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Chapter – IV: ANTIMICROBIAL ACTIVITY Antibacterial
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ANTIBACTERIAL ACTIVITY
INTRODUCTION:
The science dealing with the study of the prevention
and treatment of diseases caused by micro-organisms is known as
medical microbiology. Its sub-disciplines are virology (study of
viruses), bacteriology (study of bacteria), mycology (study of
fungi), phycology (study of algae) and protozoology (study of
protozoa). For the treatment of diseases inhibitory chemicals
employed to kill micro-organisms or prevent their growth, are
called antimicrobial agents. These are classified according to
their application and spectrum of activity, as germicides that kill
micro-organisms, whereas micro-biostatic agents inhibit the
growth of pathogens and enable the leucocytes and other defense
mechanism of the host to cope up with static invaders. The
germicides may exhibit selective toxicity depending on their
spectrum of activity. They may act as viricides (killing viruses),
bacteriocides (killing bacteria), algicides (killing algae) or
fungicides (killing fungi).
The beginning of modern chemotherapy has largely
been due to the efforts of Dr. Paul Ehrlich (1910), who used
salvarsan, as arsenic derivative effective against syphilis. Paul
Ehrlich used the term chemotherapy for curing the infectious
disease without injury to the host’s tissue, known as
chemotherapeutic agents such as antibacterial, antiprotosoal,
antiviral, antineoplastic, antitubercular and antifungal agents.
Later on, Domagk(1953) prepared an important chemotherapeutic
agent sulfanilamide.
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Classification of Antibacterial Agents:
The antibacterial agents are classified in three
categories:
(I) Antibiotics and chemically synthesized
chemotherapeutic agents.
(II) Non-antibiotic chemotherapeutic agents
(disinfectants, antiseptics and preservatives).
(III) Immunological products.
(I) Antibiotics:
They are produced by micro-organisms or they might
be fully or partly prepared by chemical synthesis. They inhibit
the growth of micro-organisms in minimal concentrations.
Antibiotics may be of microbial origin or purely synthetic or
semisynthetic [1]. They can be classified by manner of
biosynthesis or chemical structure. Structurally, they are
classified into different classes as shown in the following table.
Classification of antibiotics according to their chemical
structure (Berdy, 1974) [2].
No. Name of the group Example
1. Carbohydrate-containing
antibiotics
Pure sugars
Aminoglycosides
Orthosymycins
N-Glycosides
C-Glycosides
Glycolipids
Nojirimycin
Streptomycin
Everninomicin
Streptothricin
Vancomycin
Moenomycin
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2. Macrocyclic lactones
Macrolide antibiotics
Polyene antibiotics
Ausamycins
Macrotetrolides
Erythromycin
Candicidin
Rifamycin
Tetranactin
3. Quinones and related antibiotics
Tetracyclines
Anthracyclines
Naphthoquinones
Benzoquinones
Tetracycline
Adriamycin
Actinorhodin
Mitomycin
4. Amino acid and peptide antibiotics
Amino acid derivatives
β-Lactum antibiotics
Peptide antibiotics
Chromopeptides
Depsipeptides
Chelate forming peptides
Cycloserine
Penicillin
Bacteriacin
Actinomycins
Valinomycin
Bleomycins
5. Heterocyclic antibiotics containing
oxygen
Polyether antibiotics
Monensin
6. Heterocyclic antibiotics containing
nitrogen
Nucleoside antibiotics
Polyoxins
7. Aromatic antibiotics
Cycloalkane derivatives
Steriod antibiotics
Cycloheximide
Fusidic acid
8. Aromatic antibiotics
Benzene derivatives
Condensed aromatic antibiotics
Aromatic ether
Chloramphenicol
Griseofulvin
Novobiocin
9. Aliphatic antibiotics
Compounds containing phosphorous
Fosfomycins
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Synthetic antimicrobial agents include sulfonamides,
diamino pyrimidine derivatives, antitubercular compounds,
nitrofuran compounds, 4-quinoline antibacterials, imidazole
derivatives, flucytosine etc.
(II) Non-antibiotics:
The second category of antibacterial agents includes non-
antibiotic chemotherapeutic agents which are as follows:
(1) Acids and their derivatives: Some organic acids such as
sorbic, benzoic, lactic and propionic acids are used for
preserving food and pharmaceuticals. Salicyclic acid has
strong antiseptic and germicidal properties as it is a
carboxylated phenol. The presence of –COOH group
appears to enhance the antiseptic property and to decrease
the destructive effect. Benzoic acid is used externally as an
antiseptic and is employed in lotion and ointment. Benzoic
acid and salicylic acid are used to control fungi that cause
disease such as athlete’s foot. Benzoic acid and sodium
benzoate are used as antifungal preservatives. Mandolic
acid possesses good bacteriostatic and bactericidal
properties.
(2) Alcohols and related compounds: They are bactericidal
and fungicidal, but are not effective against endospores and
some viruses. Various alcohols and their derivatives have
been used as antiseptics e.g. ethanol and propanol. The
antibacterial value of straight chain alcohols increases with
an increase in the molecular weight and beyond C8- the
activity begins to fall off. The isomeric alcohol shows a
drop in activity from primary, secondary to tertiary.
Ethanol has extremely numerous uses in pharmacy.
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(3) Chlorination and compound containing chlorine:
Chlorination is extensively used to disinfect drinking
water, swimming pools and for the treatment of effluent
from industries. Robert Koch in 1981 first referred to the
bactericidal properties of hypochlorites. N-chloro
compounds are represented by amides, imides and amidines
wherein one or more hydrogen atoms are replaced by
chlorine.
(4) Iodine containing compounds: Iodine containing
compounds are widely used as antiseptic, fungicide and
amoebicide. Iodophores are used as disinfectants and
antiseptics. The soaps used for surgical scrubs often
contain iodophores.
(5) Heavy metals: Heavy metals such as silver, copper,
mercury and zinc have antimicrobial properties and are
used in disinfectant and antiseptic formulations.
Mercurochrome and merthiolate are applied to skin after
minor wounds. Zinc is used in antifungal antiseptics.
Copper sulfate is used as algicides.
(6) Oxidising agents: Their value as antiseptics depends on the
liberation of oxygen and all are organic compounds.
(7) Dyes: Organic dyes have been extensively used as
antibacterial agents. Their medical significance was first
recognized by Churchman [3] in 1912. He reported
inhibitory effect of Crystal violet on Gram-positive
organism. The acridines exert bactericidal and
bacteriostatic action against both Gram-positive and Gram-
negative organisms.
(8) 8-Hydroxyquinolines: 8-Hydroxyquinoline or oxine is
unique among the isomeric hydroxyquinolines, for it alone
exhibits antimicrobial activity. This attributes to its ability
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to chelate metals [4], which the other isomers do not
exhibit.
(9) Surface active agents: Soaps and detergents are used to
remove microbes mechanically from the skin surface.
Anionic detergents remove microbes mechanically; cationic
detergents have antimicrobial activities and can be used as
disinfectants and antiseptics.
(III) Immunological products:
Certain immunological products such as vaccines and
monoclonal antibodies are used to control the diseases as a
prophylactic measure.
Mode of action:
Antimicrobial drugs interfere chemically with the
synthesis of function of vital components of micro organisms.
The cellular structure and functions of eukaryotic cells of the
human body. These differences provide us with selective toxicity
of chemotherapeutic agents against bacteria.
Antimicrobial drugs may either kill microorganisms
outright or simply prevent their growth. There are various ways
in which these agents exhibit their antimicrobial activity [5].
They may inhibit,
(1) Cell-wall synthesis
(2) Protein synthesis
(3) Nucleic acid synthesis
(4) Enzymatic activity
(5) Folate metabolism or
(6) Damage cytoplasmic membrane
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Bacteriostatic dyes:
Stearn and Stearn [6] attributed the bacteriostatic
activity to triphenylmethane dyes. Fischer and Munzo [7] have
found the relationship between their structure and effectiveness
of such dyes.
A number of drugs are metal-binding agents. The
chelates are the active form of drugs. The site of action within
the cell or on the cell surface has not been established. The site
of action of oxine and its analogs has been suggested inside the
bacterial cell [8] or on cell surface [9].
Detoxification of antibacterials:
p-Aminobenzoic acid is a growth factor for certain
micro-organisms and competitively inhibits the bacteriostatic
action of sulfonamides. The metabolites identified in man are p-
amino- benzoylglucoronide; p-aminohippuric acid, p-
acetylaminobenzoic acid. 8-Hydroxyquinoline (oxine) and 4-
hydroxyquinoline are excerted as sulfate esters or glucorinides.
Bacteria:
The bacteria are microscopic organisms with
relatively simple and primitive forms of prokaryotic type. Danish
Physician Christian Grams, discovered the differential staining
technique known as Gram staining, which differentiates the
bacteria into two groups “Gram positive” and “Gram negative”,
Gram positive bacteria retain the crystal violet and resist
decolorization with acetone or alcohol and hence appear deep
violet in colour; while Gram negative bacteria, which loose the
crystal violet, are counter-stained by saffranin and hence appear
red in colour.
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These two groups of bacteria are recently classified into four
different categories as follows:
(1) The world of bacteria I: “Ordinary” Gram negative bacteria.
(2) The world of bacteria II: “Ordinary” Gram positive
bacteria.
(3) The world of bacteria III: “Bacteria” with unusual
properties.
(4) The world of bacteria IV: Gram positive filamantous
bacteria of complex morphology.
Classification of important organisms:
Class: Schizomycetes
Order Family Genus Species
Micrococeacea Staphylococcus
Micrococcus
Sarcina
S.Aureus
M.tetragenus
S.lutea
Lactobacill-
aceae
Streptococcus
Peptoatrepto-
coccus
Lactobacillus
Diplococcus
Str.pygenes
Pep.putridis
L.Acidophilus
D.pneumoniae
Neisseriaceae Neisseria N.gonorrhoeae
N.meningitidi
N.catarrhalis
Corynebacte-
riaceae
Corynebacterium
Listeria
Etysipeothrix
C.dipthheriae
L.monocytogene
L.rhusiopathiae
Eubacterials
Achromobacte-
riaceae
Alcaligenes Alc.faecalis
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Order Family Genus Species
Enterobacte-
riacea
Eschercichia
Klebsiella
Citrobacter
Cloaca
Hafnia
Serratia
Salmonella
Shigella
Proteus
K.pneumoniae
K.pneumoniae
K.aerogenes
Cit.freundii
Cl.cloacae
Haf.alvei
Ser.marcescens
Sal.typhosa
Sh.dysenterise
Pr.vulgaris
Brucellaceae Pasteurella
Fancisella
Brucella
Haemophilus
Bordetella
Morarella
Actinubacillus
P.pestis
P.pseudotuber-
culosis
F.tularensis
Br.meltitensis
Br.abortus
Br.suis
H.influenzae
H. duoreyi
Bord.pertussia
M.lacunata
A.mallei
A.lignieresii
Bacteriodacess Bacteriods
Fusobacterium
Strepto-bacillus
Sphaerophorous
Bact.fragilis
F.fusiforme
St.moniliformis
Sph.nacrophorous
Eubacterials
Bacillaceae Bacillus
Clostridium
B.anthracis
B.subtilis
Cl.tetani
Cl.welchii
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Order Family Genus Species
Pseudo-
monadaceae
Pseudomonas Ps.aeruginosa Pseudo-
monadales
Spirillaceae
Vibrio
Spirillum
V.cholerae
Sp.minus
Mycoplasm-
atales
Mycoplasma-
taceae
Mycoplasma M.pneumoniae
M.mycoids
Mycobacteri-
aceae
Mycobacterium M.tuberculosis
M.laprae
Actinomycet-
aceae
Actinomyces
Nocardia
A.israeli
A.bovis
N.modurae
Actinomyce-
tates
Streptomycet
aceae
Streptomyces Strepto.griseus
Spirochaet-
aceae
Spirochaeta
Saprospira
Nonpathogenic Spirochaeteles
Treponenat-
aceae
Borrelia
Taeponema
Leptospira
Bor.duttoni
Bor.recurrentis
Bor.vincenti
Tr.pallidum
Tr.pertenue
L.icterohaemo-
rrhagiae
(1) Staphylococcus aureus: Family (micrococcaceae):
In 1878, Koch observed micrococcus like organisms
in pus; Pasteur (1880) cultivated these cocci in liquid media.
They are Gram-positive cocci, ovoid or spheroidal, non-motile,
arranged in group of clusters; they grow on nutrient agar and
produce colonies, which are golden yellow, white or lemon
yellow in colour; pathogenic strains produce, coagulated and
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ferment glucose lactose, mannitol with production of acid,
liquefy gelation and produce pus in the lesion.
Genus: Staphylococcus:
The word staphylococcus is derived from the Greek
language (Gr. Staphylo = bunch of grapes; Gr. Coccus = a grain
or berry), while the species name is derived from Latin language
(L. aureus = golden). Staphylococcus is differentiated from
micrococcus and another genus of the same family by its ability
to utilize glucose, mannitol and pyruvate anaerobically. Cells of
staphylococci, which are slightly smaller than those of
Micrococci, are found on the skin or mucus membrane of the
animal body.
Species: Staphylococcus aureus:
Basic habital of St. aureus is the anterior naves,
though it is also a normal flora of human skin, and of the
respiratory and gastrointestinal tracts. The individual cells are
0.8 to 0.9 µ in diameter. They are oval or spherical, non-motile,
non-capsulated, non-sporulating strains with ordinary aniline
dyes and are Gram-positive, typically arranged in groups or
irregular clusters like branches of groups in pus seen single or in
pairs. They easily grow on nutrient agar; the optimum
temperature for the growth is 35ºC. They are notorious as they
cause suppurative (pyogenic or pus forming) conditions, mostitis
of women and cows, boils and food poisioning. St. aureus grows
rapidly and produce circular (1-2 mm) endive edge, convex, soft,
glistening colonies having a golden yellow pigment. St. aureus
can tolerate moderately high concentration of NaCl, hence they
can be selectively isolated on the nutrient medium containing 7.5
% sodium chloride. It is also able to ferment mannitol to organic
acid. St. aureus also produce the coagulase which is able to clot
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citrated plasma. It also produces the enzymes catalase,
hyaluronidase as well as other virulent factors like hemolysins,
leucocidins, enterotoxins and exofoliatin.
(2) Bacillus subtilis:
Genus: Bacillus:
Its members are large strength Gram-positive rods,
occurring in chains, growing aerobically and forming heat
resistant spores. Most of these organisms exist as saprophytes in
soil, water, air and on vegetation, e.g. Bacillus cereus and
Bacillus subtilis.
Species: Bacillus subtilis:
The species are differentiated by a number of criteria;
one being the size of the bacillus. There are two groups, the
large-celled, e.g. B. megaterium, B. cereus and the small-celled,
e.g. B. subtilis, B. stearothernophilus.
The following species are of special interest either in
connection with antibiotics or in tests of the effectiveness of
sterilization procedures.
Some B. subtilis strains produce an extra cellular
penicillinase. The enzyme concerned is an adaptive one and is
produced in appreciable amounts only when the organism is
grown in the presence of penicillin. B. subtilis is used as a test
organism for the efficiency of ethylene oxide sterilization. The
spores of B. pumilis are used to test the efficiency of ionizing
radiation methods of sterilization.
These organisms are found in soil, dust, hay, water,
sewage and milk. B. subtilis appear as gram positive slender rods
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with rounded ends. Organisms occur either singly or in short
chains. Organisms are non-capsulated and motile with
peritrichate flagella. They produce central or subterminal, non-
bulging, oval spores. Bacillus subtilis is a non-pathogenic
organism; however it is widely used in industries for the
production of amylase, protease and glucose dehydrogenase.
Organisms are also used for the formation of an antibiotic
subtilin.
(4) Escherichia coli: Enterobacteriaceae:
They are Gram-negative rods, motile with peritrichate
flagella or non-motile. They do not form spores. All are
sometimes (i.e. from rarely to, invariably) found in intestinal
treatment of man or lower animals.
Genus: Escherichia:
This genus comprises Escherichia coli and several
variants.
Species: Escherichia coli:
Escherichia in 1885 discovered Escherichia coli which
is a commensal of the human intestine and is found in the
sewage, water or soil contaminated by faecal matters. These are
Gram-negative rods, 2 to 4 µ, commonly seen in coccobacillary
form, which do not form any spore and have 4 to 8 paritrichate
flagella, are sluggishly motile, are facultative anaerobes and
grow in laboratory media. E. coli are generally non-pathogenic
and are incriminated as pathogens, because in certain instance
some strains have been found to produce septicemia,
inflammation of liver and gall bladder, appendix and other
infections and this species is a recognized pathogen in the
veterinary field.
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Diarrhoea: Organisms have at times found to be responsible for
summer diarrhoea especially in infants.
Bacteremic and Septicemic infections: Proteus species cause
about 11 % of cases of Gram-negative bacteria. Septicaemia,
generally occurs only in patients with serious underlying
conditions or as a complications of urinary tract surgery; but
outbreaks of septicaemia, often with meningitis may occur
among the new born in hospital.
Meningitis, chest infections, otitis media and other
nosocomial infections are caused by Proteus.
Abdominal and wound infections: Proteus is often a secondary
invader of ulcers, pressure sores (bed sores), burns and damaged
tissues.
(6) Pseudomonas aeruginosa:
Genus: Pseudomonas:
Pseudomonas is a Greek word (Gr. Pseudo = false,
Gr. Monas = a unit) while the word aeruginosa is of Latin origin
(L. aeruginosa = full of copper rust i.e. green).
Species: Pseudomonas aeruginosa:
P. aeruginosa is Gram-negative short rod with
variable length (1.5-3.0 X 0.5µm). They are motile by means of
one or two polar flagella. Organisms are non-sporulating and
non-capsulated, however, few strains possess slime layer up of
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polysaccharide. Primary habitat of P.aeruginosa is human and
animal gastro-intestinal tract, water, sewage, soil and vegetation.
It is physiologically versatile and flourishes as a saprophyte in
warm moist situations in the human environment, including
sinks, drains, respirators, humidifiers, etc. P.aeruginosa produces
several virulence factors, including exotoxin A., proteases, a
leukocidin, and phospholipase C. pseudomonas is an
opportunistic pathogen which is able to cause infections when
the natural resistance of the body is low. They are mostly related
with hospital infections and post burn infections. They also
cause infections of middle ear, eyes and urinary tracts. It is also
associated with diarrhoea, pneumonia and osteomyelitis. Due to
drug resistant nature of P. aeruginosa it causes infection in
patients receiving long term antibiotic therapy for wounds, burns
and cystic fibrosis and other illness. Approximately 25% of burn
victims develop infection which frequently leads to fatal
septicemia.
Evaluation of Antibacterial Activity:
Several standardized test procedures have been
employed for evaluating the effectiveness of antimicrobial
agents. Various ‘invivo’ screening methods [10] have been used
to evaluate the antibacterial activity. Testing in mice has become
standard. The sensitivity of bacteria to antimicrobial agents is
tested by the same methods.
The standard test to evaluate the effectiveness of
disinfectants and antiseptics was the phenol coefficient test; but
antibiotic susceptibility testing depends on the observation of its
inhibiting power on the growth and/or killing culture of micro
organisms in vitro. This test is useful to prescribe the proper
antibiotics for treating infection diseases.
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One such method was developed by Alexander
Fleming which is known as disc plate technique; later on a new
technique was developed by Foster and Woodruff. In this method
the antibiotic-impregnated filter paper strips were used on agar
plates inoculated with the test organism. Zones of inhibition
occurring around the strips were observed. In the same year
Vincent and Vincent improved the above technique by
introducing antibiotic impregnated paper disc, thus increasing
the number of antibiotics to be tested simultaneously in one Petri
dish. In 1961, a committee formed under the guidelines of WHO
to evaluate antibiotic susceptibility testing recommended a new
method known as “Bauer-Kirby Procedure”. In this method, each
item was standardized [11].
Another approach to antimicrobial susceptibility
testing is to determine the Minimum Inhibitory Concentration
(MIC) by tube dilution procedure. MIC tests are used to establish
the concentration of an antibiotic that will inhibit growth.
However, to determine whether the antibiotic is microbicidal,
another test known as Minimal Bactericidal Concentration
(MBC) test was followed. In this test serum killing power is
measured by adding a bacterium to grow in the patient’s blood is
assessed by measuring changes in turbidity.
Factors influencing inhibition zone sizes:
(1) Ingredients of culture media:
Many substances are present in culture media
affecting the zone of inhibition, common ingredients like
peptone, trypton, yeast and agar may vary in their mineral
content and many of these minerals may influence the activity of
some antibiotics. It is well known that Ca, Mg and Fe effect
sensitivity zones produced by tetracyclines and gentamycine.
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(2) Choice of medium:
Consistent and reproducible results are obtained in
media prepared especially for sensitivity testing; the plates must
be poured flat with an even depth, very thin plates being
unsatisfactory.
(3) Effect of pH:
The activity of amino-glycosides is enhanced in
alkaline medium and reduced in acidic medium; the reverse is
shown by tetracycline.
(4) Size of inoculation:
The idea of inoculums is the one which gives an even
dense growth without being confluent. In practice, satisfactory
results can be achieved by taking a loopful of a well grown
culture or a suitably made suspension of organisms and
spreading it with dry sterile swab [12].
The most notable difference between mycobacterial
and common bacterial infections lies in the host parasite
relationship. In common bacterial infections, pathogens multiply
rapidly and induce a sharp defensive reaction on the part of the
host and produce a substance which is toxic to the host. The
process is acute and of short duration. If the host can not defend
the attack of invading organism, it dies. If the host’s defenses,
whether endogenous or exogenous, are adequate, the invasion is
checked and the organisms are eliminated.
The confirmation of the antibacterial properties of
sulfonamides was provided by the clinical research of a number
of workers [13]. Monosubstitution at N1 has furnished the
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bacteriostatically most active derivatives. The character of the
substituent is of decisive influence; alkyls as well as many aryl
groups have, on the whole, a dystherapeutic effects, but a few
N1-aryl-sulfanilamides have shown excellent activity. N
1-
acylation produces derivatives which sometimes exhibit greater
activity than the parent drug in equal doses; branching of the
acyl groups reduces activity.
Among N1-heterocyclically substituent sulfanilamides,
several potent bacteriostatic drugs combine a favourable
therapeutic ratio with high activity and selectivity for important
infections. The character of the hetero ring and the substitution
of the ring in isomeric positions greatly affect the activity of the
compounds.
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EXPERIMENTAL
Materials and Methods:
The bacteriostatic property of the compounds was
tested by disc diffusion method as described by Kirby-Bauer,
which is also known as Kirby-Bauer method.
Kirby-Bauer test:
Principle:
Antimicrobial susceptibility testing with discs is a
simple and rapid method and provides a reproducible means of
testing bacterial sensitivity to various antibiotics and
chemotherapeutic agents. The test is based on the fact that for a
given antibiotic, the size of the zone of inhibition is inversely
related to the MIC (Minimal Inhibitory Concentration) of the
strain being tested when the test conditions are held constant.
There are several variations in disc diffusion test:
1. Oxford’s cylinder method
2. Agar cup method
3. Paper disc method.
Paper disc method:
Paper disc method is easy, reliable and less
cumbersome than the Oxford’s cylinder or the agar cup method.
In this method, paper disc of 4 mm size are cut from Whatman
filter paper. The discs are saturated with fixed amount of
chemicals, dried, if necessary and sterilized. Such disc when
placed on the surface of agar medium (seeded with the test
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organism) gets wet again and the chemicals can diffuse into the
medium.
Requirements:
1. Young broth culture of test organism (Inoculum)
2. Chemicals: Antibacterial solution
3. Sterile nutrient agar plate, sterile melted top/soft agar
4. Sterile filter paper discs (blank)
5. Pair of forceps, sterile pipettes
1. Preparation of Inoculum:
The selected colonies of test organism were transferred into
a tube containing 5 ml of Nutrient Broth with the help of a wire
loop. The broth cultures were incubated at 35-37ºC for 24 hours.
The test cultures were as follows:
(i) Bacillus cereus : Gram-positive rods and cocci
(ii) Staphylococcus aureus : Gram-positive cocci
(iii) Escherichia coli : Gram-negative small rods
(iv) Pseudomonas aeruginosa : Gram-negative rods and cocci
Nutrient Broth:
Peptone 3.0 gm
NaCl 1.5 gm
Meat extracts 0.9 gm
Distilled water 300 ml
pH 7.6 was maintained.
2. Preparation of Antibacterial Solution:
All the compounds were dissolved in dimethyl
formamide (DMF). Proper drug controls were used. Fig. 1 and
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Fig. 2 represent the zone of inhibition of the control and the
compound.
The compound diffused into the medium produced a
concentration gradient. After the incubation period, the zones of
inhibition were measured in mm. The tabulated results represent
the actual readings control.
3. Preparation of Nutrient Agar:
Peptone 2.5 gm
NaCl 1.5 gm
Meat extracts 0.75 gm
Agar agar powder 8.0 gm
Distilled water 250 ml
The above constituents were weighed and dissolved in
water. The sterilized medium (20.0 ml) was poured into
sterilized Petri dishes under aseptic conditions, allowing them to
solidify on a plane table.
4. Filter Paper Disc (Blank):
The paper discs of 4 mm size were cut from Whatman
filter paper (sterile).
5. Sterilized forceps and pipettes:
Sterilized pipettes were used to inoculate 0.2 ml of
test culture into sterilized melted top agar tube.
The paper discs saturated with chemical (antibacterial
solution) were placed with the use of sterilized forceps.
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Procedure:
1. 0.2 ml of test culture was inoculated into sterile melted
top agar tube which was previously cooled to 50ºC.
2. The inoculated top agar was mixed well and poured over
sterilized nutrient agar plate.
3. The top agar was allowed to solidify. 9 marks were made
using marking pen on the base of the plate regarding to
the 9 chemicals (antibacterial solutions) to be tested.
4. The forceps was sterilized by dipping it in alcohol and
flaming it.
5. A paper disc was picked up using sterilized forceps and
the disc was soaked in the chemical (antibacterial
solution) to be tested.
6. The excess of chemical was drained by touching the disc
to the sides of the tube.
7. 9 discs saturated with chemicals (antibacterial solution)
were placed on the mark made for each chemical.
8. The plate was placed in refrigerator for 30 minutes to
allow the diffusion to take place.
9. The plate was transferred in incubator at 37ºC for
overnight.
10. The growth was observed after 24 hours.
11. The diameter of the zone of inhibition was measured at
the end of the incubation period.
Interpretation:
Larger the zone of inhibition, the more effective or
potent the chemical (antibacterial solution) is for the particular
test organism.
Chapter – IV: ANTIMICROBIAL ACTIVITY Antibacterial
activity
257
Fig-1
Showing paper disc method (Agar Diffusion Technique) with
essential arrangement (diagrammatic).
Fig-2
Petridis
Heavy growth
Zone of inhibition
Paper disc
Chapter – IV: ANTIMICROBIAL ACTIVITY Antibacterial
activity
258
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