Name :asma abiib muhumed

9/7/2022
Faculty :nursing

Course :parasitology

Pc
[COMPANY NAME]
 Direct technique stool preparation

Fecal examination is indicated in diarrhoeic diseases, constipation, anorexia, jaundice, hepatitis,

lungworm infection, anemia, weakness, low milk production and in dysentery. Examination of feces is
mainly carried out for the detection of parasites, their ova/larva/oocyst, blood and other foreign
material. The fecal examination for diagnosis of parasitic infections is probably the most common
laboratory procedure performed in a veterinarian clinic. Sometimes cultural examination of feces is
performed for isolation of bacteria or virus in order to establish the etiology of diarrhea/dysentery. The
bacterial or viral antigens are also demonstrated in feces using immunological methods like immunofl
uorescence, enzyme linked immunosorbent assay and dot imrnunobinding assay. Parasitic worm eggs
from the respiratory system may be coughed into the throat and swallowed and appear in the feces.
Parasitic forms seen under the microscope have characteristic morphologic features that with a little
practice can be diagnostic for a particular parasite. Fecal examination can reveal the presence of
parasites in many parts of the animal body. This clinic will concentrate on parasites inhabiting the
digestive system. Fecal examination should be done on fresh samples. If fecal samples are used after
being in the environment for hours or days accurate reading of parasite indicators cannot be
guaranteed. Also, free- living nematodes rapidly invade a fecal sample on the ground and can confuse
diagnosis. Several grams of feces should be collected immediately after observing defecation [1].
Collection of fecal sample in domestic animals When collecting fecal samples, fi rst make certain that the
feces is from the animal in question. Secondly, secure a fresh sample that is free from rocks, soil,
bedding, and other foreign materials. Place the fecal sample in a plastic vial, glass jar, waxed cup, or
plastic bag. If the examination does not follow closely after collection, preserve the sample in a
refrigerator [2,3]. In large animals, one should wear gloves and lubricate the hands with water and
collect feces from rectum. If feces is not present in rectum, stimulate the mucosa of rectum with fi nger,
the animal will defecate within few minutes [4]. Collect 5-10 gm feces in a clear, dry glass container. If
the feces are to be collected for cultural examination, it should be collected in sterilized glass vials [5]. In
small animals like calves, sheep, goat and dogs, the feces is collected by using index fi nger. In poultry,
the cloaca1 swabs are collected for examination. If the processing of a fecal specimen must be delayed,
it may be: Refrigerated (but not frozen) for several days (not recommended for samples with live larvae
that you intend to examine using the Baermann technique) and Fixed, 10% formalin (5% formalin-saline
is better for protozoal cysts). Add fi xative tofeces at a ratio 3:1 (v:v) and mix well. Do not fi x samples
intended for use with the Baermann technique the larvae may destroy by the formalin [6].

The procedure how to take feces:

* The operator places an obstetrical sleeve on one arm

* The arm is formed into a cone and the animal’s tail held to one side with the opposite gloved hand.

* Gentle pressure is applied to the anal sphincter until penetration into the rectum is obtained.

* A fecal aliquot of suffi cient size for the intended laboratory procedure is scooped with the sleeved
hand and removed from the animal.
 * The fecal sample is placed in a separate container or the obstetrical sleeve is inverted off the arm such
that the fecal sample is trapped inside.

* Small calves, sheep, goats, and swine: restrain manually. Gently pass a gloved, lubricated fi nger
through the anus and massage the rectal wall to stimulate rectal evacuation.

* If feces are not produced, collect feces with fi nger

for examination of collected fecal sample are direct smear, fl otation, and gross examinations.

Direct fecal smear examination

The direct smear method involves mixing a very small amount of feces with a water or saline solution.
Place the mixture on a slide, overlay it with a cover glass, and examine the entire smear under a low
power microscope. Observe the mixture for worm eggs and larvae and protozoa trophozoites and cysts.
The use of a small amount of feces and the presence of fecal debris lower the reliability of this
examination method [7,8].

The fecal sample can be examined by qualitative (fl otation and sedimentation) or quantitative methods
(Mack master method) if the direct smear method interpreted negative result.

Flotation examination

Examination by the fl otation method is more complex and requires more time, but is usually more
accurate than the direct smear [8]. The feces is mixed with either saturated sugar solution, saturated
salt solution, saturated sodium nitrate solution, 41% magnesium sulfate solution or 33% zinc sulfate
solution. The fl otation method uses the principle that most fecal particles fall to the bottom of the tube
or vial. Parasite eggs and cysts rise to the top of the salt or sugar solution, which is the result of a weight
difference between feces, parasite eggs, and cysts within the solution. In pure water, parasite eggs and
cysts settle to the bottom rather than fl oat; whereas in the salt or sugar solution, they fl oat due to the
higher density of the solution [9].

Instrument and some reagent required:

● Plastic containers or two beakers

● Saturated sugar,

● Saturated salt,

● Saturated sodium nitrate,

● Magnesium sulfate a

● Zinc sulfate solutions.

● Gauze or tea strainer, double layer of cheesecloth

● Measuring cylinder

● Centerfuge

● Strirring rod

● Mortar and pistle

● Test tube

● Test tube rack

● Pipettes

● Balance

● Teaspoon

● Microscope

● Slide and cover slide The procedure how to process the fecal fl oatation is listed as following steps
[9,10]. The fl otation method involves the use of a fl otation solution that has a specifi c gravity, greater
than 1.2, such as a salt or sugar solution (Figure 6.1)  Simple fl oatation, about one gram of feces is
taken and grinded and mixed with 42 ml of saline water  Then fi lter it through a fi ne sieve or muslin
cloth or gauze in to test tube or cylinder until it form meniscus (up to top of tube).  A clean glass slide
or cover slide is placed on the mouth of test tube or cylinder. Then left it for 10- 15 minute at room
temperature without disturbance, then remove coverslide/ slide and examine under10 x of
microsocope.  In centrifugation fl oatation, the fi rst step as simple fl oatation is similar, except this
method use centrifugation. Mixed the contents and centrifuge at 1500 rpm for 5 minute, the tube is
taken out and placed without disturbance. Transfer the small amount of superfi cial contents of the tube
on a clean and dry glass slide.  Place the cover slip on a slide and examine it under a microscope and
the parasite ova may be observed under microscope as Figure 6.2. Figure 1: The step of fecal fl oatation.
A
 Concentration technique stool preparation

The number of parasitic forms of both protozoan and/or helminthic parasites) in fecal
specimens are often too low to be observed microscopically in direct wet mounts or in
stained smear preparation. In such cases, the use of the concentration technique
increases the chances of detecting parasitic organisms, thus increasing the sensitivity of
copromicroscopic technique.

The two most commonly used stool concentration techniques

are sedimentation and flotation. Sedimentation techniques are performed commonly
in general diagnostic laboratories because they are easier to perform and less prone to
technical errors.

Table of Contents

 Principle of Formal Ether Sedimentation Technique

o Materials required
 Procedure for Formal Ether Sedimentation Technique
 Quality Control
 Observation and Results
 Procedure Notes-sedimentation technique
 Limitation of Sedimentation Technique

Principle of Formal Ether Sedimentation

Technique
Sedimentation techniques use solutions of lower specific gravity than the parasitic
organisms, thus concentrating the latter in the sediment.

It takes advantage of the high specific gravity of protozoan cysts and helminth eggs
compared to water. Their natural tendency to settle out in aqueous solutions can be
accelerated by light centrifugation.

Formalin fixes the eggs, larvae, oocysts, and spores, so that they are no longer
infectious, as well as preserves their morphology. Fecal debris is extracted into the ethyl
acetate phase of the solution. Parasitic elements are sedimented at the bottom.

Materials required
 Glass container
 Guaze
 Funnel
 Centrifuge tube (15ml capacity)
 Centrifuge
 Physiological saline (0.85% w/v NaCl)
 10% buffered formalin
 Ether (ethyl acetate)
 Test tubes with stopper
 Glass rod
 Iodine
 Microscope

Procedure for Formal Ether Sedimentation

Technique
1. Wear gloves when handling stool specimens.
2. In a suitable container, thoroughly mix a portion of stool specimen about the size of a
walnut into 10mL of saline solution. Mix thoroughly.
3. Filter the emulsion through fine mesh gauze into a conical centrifuge tube.
4. Centrifuge the suspension at a relative centrifugal force (RCF) of 600 g (about 2000
rpm) for no less than 10 minutes. The suspension should yield about 0.75mL of
sediment for fresh specimens and 0.5 mL for formalinized feces.
5. Decant the supernatant and wash the sediment with 10 mL of saline solution.
Centrifuge again and repeat washing until supernatant is clear.
6. After the last wash, decant the supernatant and add 10 mL of 10% formalin to the
sediment. Mix and let stand for 5 minutes to effect fixation.
7. Add 1 to 2 mL of ethyl acetate, Stopper the tube and shake vigorously.
8. Centrifuge at 450 g RCF (about 1500 rpm) for 10 minutes. Four layers should result
as follows
1. a top layer of ethyl acetate;
2. plug of debris;
3. layer of formalin; and
4. sediment
9. Free the plug of debris from the side of the tube by ringing with an applicator stick.
Carefully decant the top three layers.
10. With a pipette, mix the remaining sediment with the small amount or remaining fluid
and transfer one drop each to a drop of saline and iodine on a glass slide. Cover with
a coverslip and examine microscopically for the presence of parasitic forms.
Quality Control
1. Check solutions with each use to be sure they are clear and free of any bacterial
contamination.
2. Run known positive specimens through the procedure to verify organism recovery.
This should be done at least two times per year.

Observation and Results

Systematically examine the entire surface of each coverslip with the 10x  objective or, if
needed for identification, higher power objectives of the microscope in a systematic
manner so that the entire coverslip area is observed. When organisms or suspicious
objects are seen, switch to higher magnification (40X) to see the more detailed
morphology of the object in question.

Eggs (ova) of various intestinal parasites

Procedure Notes-sedimentation technique

1. The sedimentation procedure can also be used to process polyvinyl alcohol (PVA)
fixed specimens. After the procedure by filing one half of a tube with the stool-PVA
mixture and add 0.85% NaCl almost to the top of the tube. Then filter the mixture
through wet gauze into a 15 mL centrifuge tube and follow the remaining standard
procedure.
2. If ethyl acetate is used, swab the insides of the tube with a cotton-tipped applicator
stick after the plug of debris is rimmed and the excess fluid is decanted. Excess ethyl
acetate in the sediment at the time smears are prepared will lead to bubbles that may
obscure parasitic forms one is attempting to observe.
3. Errors in interpretation may occur if too much or too little feces is used in the
sedimentation procedure. Adhere to the recommended formula of 0.75 mL of
sediment for fresh specimens and 0.5 mL for formalinized feces.
4. Allow the centrifuge to reach maximum speed before the time is monitored. If the
centrifugation time is too little, certain smaller parasitic forms, such as the oocysts
of Cryptosporidium species, may not reach the sediment.

Limitation of Sedimentation Technique

1. Certain parasites, such as Giardia lamblia, hookworm eggs, and Trichuris eggs may
not concentrate well from PVA-preserved specimens. The oocysts of Isospora
belli do not routinely appear in concentrates. Therefore, examination of permanently
stained smear is highly recommended.
2. With both the sedimentation and the flotation techniques, species identification may
not be possible in all cases, depending on the clarity of the forms observed.
Permanently stained smears are usually required to make the final identification,
particularly when attempting to confirm the identity of the Entamoeba histolytica.
References and further readings
1. Gracia L. Examination of Fecal Specimens. In. Diagnostic Medical Parasitology.
Washignton, DC: American Society for Microbiology.
2. Washington WJ et al., Koneman’s Color Atlas and Textbook of Diagnostic
Microbiology. Sixth Edition: Lippincott Williams & Wilkins
Related

Lab Diagnosis of Intestinal Parasitic Infections