pintu's thesis chepter-7A Antibaterialshodhganga.inflibnet.ac.in/bitstream/10603/37365/14/14...Chapter – IV: ANTIMICROBIAL ACTIVITY Antibacterial activity 237 2. Macrocyclic lactones

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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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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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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254

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.


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Fig-1

Showing paper disc method (Agar Diffusion Technique) with

essential arrangement (diagrammatic).

Fig-2

Petridis

Heavy growth

Zone of inhibition

Paper disc


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REFERENCES

1. Hand Book of Bacteriology, Robert Cruickshank, 394

(1962).

2. Essentials of medical pharmacology, K. D. Tripathi, 625

(1994).

3. J. W. Churchman, J. Exptl. Med., 16, 221 (1912).

4. A. Albert et al., Brit. J. Exptl. Pathol., 34, 119 (1958).

5. L. D. Gebbharadt and J. G. Bachtold, Proc. Soc. Exptl.

Biol. Med., 88, 103 (1955).

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