PRESERVATION OF SPECIMENS
Principle
Fecal specimens
that cannot be processed and examined in the recommended time should be placed
in an appropriate preservative or combination of preservatives for examination
later. Preservatives will prevent the deterioration of any parasites that are
present. A number of fixatives for preserving protozoa and helminthes are
available. Each preservative has specific limitations, and no single solution
enables all techniques to be performed with optimal results. The choice of
preservative should give the laboratory the capability to perform a
concentration technique and prepare a permanent stained smear for every
specimen submitted for fecal examination.
Procedure
- Wear gloves when performing this procedure.
- Add a portion of fecal material to the preservative vial to give a 3:1 or 5:1 ratio of preservative to fecal material (a grape-sized formed specimen or about 5 ml of liquid specimen).
- Mix well by stirring with an applicator stick or the “Spork” insert that is attached to the fixative vial lid to give a homogeneous solution.
- Allow to stand for 30 min at room temperature to allow adequate fixation.
- While using commercial collection system follows the manufacturer’s directions concerning shaking the vials, etc.
A. Schaudinn’s
fixative
This
preservative is used with fresh stool specimens or samples from the intestinal
mucosal surface. Many laboratories that receive specimens from in-house
patients (no problem with delivery times) may select this approach. Permanent stained
smears are then prepared from fixed material.
Advantages
1.
Designed to be used for the fixation of slides
prepared from fresh fecal specimens or samples from the intestinal mucosal surfaces
2. Prepared
slides can be stored in the fixative for up to a week without distortion of protozoan
organisms.
3.
Easily
prepared in the laboratory
4.
Available
from a number of commercial suppliers
Disadvantages
1.
Not
recommended for use in concentration techniques
2.
Has
poor adhesive properties with liquid or mucoid specimens
3.
Contains
mercury compounds (mercuric chloride), which may cause disposal problems.
B. PVA (Polyvinyl
Alcohol)
PVA is a plastic
resin that is normally incorporated into Schaudinn’s fixative. The PVA powder
serves as an adhesive for the stool material; i.e., when the stool-PVA mixture
is spread onto the glass slide, it adheres because of the PVA component.
Fixation is still accomplished by the Schaudinn’s fluid itself. Perhaps the
greatest advantage in the use of PVA is the fact that a permanent stained smear
can be prepared. PVA fixative solution is highly recommended as a means of
preserving cysts and trophozoites for examination at a later time. The use of
PVA also permits specimens to be shipped (by regular mail service) from any
location in the world to a laboratory for subsequent examination. PVA is
particularly useful for liquid specimens and should be used at a ratio of 3
parts PVA to 1 part fecal specimen.
Advantages
a.
Ability
to prepare permanent stained smears and perform concentration techniques
b.
Good
preservation of protozoan trophozoites and cyst stages
c.
Long
shelf life (months to years) in tightly sealed containers at room temperature
d.
Commercially
available from a number of sources
e.
Allows
shipment of specimens
Disadvantages
a.
Some
organisms (Trichuris trichiura eggs, Giardia lamblia cysts, Isospora
belli oocysts) are not concentrated as well from PVA as from formalin-based
fixatives, and morphology of some ova and larvae may be distorted.
b.
Contains
mercury compounds (Schaudinn’s fixative), which may cause disposal problems
c.
May
turn white and gelatinous when aliquotted into small amounts (begins to
dehydrate) or if refrigerated
d.
Difficult
to prepare in the laboratory.
C. SAF (Sodium
acetate formalin)
SAF lends itself
to both the concentration technique and the permanent stained smear and has the
advantage of not containing mercuric chloride, as is found in Schaudinn’s fluid
and PVA. It is a liquid fixative much like 10% formalin. The sediment is used
to prepare the permanent smear, and it is recommended that the stool material
be placed on an albumin-coated slide to improve adherence to the glass. SAF is
considered a “softer” fixative than mercuric chloride. The morphology of
organisms will not be quite as sharp after staining as that of organisms originally
fixed in solutions containing mercuric chloride. Staining SAF-fixed material
with iron-hematoxylin appears to reveal organism morphology more clearly than
staining SAF-fixed material with trichrome.
Advantages
a.
Can
be used for concentration techniques and stained smears
b.
Contains
no mercury compounds
c.
Long
shelf life
d.
Easily
prepared or commercially available from a number of suppliers
Disadvantages
a.
Has
a poor adhesive property. Albumin-coated slides are recommended for stained
smears.
b.
Protozoan
morphology with trichrome stain not as clear as with PVA smears. Hematoxylin
staining gives better results.
c.
More
difficult for inexperienced workers to use.
D. MIF
Merthiolate
(thimerosal)-iodine-formalin (MIF) is a good stain preservative for most kinds and
stages of parasites found in feces and is useful for field surveys. It is used
with all common types of stools and aspirates; protozoa, eggs, and larvae can
be diagnosed without further staining in temporary wet mounts. Many
laboratories using this fixative examine the material only as a wet preparation
(direct smear and/or concentration sediment). MIF is prepared in two stock
solutions that are stored separately and mixed immediately before use.
Advantages
a.
Combination
of preservative and stain (merthiolate), especially useful in field surveys
b.
Protozoan
cysts and helminth eggs and larvae can be diagnosed from temporary wet-mount
preparations.
Disadvantages
a.
Difficult
to prepare permanent stained smears
b.
Iodine
component unstable; needs to be added immediately prior to use
c.
Concentration
techniques may give unsatisfactory results.
d.
Morphology
of organisms becomes distorted after prolonged storage.
E. 5 or 10%
formalin
Formalin is an
all-purpose fixative that is appropriate for helminth eggs and larvae and
protozoan cysts. Two concentrations are commonly used: 5% which is recommended
for preservation of protozoan cysts, and 10%, which is recommended for helminth
eggs and larvae. Most commercial manufacturers provide 10%, which is most
likely to kill all helminth eggs. To help maintain organism morphology,
formalin can be buffered with sodium phosphate buffers, i.e., neutral formalin.
Advantages
a.
Good
routine preservative for protozoan cysts and helminth eggs and larvae.
Materials can be preserved for several years.
b.
Can
be used for concentration techniques (sedimentation techniques)
c.
Long
shelf life and commercially available
d.
Neutral
formalin (buffered with sodium phosphate) helps maintain organism morphology with
prolonged storage.
Disadvantage
a.
Permanent
stained smears cannot be prepared from formalin-preserved fecal specimens.
Shipment of
Specimens
Principle
In outpatient
situations, it may be necessary for a specimen to be shipped to the laboratory
for examination. Only preserved fecal specimens should be shipped, as any
delays in examination may result in deterioration of parasitic organisms. Prior
fixation also reduces the risk of infection from any etiologic agents present
in the specimen. The U.S. Postal Service regulates the shipment of clinical
specimens through the mail. It is the responsibility of the sender to
conform to these regulations.
Specimens
- Preserved fecal specimens in collection vials.
- Fecal smears for staining and examination for parasitic organisms.
- Blood smears for staining and examination for blood parasites (thin blood films should be fixed in methyl alcohol prior to shipment).
Procedure
- Place the primary container of preserved fecal material into the secondary container (metal sleeve or a sealable bag), and seal.
- Place into the mailing container, and seal.
- Label appropriately for shipment.
- Wrap glass slides in shock-absorbent material to protect from breakage, or place them in a sturdy slide container.
- Slides need not be placed in double containers for shipping.
- Place padded slides in shipping container and label appropriately. Duodenal Contents: String Test (Entero-Test Capsule)
Principle
The Entero-Test
capsule is usually administered and the string is retrieved by a physician.
This test is used to procure specimens from the duodenum that are then examined
for the presence of parasites. The Entero-Test is a gelatin capsule lined with
silicone rubber that contains a spool of nylon string and a weight. The end of
the string is taped to the back of the patient’s neck or the patient’s cheek
just before the capsule is swallowed with water. After swallowing the capsule,
the patient is allowed
to relax for 4 h. The patient is not allowed to eat during this time but is
allowed to drink a small amount of water. As the capsule dissolves, the string
unwinds and is carried by peristalsis to the duodenum, and the duodenal mucus
adheres to the string. Any Strongyloides larvae, Giardia trophozoites, or Cryptosporidium or Isospora oocysts that are present
will also adhere to the string and will be pulled up with the string when it is
removed.
The specimen can
be examined as a wet preparation or as a permanent stained smear. In rare
instances, Clonorchis sinensis eggs may be recovered. This test is a
less invasive substitute for duodenal aspiration.
Procedure
- Gloves must be worn when handling this specimen. Infectious Strongyloides larvae can penetrate the intact skin.
- Record the color of the string. Yellow bile stain indicates that the string did reach the duodenum.
- Place the specimen under the biosafety cabinet, hold the dry white end in one hand, and strip all the mucus off the string by gripping it between the thumb and index finger of the other hand and squeezing it all the way down to the end, so that the mucus goes into the screw-cap container.
- Place 1 drop of mucus on a clean slide, and cover with a coverslip (22 by 22 mm). If the mucus is very viscous, add a drop of saline before adding the coverslip.
- Store the remaining mucus in a transfer pipette placed in a labeled test tube (16 by 125 mm) so that it will not dehydrate.
- Examine the entire coverslip under low power (100X) for larvae or motile trophozoites, looking especially carefully at the mucus, where Giardia lamblia may be entangled.
- Examine the mucus under high dry power (400X), since G. lamblia may be detectable only by the flutter of the flagella rather than by motility.
- If there is enough specimen, gently smear a drop or two of patient material on two slides, and immediately immerse the slides in Schaudinn’s fixative so that permanent stained slides may be made. If the specimen is not adequate for this, place the wet mount slide in a Coplin jar containing Schaudinn’s solution after it has been read. The coverslip will float off and sink to the bottom, allowing the remaining material to be stained. The fixation and staining times are identical with those for routine fecal smears.
- If the material contains a lot of mucus or is a watery specimen, gently mix 1 or 2 drops of patient material with 3 or 4 drops of PVA fixative directly on the slide. Let the smear air dry for at least 2 h prior to staining. The fixation and staining times are identical to those for routine fecal smears.
- Place a drop of the mucus on one or more slides to be stained for Cryptosporidium and Isospora species, and then repeat the wet-mount procedure.
- Stain the Cryptosporidium and Isospora slide(s) with modified acid-fast stain, and examine as usual.
- Examine the permanent stained smear with the oil immersion lens (100X) with maximum light. Examine at least 300 oil immersion fields on each smear.