CULTURE MEDIA & CULTURE MEDIA & CULTURE METHODSCULTURE METHODS

Bacteria have to be grown (cultured) for Bacteria have to be grown (cultured) for them to be identified.them to be identified.

By appropriate procedures they have to By appropriate procedures they have to be grown separately (isolated) on culture be grown separately (isolated) on culture media and obtained as pure for study.media and obtained as pure for study.

HistoryHistory The original media used by Louis The original media used by Louis

Pasteur – urine or meat brothPasteur – urine or meat broth Liquid medium – diffuse growthLiquid medium – diffuse growth Solid medium – discrete colonies.Solid medium – discrete colonies.

ColonyColony – macroscopically visible – macroscopically visible collection of millions of bacteria collection of millions of bacteria originating from a single bacterial cell.originating from a single bacterial cell.

Cooked cut potato by Robert Koch – Cooked cut potato by Robert Koch – earliest solid mediumearliest solid medium

Gelatin – not satisfactoryGelatin – not satisfactory- liquefy at 24- liquefy at 24ooCC

AgarAgar Frau HesseFrau Hesse Used for preparing solid mediumUsed for preparing solid medium Obtained from seaweeds.Obtained from seaweeds. No nutritive valueNo nutritive value Not affected by the growth of the Not affected by the growth of the

bacteria.bacteria. Melts at 98Melts at 98ooC & sets at 42C & sets at 42ooCC 2% agar is employed in solid medium 2% agar is employed in solid medium

Types of culture mediaTypes of culture media

I.I. Based on their consistency Based on their consistency a) solid mediuma) solid mediumb) liquid mediumb) liquid mediumc) semi solid mediumc) semi solid medium

II.II. Based on the constituents/ ingredientsBased on the constituents/ ingredientsa) simple mediuma) simple mediumb) complex mediumb) complex mediumc) synthetic or defined mediumc) synthetic or defined mediumd) Special mediad) Special media

Special mediaSpecial media– Enriched mediaEnriched media– Enrichment mediaEnrichment media– Selective mediaSelective media– Indicator mediaIndicator media– Differential mediaDifferential media– Sugar mediaSugar media– Transport mediaTransport media– Media for biochemical reactionsMedia for biochemical reactions

III.III.Based on Oxygen requirementBased on Oxygen requirement- Aerobic media- Aerobic media- Anaerobic media- Anaerobic media

Solid media Solid media – contains 2% agar– contains 2% agar Colony morphology, pigmentation, hemolysis Colony morphology, pigmentation, hemolysis

can be appreciated.can be appreciated. Eg: Nutrient agar, Blood agarEg: Nutrient agar, Blood agar

Liquid media Liquid media – no agar. – no agar. For inoculum preparation, Blood culture, for the For inoculum preparation, Blood culture, for the

isolation of pathogens from a mixture.isolation of pathogens from a mixture. Eg: Nutrient brothEg: Nutrient broth

Semi solid medium Semi solid medium – 0.5% agar. – 0.5% agar. Eg: Motility mediumEg: Motility medium

Simple media / basal media Simple media / basal media - - Eg: NB, NAEg: NB, NA- NB consists of peptone, meat extract, - NB consists of peptone, meat extract,

NaCl, NaCl, - - NB + 2% agar = Nutrient agarNB + 2% agar = Nutrient agar

Complex mediaComplex media Media other than basal media.Media other than basal media. They have added ingredients.They have added ingredients. Provide special nutrients Provide special nutrients

Synthetic or defined mediaSynthetic or defined media Media prepared from pure chemical Media prepared from pure chemical

substances and its exact composition is substances and its exact composition is knownknown

Eg: peptone water – 1% peptone + 0.5% Eg: peptone water – 1% peptone + 0.5% NaCl in waterNaCl in water

Enriched mediaEnriched media Substances like blood, serum, egg are Substances like blood, serum, egg are

added to the basal medium.added to the basal medium. Used to grow bacteria that are exacting Used to grow bacteria that are exacting

in their nutritional needs.in their nutritional needs. Eg: Blood agar, Chocolate agarEg: Blood agar, Chocolate agar

Blood agar Chocolate agar

Enrichment media Enrichment media Liquid media used to isolate Liquid media used to isolate

pathogens from a mixed pathogens from a mixed culture.culture.

Media is incorporated with Media is incorporated with inhibitory substances to inhibitory substances to suppress the unwanted suppress the unwanted organism.organism.

Eg: Eg: – Selenite F Broth Selenite F Broth – for the – for the

isolation of Salmonella, Shigella isolation of Salmonella, Shigella – Alkaline Peptone Water Alkaline Peptone Water – for – for

Vibrio choleraeVibrio cholerae

Selective mediaSelective media The inhibitory substance is added to a The inhibitory substance is added to a

solid media.solid media.Eg:Eg: Mac Conkey’s mediumMac Conkey’s medium for gram negative for gram negative

bacteriabacteria TCBS TCBS – for V.cholerae– for V.cholerae LJ mediumLJ medium – M.tuberculosis – M.tuberculosis Wilson and Blair mediumWilson and Blair medium – S.typhi – S.typhi Potassium tellurite mediumPotassium tellurite medium – Diphtheria – Diphtheria

bacillibacilli

TCBSMac Conkey’s medium

Potassium Tellurite media LJ media

Indicator mediaIndicator media These media contain an indicator which These media contain an indicator which

changes its colour when a bacterium changes its colour when a bacterium grows in them.grows in them.

Eg: Eg: – Blood agarBlood agar– Mac Conkey’s mediumMac Conkey’s medium– Christensen’s urease mediumChristensen’s urease medium

Urease mediumUrease medium

Differential mediaDifferential media A media which has substances A media which has substances

incorporated in it enabling it to incorporated in it enabling it to distinguish between bacteria.distinguish between bacteria.

Eg: Mac Conkey’s mediumEg: Mac Conkey’s medium– PPeptoneeptone– LLactoseactose– AAgargar– NNeutral redeutral red– TTaurocholate aurocholate

Distinguish between lactose fermenters Distinguish between lactose fermenters & non lactose fermenters.& non lactose fermenters.

Lactose fermenters – Lactose fermenters – PinkPink colonies colonies Non lactose fermenters – colourless Non lactose fermenters – colourless

coloniescolonies

Sugar mediaSugar media Media containing any fermentable Media containing any fermentable

substance.substance. Eg: glucose, arabinose, lactose, starch Eg: glucose, arabinose, lactose, starch

etc.etc. Media consists of 1% of the sugar in Media consists of 1% of the sugar in

peptone water.peptone water. Contain a small tube (Durham’s tube) Contain a small tube (Durham’s tube)

for the detection of gas by the bacteria.for the detection of gas by the bacteria.

Transport mediaTransport media Media used for transporting Media used for transporting

the samples.the samples. Delicate organisms may not Delicate organisms may not

survive the time taken for survive the time taken for transporting the specimen transporting the specimen without a transport media.without a transport media.

Eg: Eg: – Stuart’s medium Stuart’s medium – non nutrient – non nutrient

soft agar gel containing a soft agar gel containing a reducing agentreducing agent

– Buffered glycerol saline Buffered glycerol saline – – enteric bacilli enteric bacilli

Anaerobic mediaAnaerobic media These media are used to grow anaerobic These media are used to grow anaerobic

organisms.organisms. Eg: Robertson’s cooked meat medium, Eg: Robertson’s cooked meat medium,

Thioglycolate medium.Thioglycolate medium.

BIOCHEMICAL TEST & REACTIONSBIOCHEMICAL TEST & REACTIONS They provide additional information for They provide additional information for

the identification of the bacterium.the identification of the bacterium. The tests include:The tests include:

– Oxidase testOxidase test– Triple sugar iron agar (TSI)Triple sugar iron agar (TSI)– Indole testIndole test– Citrate utilizationCitrate utilization– Urease testUrease test

OXIDASE TESTOXIDASE TEST

Detects the presence of an enzyme Detects the presence of an enzyme “oxidase” produced by certain bacteria “oxidase” produced by certain bacteria which will reduce the dye – tetramethyl-p-which will reduce the dye – tetramethyl-p-phenylene diamine dihydrochloride.phenylene diamine dihydrochloride.

Positive test is indicated by the Positive test is indicated by the development of a development of a purplepurple colour. colour.

Oxidase positive – Pseudomonas, Vibrio, Oxidase positive – Pseudomonas, Vibrio, NeisseriaeNeisseriae

Oxidase negative – Salmonella, ShigellaOxidase negative – Salmonella, Shigella

TRIPLE SUGAR IRON AGAR (TSI)TRIPLE SUGAR IRON AGAR (TSI) It is a composite media used to study different It is a composite media used to study different

properties of a bacterium – sugar fermentation, properties of a bacterium – sugar fermentation, gas production and Hgas production and H22S production.S production.

In addition to peptone, yeast extract & agar, it In addition to peptone, yeast extract & agar, it contains 3 sugars – Glucose, Lactose, Sucrose.contains 3 sugars – Glucose, Lactose, Sucrose.

The Iron salt – Ferric citrate indicates HThe Iron salt – Ferric citrate indicates H22S S production.production.

Phenol red is the indicator.Phenol red is the indicator. It is an orange red medium with a slant and a It is an orange red medium with a slant and a

butt.butt. pH of the medium – 7.4pH of the medium – 7.4

TSI REACTIONS:TSI REACTIONS:Yellow – AcidYellow – AcidPinkPink - Alkaline - Alkaline

Yellow slantYellow slant / / Yellow butt Yellow butt (A/A) – Lactose fermenters.(A/A) – Lactose fermenters. Pink slantPink slant / / Yellow butt Yellow butt (K/A) – Non lactose (K/A) – Non lactose

fermenters.fermenters. Pink slant Pink slant / / no colour change no colour change (K/K) – Non fermenters(K/K) – Non fermenters Black colour – H – H22S production.S production. Gas bubbles or crack in the medium – gas Gas bubbles or crack in the medium – gas

production.production. LF – E.coli, KlebsiellaLF – E.coli, Klebsiella NLF – Salmonella, ShigellaNLF – Salmonella, Shigella HH22S - ProteusS - Proteus

INDOLE TESTINDOLE TEST Used to detect indole production by the Used to detect indole production by the

organism.organism. They produce indole from tryptophan present They produce indole from tryptophan present

in peptone water.in peptone water. After overnight incubation, a few drops of After overnight incubation, a few drops of

indole reagent (Kovac’s reagent) is added.indole reagent (Kovac’s reagent) is added. Positive test is indicated by a pink ring.Positive test is indicated by a pink ring.

– PositivePositive indole test – indole test – pinkpink ring ring– Negative Negative indole test - indole test - yellowyellow ring ring

Indole positive – E.coliIndole positive – E.coli Indole negative – Klebsiella, Salmonella.Indole negative – Klebsiella, Salmonella.

CITRATE UTILIZATIONCITRATE UTILIZATION Done in Simmon’s Citrate medium.Done in Simmon’s Citrate medium. To detect the ability of certain bacteria to To detect the ability of certain bacteria to

utilize citrate as the sole source of carbon.utilize citrate as the sole source of carbon. Contains Sodium citrate and bromothymol blue Contains Sodium citrate and bromothymol blue

as the indicator.as the indicator. If citrate is utilized, alkali is produced which If citrate is utilized, alkali is produced which

turns the medium to blue.turns the medium to blue.– Citrate positive – Citrate positive – blue blue colour colour – Citrate negative – Citrate negative – greengreen colour colour

Positive – KlebsiellaPositive – Klebsiella Negative – E.coliNegative – E.coli

UREASE TESTUREASE TEST Done in Christensen’s urease medium.Done in Christensen’s urease medium. This test is used to detect organisms that This test is used to detect organisms that

produce urease.produce urease. Urease produced by the organisms split Urease produced by the organisms split

urea into ammonia and COurea into ammonia and CO2. 2.

– Urease positive – Urease positive – pink pink colourcolour– Urease negative – Urease negative – yellowyellow colour colour

Positive – Proteus, KlebsiellaPositive – Proteus, Klebsiella Negative – E.coli, SalmonellaNegative – E.coli, Salmonella

CULTURE METHODSCULTURE METHODS Culture methods employed depend on the Culture methods employed depend on the

purpose for which they are intended.purpose for which they are intended. The indications for culture are:The indications for culture are:

– To isolate bacteria in pure cultures.To isolate bacteria in pure cultures.– To demonstrate their properties.To demonstrate their properties.– To obtain sufficient growth for the To obtain sufficient growth for the

preparation of antigens and for other tests.preparation of antigens and for other tests.– For bacteriophage & bacteriocin For bacteriophage & bacteriocin

susceptibility.susceptibility.– To determine sensitivity to antibiotics.To determine sensitivity to antibiotics.– To estimate viable counts.To estimate viable counts.– Maintain stock cultures.Maintain stock cultures.

Culture methods include:Culture methods include: Streak cultureStreak culture Lawn cultureLawn culture Stroke cultureStroke culture Stab cultureStab culture Pour plate methodPour plate method Liquid cultureLiquid culture Anaerobic culture methodsAnaerobic culture methods

STREAK CULTURESTREAK CULTURE Used for the isolation of bacteria in pure Used for the isolation of bacteria in pure

culture from clinical specimens.culture from clinical specimens. Platinum wire or Nichrome wire is used.Platinum wire or Nichrome wire is used. One loopful of the specimen is transferred One loopful of the specimen is transferred

onto the surface of a well dried plate.onto the surface of a well dried plate. Spread over a small area at the periphery.Spread over a small area at the periphery. The inoculum is then distributed thinly over The inoculum is then distributed thinly over

the plate by streaking it with a loop in a the plate by streaking it with a loop in a series of parallel lines in different segments of series of parallel lines in different segments of the plate.the plate.

On incubation, separated colonies are On incubation, separated colonies are obtained over the last series of streaks.obtained over the last series of streaks.

LAWN CULTURELAWN CULTURE Provides a uniform surface growth of the Provides a uniform surface growth of the

bacterium.bacterium. UsesUses

– For bacteriophage typing.For bacteriophage typing.– Antibiotic sensitivity testing.Antibiotic sensitivity testing.– In the preparation of bacterial antigens and In the preparation of bacterial antigens and

vaccinesvaccines.. Lawn cultures are prepared by flooding the Lawn cultures are prepared by flooding the

surface of the plate with a liquid suspension surface of the plate with a liquid suspension of the bacterium.of the bacterium.

Antibiotic sensitivity testing

STROKE CULTURESTROKE CULTURE Stroke culture is made in Stroke culture is made in

tubes containing agar slope tubes containing agar slope / slant./ slant.

Uses Uses – Provide a pure growth of Provide a pure growth of

bacterium for slide bacterium for slide agglutination and other agglutination and other diagnostic tests.diagnostic tests.

STAB CULTURESTAB CULTURE Prepared by puncturing a suitable Prepared by puncturing a suitable

medium – gelatin or glucose agar with medium – gelatin or glucose agar with a long, straight, charged wire.a long, straight, charged wire.

UsesUses– Demonstration of gelatin liquefaction.Demonstration of gelatin liquefaction.– Oxygen requirements of the Oxygen requirements of the

bacterium under study.bacterium under study.– Maintenance of stoke cultures.Maintenance of stoke cultures.

Gelatin liquefaction Oxidation – Fermentation medium

POUR PLATE CULTUREPOUR PLATE CULTURE Agar medium is melted (15 ml) and cooled to Agar medium is melted (15 ml) and cooled to

4545ooC.C. 1 ml of the inoculum is added to the molten 1 ml of the inoculum is added to the molten

agar.agar. Mix well and pour to a sterile petri dish.Mix well and pour to a sterile petri dish. Allow it to set.Allow it to set. Incubate at 37Incubate at 37ooC, colonies will be distributed C, colonies will be distributed

throughout the depth of the medium.throughout the depth of the medium. UsesUses

– Gives an estimate of the viable bacterial count in a Gives an estimate of the viable bacterial count in a suspension.suspension.

– For the quantitative urine cultures.For the quantitative urine cultures.

LIQUID CULTURESLIQUID CULTURES Liquid cultures are inoculated by touching Liquid cultures are inoculated by touching

with a charged loop or by adding the with a charged loop or by adding the inoculum with pipettes or syringes.inoculum with pipettes or syringes.

UsesUses– Blood cultureBlood culture– Sterility testsSterility tests– Continuous culture methodsContinuous culture methods

DisadvantageDisadvantage– It does not provide a pure culture from It does not provide a pure culture from

mixed inocula.mixed inocula.

Blood culture bottles

ANAEROBIC CULTURE METHODSANAEROBIC CULTURE METHODS Anaerobic bacteria differ in their requirement Anaerobic bacteria differ in their requirement

and sensitivity to oxygen.and sensitivity to oxygen. Cl.tetani is a strict anaerobe – grows at an Cl.tetani is a strict anaerobe – grows at an

oxygen tension < 2 mm Hg.oxygen tension < 2 mm Hg.Methods:Methods:

– Production of vacuumProduction of vacuum– Displacement of oxygen with other gasesDisplacement of oxygen with other gases– Chemical methodChemical method– Biological methodBiological method– Reduction of mediumReduction of medium

Production of vacuum:Production of vacuum: Incubate the cultures in a vacuum Incubate the cultures in a vacuum

desiccator.desiccator.

Displacement of oxygen with other gasesDisplacement of oxygen with other gases Displacement of oxygen with hydrogen, Displacement of oxygen with hydrogen,

nitrogen, helium or COnitrogen, helium or CO22.. Eg: Candle jarEg: Candle jar

Chemical methodChemical method Alkaline pyrogallol absorbs oxygen.Alkaline pyrogallol absorbs oxygen.

McIntosh – Fildes’ anaerobic jarMcIntosh – Fildes’ anaerobic jar Consists of a metal jar or glass jar with a Consists of a metal jar or glass jar with a

metal lid which can be clamped air tight.metal lid which can be clamped air tight. The lid has 2 tubes – gas inlet and gas outletThe lid has 2 tubes – gas inlet and gas outlet The lid has two terminals – connected to The lid has two terminals – connected to

electrical supply.electrical supply. Under the lid – small grooved porcelain Under the lid – small grooved porcelain

spool, wrapped with a layer of palladinised spool, wrapped with a layer of palladinised asbestos.asbestos.

Working:Working: Inoculated plates are placed inside the jar and Inoculated plates are placed inside the jar and

the lid clamped air tight.the lid clamped air tight. The outlet tube is connected to a vacuum The outlet tube is connected to a vacuum

pump and the air inside is evacuated. pump and the air inside is evacuated. The outlet tap is then closed and the inlet tube The outlet tap is then closed and the inlet tube

is connected to a hydrogen supply.is connected to a hydrogen supply. After the jar is filled with hydrogen, the electric After the jar is filled with hydrogen, the electric

terminals are connected to a current supply, terminals are connected to a current supply, so that the palladinised asbestos is heated.so that the palladinised asbestos is heated.

Act as a catalyst for the combination of Act as a catalyst for the combination of hydrogen with residual oxygen.hydrogen with residual oxygen.

Gaspak Commercially available disposable

envelope. Contains chemicals which generate H2

and CO2 on addition of water. Cold catalyst – in the envelope Indicator is used – reduced methylene

blue.– Colourless – anaerobically– Blue colour – on exposure to oxygen

Biological methodBiological method Absorption of oxygen by incubation Absorption of oxygen by incubation

with aerobic bacteria, germinating with aerobic bacteria, germinating seeds or chopped vegetables.seeds or chopped vegetables.

Reduction of oxygenReduction of oxygen By using reducing agents – 1% By using reducing agents – 1%

glucose, 0.1% Thioglycolateglucose, 0.1% Thioglycolate

THANK YOUTHANK YOU