Purpose:
Microbiological surveillance of OT by surface swab method for
anaerobic infection and identify the clostridial spores (Clostridium tetani).
Principle:
Infection control by environmental monitoring
plays a significant role in reducing the morbidity and mortality of patients
who affected the hospital-associated infections. The regular monitoring of
pathogens in the OTs is one of the preventive measures for post-operative
infections. Clostridium tetani is one of the major threatening
anaerobic bacteria of OTs as Clostridial spores easily spread into the air.
Regular monitoring of OTs for screening of clostridial spore by inoculating in
Thioglycollate broth for anaerobic culture should be performed. This broth
supports the growth of anaerobic and fastidious microorganisms. It
contains many nutritive factors, including casein, yeast, beef extracts, and
vitamins to enhance the growth of the most medically important bacteria.
This agar is supplemented with reducing agent thioglycolic acid to create an
anaerobic environment deeper in the tube which allows anaerobic bacteria to
grow.
Type of container and additives:
1. Sterile Cotton
swabs
Required equipment and reagents:
Equipment: Sterile Cotton swab,
laminar airflow, Incubator, Gaspak, anaerobic jar.
Reagents: Thioglycollate broth, Spore stains kit.
Environmental and safety controls:
Observe all precautions for handling any biological specimens.
Wear facemask and disposable gloves while transferring and
handling the specimen.
All cultures should be considered as biohazards and hence should
be disposed of in respective color-coded bags only.
Anaerobic culture procedure:
1. Sterile
swabs moistened with sterile distilled water and rub the surface of the
different sites.
2.
Inoculate the swabs into a tube of Thioglycolate broth and incubate at 37°C for
72h in Gaspak anaerobic jar.
3. After
incubation, the results will be reported as the present or absence of
clostridium spores after endospore staining (Schaeffer-Fulton method).
a. Add malachite
green stain solution and steam for 5 minutes, keeping the paper moist and
adding more dye as required. Alternatively, the slide may be steamed over a
container of boiling water.
b. Wash the slide
in tap water.
c. Counterstain
with 0.5% safranin for 30 seconds.
d. Examine the
slide under a microscope for the presence of endospores. Endospores are bright
green and vegetative cells are brownish-red to pink.
4. Examples of positive
endospore staining: Clostridium perfringens, C. botulinum,
C. tetani, Bacillus anthracis, Bacillus cereus, Desulfotomaculum spp,
Sporolactobacillus spp, Sporosarcina spp,
Interpretation:
If presence of turbidity, make a smear and perform endospore
staining. Look especially for organisms morphologically suggestive of
clostridia (Clostridium tetani).
In the absence of detection of bacilli suggestive of Clostridium
tetani, hold culture for 7 days and report as negative if no turbidity.
Reporting of
test results:
Absence of Clostridium spores: Only brownish-red to
pink vegetative cells were seen.
Presence of Clostridium spores: Green endospores located
in the terminus (drumstick appearance) of the cells were observed. Suggestive
of Clostridium tetani.
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