bacterio ogy

8/20/2019 Bacterio Ogy

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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 forbacterial 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,

, !g) and accessory growth factor (riboflavin)

d) Meat Extract- contains protein degradation products, carbohydrates and

inorganic salts.

e) Blood / Serum- "-#$% 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 &-'%-used to solidify culture media because of its high gelling strength

- niue character melting at *+$C and solidifying at &$C

Characteristics of an ideal culture media

i) !ust give satisfactory growth from single inoculum

ii) hould give a rapid growth

iii) easonably cheap

iv) /asily reproduciblev) /nable to demonstrate all characters

Types of culture media


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Based on physical state

Solid Media

-media are solidifedby incorporating agarat 1.5-2% agar conc

-produce pure cultureor identifcation andAST

Semi-solid media

-prepared by adding0.4-0.5% agar toliuid media

-used or transportmedia! o rmotility andbioc"emical test

Liquid Media

-most commonlyused as enric"mentmedia #"en organimare e#

specimen containingin"ibitory substanceli$e antibiotic getsdiluted

Based on nutritional factor

! Simple / "asic Media

• simplest media that supports growth of micro-organism that don0t reuire

special nutrition

• e.g- peptone water (#% peptone 1 $."%NaCl)

• nutrient broth- (#% meat extract 1 peptone water)

• nutrient agar (&.'% agar 1 Nutrient broth)

#! Complex media

• media containing additional ingredients for growth of bacteria reuiring

special media are complex media

• all media are complex media except simple media

$! Synthetic / %efined Media

• prepared from pure chemical substances

• here, 2nown chemical substances are added separately for preparation of

product of 2nown composition


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&! Special media

-it is of & 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-yol2 agar

"! Enrichment Media

- 'i(uid media containing special substance which favors growth and multiplication

of specific bacteria

e.g.

• 3etra thionate broth 4 for salmonella

• elenite 5-broth 4 for salmonella and shigella

• Al2aline peptone water 4 for vibrio cholera

• Chriestense0s urea broth 4 for proteus spp

• 3hyoglycollate broth 4 for anaerobic bacteria

• oberson0s coo2ed meat media 4 for anaerobic bacteria

)! Selecti*e 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. /.g.

• !annitol salt agar (!A) 4 taph aureus• 3hayer !artin agar 4 Neisseria spp

• Al2aline peptone water and 3C6 agar 4 vibrio spp

• (salmonella shigella) agar- salmonella

• Cetrimide agar 4 7seudomonas spp

+! ,ndicator Media


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- contains certain indicators (neutral red 8 636) or reducing substance (potassium

tellurite)

e.g.

• !ac Con2ey agar 4 neutal agar

• 39 4 phenol agar• :ilson and 6lair medium 4 sulphite

• 3C6 4 bromothymol blue and thymol blue

! %ifferential Media

-differentiate bacteria in at least & group

• e.g. !ac con2ey agar

.! Transport Media

- emi- 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 6lair medium and 6ile peptone medium 4 ;. choleriae•


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-1ichrome 2ire

=ength of nichrome wire 4 >cm

9nternal diameter of loop 4 &mm

3hic2ness of wire 4 &>8&? standard wire gau@e (swg)

#) Strea3 Method 4surface plating!- routinely used method

&) 'a2n or carpet culture method 4for A3 and bacteriophage

') Stro3e culture- done for agar slope) Sta" culture 4 to demonstrate gelatin liuefication, oxygen reuirement and

maintenance of culture

") Pour plate culture- to estimate viable bacterial count in suspension, for bacterial

count in urine culture

>) S2eep plate method- for studying micro-organism

Method of Anareo"iosis


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A) By displacement of oxygen

B) By use of anaerobic chamber for oxygendisplacement

C) Oxygen absorption by chemical method

) By biological method

!) By incorporating reducing agents in culturemedia

A!By displacement of oxygeni) Culti*ation in *accum 4 done in dissicator and is unsatisfactory

ii) %isplacement of oxygen "y inert gas (hydrogen and nitrogen)

a) 6y repeated evacuation and re-filling sealed ar loaded with inoculated

media with inert gas li2e hydrogen and nitrogen gas

b) 6y use of candle ar- is ineffective but widely used method

B! By use of anaero"ic cham"er for oxygen displacement

-it is performed by use of Mc ,ntosh and 5ilde6s 7ar

-it is most dependable and widely used method

Principle8 - spongy palladium or platinum 2ept inside the ar acts as a cataly@ing

agent that causes slow combination of hydrogen and nitrogen to form water.

A"out Mc ,ntosh and 5ilde6s 7ar-+B" in stout8metal ar with tight fitting metal lid

-consists of & terminals at lid

9nlet- to introduce gas i.e.


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Catalyst- is a capsule containing alumina pellets coated with palladium

(palladinised alumina) suspended under lid by staut wires.

9ndicator- red methylene blue

-in aerobic condition blue colour

-in anaerobic condition colourless

Procedure-color plates inoculated with specimen are 2ept inside ar along with an indicator

-lid is screwed tight outlet tube is connected to vaccum pump and #8' rd of air is

removed from ar

-it reduces pressure of ar to #$$mm of $

mm of


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- 9s slow and ineffective method

- 9ncubating anaerobic 1 anaeriobic organism resulting in anaeriobic

condition

- & agar plates i. aeriobic bacteria (7seudomonas aerioginosa)

ii. anaeriobic bacteria

-they are place one after another, sealed and incubated at '?$c

E! By incorporating reducing agents in culture media

-oxygen is reduced by

#% glucose

$.$"% cysteine

$.#% thioglycollate broth

$.#% ascorbic acid

Coo2ed meat pieces-& widely used anaeriobic liuid culture media are

,! Thioglycollate "roth (Nutrient broth 1 #% thiglycollate)

,,! ation

test

.= ?rease test

= 'itmus Mil3


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%ecolori>ation test

0= Aesculin hydrolysis

test

= Triple sugar iron

agar

#= Phenyl alaninedeamination 4P%A! test

$= 9xidation

5ermentation test

&= M


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$$= ;AP test

$&= '%C test

$)= E,EC test 4Sereny

test!

$+= Bi3en test

$= Ele36s ;elPrecipitation

test

! 9xidase test

- 3o identify organisms producing cytrochrome oxidase

- Gone by oxidase reagent ( Tetra methyl P-phenylene diaminedihydrochloride!

Principle- a colony of test organism is smeared on a filter paper soa2ed with few

drops of oxidase reagent. 3his reagent seems as an artificial substrate donating

electrons. 3herby, organisms producing cytochrome oxidase are oxidi@ed to deep

purple colour.

-all oxidase positive are catalase positive but not all catalase positive are oxidasepositive.

-it must not be done from C


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Al2aligenes

1esseria

Campylobacter spp

#! Catalase test

- 3o differentiate catalse producing bacteria (staph) from non-catalseproducing bacteria (strepto)

- 9t uses '%


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- Gone in GNAse plate and #mol8l


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ovac0s reagent

ed ring

Positi*e ( PM is @ery @ery Emportant Person )

- E.coli

- Proteus vulgaris

- Proteus rettgeri

- Morgenella morgani

- @. cholera

- @. parahaemolyticus

1egati*e (FP!

- Flebsiella (except . xitoca)- Proteus mirabilis

! Citrate utili>ation test

- 3o identify enterobateria

- ses Simmon6s Citrate Agar that contains

•odium citrate as a sole source of carbon and energy for growth

•Ammonium salt as nitrogen

•6romothymol blue (BTB) as indicator

Principle- when test organism is cultured in a medium, organism uses sodium

citrate and ammonia salt producing carbondioxide as byproduct.

3hus, produced carbondioxide combines with sodium and water to form sodium

carbonate, an al2aline product, which changes colour of medium from green to

blue.

Positi*e (FP is Student of PEC)

- F. pneumoniae

- P. mirabilis

- Providencia

- Enterobacter

- Citrobacter

- Serratia


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1egati*e (EMbaSS)

- E. coli

- M. morganni

- S.typhi

- Shigella- . enterocolitica

.! ?rease test

- 3o differentiate enterobacteria producing urease en@yme

- ses Christensen6s ?rea Broth that contains

• rea

• Phenol red as indicator

Principle 4 test organism is cultured in a medium. After overnight incubation, iforganism is urease producing en@yme will hydroly@e urea to give ammonia and

carbondioxide.

3he formation of ammonia al2alini@es the medium which is detected by change in

colour of indicator from light orange (p< >.+) to magenta colour (p< +.#).

Positi*e 4:aPP MF!

- Proteus spp

- Providencia spp

- . enterocolitica

- Flebsilla spp

- Morganella spp

- :. pylori

1egati*e 4ESS!

- E.coli

- Salmonella spp

- Shigella spp

! 'itmus Mil3 %ecolori>ation test

- 5or identification of enterococci

- 9s based on the ability of most strains of /nterococci spp to reduce litmus

mil2 by en@yme action


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- Gone on =itmus !il2 !edium that contains

•2immed mil2 powder

• =itmus as indicator

Principle- A heavy inoculum of test organism is incubated at '" - '?$c for hr in a

tube containing litmus mil2 medium. 3hen, reduction of litmus mil2 is indicated by

change in colour of medium from mauve to white or pale yellow.

0! Aesculin hydrolysis test

- 3o differentiate enterococci from streptococci

Principle 4 it is used to determine the ability of an organism to hydroly@e the

glycoside, aesculin to aesculetin and glucose in presence of #$ 4 $ % bile.

6ile inhibits growth of most gram 1ve cocci other than enterococcus spp.

Aesculetin combines with ferric ions in medium to form a dar2 brown or blac2

phenolic complex

! Triple sugar iron agar- 3o identify enterobacteria by their specific reaction on slant

Principle 4 39 reaction are based on the fermentation of lactose and glucose

(dextrose) and production of


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9f no fermentation occurs (e.g obligate aerobes), slant and butt remains

red. ( 9f organism produces :#S, which will react with ferrous sulfate (present in

medium) to form ferrous sulphide, which appears as "lac3 ppt in "utt.

9f organism forms gas during fermentation, it will show the butt either as

"u""les or as a crac3 in agar.

Slant/But

t

;as :#S Possi"le organism

/ 1 - /.coli, 2lebsiella spp, and enterobacter

/ 1 1 7roteus vulgaris


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$! 9xidation 5ermentation test

- To differentiate those organism that oxidi>e car"ohydrate (aerobic

utili@ation) from those organism that ferment car"ohydrate (anaerobic).

- Gone in semi - solid ($.'%) 9/f media 4also 3no2n as :ung and 'eifson6s

media! that contains

• BTB (bromothymol blue) as an indicator

Principle 4 A heavy inoculum of test organism is stab in two tubes containing 8f

media

- Tu"e st- 9t is covered with paraffin wax or melted wax immediately after

inoculation.

- Tu"e #nd 4 it is not covered with wax- After overnight incubation, organism are detected by colour change of

bromothymol blue from green to yellow

9f colour of both tube change to yellow, organism will be fermentative.

9f colour of sealed tube only change to yellow, organism will be oxidative.


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5or M< test 4 test organism is cultured in a buffered glucose phosphate broth.

After overnight incubation, bacteria if fermentative produces sufficient amount

of acid that ma2es medium acidic.

3hus, shift of p< of medium is detected by change in colour of methyl red from

red to yellow.

5or @P test 4 test organism is cultured in buffered glucose phosphate broth.After overnight incubation, bacteria motabolise glucose producing pyruvic acid.

3hus, produces pyruvic acid are converted to acetoin or its reduced product &,' 4

butanediol which is then converted to pin2 coloured compound by action of

creatine.

All M< G*e are @P D*e and *ice *ersa.

)! CAMP test 4ChristieH At3insH Munch-Petersen test!

- 3o identify 'ancefied ;roup B I-Sterptococci based on their formation of

substance, CAMP factor.

Principle- 3est is performed by strea2ing 2nown I- lytic taph. aureus across )-

0 "lood agar plate at center.

3hen, test organism is inoculated at right angle to it (with in & cm) but not

touching taph strea2.

After overnight incubation, CA!7 factor produced by trep. agalacticae reacts

with I- lysine produced by staph resulting in production of enlarged, arrow head

shaped area of haemolysis.

+!


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3hen, an inoculum of Jroup 6 I- haemolytic streptococci is inoculated at right

angle to it (within &cm but not touching to the #st strea2).

After overnight incubation, phospholipase produced by organism reacts with I-

haemolysin of staph resulting in inhibition of hemolysis.

An arrow of no haemolysis is formed at unction of organism. 3hus, as arrow ofhaemolysis pointing from streptococcus to test organism is formed.

! 1agler


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&$) Luellung eaction

) 3uberculin 2in test

&&) Nitrate eduction test

&') Niacin test (nicotinic test)

&) !odified 7etroff !ethod

&") =epromin test

&>) ;on-7iruet test

&?) Gic2 test

&+) 7M (7yrrolidonyl test)

&*)


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'$) chult@ 4 Charlton reaction

'#) 5rancis test

'&) N7J test

'') JA7 test

') =GC test

'") /9/C test (ereny test)

'>) 6i2en test

'?) /le20s Jel 7recipitation test

'+) Complement 5ixation test


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ASTmet"od

%iscdi&usion

stro$es$irbybaurer

%ilution

brot"dilution

agardilution

susceptibility

Anti"iotic Sensiti*ity Testing 4AST!

-is used to select effective anti-microbial drugs.

- is useful essentially in following conditions

i. 7atients getting prolonged treatment for serious infectiously e.g

endocarditis

ii. Neonates and infants with serious infections

iii. 7atients not responding to therapy

iv. 3o chec2 patient complain

v. Certain antibiotics (e.g aminoglycerides)

i.e. uccessful concentration reuired for successful treatment and in case of

concentration toxic to patient is reuired to monitor for prevention of toxicity to

patient also, to find effective therapeutic concentration of drug.

Techni(ues

-can be done by & principle methods


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A!%isc diffusion Method

- 9t is routinely used method that uses anti-microbial disc of different

concentration.

- 3hen, 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.- one of inhibition or resistance to anti-microbial drug disc is related to

susceptibility.

- 3est is done on media such as

a. !uller


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- !c 5arland is a barium sulphate F #"$ million bac conc. 8 ml

(#% barium chloride ($.$"ml) 1 #%


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a) MicrodilutionBroth test

-it uses 0.05 - 0.1 mlof total brot"

'olume

-is performed inmicro tritre format.

b) MacrodilutionBroth test

-it uses about 1.0mlof total brot"

'olume

-is performed instandred test tubes

After overnight incubation, !9C and !6C is recorded

!9C is useful where therapeutic dose is reuired to accurately regulated (e.g.

bacterial endocarditis) and in drug sensitivity of slow growing bacteria (e.g

mycobacteria).

Antibiotic conc (Og8ml)

#$$ "$ &" #&." >.& '.# #.> $.+ o. $

()A brot"

ii! Agar %ilution Suscepti"ility test

-9t is done by adding serial dilution of anti-microbial agent to agar agent to agar

medium. 3hen, a standardi@ed suspension of test organism is inoculated

- After overnight incubation, !9C and !6C is recorded


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-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 utili@es a commercially available

non 4 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 !


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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.9. 9nhibition of cell wall synthesis

99. 9nhibition of protein synthesis

999. 9nhibition of bacterial nucleic acid synthesis

,nhi"ition of cell 2all synthesis

-inhibition is due to I-lactamase agent that contains a I-lactam ring by binding to

penicillin binding protein (767s)

-e.g.

%rug Example Effect

Penicillin AmpicillinD Amoxicillin 5or gram positive and

membered ring

!ainly used for serious

infection caused by gram

negative bacteria

;lycopeptides ;ancomycin 6actericidalD for seriousinfective endocarditis

and septicaemia by gram

positive bacteria.

Car"apenems 6road spectrum of action

,nhi"ition of protein synthesis and impairment of function ri"osomes


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i! ,nhi"itors of $0s su"units of ri"osome

Aminoglycoside

s

;entamicinH

streptomycineH

neomycineH

ami3acine

Bacteriostatic

?sed for gram negati*e "acteria

and treatment of sepsis

Tetracyclines Tetracycline Broad spectrum anti"ioticH

effects against cocci and "acilli

Mostly for ric3ettsial and

chlamydial infection

Bacteriostatic

ii! ,nhi"itors of )0s su"unit of ri"osome

Chloramphenicol -"acteriostatic "road spectrum

drug-"inds to peptidyl transferase

component of )0s ri"osomal

su"unit and thus "loc3s peptide

elongation resulting in premature

termination of peptide chain

Macrolides Erytromycin -"acteriostatic agent

- "inds to ri"osomal )0simmo"ili>e peptidyl t


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-%1A synthesis inhi"itors

a! 1o*o"iocin- interferes with synthesis of GNA gyrase

"! uinolones- are also GNA gyrase inhibitorse.g. nalidixic acid- for variety of gram negative bacteria except pseudomonas

-for 39

-is #st generation drug

5lurouinolones- for gram positive and gram negative bacteria

-is &nd generation drug

e.g. ciprofloxicine, norfloxacine, ofloxacine

c! 1itroimida>ole- is an anti-flagellate -organism sensitive to metro utili@es low redox potential compounds

that reduce "-nitro group of imida@ole, producing metabolities that apparently

al2ylate GNA and inhibits GNA synthesis

e.g. mertonida@ole

-

bacterio ogy

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