Specimen Collection For Fecal Samples

is the MOST COMMON, (95%) of parasite specimen submitted

in the laboratory.

Stool or Fecal sample

is the most common procedure performed

in parasitological examination.

O&P or Ova and Parasite / Fecal Analysis

Other specimen types

- Sputum

- Urine

- Genitalia saline wet swabs

- Tissue / skin snips

- Blood

Sample container for collection of stool sample

Clear/Transparent, Disposable, Water-tight/Leak-proof, Wide-mouthed, Tight-fitting lid, Sterile

Sample container must include the following labeled information:

- Patient's name, age & gender, and identification number (if available)

- Date and time of sample collection

Amount needed for a stool sample

2-5g (size of a walnut); at least 10cc or 1/3 full of container for watery stool

Amount needed for preparing of stool sample

10g

Sampling / Number of Specimen for a typical stool collection

Minimum of 3 specimens, (or 1 sample to be collected every other day in 10 days)

In case of Amebiasis, minimum of ____________________ is required to be submitted for __________________

6 specimens in 14 days / 2 weeks

Infant's stool is scrapped-off from the diaper

Cammidge Method

Diarrheic specimens may be collected in ________________

clean bedpans

Specimens may be passed directly into ______, ___________, _______________ and a portion may be transferred to a specific container.

clean; dry paper cartons; wax paper(wax based cardboard);

a kind of laxative used for sampling on special conditions

carthatic substances (Magnesium sulfate or Fleet's Phospho-soda)

Medications / Substances that results to unsatisfactory specimen samples

- Barium, Bismuth, or Mineral oils

- Anti-malarial Drugs or antibiotics

Fecal samples in this case, must be collected prior to therapy or not until 5-7 days after completion of therapy

Barium, Bismuth, or Mineral oils

Collection of specimens should be delayed for 2 weeks following therapy

Anti-Malarial or antibiotics

Fecal specimen contaminants

- Urine / Toilet water

- Soil

- Toilet Paper

may destroy protozoal or amebic trophozoites and some parasites

Urine / Toilet water

contains free-living protozoans and may be confused with human parasites

Toilet bowl water

example of free-living protozoan in toilet bowl water

paramecium

May accidentally introduce free-living microorganisms and interfere and inhibit the identification and differentiation of parasitic microorganisms.

Soil

It may mask parasites or make examination of the sample more difficult.

Toilet Paper

Specimen Handling and Safety

- Each specimen represents a potential source of infection (bacteria, viruses, fungi, and parasites)

-Gloves and protective coats are necessary while handling fecal samples

- Universal Precautions as outlines by the OSHA for specimen handling must be enforced and exhibited at all times

Safety precaution should include awareness of the following:

- proper labeling of fixatives

- specific areas designated for specimen handling (biological safety cabinets may be necessary under certain circumstances)

- acceptable discard policies

- appropriate policies for no eating, drinking, or smoking, etc., within the working areas; and, if applicable, correct techniques for organism culture and/or animal inoculation

Generalized processing protocol for liquid stool sample

Must be examined within 30 minutes of passage

Generalized processing protocol for semi-formed / soft samples

Must be examined within 1 hour after passage

Generalized processing protocol for formed stool samples

Must be held within 24 hours following collection

Soft and liquid stools potentially carry __________

amebic trophozoites, helminth eggs and larvae

Formed stool samples potentially carry __________

parasitic cysts, helminth eggs and larvae

Semi-formed stool samples may exhibit ___________

a mixture of protozoan cysts and trophozoites

General Importance of Fecal Examination

- Detects the presence of pathogenic parasite/s

- Detects evidence of malfunction of some parts of the gastrointestinal tract, liver or pancreas

- Detect gastrointestinal bleeding

- To investigate for steatorrhea

- Used as a clue in medical or surgical diagnosis

fat in the feces

Steatorrhea

Stool sample processing can be routinely categorized into:

Macroscopic and Microscopic Examination

gross examination of the fecal samples which normally includes the physical characteristics of the specimen

Macroscopic examination

In a macroscopic examination, only ________________________ fecal samples are accepted or viable.

Fresh and unpreserved

Macroscopic examination is processed according to

- Form / Consistency

- Color

- Odor

normal color of feces

brown

Fecal odor ranges from ______________________

foul to offensive

used as confirmatory procedure

Microscopic Examination

Methods under microscopic examination

- Direct Fecal Examination / Direct Wet Mount

- Concentration Methods

- Staining Methods

A microscopic examination that enhances visualization of the sample

Staining method

Chemical Examinations

- FOBT (Fecal Occult Blood Test)

- Benzidine Test

- Guaiac's Test

- Hematest Tablet

most sensitive chemical examination, that is also prone for false positive

Benzidine Test

least sensitive chemical examination, has lower chance of false positive and is most preferred. Plant-based

Guaiac's Test

Other Examinations

- Thick and Thick Blood Film

- Immunologic Examinations

Rice water stool

Cholera

Pea soup stool

Typhoid fever

Flattened / ribbon-like or known as pipestem stool

Spastic colitis, cancer, ulcer, tumor

Bulky / Frothy stool

Bile duct obstruction, pancreatic disorders, abundant artifacts

Butter-like stool

Cystic Fibrosis, celia disease, pancreatic problems, very high-fat diet

Hard and scybalous stool

Constipation

A medical tool / atlas designed to classify fecal consistencies into seven (7) groups

Bristol Stool Chart

Separate hard lumps consistency and appearance; rabbit droppings

Type 1: severe constipation

Sausage-shaped but lumpy; bunch of grapes

Type 2: mild constipation

Sausage-like but with surface cracks; corn on cob

Type 3: normal

Sausage-shaped or snake-tail appearance, usually smooth / soft; sausage

Type 4: normal

Soft blobs with clear-cut edges; chicken nuggets

Type 5: lack of fiber

Fluffy pieces with ragged edges, mushy stool; porridge

Type 6: inflammation

Water, entirely liquid consistency, no solid constituents; gravy

Type 7: inflammation and diarrhea

Normal stool color; may range from light or dark due to hydrobilirubin (urobilirubin or stercobilin)

Brown

Abnormal/other variations in stool color

Yellow, Red, Black/Tarry (melena), Dark Red/Chocolate Brown, Green, Clay/Putty/Tea Color, Gray, Mucus/Blood-streaked mucus

Color due to milk diet, cornmeal, unchanged bilirubin, santorin and senna, and starchy foods (abundance of starch in stool-amylorhea)

Yellow

Lower GIT bleeding, food coloring, rifampin

Red

Upper GIT bleeding, iron, bismuth, charcoal

Black/Tarry (melena)

Upper GIT (converted into acidhematin by HCl), increased intake of coffee, chocolates consumption,

Dark red/Chocolate brown

unchanged biliverdin, increased green vegetable diet, calomel, diarrhea in children and faulty carbohydrate digestion

Green

may appear in cases of meconium and porphyrins

Dark green color

due to absence of urobilin in obstructive jaundice, steatorrhea in pancreatic disease, barium meal in x-ray examination, intestinal tubercolosis, blockage of common bile ducts (pale and greasy stool)

Clay/Putty(alcoholic)/Tea Color

due to cocoa and chocolates

Gray

Dysentery, colitis, malignancies, constipation

Mucus/Blood-streaked mucus

Normal odor

peculiar offensive odor but not excessively foul ( due to indole and skatole)

Infants: fatty acids (milk)

Adults: steatorrhea gas formation, fermentation, unabsorbed fatty acids

high acidity

Sour/Rancid

ulcerate and malignant tumor of the lower bowel; alkaline stools (amoy patay) , putrefaction of undigested proteins

Putrid/Extremely foul

mainly caused by increased in meat diet (bacterial putrefaction), causing increased tryptophan residues

Foul smelling stool

Excess carbohydrates in the diet

Strongly acidic stools

Excess protein in the diet

Strongly alkaline stools

Direct wet mount methods

- Normal Saline Solution mount (NSS mount)

- Iodine mount (D' Antoni's/Lugol's)

- Kato Thick Smear (KTS)

- Simplest and most frequently utilized method for examination.

- smear should not be too thick nor too thin

Principle:

- To provide quick diagnosis of heavily infected specimen; to check for organism motility (protozoan trophozoites); to evaluate organisms that might not be seen from the permanent stain methods

Direct Fecal Examination / Direct Wet Mount

- Observation of protozoan trophozoites motility

- Cysts appear glistening and refractile

- Doesn't demonstrate glycogen vacuole and nuclei

Normal Saline Solution mount / NSS mount

Reagent used in NSS mount

(0.85% NaCl) + 2g of stool (pea-sized)

Advantages:

- Glycogen vacuole becomes prominent

- Chromatoidial bars are fully seen

- Enhance details of protozoan cysts

Disadvantages:

- Kills the trophozoites and helminth larvae

- Overstains the larvae

- (Lugol's/D' Antoni's Iodine) + 2mg of stool

Iodine mount (D' Antoni's/Lugol's)

Advantages:

- Used for large scale examination

Disadvantages:

- Unsuitable for diarrhea stools

- Cannot demonstrate protozoan cyst, helminth eggs and larvae

Kato Thick Smear (KTS)

Developers of KTS

Kato and Miura in 1954

- Used for low intensity of parasitism

- Used in detection of small number of parasites not detected using DFS

- Based on specific gravity; remove fecal debris (more parasite yield)

Principle:

- To concentrate parasites present in the specimen, either through sedimentation or flotation methods; specifically designed to allow recovery of protozoan cysts, coccidian oocysts, microsporidian spores, helminth eggs and larvae

Concentration methods

Concentration methods

- Sedimentation method

- Flotation methods

- more efficient and easier to perform

- uses low specific gravity reagent

Sedimentation method

Sedimentation methods

- Formalin Ether Concentration Technique

- Acid Ether Sedimentataion Technique

- Simple Sedimentation

- Merthiolate-Iodine Formalin Concentration Technique

- Acid-Sodium Sulfate Tritan

- KOH Concentration

- Knott's Concentration Technique

- uses fresh or Polyvinyl Alcohol preserved specimen

- more fecal debris are left

Formalin Ether Concentration Technique

Uses formed stool, not applicable for protozoan cysts, applicable for helminth eggs and larvae

Acid Ether Sedimentataion Technique

used in cyclospora spp

KOH Concentration

used in microfilariae

Knott's Concentration Technique

- allows separation of helminth eggs and larvae, and protozoan cyst using liquid of higher specific gravity but some eggs are dense and will not float

- parasitic elements are recovered on the surface and some parasites and fecal debris at the bottom

Flotation methods

Flotation methods

- Modified Sheather's Sugar Flotation Technique

- Willie Brine

- Lane's Direct Centrifugal Flotation Technique

- Magnesium Sulfate Technique

- Zinc Sulfate Flotation Technique

- most commonly used flotation technique

- uses ZnSO4 solution

- some helminth eggs are dense and will not float

Zinc Sulfate Flotation Technique

Zinc Sulfate Flotation Technique should be done within __________

5-30 minutes

- Primarily used for detailed structural and morphology features of parasites

Principle:

- To provide contrasting colors for the background debris and parasites present

Staining methods

- D' antonis

- 1% isotonic eosin solution and 2% brilliant cresyl blue

- Eosin in saline

- Buffered methylene blue

Temporary staining

- confirms morphology of protozoan cysts and trophozoites

- Not suitable for helminth eggs and larvae

- Wheatley (modification of trichrome stain)

- IronHematoxylin stain

- Modified Acid-Fast stain (Kinyoun's)

- Modified Iron Hematoxylin (Carbol Fuschin)

Permanent staining

Fixatives used in permanent staining

Polyvinyl alcohol and sodium-acetate formalin

Preservation Categories

- Physical

- Chemical