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ISOLATION, IDENT1FICATION AND SENSITIVITY
T 83THW OP 3E3S FLORA FR(M KENYATTA
B03PITAL 3LAPP \NB
.
K!
BY
, ,.ui ftj.B-iIL/i ( h i
3)
A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT
FOR THE AWARD OF THE BACHALOE OF PHARMACY DEGREE
DEPARTMENT 0? PHARMACY
FACULTY OF MEDICINE
UNIVERSITY OF NAIROBI
NAIROBI, KENYA.
T3
JUNA4L 19 80
university of NAIROBI Library
■III.
0392015 4
/jy
(ii)
ISOLATION f IDENTIFICATION AND SENSITIVITY
TH .Jf4» OF 3KIN FLORA FROM KENT ATT A
HOSPITAL STAFF AND STUDENTS
BY
UNDER THE SUPERVISION OF
MR. A.L. PALEKAR
FACULTY OF MEDICINE
DEPARTMENT OF PHARMACY
UNIVERSITY OF NAIROBI
(iii)
ti ri.JP
A g- A .A.if
This piece of work is affectionately dedicated to:
My Parents Mr. & Mrs. Salim.
My Twin 3i3ter Jamnia and also to my very
special friends rialixaa* Salim and Amin.
Praise be to Allah for giving me
strength and determination to start
and finish this project in time.
I wish to say special thanks to:
Who supervised me during
Hr. A.L. Palekar
the entire period this project
#
• »•****#
ft * * * Sr & *.'
* A * 4
«• «
* * *
was carried out.
I am
particularly grateful to him
for reading through the 3cript
and quidlng me in any
alterations and corrections
that had to be made.
Technical Staff of the -
My appreciation particularly to
Department of Phnroacy
th038 ln tha Aerobiology/
phamaceutics section for then
continous assistance without
'whose help the experimental 'work
would not have be on finished in
time.
Special thanks for her tireless
Miss Mwarl
assistance and kindness.
Department of Pharmacy -
From whom I collected the akin
staff and students
microorganism.
Mias hunice Iiuthoni
-
For her fantastic, flawless
typing.
(v)
JLJm-JS-J... JL-Z -3
BAGS
SUMMARY.... ..................................1
INTRODUO?10N.................................. 2
EXPERIMENTAL................. ........... 16
R E S U L T S .......................................21
DISCUSSION.................................... 29
CONCLUSION....................................50
REFERENCES
31
1
3
U
H
M
A
S .T
The purpose of this research project was to
isolate ;Acroorganisras from skin surface and identify
them using various microbiological techniques.
There
after their sensitivity was checked against commonly
used chemotherapeutic agents.
The skin flora
specimens were collected from Kenyatta National
hospital (Department of Pharmacy), staff and students.
As such results obtained refer to this locality.
Hence
variations may occur if the research was carried elsewhere.
Cultures from specific parts of the body
(Pore-head, arm, and leg) were done on agar plates.
Different colonies obtained were identified and their
sensitivity checked.
Prom the results obtained, it
was found that:(i)
The microorganisms were gram-positive
(ii)
A majority of these were coagulase positive
(iii)
Some were found to be catalase positive
while others were catalase negative.
(iv)
The microorganisms were found to be most
sensitive to Gentamycin and least sensitive
to 3ulphafurazole.
2
2
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1
a
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a
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s
..Bacteria have the following general structure.
'
srnowM i t %:9 b© j.mta©#p®ia in salso aa4
Io '..
p- ••;*
n>raxnhaeh described two- speciesi afxgn
-•4
jM t «*tj& alv,:%
fuIianeXXe in. 1930 introduced the
alau-siideation o f staph .based on differences. in :
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composed of (i) Proteins (ii) Lipids (ill) Carbohydrates
and (xv) nucleic acids.
■ H-.j ,
la.^eriftL.g.eiA Chemi.stiy
, ^TM::}0$Xt XV B QV-r<
Under all conditions of growth, protein is a raj or
component of the bacterial cell.
However, the existance
of certain classes of conjugated proteins such as
phosphoproteins and glycoproteins have not been
established in bacteria.
Lipoprotein exists in cell
Membrane of the bacteria {Hcquillen I960),
lipids have been extracted from bacteria.
Simple
In the main,
these are fatty acids, although a few neutral compounds
Lave been obtained as well.
Carbohydrates occur as
intermidiates in biochemical reactions, as intracellular
storage material, as a portion of nucleic acid, as part
of the cell wall and as extracellular capsules.
DUA of
bacteria is similar in composition to that of higher animals
and plants.
Bacterial RHA contains the eonvectional
components of ribose, adenine quanin, cytosine and uracil
together with minor amounts of unusual bases.
4
However in terms of human disease, the most important genetic
changes are those leading to drug resistance.
•*#*»9**>**N!»~***m‘
a-.JI
s-'.vwv?
s --^ «MK*)Rac
10 PBYJIOAI, “ias *oih;t;
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Staph are among the most resistant of the non-sparing,
bacteria and cati survive many adverse n m r
n rental
conditions enroute from host to host they highly' resis­
tant to light, etreiaes of temperature and drying.
They
are roarkedly resistant to phenols and many other
Dcidin
te leucocytes
'WQ
disinfectants.
Cellular Antigent3:
»>peeies-specific polysaccharide A and 3 are two
cell wall antigens which determine the immunological
specificities of staph, aureus and staph. Alfeus respectively.
A - polysaccharide from staph aureus has been shown to be
composed of reactive residues of N-acetyl glucosamine i.e.
Teichoic acid.
B - polysaccharide on the other hand was
found to be a polyglycerol-phospho Teichoic acid.
Product
Coagulase
Activity
Other properties
dnsyrne that clots blood
plasma
Staphylokinase
Snsyme that degrades
fibrin clot
Haemolysin3
All are haemolytic
(Toxins)
Harrow haemolytic
Disrupts mammalian
(Alphan)
spectrum
cells, Is
Jertnonec to ti zing,
- • lethal
Product
(Beta) B
Activity
Other proptertiea
Phospholipase, Narrow
haenolytic spectrum
(Qamiaa) &
Broad iiaemolytic
Uytotoxic to
(delta)
spectrum
mammalia cells
Disrupts
mammalian cells
Leukocidin
Kills leucocytes
Two -(interacting
proteins and
heat labile
Eaterotoxin
Hyalvaronidase
Exfoliation of
produced by phage
Infant skin
group II organisms
Enzyme that degrades
hyaluronic acid
Lipase
Enzyme that degrades
lipids
Proteinase
Enzyme that degrades
proteins
Penicillinase
Enzyme that splits the
3-lactam ring
.
PATHOGiafSCITY
Mo3t normal human beings carry large numbers of
staphylococci and related organisms both in the nose and
on the skin.
She relatively non-pathogenic opportunistic
staph, epidermidis is almost always found among the normal
flora of the skin and mucous membranes of the G.I.X. and
respiratory tract.
Whereas the pathogenic staph, aureus is
a transient, temporary member of the microb'al flora.
Sven though staph aureus is often carried by healthy
individuals, under certain circumstances it causes severe
infection and can kill its host.
Because of its frequent
presence on the body 3urface3, it is in a position to invade
whenever defenses are slightly impaired.
Consequently it is
the mo3t common cause of both traumatic, and surgical wound
infection and superficial 3kin infections.
Hature of lesiona:
?he hallmark of staph, diseases is
aupp: ioation. Once virulent staph, gain a foothold in deeper
tissues if the body, their multiplication causes necrosis
and eventual abscess formation.
Ituch of the localised tissue
damage that results ia irreversible and therefore leads to
permanent scarring.
Only in unusual severe infections do
the organisms break through the localising barrier of the
lesions and invade the lymphatics and blood stream.
Mechanism of pathogenisitv:
Many of the toxins and other
substances produced by staph,
its pathogenisity.
ureusprobably contribute to
The precise contributions of these are
not known but vary from one infection to another.
The outer layer of the cell wall of staph, aureus
contains a substance called protein A.
demonstrated to be antiphagocytic.
This has been
It has the unique ability
to interact with, the Fc region of lg G, thereby interfering
with opsarization involving cytophilic antibodies.
Specific-phage receptor-sites are present on the surface of
staph, aureus cells.
Of the Fom main groups identifiable
-
by phage typing, strains of group I and III are most often
responsible for hospital infection with antibiotic resistant
staph.
Most strains produce a leukocidin, which apparently
acts on the agtoplasmic membrane of many cell types
including human neutrophiles and macrophages causing lysis
of the cells.
X-taxins of virulent staph, is highly toxic for human
macrophages (but not neutrophiles), epithelial cells.
causes necrosis of skin.
It
Fnterotoxi.n, is the major factor
in the pathogenesis of staph food poisoning.
CraOTHXRAPY
Chloraraphenical
Chemistry
Chloramphenicol is a crystalline, neutral and stable compound
with the following structure:
-
%
It is highly soluble in alcohol and poorly soluble in water.
However, its succinate ester i3 highly soluble in water and
ia hydrolysed in tissues with the liberation of free
chloramphenicol.
Antimicrobial action
Chloramphenicol i3 a potent inhibitor of protein
synthesis and has little effect on other metabolic functions.
It acts on the 50 > units of ribosomes and interferes markedly
with the i corporation of amind acids into newly formed peptide.
Chloramphenicol is bacteriastatic for many bacteria.
action is reversible with removal of the drug.
Its
Host gram
positive bacteria are inhibited by chloramphenicol in
concentration 1 - 1 0 % / ml and gram negative bacteria in cone.
0.2-5mg/ial.
Resistance
In most bacterial species, large population of
chloramphenicol susceptible cells contain occasional resistant
mutants.
These mutants are usually only 2-4 times more
resistant than the parent populations.
Consequently they
emerge slowly in treating the individual.
The precise
mechanism of chloramphenicol resistance in bacteria mutants
has not been established.
However, it may be associated with
the presence of bacteria ensymes which acetylate the drug
clinical uses*
Chloramphenicol is a possible drug of choice in ly a few
types of infections (1) Symptomatic salmonella infection
9
(2) Haemophilus influenza Meningitis
(3) Occasional bacterimia caused by gram
negative bacteria
Adverse Reaction - (1)
Gastroenteritis
(2)
Bone-marrow disturbanc e3
(3)
Toxicity in new b o m s
Chemistry:
Free tetrcyclinea are crystalline, amphotecic
substances of low solubility.
They aro available as their
hydrochloride which are more soluble giving acidic solutions.
Tetrcyclines combine firmly with divalent metal roins, and
this chelation can interfer with its absorption and therefore
its activity.
Antibiotic activity:
Tetracyclines are bacteriastatic for many gran
and gran negative bacteria.
ositive
They are effective inhibitors
of phosphorylation of protein synthesis; they appear to
inhibit the binding of Aminocrcyl -t KNA to 30s units of
ribosomes.
The basis of their selective action on different
organisms is not understood.
Resistance:
In vitro resistance to tetracycline develop slowly in
a atep-wive manner.
Bacteria resistance to one tetracyoline
usually show resistance to others in the group.
retraoycline reelstance organisms are likely to be acquired
frora other individual in a population where tetracycline
has been widely used.
Clinical use.
Tetracyclines are the moot typical "Iroud
Spectrum’’ antibiotics.
Tfcey are effective against a v riety
of microorganisms and for this reason, are often U 3 e d
indiscriminately.
They may be the drug of choice in cholera
mycoplastica pheumonia.
They are Useful in mixed bacterial
infection related to the respiratory tract especially
sinusitis and bronchitis, have been used in skin infection
particularly ache.
verse reactions:
(a)
G.I.T. disturbances
(b)
Liver, kidney local tissue toxicity
(o)
ihotosensitization
■
Chemistry;
Streptomycin is a triacidic base consisting of
streptidine, streptose and N-methy1-1-glucosamine.
/
Seve.al salt3 have been prepared from streptomycin, the
sulphate being the most widely used.
It i3 a white
substance with a bitter taste, quite soluble in water and
insoluble in alcohol.
-
11
-
Antimicrobial ..ctivity:
A number of mechanisms have been postulated to account
for the antimicrobial activity of streptomycin.
Experimentally,
it can inhibit (1 ) an oxalo-acetate-pynivate condensation
•
...
reaction carried out b y some organisms;(2 ) it can alter the
permiability of the microbacteria cell membrane; (3 ) and
it can cause inhibition of p o l y p e p t i d e synthesis and
misreading of the genetic m e 3 3 age.
This latter mechanism
is currently believed to form the basis of the antimicrobial
action of aminoglycosides in vivo.
Streptomycin attaches
to a protein of the 30s ribosome unit and inhibit its
function in protein 3yntheis.
Resistance:
:
;
Streptomycin resistance occur when a mutation alters
or eliminates the binding site for the drug on the ribosome.
Clinical U s e ;
9
Used in Tuberculosis, in systemic and urinary tract
infections, enterococcal infection (aa a combined treatment.
Adverse effects:
Jtreptomycin cause allergy, vertigo and
loss of balance.
In large doae 3 , it exerts anephrotoxic
'
effect.
m m m i
Chemistry:
Kanamycin lias the following formula ^ 3.8 ^ 3 6^ 4 ° H
,in(1
consists of 3 -U-glucosamine, 6 - 0 -glucosamine linked to
doxystreptamine.
Kanamycin sulphate is a white powder which
is readily soluble in water and quite stable at 20°C.
op
U
A fry
Antimicrobial a tivity:
Kanamycin is bacteriaeidal in concentrations 1-lCtag/ial
for many gram positive and gram negative bacteria
particularly in alkaline medium.
Proteins are often
susceptible but p s e u d o m o n a s and streptococci are quite
resistant.
Kanamycin have sane antimicrobial action as
streptomycin.
Resistance;
dtrains of staph and K.coli may develop i*esistance
to kanamycin.
She situation with regard to staph varies
widely from region and does not seem to be related solely
to the frequency with which the drug is used.
Clinical a b d i c a t i o n :
Kanamycin i3 a drug effective against a wide range of
gran negative bacteria and has some use in the treatment of
Tuberculosis.
Its clinical application i3 confined to those
patient3 who have serious infections due to gram negative
organisms and in certain cases of T.3.
iide Effects:
Kanamycin cause Hypersensitivity, neuroto­
xicity (cranial nerve damage and curare-like effect) and
iaphrotoxicity.
■J-KITAKYCIH:
Gentamycitt 13 soluble in water and resulting solutions
are stable weeks.
Antimicrobial ac ti vit.y:
Gentamycin i3 bacteriaeidal at concentrations of 0.5”
5fflg/ml.
The activity is enhanced at alkaline pH. Hentamycin Gulphate
lQxag/ml inhibit most strains of staph, and calif arms in
vitro,
fhe pacterieidial action i3 probably due to
inhibition of protein synthesis, perhaps by causing a
\
misreading of the M R M message, at the 30s unit of ribosome,
ftaaiatance;
'Gentamycin resistance bacteria appear to alter
ribosome protein.
Microorganisms made resistant to
gentamycin are usually cross-resistant to Kanamyciakeomycin, but tne reverse i3 not the case. Some bacteria
inactivate gentamycin by phosphorylation, and this property
can be transmitted by resistant transfer factors (RTF) from
one bacteria species to another.
Clinical a m i l e t i o n :
The main indication for the use of gentamycin is the
.urinary tract infection due to sensitive organisms;
meningitis or septicaemia caused by gram negative organism
can be successfully treated.
bide effects:
The chief toxic effect of Gentamycin is on
the eight cranial nervd.
Both vestibular and auditory
branches may be affected, but the former is more susceptible.
Urticaria and transient elevation of serum transaminases
has been described.
3ULF0N G .IDE
* fiMLdtr.y:
These are generally white, odourless, bitter tasting
crystalline powders which are much soluble in alkaline than
acidic pH
14
Moat sulphonamide can be prepared aa sodium aalta which are
moderately soluble and these are used for intravenous
administration.
Mlt ini era bia.1 activity:
The action i3 sulp- o .amide is bacteriatatic and is
neversible with the removal of the drug or in the presence
of excess of Para-amino-benzoic acid.
Susceptible
microorganisms require extracellular para-amino-benzoic acid
to form folic acid, an essential step in the production of
purines.
Sulphonaaide can enter into the reaction in place
of PABA, compete for t e enzyme involved, and form non­
function analogues of folic acid.
A3 a result, further growth
of the microorganism is prevented.
Resistance:
Animal cells are unable to synthesis folic acid but
depend on exogeneous sources and for this reason are not
susceptible to sulphonamide action.
Other cells which p m
co
a large excess of PABA are resistant to sulphonamide; and
still others may actually destroy sulphonamide.
Sulphonamide
resistant mutants occur in most susceptible bacteria
populations and tend to emmerge under suitable selected pressure
The wide-spread therapeutic use of sulphonamide against
gonorrhoea has resulted in the establishment of sulphonaciide
resistant strains.
Most recently, sulphonamide resistant
meningococci have appeared.
It should be specifically
mentioned that rickettsiae are not only inhibited by
sulphonaaide but are actually stimulated in their growth.
Clinical U s e :
The number of antibacterial agents is now so great
that sulphonamide are first choice in lew situation.
Kiningococcil infection being the clearest indicator for
their
U3e.
This includes those forms of diseases with
acute and chronic septicaemia, whether meningitis is present
or not.
In epidemics these drugs may be used prophylactically.
In circumstances where the choice of agent is limited,
usually because of cost, 3ulphonamide continue to have an
important role, a3 they possess the great advantage of
cheapness and ease of administration.
’
.
-
16
EXPMIilEH?
Materials:
X.
(1)
Nutrient Agar, (oxoid Ltd.)
-
l.Og.
Yeast Extract (oxoid L20)
-
2•Og .
Peptone (oxoid L37)
-
5.0g.
Naci
-
5.0g.
Agar No.3 (oxvoid L13)
- 15.Og.
Lab. leraco powder
(oxoid L 29)
pH (7.4) Dio tilled water to lOOOnil.
Method of Preparation:14.Og of Nutrient agar was weighed and boiled in 500ml.
distilled water with stirring to dissolve.
then autoclaved at 121°c for 15 minutes.
The solution was
After which it was
removed covered and kept in the regrigerator until required.
(2)
Gram stain - composed of:
(i)
Methyl violet - solution
(ii)
Iodine solution - 2 g KI and Ig Iodine.
(iii) Safranin
(iv)
0.5g in 100ml water
- 0.25g in 100ml water
Alcohol 955®
Preparations:
(i)
Me thjrl violet solution:
0.25i of methylviolet was dissolved in 50ml of distilled
water, poured into reagent bottles and stored in a
cool place.
(ii)
Potassium iodide Solution:
lg of Potassium Iodide and 0.5g of Iodine were
mixed and dissolved in Idol, of distilled water.
Volume was made to 50ml. with distilled water and
packed in reagent bottles.
^
Oaframin Solution;
0.25g of safranin was ground in a morto'r with 5.0ml.
of 95$ ethanol and volume made to 50m l « with
distrilled water, and packed in reagent bottles.
A p p a ra tu s :
(1 )
Glass - Petri dishes and Reagent bottles.
(2 )
Cotton wool and applicator sticks.
(3)
(4)
Beaters and 3tirring rods.
■
:w .4.4
>iHs9 *■*&. ~
Bunsen burner.
(5)
Flasks and flask brushes.
(6 )
Microscope and Mcro-slides.
(7)
Autoclave.
(8 )
Oven.
(9)
Refrigerator.
-r«3F*d-
Preparation of Cotton
Arabs:
These were prepared from ootton
sticks.
ol and applicator
Were then double wrapped in seta of threes and
autoclaved at 115°C for 30 minutes.
After the allocated
times were removed and stored in a cupboard.
-
d-S
-
The glasa-petri-dishes were washed with detergent,
rinsed with distilled water and dried in a hot-air-oven.
These were then wrapped in autoclaving paper packets and
sterilized in an autoclave at 121°G for 15 min.
'These can be catergorised into 3 sections:
(i)
isolation of the microorganisms and colony counting
(ii) Identification.
(iii) Sensitivity testing.
(1)
Isolation
The microorganisms under test, being skin flora were
collected from throe sites of body surface i.e.
(i )
Pore-head
(ii)
The arm
(iii) The leg.
Using sterile cotton-swabs soaked in normal saline,
swabs were made from the three parts of the body.
Inoculated onto sterile agen plates which have been
marked with three crossing lines.
The three sections
were designated with the following letters
H
for
head
A
for
Arm
L
for
Leg
The above plates were then incubated for 46 hours
in an incubator at 37 °C.
10
19
(1)
-Jo lon.v Count i n **:
,
Alter incubation, discrete colonies were*
obtained.
These were counted using "surface
colony count*1 technique.
(2)
Identxxication:
This was done under the following aubheadin ;ss-
(a)
(a)
Gram-staining and Microscopy
(b)
Goagulaae test
(c)
Oatalase Test
Gran-staining and Mi erascony
Smears of different colonies from the
three sections of the ager plate were aade
outo microscope-slides.
and then flame fixed.
These were air dried
After which the
following staining technique was done:Slides were stained with Methyl violet
solution for 20 seconds.
Then washed off with
tap-water and stained with Iodine solution for
one minute.
The iodine solution was washed
off with 95h alcohol, leaving the alcohol on
the slide for a few seconds.
was done wit;
Counter staining
3afranin and left for 20 seconds
The gran-stained slides containing smears of
different colonies were identified under an oilimiaer3ion lens.
10
m
Slide-coagulese test was done
Culture from one colony was emulsified in a drop of water
on a slide.
This bacterial suspension was then st*rjpe4 with
a drop of plasma.
Then observed under a mi roscope for the
presen t or absence of coagulation.
Catalase testingt
A drop of 30j& hydrogen peroxide was placed on a slide
smeared with microorganisms,
tvA&ition of gas or
effervescence was recorded*
After the above identification tests, subcutures of the
different colonies were done in nutrient broth.
Then
incubated at 37°C for 4d hours in an incubator.
■
Sensitivity testing:
Standardized commercial r.ast-rings KGL 2/2 were used.
These contained known amounts of antimicrobial agents.
Outo sterile agar plates seeded with the microorganisms were
placed the mast-rings.
a
37 2 far 24 hours.
surround the rings.
The plates were then incubated at
Upon incubation clear oonee wehe seen to
Zones of inhibition ware measured in
—
j*
21
-
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ji ;ou>.;ion Aim
con jlu~>ion
Results obtained from Gram-stain, and coagulase tests
complied with the ones found for staphyloccoci.
particular staph, aureus as these were
positive.
Prom sensitivity
In
found to be eoagulaae
testing results,
it was found
that the microorganisms wore most sensitive to Gentamyein
and least sensitive to sulphafurazole.
However, because
many chemotherapeutic agent3 were used, the results can be
represented as followss-
GEIfTAMYCIN
most- sftasitive
KANAMYC1M
STREPTOMYCIN
order of
CHLORAMPHENICOL
decreasing
AMPICILLIN
sensitivity
TETRACYCLIN
CO—TRIMAZOLE
SULPHAFURAZOLE
(least sensitive)
The above results refer only to specimens collected in
Kenyatta National Hospital.
Resistance to the commonly used antibacterial agents
like Ampicillin, tetracycline and chloramphenicol could be
due t o :
(i)
Development of a resistant staphylococcal strain.
(ii5
Degradation of the antibacterial agents by the
microorganisms.
p
(iii)
People from whom the specimens were collected, had
been in contact with.these antibacterial agents,
therefore the microorganisms might have acquired
resistance.
Although Uentamycin has been found to be more effective
against staph aureus; it should not be taken as the drug of
choice.
This i3 because it has a drastic 3ide effect; it
affects the vestibular branch of the 8th cranial nerve.
.
However, auditory damage is rare.
Hence, other medications
should be used as first line of treatment in staphylococci
skin infection.
These include using hot water for cleaning
body surfaces (i.e. face, a m
etc.)
The heat or steam will
cause the staphylococci infected pores to rupture and
therefore degrade the organisms.
Use of soaps and antiseptics
is also of importance a3 some of these are bactericidal.
Antibiotics should be given as the last line of medication.
3a
R 3 P E R 3 n 0 E J
1.
Bailey W.R. in "Diagnostic Microbiology", 2nd Edn.
P IS, 22, 31 (1966).
2.
Churchill L. in "Medical Microbiology", 13th Edn,
p. 236 - 255 Vol.1 (1978).
3.
Collins C.H. & Lyne P.M., "Microbiological Methods
4th Edn., p. 1 0 9 .
t*.
4.
Oruickshank R. in "Medical Microbiology",
12th Edn., pg.
5.
236 - 255 (1973).
Davis, Dulbecco, Eisen, G .rf. in "Microbiology"
P 21, 122 301 (1970).
6.
Ernest J., Joseph
L.M.;
Edward A.A. in "Medical
Microbiology" p 161 (1970).
7.
Frederick H.M; Ernest J; Alan G. in "Medical Pharmacology"
p 467, 475 494 (197©).
8.
Hillas 3. in "Antibiotics in clinical practice",
p 20, 20 61* 86 (1972).
9.
Jawetz E . , in "Review of Medical Microbiology"
13th Edn., p 122. (1978).
10.
Lawrence P.G., Prancia 0. "Antibiotic and Chemotherapy"
p 70, £8 115 147 (1971).
11.
Meynell M. in "Theory and Practice in Experimental
Bacteriology", p 35, 125, (1930).
12.
Myruik P.W. in "Fundamentals of Medical Bacteriology
and Mycology" p 153 (1974).
a
'’"'ersity o f
l IX*A »v
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