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Journal of Clinical and Diagnostic Research. 2012 May (Suppl-1), Vol-6(3):503-509 503
Kotgire Santosh A. Microbiological Stool Examination www.jcdr.net
7. Shigella species It is endemic in many parts of the tropical and sub-tropical areas. It
is transmitted by the faeco-oral route. The cysts which contain four
S.dysenteriae, S.flexneri, S.bodyii and S.sonnei cause bacillary nuclei are indicative of an infective stage in humans. It produces
dysentery. The dysentery which is caused by the Shigella species amoebic dysentery which is characterized by large, flask shaped
is referred to as shigellosis. The Shigella spp causes at least 50% ulcers. It may get complicated into amoebic liver abscess and
of the cases of bloody diarrhoea in young children and adults in the amoebic lung abscess [7].
developing countries. S.dysenteriae type 1 is particularly virulent,
causing epidemic and endemic dysentery. It is highly infectious and 2. Giardia lamblia
resistant to the commonly available antibiotics [2].. The infection with Giardia lamblia may lead to explosive watery
8. Salmonellae diarrhoea, foul smelling stools and steatorrhoea. In endemic
areas, young children are more frequently infected than the adults,
Acute gastroenteritis which is caused by the Salmonella spp is particularly those who are malnourished. It produces a severe form
characterized by self limiting fever and diarrhoea. The incubation of infection in immuno-compromised individuals, particularly in
period is 12-36 hours. A large majority of the outbreaks are caused those with AIDS [7].
by, S.typhimurium and S .enteritidis. The human infection is usually
caused by the consumption of animal foods or food products. 3. Coccidian parasites
The infection leads to salmonella food poisoning and sometimes Cryptosporidium parvum, Isospora. Belli and Cyclospora have
septicaemia may develop [2]. been recognized as important agents which are responsible for the
9. Campylobacter jejuni watery diarrhoea in immunocompromised patients, particularly in
those with HIV-AIDS [7].
C.jejuni is the common cause of enteritis in the developing coun
tries. It occurs in the intestinal flora of many animals, especially 4. Balantidium coli
poultry, which probably serves as the major source of the infections It is an uncommon parasite which is found in humans. It commonly
in humans. The milk and waterborne outbreaks of diarrhoea have infects pigs and it has a world-wide distribution. It produces
also been documented. The disease is sometimes associated with dysentery which is transmitted by the ingestion of infective cysts
vomiting and bloody mucoid stools [1,2]. in food and water or from hands which are contaminated with pig
10. Yersinia enterocolitica. faeces [7].
504 Journal of Clinical and Diagnostic Research. 2012 May (Suppl-1), Vol-6(3):503-509
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Journal of Clinical and Diagnostic Research. 2012 May (Suppl-1), Vol-6(3):503-509 505
Kotgire Santosh A. Microbiological Stool Examination www.jcdr.net
The simplest way of examining a bacterial suspension for motile Thiosulphate citrate bile salt sucrose (TCBS) agar
bacteria is by doing a wet mount. Place a small drop of suspension
This is an excellent, selective medium for the primary isolation
on a slide, cover it with a coverslip and examine microscopically for
of V.cholerae. Prior enrichment in alkaline peptone water is
motile organisms by using the 10X and the 40X objectives. Also
recommended unless the specimen contains large number of Vibrio
make sure that the iris diaphragm of the condenser is sufficiently
bacteria in the acute stage. On TCBS, Vibro produces large yellow
closed, to give a good contrast [2].
coloured colonies because of sucrose fermentation [10,11].
Type of motility Organisms Sorbitol MacConkey’s agar
Sluggish E.coli
This MacConkey’s agar contains sorbitol instead of lactose. E.coli
Actively motile Salmonella spp 0157 produces colourless colonies on this medium because it
Dartingly motile Vibro cholera does not ferment sorbitol. Most of the other E.coli strains and other
Tumbling Campylobacter spp enterobacteria ferment sorbitol and produce pink colonies. So, this
Falling leaf Giardia lamblia medium is useful for screening 0157 E.coli [10,11].
Actively motile with pseudopodia Entamoeba histolytica Slide agglutination test
[Table/Fig-1]: Types of motility
The isolation of Salmonella and Vibrio can be done by doing the
Hanging drop preparation slide agglutination test. A loopful of growth from the culture is
emulsified in two drops of saline on a slide. One emulsion acts as
Placing a drop of suspension on a cover glass and inverting this a control to show that the strain is not autoagglutinable. In case
over a cavity slide or a normal slide which is supported on a ring of of Salmonella, the ‘o’ antiserum is added to one drop of bacterial
plasticine can also be used for observing motile organisms [1,2]. emulsion on the slide. A prompt agglutination denotes the presence
Basic fuschin smear of the Salmonella group [8,9].
Make a thin smear of the specimen on a slide, stain it with Basic Salmonella spp ‘o’ antiserum ‘H’antiserum
fuschin and examine the slide by using a 100X objective. This has S. typhimurium 4-B i
been shown to be a sensitive method for the presumptive diagnosis S..enteritidis 9-D d
of Campylobacter spp. It appears as small, delicate, spiral curved [Table/Fig-3]: Slide agglutination test
bacteria or s-shaped forms [10,11].
Similarly for V.cholera, the cholera o subgroup I serum is tested. If it
Culturing the specimen is found to be positive, the agglutination may be repeated by using
MacConkey’s Agar the specific Ogawa and Inaba sera for serotyping.
Gas Acid
Bacteria Motility formation formation Indole test MR test VP test Citrate Urease H2S
Escherichia + + + + + _ _ _ _
Salmonella spp + _ + - + _ + _ +
Shigella spp - _ + d + _ _ _ _
Vibrio + _ + + _ _ _ _
506 Journal of Clinical and Diagnostic Research. 2012 May (Suppl-1), Vol-6(3):503-509
www.jcdr.net Kotgire Santosh A. Microbiological Stool Examination
Stool examination for parasites [7,12] • Rest the tube on a stand. Four layers now become visible; the
top layer consists of ether, the second layer is a plug of debris,
1. Saline wet mount: It is used to detect worms, bile stained
the third layer is a clear layer of formalin and the fourth layer is
eggs, larvae, protozoan trophozoites and cysts. In addition, it
the sediment [Table/Fig-2].
can reveal the presence of RBCs and WBCs.
• Detach the plug of debris from the side of the tube with the
2. Iodine wet mount: It is used to stain the glycogen and aid of a glass rod and pour off the liquid, leaving only a small
nuclei of the cysts. A cyst is appreciated better in an iodine amount of formalin for the suspension of the sediment.
preparation, but the motility of the trophozoite is inhibited in • With a pipette, remove the sediment and mix it with a drop of
the iodine preparation. iodine. Examine under the microscope.
Procedure The saturated salt flotation technique [7,8,9]
• Place a drop of saline on the left half of the slide and one drop • Place about one millilitre of faeces in a container which is flat
of iodine on the right half. bottomed and which has a diameter of less than 1½ inches
• With an applicator stick, pick up a small portion of the and a capacity of about 15-20 ml.
specimen (equivalent to the size of a match head) and mix it • Add a few drops of saturated salt solution (specific gravity
with a saline drop. 1.200) and stir it to make a fine emulsion.
• Similarly, pick up a similar amount and mix with a drop of • Add more salt solution so that the container is nearly full,
iodine. stirring the solution continuously.
• Put the cover slip separately on both and examine under the • Remove any coarse matter which floats up.
microscope. • Place the container on a levelled surface. Do the final filling by
• The ova, cysts, trophozoites and adult worms can be identified using a dropper until a convex meniscus is formed.
as per their characteristic features. • A glass slide 3”x 2” is carefully laid on the top of the container
so that the centre is in contact with the fluid.
Concentration techniques [7,8,13,14]
• This preparation is allowed to stand for 20 minutes after
If the number of parasites in the stool specimens is low, the exam which the glass slide is quickly
ination of a direct wet mount may not reveal them and hence the lifted, turned over smoothly to
stool should be concentrated. Eggs, cysts and larvae can be avoid the spilling of the fluid and it
recovered after the concentration procedure, whereas trophozoites is examined under the microscope
can get destroyed during this procedure. This makes a direct wet after putting a coverslip on it.
mount examination obligatory as the initial phase of the microscopic
Staining procedures [12,13]
examination.
The modified acid fast staining is a
The concentration procedures can be grouped under 2 categories:
simple and effective method for the
[Table/Fig-4]: Modified AFB
1. Sedimentation procedures: In which the eggs and cysts stain showing ooyst of C. diagnosis of coccidian parasites
settle down at the bottom. parvum in the stool sample by using
2. Flotation procedures: In which the eggs and cysts float at the 1% H2SO4 as a decolourizer.
surface due to the specific gravity gradient. In unstained wet preparations, the small oocysts of C.parvum
are difficult to differentiate from those of yeasts and other small
The basic disadvantage of the sedimentation technique is that the
spherical structures which are found in faeces.
examination of the sediment is often difficult due to the presence
of excessive faecal debris that may mask the presence of the The oocyst of C.parvum
parasites. The basic disadvantage of the flotation technique is that Small, round to oval pink red stained bodies which measure
not all eggs and cysts float in the flotation procedures. 4-6µm.
Two commonly used concentration techniques are the formalin- The oocyst of Isospora belli
ether and the saturated salt solution techniques.
It is oval, measuring 20-22 µm in diameter and it usually shows a
The formal ether sedimentation technique [7,8,15] granular zygote. Occasionally, the oocyst may show two sporocysts
Procedure (each with four sporozoites).
Journal of Clinical and Diagnostic Research. 2012 May (Suppl-1), Vol-6(3):503-509 507
Kotgire Santosh A. Microbiological Stool Examination www.jcdr.net
Hookworm egg
[Table/Fig-15]: Eggs of
[Table/Fig-8]: Trophozoite B- Pink red coloured oocyst of I.belli They are oval and ellipsoid and they Taenia spp
of G. lamblia showing a zygote measure 60x40 µ. Their shells are
thin walled, smooth and colourless. Their internal cleavages are
Morphological features of the common well developed at the 4-8 cell stage, which pull away from the shell,
parasites/eggs/ova/cysts leaving an empty space.
It measures 12-60 µ, it is asymmetric, it It is a planoconvex, elongate and an asymmetric egg which meas
shows purposeful directional motility and ures 55 x 26 µ. Its shell is thin and smooth. Fully developed larvae
it has a single spherical nucleus, a single are seen in the eggs.
[Table/Fig-9]: Cyst of
central karyosome and delicate and G. lamblia Whipworm egg (Trichuris trichura)
evenly distributed chromatin.
It is elongate and barrel shaped, with polar hyaline plugs. It
Entamoeba histolytica cyst measures 54-22 µ. Its shells is yellow to brownish and its plugs
It is spherical and it measures 10- are colourless.
20 µ. It is a mature cyst with four Tapeworm egg (Taenia spp)
nuclei, with a compact, centrally
It is spherical, it measures 31-43µ and it has a thick shell with
located karyosome; the chromatin
prominent radial striations. An embryonated oncosphere which
is delicate. Some cysts may have
possesses 3 pairs of hooklets within the shell is diagnostic of the
chromatoid bars. This is the infec-
genus. Species identification on the basis of morphology is not
tive stage of the parasite
possible.
[Table/Fig-10]: Fertile egg of Giardia lamblia trophozoite It
round worm measures 9-21x5-15 µ and is pear References
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AUTHOR(S): NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING
1. Dr. Kotgire Santosh A. AUTHOR:
Dr.Santosh Kotgire
PARTICULARS OF CONTRIBUTORS:
Assistant Professor, Department of Microbiology,
1. Assistant Professor
Dr. Ulhas Patil Medical College, Jalgaon, Maharashtra, India.
Department of Microbiology, Dr. Ulhas Patil Medical
Phone: +919922867558
College, Jalgaon, Maharashtra, India.
E-mail: santosh_kots2001@yahoo.com
Journal of Clinical and Diagnostic Research. 2012 May (Suppl-1), Vol-6(3):503-509 509