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 etcc) 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 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

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 fewspecimen containing inhibitory substance like antibiotic gets diluted

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 culture5) 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 i) Cultivation in vaccum – done in dissicator and is unsatisfactory

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

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 colourlessProcedure -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 1/3rd 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 methodi)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 370c

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 test2. Catalase test3. Coagulase test4. DNAase test5. Bile Solubility test 6. Indole test 7. Citrate utilization

test8. Urease test9. Litmus Milk

Decolorization test

10.Aesculin hydrolysis test11. Triple sugar iron

agar12. Phenyl alanine

deamination (PDA) test

13. Oxidation Fermentation test

14. MR-VP test 15. CAMP test16. Reverse CAMP

test17. Nagler Reaction 18. Satellitism test19. X and V factor 20. Quellung Reaction21. Tuberculin Skin

test22. Nitrate Reduction

test23. Niacin test

(nicotinic test)24. Modified Petroff

Method25. Lepromin test26. Von-Pirquet test27. Dick test28. PYR (Pyrrolidonyl

test)29. Hippurate

Hydrolysis test30. Schultz – Charlton

reaction 31. Francis test32. ONPG test33. GAP test34. LDC test35. EIEC test (Sereny

test)36. Biken test37. 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 α-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-P-

dimethyl 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 - 370c 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/Butt

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 enterocolitica, V. cholera, V. parahaemolytics

R/Y + + Proteus mirabilis, salmonella spp (except S. typhi)

R/Y + - 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 β-Sterptococci based on their

formation of substance, CAMP factor.

Principle- Test is performed by streaking known β- lytic Staph. aureus across 5-10% 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 β- 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 β- 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 β-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 (α-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 (α-toxin) producing opacity.

While on other half of plate, no opacity is produced due to neutralization of α-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. Patients getting prolonged treatment for serious infectiously e.g. endocarditis

ii. Neonates and infants with serious infectionsiii. Patients not responding to therapy iv. To check patient complain v. 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

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 usedb. Chocolate agar – for fastidious bacteria e.g. Haemophilusc. Blood agar – for S. pyogensd. 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

AST method

Disc diffusion

strokes kirby baurer

Dilution

broth dilution agar dilution susceptibility

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 anti-microbial at which the isolate is completely inhibited.

MBC (minimum bactericidal concentration) – lowest concentration of anti- microbial 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

anti-microbial agent- Is further divided into 2 types

- 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 (µg/ml)

100 50 25 12.5 6.2 3.1 1.6 0.8 o.4 0

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

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

-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 µg/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 synthesisII. Inhibition of protein synthesis III. Inhibition of bacterial nucleic acid synthesis

i) Inhibition of cell wall synthesis-inhibition is due to β-lactamase agent that contains a β-lactam ring by binding to penicillin binding protein (PBPs)-e.g.

Drug Example Effect i. Penicillin Ampicillin; Amoxicillin For gram positive and

Haemophilus influenza

Flucloxacillin; cloxacillin

for staph

Benzyl penicillin For strepto, pneumococcus, clostridia.

Phenoxymethyl penicillin

ii. Cephalosporins

Bactericidal and have 6 membered ringMainly used for serious infection caused by gram negative bacteria

iii. Glycopeptides

Vancomycin Bactericidal; for serious infective endocarditis and septicaemia by gram positive bacteria.

iv. Carbapenems

Broad spectrum of action

ii) Inhibition of protein synthesis and impairment of function ribosomes

i) Inhibitors of 30s subunits of ribosomeAminoglycosides

Gentamicin, streptomycine,

BacteriostaticUsed for gram negative bacteria and treatment of

neomycine, amikacine

sepsis

Tetracyclines Tetracycline Broad spectrum antibiotic, effects against cocci and bacilliMostly for rickettsial and chlamydial infectionBacteriostatic

ii) Inhibitors of 50s subunit of ribosomeChloramphenicol

-bacteriostatic broad spectrum drug-binds to peptidyl transferase component of 50s ribosomal subunit and thus blocks peptide elongation resulting in premature termination of peptide chain

Macrolides Erytromycin -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 hypersensitivity

Lincosamides Clindamycin -binds to ribosomal 50s subunit and therby block protein elongation

iii) Inhibition of nucleic acid synthesis

-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 synthesise.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.