Bacterial Culture

-is a gold standard method for demonstration of organism using culture media
Culture Media
-it gives an artificial environment stimulating natural condition necessary for
bacterial growth
Basic constituents of culture media
a) Water- is hydrogen and oxygen source
b) Electrolytes-e.g. NaCl etc
c) Peptone- is a complex mixture of partially digested protein
-it consists of proteases, polypeptide, AAs and inorganic salts (phosphorus,
K, Mg) and accessory growth factor (riboflavin)
d) Meat Extract- contains protein degradation products, carbohydrates and
inorganic salts.
e) Blood / Serum- 5-10% defibrinated sheep blood is used
- used for enriching culture media
f) Agar- obtained for sea weeds
-its chief constituent is long chain polysaccharide
- Concentration used 2-3%
-used to solidify culture media because of its high gelling strength
- Unique character melting at 980C and solidifying at 420C
Characteristics of an ideal culture media
i) Must give satisfactory growth from single inoculum
ii) Should give a rapid growth
iii) Reasonably cheap
iv) Easily reproducible
v) Enable to demonstrate all characters
Types of culture media

Based on physical state

Solid Media
-media are solidified
by incorporating agar
at 1.5-2% agar conc
-produce pure culture
for identification and
AST

Semi-solid media
-prepared by adding
0.4-0.5%
agar to
liquid media
-used for transport
media, fo rmotility and
biochemical test

Liquid Media
-most
commonly
used as enrichment
media when organim
are few
specimen containing
inhibitory substance
like antibiotic gets
diluted

Based on nutritional factor

1) Simple / basic Media
simplest media that supports growth of micro-organism that don't require
special nutrition
e.g- peptone water (1% peptone + 0.5%NaCl)
nutrient broth- (1% meat extract + peptone water)
nutrient agar (2.3% agar + Nutrient broth)
2) Complex media
media containing additional ingredients for growth of bacteria requiring
special media are complex media
all media are complex media except simple media
3) Synthetic / Defined Media
prepared from pure chemical substances
here, known chemical substances are added separately for preparation of
product of known composition

4) Special media
-it is of 2 types
a) Enriched Media
-solid media containing a specific nutritive substance such as blood serum, or egg
- used for fastidious organism
e.g- blood agar, chocolate agar, egg-yolk agar
b) Enrichment Media
- Liquid media containing special substance which favors growth and multiplication
of specific bacteria
e.g.

Tetra thionate broth for salmonella

Selenite F-broth for salmonella and shigella
Alkaline peptone water for vibrio cholera
Chriestense's urea broth for proteus spp
Thyoglycollate broth for anaerobic bacteria
Roberson's cooked meat media for anaerobic bacteria

5) Selective Media
- is a solid media that contains substance (e.g bile salts, dyes or antibiotics) that
allows the growth of particular bacteria by suppressing other.
-used for culturing specimen from sites having normal microbial flora. E.g.

Mannitol salt agar (MSA) Staph aureus

Thayer Martin agar Neisseria spp
Alkaline peptone water and TCBS agar vibrio spp
SS (salmonella shigella) agar- salmonella
Cetrimide agar Pseudomonas spp

6) Indicator Media

- contains certain indicators (neutral red / BTB) or reducing substance (potassium

tellurite)
e.g.

Mac Conkey agar neutal agar

TSI phenol agar
Wilson and Blair medium sulphite
TCBS bromothymol blue and thymol blue

7) Differential Media
-differentiate bacteria in at least 2 group
e.g. Mac conkey agar
8) Transport Media
- Semi- solid media containing ingerdiants to prevent overgrowth of commensals
and ensure survival of pathogens when specimens cannot be cultured immediately
afer collection .
-used for transporting specimen

e.g. Cary Blair medium and Bile peptone medium V. choleriae

Hug mucin H. pylori
Stuart's transport medium gonococci
Amins transport medium N. gonorrhea (for swab culture)

9) Sugar Media contains 1% sugar in peptone water along with an indicator

e.g Hiss's serum sugar for pneumococcus
10) Anaerobic Media for cultivation of anaerobic bacteria
e.g- Robertsons Cooked meat media

Methods of incoculation by using Nichrome wire

-Nichrome wire
Length of nichrome wire 6cm
Internal diameter of loop 2mm
Thickness of wire 26/27 standard wire gauze (swg)
1) Streak Method (surface plating)- routinely used method

2) Lawn or carpet culture method for AST and bacteriophage

3) Stroke culture- done for agar slope

4) Stab culture to demonstrate gelatin liquefication, oxygen requirement and
maintenance of culture
5) Pour plate culture- to estimate viable bacterial count in suspension, for bacterial
count in urine culture
6) Sweep plate method- for studying micro-organism

Method of Anareobiosis

A) By displacement of oxygen
B) By use of anaerobic chamber for oxygen
displacement
C) Oxygen absorption by chemical method
D) By biological method
E) By incorporating reducing agents in culture
media

A) By displacement of oxygen
i)
Cultivation in vaccum done in dissicator and is unsatisfactory
ii)
Displacement of oxygen by inert gas (hydrogen and nitrogen)
a) By repeated evacuation and re-filling sealed jar loaded with inoculated
media with inert gas like hydrogen and nitrogen gas
b) By use of candle jar- is ineffective but widely used method

B) By use of anaerobic chamber for oxygen displacement

-it is performed by use of Mc Intosh and Filde's jar
-it is most dependable and widely used method
Principle: - spongy palladium or platinum kept inside the jar acts as a catalyzing
agent that causes slow combination of hydrogen and nitrogen to form water.
About Mc Intosh and Filde's jar
-8*5 in stout/metal jar with tight fitting metal lid
-consists of 2 terminals at lid
Inlet- to introduce gas i.e. Hydrogen in jar
Outlet- for vaccum valve; to take out air
Electrical terminals
-consists of

 Catalyst- is a capsule containing alumina pellets coated with palladium

(palladinised alumina) suspended under lid by staut wires.
Indicator- red methylene blue
-in aerobic condition blue colour
-in anaerobic condition colourless
Procedure
-color plates inoculated with specimen are kept inside jar along with an indicator
-lid is screwed tight outlet tube is connected to vaccum pump and 1/3 rd of air is
removed from jar
-it reduces pressure of jar to 100mm of Hg noted by vaccum gauze
-then, inlet tube is connected to hydrogen supply and pressure is increased to 760
mm of Hg
-electrical terminals are switched on that heats the crystals
C) Oxygen absorption by chemical method
i) Pyrogollol (Buchner)
- Is an alkaline pyrogallol for anaerobiosis which absorbs oxygen
- Is done in large tube i.e Bunchner's tube containing solution of NaOH,
pyragollic acid is added
- Tube is then placed inside an air tight jar loaded with inoculated plates
and tubes.
ii) Mixture of powdered chrominium and H2SO4
- Chrominium and H2SO4 reacts in presence of oxygen producing
chromous sulfate that creates anaeriobic condition
iii)Gas pack
- Is a simple and effective technique for anaeriobiosis
- A commercial product is available in the form of a disposable product
of aluminium foil containing pallets of (i. sodium borohydride and cobalt
chloride & ii. Citric acid and sodium bicarbonate)
- Chemicals + water (10ml)= hydrogen + CO2
- Hydrogen + oxygen + catalyst (alumina pellets coated with palladium)
creates anaeriobiosis
- Incubated at 370c with culture palte
D) By biological method

- Is slow and ineffective method

- Incubating anaerobic + anaeriobic organism resulting in anaeriobic
condition
- 2 agar plates i. aeriobic bacteria (Pseudomonas aerioginosa)
ii. anaeriobic bacteria
-they are place one after another, sealed and incubated at 37 0c
E) By incorporating reducing agents in culture media
-oxygen is reduced by
1% glucose
0.05% cysteine
0.1% thioglycollate broth
0.1% ascorbic acid
Cooked meat pieces
-2 widely used anaeriobic liquid culture media are
I) Thioglycollate broth (Nutrient broth + 1% thiglycollate)
II) Robertson's Cooked meat media (cooked meat broth)
(Nutrient broth + fat free minced cooked meat of ox heart)
- Prevents growth of strict anaerobes and preserves delicate organism

Biochemical Test of Bacteria

Biochemical Test
1. Oxidase test
2. Catalase test
3. Coagulase test
4. DNAase test
5. Bile Solubility test
6. Indole test
7. Citrate
utilization
test
8. Urease test
9. Litmus

Milk

10.

Decolorization test
Aesculin
hydrolysis

test
11. Triple

sugar

agar
12. Phenyl

iron
alanine

deamination (PDA) test

13. Oxidation
Fermentation test
14. MR-VP test
15. CAMP test
16. Reverse CAMP test
17. Nagler Reaction
18. Satellitism test
19. X and V factor
20. Quellung Reaction
21. Tuberculin Skin test
22. Nitrate
Reduction
test
23. Niacin test (nicotinic
test)
24. Modified

Petroff

Method
25. Lepromin test
26. Von-Pirquet test
27. Dick test
28. PYR
(Pyrrolidonyl
test)
29. Hippurate Hydrolysis
test
30. Schultz

reaction
31. Francis test
32. ONPG test

Charlton

33. GAP test

34. LDC test
35. EIEC test

(Sereny

test)
36. Biken test
37. Elek's Gel
Precipitation
test

1) Oxidase test
- To identify organisms producing cytrochrome oxidase
- Done by oxidase reagent (1% Tetra methyl P-phenylene

diamine

dihydrochloride)
Principle- a colony of test organism is smeared on a filter paper soaked with few
drops of oxidase reagent. This reagent seems as an artificial substrate donating
electrons. Therby, organisms producing cytochrome oxidase are oxidized to deep
purple colour.
-all oxidase positive are catalase positive but not all catalase positive are oxidase
positive.
-it must not be done from CHO containing media as acidity inhibits the enzyme
-not from media containing nitrate, it may give unreliable test result
e.g. Positive Organism (Very Handsome BAP BHANCin)

Pseudomonas spp
Aeromonas
Vibrio spp
Brucella spp
Branhamella spp
H. pylori
Hemophilus spp (except H. ducri)

 Alkaligenes
Nesseria
Campylobacter spp
2) Catalase test
- To differentiate catalse producing bacteria (staph) from non-catalse
producing bacteria (strepto)
- It uses 3% H2SO4
Principle- catalase produced by bacteria acts as a catalyst in breakdown of
hydrogen peroxide to water and oxygen which is indicated by bubble formation.

3) Coagulase test
- To identify staph aureus from other staph
- Plasma is used except citrated plasma
Principle coagulase if present in bacteria, causes plasma to clot by converting
fibrinogen to fibrin (10 sec)
-2 types of coagulase are produced by most strain of staph aureus
i) Free coagulase
-done in a test tube
-converts fibrinogen to fibrin by activating coagulase reacting factor (CRF)
ii) Bound coagulase
-done in slide
-converts fibrinogen to fibrin directly

4) DNAase test
- To identify staph aureus from other staphylococcus
- Used when plasma unavailable for coagulase test

- Done in DNAse plate and 1mol/l HCl

Principle- DNAase hydrolyses DNA. When test organism is cultured in a medium
containing DNA then after overnight incubation colonies are tested for DNAase
by flooding plate with weak HCl.
The acid precipitates unhydrolysed DNA. DNAase producing colonies are
therefore surrounded by clear due to DNA hydrolysis.
5) Bile Solubility test
- To differentiate S. pneumoniae (soluble) from other -haemolytic
streptococcus (insoluble)
Principle- A heavy inoculum of test organism are emulsified in physiological saline
that gives turbid suspension.
Then, when bile salt, sodium deoxycholate is added it dissolves S. pneumoniae by
clearing turbidity within 5-10 min.
While S. viridians are insoluble in bile salt and therefore there is no clearing of
turbidity.
6) Indole test
- To identify enterobacteria producing tryptophanase enzyme producing
bacteria
- It is done by Kovac's or Ehrlich's reagent which contains 4-Pdimethyl amino benzaldehyde (DAB)
- Uses SIM (Sulphide Indole Motility Medium) or 1% Tryptophan broth
Principle- Test organism is cultured in SIM or tryptophan medium. After
overnight

incubation,

organism

containing

tryptophanase

hydrolyses

AA

tryptophan into indole, pyruvic acid and ammonia.

Thus, produced indole reacts with Kovac's reagent producing red coloured
compound in surface (red ring)
Tryptophan + H2O tryptophanase

indole + pyruvic acid + ammonia

Kovac's reagent
Red ring
Positive ( PM is Very Very Emportant Person )
- E.coli
- Proteus vulgaris
- Proteus rettgeri
- Morgenella morgani
- V. cholera
- V. parahaemolyticus
Negative (KP)
- Klebsiella (except K. Oxitoca)
- Proteus mirabilis
7) Citrate utilization test
- To identify enterobateria
- Uses Simmon's Citrate Agar that contains
Sodium citrate as a sole source of carbon and energy for growth
Ammonium salt as nitrogen
Bromothymol blue (BTB) as indicator
Principle- when test organism is cultured in a medium, organism uses sodium
citrate and ammonia salt producing carbondioxide as byproduct.
Thus, produced carbondioxide combines with sodium and water to form sodium
carbonate, an alkaline product, which changes colour of medium from green to
blue.
Positive (KP is Student of PEC)
- K. pneumoniae
- P. mirabilis
- Providencia
- Enterobacter
- Citrobacter
- Serratia

Negative (EMbaSSY)
- E. coli
- M. morganni
- S.typhi
- Shigella
- Y. enterocolitica
8) Urease test
- To differentiate enterobacteria producing urease enzyme
- Uses Christensen's Urea Broth that contains
Urea
Phenol red as indicator
Principle test organism is cultured in a medium. After overnight incubation, if
organism is urease producing enzyme will hydrolyze urea to give ammonia and
carbondioxide.
The formation of ammonia alkalinizes the medium which is detected by change in
colour of indicator from light orange (pH 6.8) to magenta colour (pH 8.1).
Positive (HaPPY MK)
- Proteus spp
- Providencia spp
- Y. enterocolitica
- Klebsilla spp
- Morganella spp
- H. pylori
Negative (ESS)
- E.coli
- Salmonella spp
- Shigella spp
9) Litmus Milk Decolorization test
- For identification of enterococci
- Is based on the ability of most strains of Enterococci spp to reduce litmus
milk by enzyme action

- Done on Litmus Milk Medium that contains

Skimmed milk powder
Litmus as indicator
Principle- A heavy inoculum of test organism is incubated at 35 - 37 0c for 4 hr in a
tube containing litmus milk medium. Then, reduction of litmus milk is indicated by
change in colour of medium from mauve to white or pale yellow.

10) Aesculin hydrolysis test

- To differentiate enterococci from streptococci
Principle it is used to determine the ability of an organism to hydrolyze the
glycoside, aesculin to aesculetin and glucose in presence of 10 40 % bile.
Bile inhibits growth of most gram +ve cocci other than enterococcus spp.
Aesculetin combines with ferric ions in medium to form a dark brown or black
phenolic complex

11) Triple sugar iron agar

- To identify enterobacteria by their specific reaction on slant
Principle TSI reaction are based on the fermentation of lactose and glucose
(dextrose) and production of H2S.
Bacteria that ferment any of 3 sugars (glucose, lactose, sucrose) in medium will
produce byproducts, usually acids, which will change the colour of indicator
phenol red to yellow colour.
While the positions of colour change, either in butt, slope or both distinguish
the bacteria.
If only glucose is fermented, acid produced in butt will turn it yellow but
insufficient acid products are formed to affect methyl red in slant. (R/Y)
If sucrose or lactose or both are fermented, sufficient fermentation
products are formed to turn both butt and slant yellow. (Y/Y)

 If no fermentation occurs (e.g obligate aerobes), slant and butt remains

red. (R/R)
If organism produces H2S, which will react with ferrous sulfate (present in
medium) to form ferrous sulphide, which appears as black ppt in butt.
If organism forms gas during fermentation, it will show the butt either as
bubbles or as a crack in agar.

Slant/But

Gas

H2S

Possible organism

Y/Y

E.coli, klebsiella spp, and enterobacter

Y/Y

Proteus vulgaris

R/Y

Shigella spp, Morganella spp, Providencia spp,

yersinia
R/Y

enterocolitica,

V.

cholera,

V.

parahaemolytics
Proteus mirabilis, salmonella spp (except S.

R/Y

typhi)
S. paratyphi, Providencia alcalifaciens

R/Y

S. typhi

R/R

No enterobacteriaceae family

12) Phenyl alanine deamination (PDA) test

- To identify Proteus spp that produce enzyme deaminase
- Done in phenyl deaminase medium
Principle It is based on the ability of organism to convert AA phenyl alanine, to
phenyl pyruvic acid by oxidative deamination.
Test organism is inoculated on phenyl deaminase medium. After overnight
incubation, phenyl pyruvic acid is produced which is detected by addition of few
drop of 10% Ferric Cholride ions, that gives green colour on surface of culture.
Phenylalanine deaminase

ammonia + phenyl pyruvic acid

13) Oxidation Fermentation test

- To differentiate those organism that oxidize

carbohydrate (aerobic

utilization) from those organism that ferment carbohydrate (anaerobic).

- Done in semi - solid (0.3%) O/f media (also known as Hung and Leifson's
media) that contains
BTB (bromothymol blue) as an indicator
Principle A heavy inoculum of test organism is stab in two tubes containing O/f
media
- Tube 1st- It is covered with paraffin wax or melted wax immediately after
inoculation.
- Tube 2nd it is not covered with wax
- After overnight incubation, organism are detected by colour change of
bromothymol blue from green to yellow
If colour of both tube change to yellow, organism will be fermentative.
If colour of sealed tube only change to yellow, organism will be oxidative.
Result
Acid production (on both tube) E.coli and staph aureus
Acid production (on sealed tube only) Ps. Aeruginosa and Micrococcus spp.
No Acid production (on both tube) Brodetella, Borellia

14) MR-VP test (Methyl red Voges Prokauer test)

- To distinguish two type of organism
i. Organism that produce sufficient amount of acid (lactic acid, acetic acid,
formic acid, ethanol)
ii. Organism that produce acetoin and 2,3 butanediol from pyruvic acid fromed
after metabolism of glucose.
-Done on glucose phosphate broth that contains methyl red as an indicator.
Principle

 For MR test test organism is cultured in a buffered glucose phosphate broth.

After overnight incubation, bacteria if fermentative produces sufficient amount
of acid that makes medium acidic.
Thus, shift of pH of medium is detected by change in colour of methyl red from
red to yellow.
For VP test test organism is cultured in buffered glucose phosphate broth.
After overnight incubation, bacteria motabolise glucose producing pyruvic acid.
Thus, produces pyruvic acid are converted to acetoin or its reduced product 2,3
butanediol which is then converted to pink coloured compound by action of
creatine.
All MR +ve are VP ve and vice versa.
15) CAMP test (Christie, Atkins, Munch-Petersen test)
- To identify Lancefied Group B -Sterptococci based on their formation of
substance, CAMP factor.
Principle- Test is performed by streaking known - lytic Staph. aureus across 510% blood agar plate at center.
Then, test organism is inoculated at right angle to it (with in 2 cm) but not
touching Staph streak.
After overnight incubation, CAMP factor produced by Strep. agalacticae reacts
with - lysine produced by staph resulting in production of enlarged, arrow head
shaped area of haemolysis.

16) Reverse CAMP test

- Is an alternative to Nagler test
- to identify Cl.perfringes
Principle- Test organism is inoculated on anaerobic blood agar plate with single
streak.

Then, an inoculum of Group B - haemolytic streptococci is inoculated at right

angle to it (within 2cm but not touching to the 1st streak).
After overnight incubation, phospholipase produced by organism reacts with haemolysin of staph resulting in inhibition of hemolysis.
An arrow of no haemolysis is formed at junction of organism. Thus, as arrow of
haemolysis pointing from streptococcus to test organism is formed.

17) Nagler Reaction

- To identify Clostridium perfringes
- Done on Lactose egg yolk milk agar that contains
Lecithin to observe Lecithinase- C activity (-toxin)
Lactose to differentiate lactose fermenter from non-lactose fermenter.
Neutral red- as an indicator
Principle- test is performed by covering one half of plate with specific antibiotic
serum which will inactivate lecithinase.
Then, test organism at right angle to center line so that inoculum passes from
antitoxin free half of plate to antitoxin covered half.
After incubation, Cl.perfringes produces in a medium containing lecithin sue to
lecithinase c activity (-toxin) producing opacity.
While on other half of plate, no opacity is produced due to neutralization of toxin by antitoxin.

18)

Satellitism test

19)

X and V factor

20) Quellung Reaction

21)

Tuberculin Skin test

22) Nitrate Reduction test

23) Niacin test (nicotinic test)

24) Modified Petroff Method

25) Lepromin test

26) Von-Pirquet test

27) Dick test

28) PYR (Pyrrolidonyl test)

29) Hippurate Hydrolysis test

30) Schultz Charlton reaction

31)

Francis test

32) ONPG test

33) GAP test

34) LDC test

35) EIEC test (Sereny test)

36) Biken test

37) Elek's Gel Precipitation test

38) Complement Fixation test

Antibiotic Sensitivity Testing (AST)

-is used to select effective anti-microbial drugs.
- is useful essentially in following conditions
i.
ii.
iii.
iv.
v.

Patients

getting

prolonged

treatment

for

serious

infectiously

e.g.

endocarditis
Neonates and infants with serious infections
Patients not responding to therapy
To check patient complain
Certain antibiotics (e.g aminoglycerides)

i.e. Successful concentration required for successful treatment and in case of

concentration toxic to patient is required to monitor for prevention of toxicity to
patient also, to find effective therapeutic concentration of drug.
Techniques
-can be done by 2 principle methods

AST
method

Disc
diffusion

strokes

Dilution

kirby
baurer

broth
dilution

agar
dilution
susceptibili
ty

A) Disc diffusion Method

- It is routinely used method that uses anti-microbial disc of different
concentration.
- Then, anti-microbial diffuses from disc into the medium and growth of test
organism is inhibited at distance disc that is related to susceptibility of the
organism.
- Zone of inhibition or resistance to anti-microbial drug disc is related to
susceptibility.
- Test is done on media such as
a. Muller Hinton Agar (MHA)- commonly used
b. Chocolate agar for fastidious bacteria e.g. Haemophilus
c. Blood agar for S. pyogens
d. Wilkin's chalgren agar for anaerobic bacteria

Diameter of antibiotic disc 6mm

Disc distance from edge of plate 15mm
Disc to disc distance 25mm
No. of disc not more than 6
Diameter of petri disc 90cm
Depth of agar in petri disc 4mm
Amount of agar in petri disc approx. 25ml

Types of disc diffusion method

a) Strokes method
-both

test and control organism are inoculated on same plate

- Then, zone size of test organism is compared with that of control organism
- Not as highly standardized as Kirby bauer method
b) Kirby Bauer method
- is recommended by WHO
-test and control organism are placed separately
-standard inoculum (turbidity of test) is matched with 0.5 Mc Farland

- Mc Farland is a barium sulphate = 150 million bac conc. / ml

(1% barium chloride (0.05ml) + 1% H2SO4 (9.95 ml))
-then according to the size of zone of inhibition, organism is recommended as
Resistant- it implies that the drug is resistant (i.e no response) to that
organism.
Intermediate- it implies that drug is likely to respond to treatment when
drug is used in larger doses than normal or when drug is concentrated at site
of infection.
Susceptible- it implies that drug is responding to treatment.
B) Dilution method
-it measures
MIC (minimum inhibitory concentration) the lowest concentration of antimicrobial at which the isolate is completely inhibited.
MBC (minimum bactericidal concentration) lowest concentration of antimicrobial required to kill bacteria
- It is of 2 types
i)

Broth Dilution test

- It involves addition of isolate to a broth containing serial dilution of antimicrobial agent
- Is further divided into 2 types

a) Microdilution
Broth test
-it uses 0.05 - 0.1 ml
of total broth
volume
-is performed in
micro tritre format.

b) Macrodilution
Broth test
-it uses about 1.0ml
of total broth
volume
-is performed in
standred test tubes

- After overnight incubation, MIC and MBC is recorded

- MIC is useful where therapeutic dose is required to accurately regulated (e.g.
bacterial endocarditis) and in drug sensitivity of slow growing bacteria (e.g.
mycobacteria).
Antibiotic conc (g/ml)
100

50

25

12.5

6.2

3.1

1.6

0.8

o.4

MHA broth

ii) Agar Dilution Susceptibility test

-It is done by adding serial dilution of anti-microbial agent to agar agent to agar
medium. Then, a standardized suspension of test organism is inoculated
- After overnight incubation, MIC and MBC is recorded

-is convenient when several strains of bacteria are to be tested at same time.
C) E- test
-is a modification of gradient diffusion test which utilizes a commercially available
non porous plastic (non- absorbent plastic) strip.
-plastic strips contain a gradually decreasing concentration of a particular
antibiotic.
-strip also display a numerical scale that indicates antibiotic concentration
contained there in
-is applied onto inoculated MHA plates
-after overnight incubation, a symmetrical inhibition elipse (elliptical shaped zone)
is formed
-then MIC is read directly from scale in g/ml at point where a clearly defined
growth or inhibition edge intersects the strip.

Anti-microbial Agents
Definitions
a) Antibiotics-substance produced by micro-organism that inhibits the growth
of other micro-organism
b) Anti-bacterial agent- any compound (natural, synthetic or semi-synthetic)
that is active against microorganism
c) Bacteriostatic Agent- that inhibits

growth

of

micro-organism

e.g.

sulphonamides, chloramphenicol.
d) Bactericidal Agent- that kills microorganisms and kill only the growing
organisms e.g. penicillin, streptomycin
e) Narrow spectrum drug- active against a limited variety of bacteria either
gram positive or gram negative.
f) Broad spectrum drug- active against both gram positive and gram negative
bacteria.

Mechanism of Action
-for therapeutic purposes, an anti-microbial drug should exhibits selective
toxicity i.e. drug is harmful to parasite but not to host in concentration tolerated
by host.
- Anti-bacterial agents can be grouped by their mode of action i.e.
I. Inhibition of cell wall synthesis
II. Inhibition of protein synthesis
III. Inhibition of bacterial nucleic acid synthesis
i)

Inhibition of cell wall synthesis

-inhibition is due to -lactamase agent that contains a -lactam ring by binding to
penicillin binding protein (PBPs)
-e.g.
Drug

Example

Effect

Penicillin

Ampicillin; Amoxicillin

For gram positive and

Haemophilus influenza

Cephalosporins

Flucloxacillin; cloxacillin for staph

Benzyl penicillin
For
strepto,
Phenoxymethyl penicillin pneumococcus, clostridia.
Bactericidal and have 6
membered ring
Mainly used for serious
infection caused by gram

Glycopeptides

Vancomycin

negative bacteria
Bactericidal; for serious
infective

endocarditis

and septicaemia by gram

positive bacteria.
Broad spectrum of action

vii.

Carbapenems

ii)

Inhibition of protein synthesis and impairment of function ribosomes

i)

Inhibitors of 30s subunits of ribosome

Aminoglycoside

Gentamicin,

streptomycine,

Tetracyclines

Bacteriostatic
Used for gram negative bacteria

neomycine,

and treatment of sepsis

amikacine
Tetracycline

Broad

spectrum

antibiotic,

effects against cocci and bacilli

Mostly
for
rickettsial
and
chlamydial infection
Bacteriostatic
ii)
Inhibitors of 50s subunit of ribosome
Chloramphenicol
-bacteriostatic broad
drug
-binds

to

component

peptidyl
of

spectrum

transferase

50s

ribosomal

subunit and thus blocks peptide

elongation resulting in premature
Macrolides

Erytromycin

termination of peptide chain

-bacteriostatic agent
binds
to
ribosomal

50s

immobilize peptidyl tRna leading

to

premature

termination

of

peptide chain
-to treat streptococcal infection,
respiratory

infection,

non-

specific urethritis
-2nd line of drug for penicillin
Lincosamides

Clindamycin

hypersensitivity
-binds to ribosomal 50s subunit
and

therby

elongation
iii)

Inhibition of nucleic acid synthesis

block

protein

-DNA synthesis inhibitors

a) Novobiocin- interferes with synthesis of DNA gyrase
b) Quinolones- are also DNA gyrase inhibitors
e.g. nalidixic acid- for variety of gram negative bacteria except pseudomonas
-for UTI
-is 1st generation drug
Fluroquinolones- for gram positive and gram negative bacteria
-is 2nd generation drug
e.g. ciprofloxicine, norfloxacine, ofloxacine
c) Nitroimidazole- is an anti-flagellate
-organism sensitive to metro utilizes low redox potential compounds
that reduce 5-nitro group of imidazole, producing metabolities that apparently
alkylate DNA and inhibits DNA synthesis
e.g. mertonidazole
-RNA synthesis inhibitors
-binds to DNA dependent RNA polymerase and inhibits initiations of RNA
synthesis
-bactericidal for M.tuberculosis and also used for leprosy treatment.