Materials & Methods
Materials
&
Methods
45
Materials & Methods
ETHICAL CONSIDERATION
This research was approved by the Institution Review Committee (IRC) & the
Ethical Review Committee (ERC). Before enrollment in this research, written
consent was taken from the parents and/ or legal guardian of the children. The
risk and benefit had been interpreted in the consent form.
TYPE OF STUDY- Prospective cross sectional study..
SOURCE OF DATA-Stool samples sent to microbiology laboratory for
routine parasitic examination were included.
STUDY PERIOD-6 months from January2019 - June2019
PLACE
Integral Institute of Medical Sciences & Research, Lucknow.
COLLECTION OF DATA:
The cases were studied as per the pro forma enclosed. A detailed history
including age, sex. Samples were collected and processed in the department of
microbiology, Integral Institute of medical Sciences & Research, Lucknow.
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Materials & Methods
SAMPLE COLLECTION:-
Stool samples were collected from each patient into a wide mouthed clean, dry, properly
labelled disposable plastic container with tight fittings.
SAMPLE SIZE ESTIMATION:-
The minimum sample size required for prevalence based study is given by
n=z2P.Q/d2
where, n=number of samples
z=1.96(Obtained from table normal distribution
P= prevalence
Q=(1-p)
d=specified absolute precision
P= 0.76 ( Sahai&jamali., Indian journal of basic and applied medical
research, March 2015: vol.-4, Issue -2, P.560-567).
d=5(absolute error)
Then, n=(1.96)2*0.824/0.0025=222
N=222
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Materials & Methods
INCLUSION CRITERIA:-
All the outdoor patients and the indoor patients who were admitted at Integral
Institute of Medical Sciences and Research, Hospital Lucknowand whose stool
samples were sent to the microbiology laboratory for routine parasitic
examination.
EXCLUSION CRITERIA:-
Patients and patients guardians who were not willing to give consent to
participate inthe study.
History of Anti-parasitic medication in past four weeks.
STATISTICAL ANALYSIS:-
Data collected wasanalysed using statistical package for the Social Sciences
(SPSS) Software and result were interprete
48
Materials & Methods
LABORATORY EXAMINATION FOR DETECTION OF
INTESTINAL PARASITES
MACROSCOPIC EXAMINATION: -
Faeces were examined for its consistency, colour, odour and presence of blood or
mucus adult intestinal Helminths e,g. Ascaris lumbricoides and enterobius
vermicularis or segments of tapeworms may be seen in the stool
specimens.(Mackie & McCarteny 14ed. )
MICROSCOPIC EXAMINATION:-
The stool sample was examined microscopically using normal saline & lugols
iodine mount on grease free slides. (Mackie & McCartney 14 ed.)
[A] SALINE WET MOUNT
Saline wet mount is useful for live motile trophozoites of parasites. (Paniker 7 ed.)
MATERIAL REQUIRED:
Glassslide,
coverslip,
Wooden applicator
Grease pencil
Microscope.
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Materials & Methods
PROCEDURE
1- For a direct smear preparation 2-3mg of faecal sample are picked up using a
wooden stick.
2- It was mixed with drop of normal saline on a clean glass slide.
3- Then preparation should be covered with a cover slip.
4- Then observed under microscope.
5- Saline wet mount are used for the identification of thetrophozoites (Mackie
& McCartney).
Fig 15-Appratus and reagents used for wet mount preparation of
stool samples
50
Materials & Methods
IODINE WET MOUNT
MATERIAL REQUIRED :
Glass slide
lugols iodine
Coverslip
Wooden applicator
Grease pencil,
Microscope.
PROCEDURE:
Faecal sample 2-3mg will be taken with a wooden stick and will be placed
on a slide.
A drop of lugols iodine is added.
Then cover with coverslip and observed under microscope.
Iodine wet mount are used for identification of cyst (Mackie & McCartney).
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Materials & Methods
MODIFIED ACID FAST STAINING: ( MACKIE & McCARTNEY
14ed. Ch. No.45)-
Modified acid – fast staining techniques were used for determination of
protozoal parasites (Cryptosporidium, Isopora and Cyclospora)
MATERIAL REQUIRMENT:
1-Carbol fuchsin – 4gm basic fuchsin will be dissolved in 20 ml 95%
ethanol and solution add in 100ml distilled water.
2- Decolouriser – 5 % sulphuric acid.
3- Counter stain – 0.3 % malachite green in distilled water .
PROCEDURE:
1-A smear were prepared on a numbered glass slide, dry and fix by flaming.
2-Smear was covered with primary stain (Carbolfuchsin) for 5 minutes. No
intermitted heating is required.
3-Smear were washed under the running tap water.
4-Smear were covered with acid alcohol decolorizer for2-3 minutes. After
the decolourization smear will be washed under the running tap water.
5-Smear were covered with counter stain (malachite green ) for one minute.
When smear were stained with counter stain, smear wash under tap water.
After the whole procedure, smear were examined under the microscope.
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