stain for Coccidian cysts.
trophozoites)
Semi-formed stool samples: Evaluated within 1 hour MODIFIED POLYVINYL ALCOHOL (PVA)
(presence of trpphozoite and cysts) - Alternative fixative to Mercury-based PVA by using Copper
Formed samples: Can be held within 24 hours Sulfate and Zinc Sulfate
Guideline are not met: the samples must be preserved - Can be used for concentration methods and permanent
smears
FIXATIVE AS PRESERVATION - Do not provide the same quality of preservation of protozoan
- Ideal sample: Freshly collected stool sample morphology
- Substances that preserve the morphology of protozoans and
prevent development of helminth eggs and larvae
ALTERNATIVE SINGLE-VIAL SYSTEM
- Ratio is 3:1 (3 parts fixative and 1 part of stool)
- Single vial systems are free of Formalin and Mercury
TYPES OF FIXATIVE: - Used for concentration techniques and permanent smears
1. Formalin - Used in fecal immunoassays
2. Polyvinyl Alcohol (PVA) - Disadvantage:
3. Sodium Acetate Formalin (SAF) 1. Do not provide the same quality as Mercury-based
4. Modified Polyvinyl Alcohol fixative
5. Alternative Single Vial Systems 2. Organism identification will be more difficult
FORMALIN PROCESSING
- All-purpose fixative for helminths and protozoans MACROSCOPIC
- 2 concentrations: - Consistency (degree of moisture)
5% - protozoan cysts
- Color (normal: brown)
10% - helminth eggs and larvae
- Gross abnormalities (adult worms, proglottids, pus and
Used for routine direct examinations and concentration
techniques but NOT for permanent smears. mucus)
4
, CLINICAL PARASITOLOGY LABORATORY
- Ethyl Acetate is added to a saline-washed formalin-fixed
sample.
- Parasites heavier than the solution settles in the
sediment of the tube.
- Fecal debris are lighter and rises to the upper layer of the
tube.
ADVANTAGES: DISADVANTAGES:
Easy to prepare More fecal debris
Good recovery of parasites Challenging to the
COLOR: INDICATIONS: microscopist.
Brown – color caused by
chemical changes in bilirubin (a Zinc Sulfate Flotation
pigment resulting from the - Principle: Specific gravity
breakdown of red blood cells). - Debris sinks at the bottom of the tube
All shades of brown are - Parasites are lighter thus rises on the tube of the tube
considered normal. - Zinc sulfate (1.18-1.20) is used as the concentration
Green – green vegetable such as solution.
spinach, are common caused of
ADVANTAGES: DISADVANTAGES:
green stool.
More fecal debris is removed Some helminths are dense
Red – can indicate bleeding of
Cleaner preparation, easier
the lower digestive tract or
for microscopic examination
rectum.
Yellow – can be caused by an
infection known as Giardia or PERMANENT STAINS
can indicate bowel - Final procedure in O & P examination
hyperactivity. - Microscope slides that contains a fixed sample that has
Blue – can indicate illness in been allowed to dry and stained
babies or can be the result of - Critical portion of the O & P examination
eating foods that contain blue
dyes Sample of choice is PVA-prepared sample. SAF samples can
White – clean caused by a lack be used but the stain must be Iron Hematoxylin.
of bile (produced in the liver) Slides can be prepared from fresh samples but must not be
and by certain medications. allowed to dry and place immediately into a fixative.
Black – may be caused by Two common stain:
bleeding in the upper digestive Wheatley Trichrome
tract.
Iron Hematoxylin
MICROSCOPIC WHEATLEY IRON SPECIALIZED
1. Direct methods TRICHROME HEMATOXYLIN STAINS
2. Concentration techniques Most widely used Excellent Modifies acid-fast
3. Permanently stained smears permanent stain. morphology of stain
intestinal (Cryptosporidium,
DIRECT WET PREPARATION protozoa. Isospora,
- Direct Wet Mount Cyclospora spp.)
- Slide made by mixing a small portion of unfixed stool with Long shelf life and Nuclear detail are Do not detect
saline or Iodine easy to perform. stained clearer oocysts and spores
- To detect the presence of motile protozoan trophozoites and sharper. (Coccidian/Sporidia)
(fresh samples) Distinct color Modified Iron
- Direct Saline Wet Preparation difference Hematoxylin
between the (incorporates with
- Direct Iodine Wet Preparation
cytoplasmic and Carbolfuschin to
nuclear detect acid-fast
structures. parasites;
combined with SAF-
preserved fecal
samples).
STOOL SCREENING METHODS
- Rapid methods (obtained as kits that contain monoclonal
antibody).
- Commercial antibody is used to detect antigens in patient’s
samples.
- Enzyme Immunoassay, DFA & Membrane Flow Cartridge
CONCENTRATION TECHNIQUES
Technique.
Formalin-Ethyl Acetate Sedimentation
- E. histolytica, G. intestinalis & Cryptosporidum spp.
- Principle: Specific gravity
- Highly sensitive and specific but detects one pathogen at a
time.
4
