BACTERIAL FLORA ASSOCIATED WITH WHITE LABORATORY COATS OF PHYSICIANS AT FEDERAL MEDICAL CENTRE, ABAKALIKI.
A RESEARCH PROJECT SUBMITTED TO THE DEPARTMENT OF
APPLIED MICROBIOLOGY
ABSTRACT
RELEVANCE
OF THE RESEARCH WORK
The increasing rate of hospital acquired infections especially those caused by multi resistant Staphylococcus aureus are causing
certain apprehension. White laboratory coats are used as a means of preventing
cross contamination between patient and their physicians. But that role is
being questioned now with several evidence that shows that the white laboratory
coats are contaminated with nosocomial microbes even the multi drug resistant
ones.
Hence this work will help to assess
the white laboratory coats of physicians in this part of the world (usingphysicians in Federal Medical Centre, Abakaliki as case study) for similar contamination
and also assess the susceptibility profile of any organisms found.
AIMS
AND OBJECTIVES
This work has the following aims and
objectives
1. To assess the level of
microbial contamination of the white laboratory coats of physicians in Federal
Medical Centre, Abakaliki.
2. To determine the type of microbial contamination and the area of the coat that is mostly contaminated.
3. To assess the susceptibility of
the isolates to various antibiotics used normally in the hospital.
4. To evaluate the relationship
between white laboratory coat contamination and knowledge, attitudes and
practice of physicians.
MATERIALS
AND METHODS
STUDY
AREA
This research work was conducted at
the Federal Medical Centre, an urban hospital and one of the two tertiary
health institutions in Abakaliki, Ebonyi State (South-Eastern Nigeria).
This research was conducted from October to November, 2011.
ETHICAL
CONSIDERATION
The information gotten from this
research are treated with utmost confidentiality and used solely for the
purpose of this research.
The physicians that participated in
this research did so willingly and the research was done in accordance with the
ethical guidelines of medical research.
STUDY
POPULATION
The study population include
physicians from the Federal Medical Centre, Abakaliki.
The physicians were drawn from
different specialties and units, which includes the medical specialties,
surgical specialties, accident and emergency and other specialties. They also
come from different grades that include House officers, Registrars, Consultants
and other grades.
TYPES
OF MEDIA USED
The types of media used for the
isolation bacteria from the white laboratory coat includes:
a. Blood agar (oxide U.K.)
b. Mac-Conkey Agar
c. Chocolate Agar
d. CLED
e. Oxidase paper (reagent)
for biochemical test.
They were
prepared using manufacturer’s directions
STERILIZATION
OF MATERIALS
All the glass materials used during
the course of this work were sterilized by autoclaving at the standard time of
15 minutes at 121oC. The used apparatus includes:
·
Swab (moistened)
·
Glass Petri dish
·
Plates
·
Glass slide
·
Bijou bottle
·
Test tubes
·
Syringe
·
Glass pipette
·
Wire loop
·
Light microscope
SAMPLE
COLLECTION TECHNIQUE
Samples were collected from
physicians of different grades and specialties from Federal Medical Centre. The
numbers of coats each physician had, the time that their coats has been in use,
how often their coats are washed, the type of cleaning agent used, where the
coats are stored after work and the actual usage of the coats and hand washing
practice were determined by direct questioning.
The usage of a white laboratory coat
was defined roughly as the percentage of time the coat was worn while the physicians
were on duty.
Two samples were collected from each
physician: a saline moistened swab of the cuff and a saline moistened swab of
the lower front pocket of the coat. These sites were chosen because they are
the place where microbial contamination was thought to be greatest as these are
the most heavily used areas on a white laboratory coat (Loh et al., 2000) and (Wong et al., 1991). The samples were
appropriately labelled and then carried to the laboratory for analysis. Ten
clean washed white laboratory coats were used as control.
LABORATORY
ANALYSIS
The samples were cultured directly
on blood agar, chocolate agar, Mac Conkey agar and CLED. After 18 hours of
aerobic incubation at 37oC, the plants were examined for the total
microbial count (in CFU/Cm2) and the presence of possible pathogens
in particular S. aureus, Enterococci and gram-negative bacilli. Grams staining and
subsequent microscopy were used to examine the morphology and grams reaction of
the organisms. Various biochemical tests were used in the characterization and
identification of the isolates antimicrobial sensitivity were determined by the
modified Kirby Bauer method (Cheesbrough, 2006).
MEDIA
PREPARATION
The following steps were taken in
preparation of the media as outlined by Okereke and Kanu (2004).
1.
I weighed and dissolved the
nutrient agar base according to the manufacturer’s instruction.
2.
I dispensed the media in 14ml
in bijou bottles and sterilized in an autoclave at 121oC for 15
minutes.
3.
I also autoclaved the Petri
dishes at the above temperature and pressure for 15 minutes.
4.
I allowed the media to cool to
about 50oC and using a sterile syringe I dispensed 1ml of sterile
blood into the sterile plates.
5.
I aseptically unscrewed the cap
of the bottle flames the mouth of the bottle for few seconds and poured into
the Petri dishes
6.
I them mixed properly and
allowed to gel.
INOCULATION
AND INCUBATION
Before inoculation of the media, I
made sure that the surface of the media was dry. Then I inoculated using the
steps listed by Cheesbrough (2006).
1.
Using a swab of the specimen I
applied the inoculums to a small area of the plate and then spread to other
parts of the media by a zigzag streaking method.
2.
After this, I inverted the
plates and labelled, then I incubated at 37oC for 18hrs.
The plates were
inverted so as to prevent any condensation from falling onto the cultures.
CHARACTERIZATION AND IDENTIFICATION OF THE
ISOLATES
After the primary inoculation and
incubation the various colonies were then examined for the colony
characteristics like colour, size, edge/margin, elevation, form, translucency,
texture, and haemolysis. These were recorded. Then, the various colonies were
then sub-cultured so as to obtain the pure culture, which were subjected to further
analysis for identification (Cheesbrough, 2006).
WET
MOUNT (MOTILITY TEST)
Wet mounting is mainly used to
examine culture and specimen for motile bacteria (Cheesbrough, 2006). The
method used was that by Cheesbrough using transmitted light microscopy
(Cheesbrough, 2006).
The steps include the following
1.
I placed a drop of distilled
water on a clean glass slide and emulsified a colony of the organism in it.
2.
I then covered it with a cover
slide and then viewed using the 10x and 40x objectives.
GRAMS
STAINING
The staining technique introduced by
Christian Gram in 1884 is very important in Bacteriology. It is used in the
recognition and identification of bacteria. It has a very wide application
because it distinguishes nearly all bacteria as Gram positive and Gram negative
according to whether or not they resist decolourization of crystal violet by
acetone.
Principle: Differences in
Gram reaction between bacteria is thought to be due to differences in the
permeability of the cell wall of Gram positive and Gram-negative organisms
during the straining process (Cheesbrough, 2006). The technique I used was the
one listed by Cheesbrough (Cheesbrough, 2006) and it includes:
1.
I prepared a smear from the
pure culture, dried it and hen heat fixed.
2.
I covered the smear with
crystal violet stain for 60 seconds.
3.
I washed off the stain with
clean water.
4.
I tipped off the water and
covered the smear with Lugols iodine for 60 seconds.
5.
I washed off the iodine with
clean water.
6.
I decolorized rapidly with
acetone alcohol and then washed immediately with clean water.
7.
I covered the smear with
neutral red stain for 2 minutes.
8.
I washed off the strain with
clean water.
9.
I wiped the back of the slides
and placed in the draining rack to dry.
10.
I ten examined microscopically,
first with the 40x object to check the straining and then with the 100x oil
immersion objective Gram-positive organisms appear dark purple while
Gram-negative ones appeared red.
COAGULASE
TEST
This test used to identify S. aureus, which produces the enzyme
coagulase.
Principle: Coagulase
causes plasma to clot by converting fibrinogen to fibrin. The procedure of the
test is as follow (Cheesbrough, 2006).
1.
I placed a drop of distilled
water on each end of a glass slide.
2.
emulsified a colony of the test
organism in each of the drop to make two thick suspensions and
3.
Added a loopful of plasma to
one of the suspensions and mixed gently.
4.
I examined for clumping in 10
seconds
CATALASE
TEST
This test is used to differentiate
those bacteria that produce the enzyme catalase such as staphylococci from non-catalase producing bacteria such as streptococci.
Principle: catalase acts
as a catalyst in the break down of hydrogen peroxide to oxygen and water.
The procedure I took was as
described by Cheesbrough (Cheesbrough, 2006).
1.
I poured 2ml of hydrogen
peroxide solution into a test tube.
2.
Using a sterile wooden stick, I
removed several colonies of the test organisms and immersed it in the hydrogen
peroxide solution.
3.
I then check for immediate
bubbling. Immediate bubbling showed positive result while absence of bubble
showed negative result.
OXIDASE
TEST
This test is used to assist in the identification of organisms that
produce the enzyme cytochrome oxidase like Pseudomonas
spp.
Principle: The oxidase
produced oxidizes the phenylendediamine in the reagent to a deep purple colour
(Cheesbrough, 2006).
METHOD
1.
A piece of filter paper was
placed in a clean Petri dish and 2 drops of freshly prepared oxidase reagent
was added to it.
2.
Using a piece of stick I remove
a colony of the test organism and smeared it on the filter paper.
3.
Then I checked for the
development of a blue purple colour within a few seconds.
ANTIBIOTICS
SUSCEPTIBILITY TEST
This was carried out using the Kirby
Bauer method stated by (Ugbogu, 2004) in: Laboratory Guide for Microbiology (WHO,
2003).
1.
The agar plate was prepared
according to manufactures directives and pour in 25ml amount into Petri dishes.
2.
Using a sterile wire loop, I
collected colonies of the test organism and emulsified in 3ml of sterile
physiological saline.
3.
I them matched the turbidity of
the suspension to the turbidity standard.
4.
Using a sterile swab I
inoculated the agar plate, removing excess fluid by pressing and rotating the
swab against the slide of the tube.
5.
I then placed the disc on the
inoculated plates making sure that the diseases are correctly placed.
6.
Within 30 minutes of applying the
discs I inverted the plates and incubated it aerobically at 35oC for
18 hours.
7.
After this I then measured the
zone of inhibition.
STATISTICAL
ANALYSIS
Baseline comparism were assessed
using chi-square (X2) test analysis were appropriate and statistical
significant was reached at P > 0.05.