EXERCISE NO. 3 COLLECTION AND PRESERVATION OF STOOL SPECIMEN Flashcards
refers to the egg stage
ova
most common procedure performed in the area of parasitology
O & P
are examined for the presence of intestinal parasites
Stool specimens
several factors due to (?) may grossly affect accurate diagnosis
improper specimen collection, transport and preservation
Diagnosis of intestinal parasitosis relies largely on (?) of stool specimen.
macroscopic and microscopic examination
As in all areas of laboratory testing, the quality of results is dependent on the (?)
appropriate collection of specimen
protozoan forms
trophozoites and cysts
Helminth stages
eggs, larvae, proglottids, and adult worms
A stool specimen should be (?) a patient’s intake of drugs or (?) intake
examined before
collected a week after
a. Certain medications such as (?); and substances such as (?) (as x-ray contrast medium) may leave (?) which can interfere with identification of parasites.
anti-diarrheal, antacids, anti-malarial agents
bismuth and barium
crystalline residues
b. (?) such as castor oil, mineral oil, or suppositories also interfere with the
examination as they (?) of protozoan trophozoites, and (?) of select parasites
Oily laxatives
retard the motility
distort morphology
c. (?) that affect the normal gastrointestinal flora usually decreases the number of protozoans for several weeks (i.e. 2 weeks), since they feed on intestinal bacteria
Antibiotics
The (?) is important to prevent accidental spillage of the specimen and to maintain moisture within the specimen. Integrity of the morphology of certain parasites are affected by (?)
fit of the lid
desiccation
a. (?) may destroy protozoans especially the motile stages.
Urine
b. (?) as they may contain free-living organisms that can be mistaken for human parasites, thus, complicate diagnosis of infections
Toilet water and/or soil
Routine procedure usually requires:
a thumb-sized specimen of a formed stool, or 1/2 teaspoon or 5-6 tablespoons of watery specimen.
For routine examination for parasites before treatment, a series of (?) is considered minimum for adequate examination
3 fecal
samples
Fecal samples should be collected on separate days, if possible every other day, or within a (?). This is since many parasites do NOT appear in the fecal sample in consistent numbers on a daily basis, thus collection on alternate days is likely to yield a higher percentage of positive samples.
10-day period
To ensure the recovery of parasitic organisms that are passed intermittently and in fluctuating numbers, the examination of a minimum of (?) collected over a 7- to 10-day period is recommended. Particularly, (?) collected from normal bowel movement and (?) collected after catharsis/purge.
three specimens
2 specimens
1 specimen
are prescribed in order to stimulate some “flushing action” within the GIT, possibly allowing one to obtain more organisms for recovery and identification
Cathartics
Cathartics such as (?) are preferred.
saline, magnesium sulfate, or Fleet’s Phospho oda
should NOT be used since they retard the motility of trophozoites and distort the morphology of the parasites
Oil-based cathartics
For obvious reasons, the use of cathartics would be contraindicated if the patient already has
diarrhea or dysentery
When a patient is suspected of having intestinal amoebiasis, 6 specimens is
recommended (however, is rarely requested); collected on separate days or within 14-day period:
3 specimens collected from normal bowel movement
3 specimen collected after catharsis/purge
For post-therapy examinations, (?) are also recommended, and collected as outlined above.
3 specimens
For protozoan infection, stool specimen must be checked (?) after therapy.
3–4 weeks
As for Taenia infection, fecal sample must be examined (?) after therapy
5–6 weeks
The (?) are most relevant for the recovery and identification of intestinal protozoa
time limit recommendations
(?) must be carried out within 30 minutes of passage (NOT from the time it reaches the laboratory).
Examination of liquid stool
are mandatory for the recovery of motile trophozoites which are normally found in cases of diarrhea.
Fresh specimens
During this time, the gastrointestinal tract contents are moving through the system too rapidly for (?) to occur. Once the stool specimen is passed from the body, the (?) do NOT encyst but may disintegrate if not examined or preserved within a short time after passage.
cyst formation
trophozoites
(?) must be examined within 1 hour of passage whereas, formed stool be examined at any time within 24 hours after passage as immediate examination is not critical. This is since (?) which due to their cyst wall are more resistant to disintegration are found more commonly in formed stools and most helminth eggs and larvae will survive for extended periods.
Soft/Semi-formed stool
protozoan cysts
Although (?) are preferred for examination, if general time recommendation is NOT possible, preservatives should be used.
freshly passed stools
Fecal sample should be submitted promptly to the laboratory
Correctly labelled with the following minimal information
i. Patient’s name and identification number, age, sex
ii. Date and time of specimen collection
iii. Requesting physician
iv. Presumptive diagnosis
Accompanied by a (?) indicating which laboratory procedures are to be performed
request form
Successful diagnosis of intestinal parasitic diseases requires (?) stool specimens.
“fresh”
Therefore, when examinations must be delayed, it is important to preserve the integrity of the specimen by placing it in a proper (?) either immediately after passage by the patient or as soon as the specimen arrives in the laboratory
preservative or fixative medium
Reasons for a lag time between specimen passage and examination in the lab include:
- Transit distance or time for the specimen to reach the facility.
- Workload in the laboratory
When a delay in the examination of the stool specimen is expected, preservation is employed primarily for the following purpose:
- To maintain protozoan morphology.
- To prevent continued development of some helminth eggs and larvae
Generally speaking, preservation of stool specimen can be carried out by either of the following methods
A. Refrigeration
B. Chemical Preservation
is carried out at 3 – 5 oC.
When employed, this method generally preserves protozoan cysts , and helminth eggs and larvae however, trophozoites are killed.
Also, prolonged refrigeration can bring about
desiccation
Note however that although (?) is proven to be the most convenient method of preservation, fecal specimen should never be incubated nor frozen since these may parasitic forms deteriorate rapidly
stool preservation
Depending on their availability in the laboratory, several (?) have been proven effective in preserving the integrity of stool specimen for detection of ova and parasites.
chemical agents
Although each of these chemical agents present with both advantages and disadvantages, some of them may be considered superior over the other depending on the (?) to be performed on the preserved or fixed stool specimen
parasitologic technique
For efficient stool preservation, fecal samples must be adequately mixed with selected preservative in a proportion of
1 part stool to 3 parts preservative
2 concentrations commonly used:
5 % - recommended for preservation of protozoan cysts
10% - for helminth eggs and larvae
Formalin
Formaldehyde (USP): 50 ml 100 ml
Saline (0.85% NaCl): 950 ml 900 ml or distilled water
Formaldehyde is normally purchased as 37-40% solution; however, for dilution, it should be considered to be 100%
Formalin
Buffered Formalin:
1 liter of 5% or 10% formalin
0.8 g of phosphate buffer salt mixture
- 610 g Na2HPO4
- 0.15 g NaH2PO4
Formalin
- An all-purpose fixative appropriate for helminth eggs and larvae, and for protozoan cysts, oocysts, and spores.
Formalin
- Easy to prepare, and has a long shelf life.
Formalin
- Suitable for concentration procedures.
Formalin
- Suitable for acid-fast, safranin and chromotrope stains.
Formalin
- Compatible with immunoassay detection kits
Formalin
- Not suitable for some permanent stained smears like trichrome.
Formalin
- Inadequate preservation of the morphology of protozoan trophozoites.
Formalin
- Can interfere with polymerase chain reaction after extended fixation time
Formalin
is prepared in 2 stock solutions and mixed immediately before use
MIF (Merthiolate-Iodine-Formaldehyde
Components:
Solution I (stored in brown bottle)
Distilled water 50 ml
Formaldehyde (USP) 5 ml
Thimerosal (tincture of merthiolate,1:1,000) 40 ml
Glycerin 1 ml
Solution II – Lugol’s solution (good for several weeks in tightly stoppered brown bottle)
Distilled water 100 ml
Potassium iodide crystals 10 g
Iodine crystals 5g
MIF (Merthiolate-Iodine-Formaldehyde)
Procedure: Combine 9.4 ml of solution I with 0.6 ml of solution II
MIF (Merthiolate-Iodine-Formaldehyde)
if undisturbed within 24 h, form 3 well-defined layers
MIF-preserved specimen
clear orange fluid, consists mainly of formalin, merthiolate and water; it does NOT trap eggs and protozoa
Upper
thick, pale orange or creamy yellow, usually 1-2 mm thick; may trap some protozoa and helminth eggs
Interface
consists of deeper staining particulate matter; eggs and protozoa are found throughout this layer
Bottom
- Good stain preservative for most kinds and stages of parasites found in feces.
MIF (Merthiolate-Iodine-Formaldehyde)
- Easy to prepare, and has a long shelf life.
MIF (Merthiolate-Iodine-Formaldehyde)
- Useful for field surveys.
MIF (Merthiolate-Iodine-Formaldehyde)
- Suitable for concentration procedures
MIF (Merthiolate-Iodine-Formaldehyde)
- Not suitable for some permanent stained smears like trichrome.
MIF (Merthiolate-Iodine-Formaldehyde)
- Inadequate preservation of the morphology of protozoan trophozoites.
MIF (Merthiolate-Iodine-Formaldehyde)
- Iodine interferes with other stains and fluorescence.
MIF (Merthiolate-Iodine-Formaldehyde)
- Iodine may cause distortion of protozoa
MIF (Merthiolate-Iodine-Formaldehyde)
Components:
Sodium acetate 1.5 g
Acetic acid, glacial 2.0 ml
Formaldehyde,
37-40% solution 4.0 ml
Distilled water 92.0 ml
SAF (Sodium Acetate-Acetic Acid-Formalin)
- Mix equal parts of egg white and glycerin.
- Place 1 drop on microscope slide, and 1 drop of SAF-preserved fecal sediment.
- After mixing, allow the smear to dry at room temperature for 30 min prior to staining
Mayer’s Albumin
- Suitable for both concentration procedures and permanent stained
smears.
SAF (Sodium Acetate-Acetic Acid-Formalin
- Easy to prepare or commercially available from a number of suppliers, and has a long shelf life.
SAF (Sodium Acetate-Acetic Acid-Formalin
- Compatible with immunoassay detection kits.
SAF (Sodium Acetate-Acetic Acid-Formalin
- Does not contain mercury compounds
SAF (Sodium Acetate-Acetic Acid-Formalin
- Poor adhesive properties (requires additive, e.g., albumin-glycerin, for adhesion of specimens to the slides).
SAF (Sodium Acetate-Acetic Acid-Formalin
- Protozoan morphology with trichrome stain not as clear as with PVA or Shaudinn’s smears.
Hematoxylin staining gives better results.
SAF (Sodium Acetate-Acetic Acid-Formalin
- May be a bit more difficult to use
SAF (Sodium Acetate-Acetic Acid-Formalin
- More difficult for inexperienced workers to use
SAF (Sodium Acetate-Acetic Acid-Formalin)
Components:
Mercuric Chloride, Saturated Aqueous Solution:
Mercuric chloride 110 g
Distilled water 1,000 ml
Schaudinn’s Fluid
Procedure:
1. Use a beaker as water bath.
2. Boil until mercuric chloride is dissolved.
3. Let stand several hours until crystals form
Schaudinn’s Fluid
II. Schaudinn’s Fixative (Stock Sol’n): Mercuric Chloride, sat. aq. 600 ml
Ethyl alcohol, 95% 300 ml
Schaudinn’s Fluid
*Immediately before use, add 5 ml glacial acetic acid per 100 ml stock solution
Schaudinn’s Fluid
- Provides excellent preservation of morphology of protozoan trophozoites and cysts.
Schaudinn’s Fluid
- Fixative for smears prepared from fresh fecal specimens or samples from intestinal mucosal surfaces.
Schaudinn’s Fluid
- Easy preparation of permanent stained smears.
Schaudinn’s Fluid
- Easily prepared in the laboratory and available from a number of commercial suppliers
Schaudinn’s Fluid
- Not generally recommended for use in concentration procedures.
Schaudinn’s Fluid
- Contains mercuric chloride – presents disposal problem.
Schaudinn’s Fluid
- Inadequate preservation of helminth eggs and larvae, coccidian and microsporidia.
Schaudinn’s Fluid
- Poor adhesive properties with liquid or mucoid specimens to the slides
Schaudinn’s Fluid
Components:
PVA 10.0 g
Ethyl Alcohol, 95% 62.5 ml
Mercuric chloride, sat. aq. 125.0 ml
Acetic acid, glacial 10.0 ml
Glycerin 3.0 ml
PVA (Polyvinyl Alcohol)
Procedure:
1. Mix the liquid ingredients in a beaker, then add the PVA powder.
2. Cover and allow PVA to soak overnight. Heat the solution slowly to 75oC.
3. When this temperature is reached, remove the beaker and swirl the mixture for 30 secs until a homogeneous, slightly milky solution is obtained
PVA (Polyvinyl Alcohol)
- Provides excellent preservation of morphology of protozoan trophozoites and cysts.
PVA (Polyvinyl Alcohol)
- Easy preparation of permanent stained smears.
PVA (Polyvinyl Alcohol)
- Long shelf life (months to years) tightly sealed containers at room temperature.
PVA (Polyvinyl Alcohol)
- Preserved samples remain stable for several months.
PVA (Polyvinyl Alcohol)
- Allows the specimen to be shipped to any laboratory for subsequent examination
PVA (Polyvinyl Alcohol)
- Inadequate preservation of helminth eggs and larvae, coccidian and microsporidia.
PVA (Polyvinyl Alcohol)
- Contains mercuric chloride which may cause disposal problems.
PVA (Polyvinyl Alcohol)
- Difficult to prepare in the laboratory.
PVA (Polyvinyl Alcohol)
- May turn white and gelatinous when it begins to dehydrate or when refrigerated.
PVA (Polyvinyl Alcohol)
- Less suitable for concentration procedures.
PVA (Polyvinyl Alcohol)
- Cannot be used with immunoassay detection kits
PVA (Polyvinyl Alcohol)
- Stool O and P is a routine laboratory procedure performed for diagnosis
of intestinal parasitosis
TRUE
- Laxatives such as mineral oil, castor oil, and suppositories may cause the
formation of crystalline residues that deter proper parasite identification
FALSE
- One may directly scoop up stool from the toilet bowl and submit it for
examination
FALSE
- A thumb-sized specimen of formed stool is considered sufficient to
perform routine parasitologic procedures such as O and P.
TRUE
- Ideally, a single specimen is sufficient to conclude whether or not a
patient has intestinal parasitosis
FALSE
- After treatment, a patient is no longer required to submit a stool sample
for examination
FALSE
- With the exception of oil-based ones, cathartics allow higher yield of
parasites, hence better diagnosis of parasitosis
TRUE
- Information on the specimen container should match those that are
found in the requisition form during submission of a fecal sample
TRUE
- When delay in examination or long transit is expected, stool samples
need not be preserved
FALSE
- When appropriate chemical preservatives are unavailable, stool
samples may also be stored in a laboratory refrigerator
TRUE
