CULTURE MEDIA & CULTURE MEDIA & CULTURE METHODSCULTURE METHODS

Culture Media

- Bacteria and other microbes have particular requirements for growth- When they reside in and on our bodies or in the environment, they harvest their food from us or from the environment- When we grow bacteria in lab, we are essentially creating a captive environment for bacteria – like a bacteria zoo. So we must provide the bacteria we grow in lab with all of the materials that they need to grow

•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 Pasteur The original media used by Louis Pasteur – urine or meat broth– 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 originating from a single bacterial cell.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 2% agar is employed in solid medium medium

•After sterilization, the media is poured into sterile Petrie plates

•Agar is liquefies at 100 C and solidifies at 40 C•After autoclaving the media(in tube) for 20 minutes, the tubes are placed in a slanted position to allow the agar to solidify. These tubes are called slants

•Microorganisms grow on the surface of agar plates and slants

How is media made?• Media is measure - a specific quantity of dry

powdered nutrient media, add water and check the pH(7).

• Dispense the media into bottles (flask, tube), cap it and autoclave. The autoclave exposes the media to high temperature (121°C) and pressure (15 psi) for 20 minutes.

• Once the media is autoclaved it is sterile• (all micro-organism forms killed)

Aseptically pouring agar plates

•Using a marker, label To write information on culture tubes or Petri plates (the name of the microorganism you are growing, your group symbol)

• All labeling is done on the bottom of the agar plate

1. Initials2. Date

(mm/dd/yy)3. Code # or letter

Environmental sampling

Surface samples are normally taken using sterile swabs

Normal Flora Samples

• Important to remember that microbes are (everywhere)!

• We are inhabited (covered) by many different bacteria. .

• Most of the symbiotic relationships that we have with microbes are beneficial to both the microbe and us!

• In today's lab we will examine normal flora (hand. hair. skin)

Applying oral sample to surface of agar.

• Place all inoculated material in incubator Culture tubes should be stored upright in plastic beakers, while Petri plates should be incubated upside down ‑(lid on the bottom )

Agar plates are stored upside down to prevent condensation.

•These plates are incubated at •37° C for 24 hours and then stored at refrigerator until next 24 hours or a week when we will observe for results.

Typical environmental sampling results

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 can Colony morphology, pigmentation, hemolysis can

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

Liquid media ( Broth)Liquid media ( Broth)– 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: Nutrient Broth, Nutrient Eg: Nutrient Broth, Nutrient AgarAgar

- Nutrient Broth consists of - Nutrient Broth consists of peptone, meat extract, NaCl, peptone, meat extract, 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, Substances like blood, serum, egg are added to the basal egg are added to the basal medium.medium.

•Used to grow bacteria that are Used to grow bacteria that are exacting in their nutritional exacting in their nutritional needs.needs.

•Eg: Blood agar, Chocolate agarEg: Blood agar, Chocolate agar

Blood agar Chocolate agar

• This media is differential because:

• Certain bacteria produce enzymes (hemolysins…) that act on the red cells to produce either:

• Beta hemolysis: Enzymes lyse the blood cells completely, producing a clear area around the colony.

• Alpha hemolysis: Incomplete hemolysis produces a greenish discoloration around the colony

• Gamma hemolysis: No effect on the red cells.

Enrichment media Enrichment media •Liquid media used to isolate Liquid media used to isolate pathogens from a mixed culture.pathogens from a mixed 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 isolation – for the isolation

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

Vibrio choleraeVibrio cholerae

Selective mediaSelective media•culture medium that allows the growth of one type of organisms, while inhibiting the growth of other organisms

•The inhibitory substance is added to a solid media.The inhibitory substance is added to a solid media.Eg: Eg: Mac Conkey’s mediumMac Conkey’s medium for gram negative bacteria for gram negative bacteria•Thiosulphate Citrate Bile Salt Sucrose Agar Thiosulphate Citrate Bile Salt Sucrose Agar – for V. – for V.

choleraecholerae•Lowenstein Jensen mediumLowenstein Jensen medium – M. tuberculosis – M. tuberculosis•Wilson and Blair mediumWilson and Blair medium – S. typhi – S. typhi•Potassium tellurite mediumPotassium tellurite medium – Diphtheria bacilli – Diphtheria bacilli•EMB (Eosin Methylene Blue)•dyes inhibit Gram (+) bacteria. Selects for Gram (-) bacteria

TCBSMac Conkey’s medium

Potassium Tellurite media LJ media

Indicator mediaIndicator media•These media contain an indicator These media contain an indicator which changes its colour when a which changes its colour when a bacterium grows in them.bacterium 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 incorporated in A media which has substances incorporated in it enabling it to distinguish between bacteria.it enabling it to distinguish between bacteria.

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

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

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

MacConkey's•MacConkey’s is both a selective & differential media.

•MacConkey’s is selective media because it inhibits the growth of some organisms [Gram positive bacteria].

2. MacConkey’s is differential media

•- “lactose fermenters” bacteria will grow in red colonies while” non-lactose fermenters” will be colorless and clear.

left: no lactose fermentationright: lactose fermentation

MacConkey Agar

So if there are colonies of bacteria growing on MacConkey’s, it’s understood that they are Gram-If those colonies are colorless, they are not lactose fermenters. If the colonies have a pinkish appearance, they are lactose fermenters

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 for the detection of gas by the bacteria.bacteria.

Transport mediaTransport media•Media used for transporting the Media used for transporting the samples.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 – enteric bacilli bacilli

Anaerobic mediaAnaerobic media

•These media are used to grow anaerobic organisms.These media are used to grow anaerobic 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 They provide additional information for the identification of the for the identification of the bacterium.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 development Positive test is indicated by the development of a 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 REACTIONSTSI REACTIONS::

Yellow – AcidYellow – AcidPink - AlkalinePink - 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 production.Gas bubbles or crack in the medium – gas 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 Contains Sodium citrate and bromothymol blue as the indicator.blue 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 Culture methods employed depend on the purpose for which they are intended.the 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 preparation of To obtain sufficient growth for the preparation of

antigens and for other tests.antigens and for other tests.• For bacteriophage & bacteriocin susceptibility.For bacteriophage & bacteriocin 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 methodsCulture methods include: 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 culture Used for the isolation of bacteria in pure culture from clinical specimens.from clinical specimens.

•Platinum wire or Nichrome wire is used.Platinum wire or Nichrome wire is used.•One loopful of the specimen is transferred onto One loopful of the specimen is transferred onto the surface of a well dried plate.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 The inoculum is then distributed thinly over the plate by streaking it with a loop in a series of plate by streaking it with a loop in a series of parallel lines in different segments of the plate.parallel lines in different segments of the plate.

•On incubation, separated colonies are obtained On incubation, separated colonies are obtained over the last series of streaks.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 vaccinesIn the preparation of bacterial antigens and vaccines..

•Lawn cultures are prepared by flooding Lawn cultures are prepared by flooding the surface of the plate with a liquid the surface of the plate with a liquid suspension of the bacterium.suspension of the bacterium.

Antibiotic sensitivity testing

STROKE CULTURESTROKE CULTURE •Stroke culture is made in tubes Stroke culture is made in tubes containing agar slope / slant.containing agar slope / slant.

•Uses Uses •Provide a pure growth Provide a pure growth of bacterium for slide of bacterium for slide agglutination and agglutination and other diagnostic tests.other diagnostic tests.

STAB CULTURESTAB CULTURE•Prepared by puncturing a suitable medium – Prepared by puncturing a suitable medium –

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

•UsesUses•Demonstration of gelatin Demonstration of gelatin liquefaction.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 45Agar medium is melted (15 ml) and cooled to 45ooC.C.•1 ml of the inoculum is added to the molten agar.1 ml of the inoculum is added to the molten 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 Liquid cultures are inoculated by touching with a charged loop or by touching with a charged loop or by adding the inoculum with pipettes or adding the inoculum with pipettes or syringes.syringes.

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

•DisadvantageDisadvantage• It does not provide a pure culture from mixed inocula.It does not provide a pure culture from mixed inocula.

Blood culture bottles

ANAEROBIC CULTURE METHODSANAEROBIC CULTURE METHODS•Anaerobic bacteria differ in their Anaerobic bacteria differ in their requirement and sensitivity to oxygen.requirement and sensitivity to oxygen.

•Cl.tetani is a strict anaerobe – grows at Cl.tetani is a strict anaerobe – grows at an oxygen tension < 2 mm Hg.an 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 Displacement of oxygen with other gasesgases

•Displacement of oxygen with Displacement of oxygen with hydrogen, nitrogen, helium or COhydrogen, nitrogen, 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 metal Consists of a metal jar or glass jar with a metal lid which can be clamped air tight.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 spool, Under the lid – small grooved porcelain spool, wrapped with a layer of palladinised asbestos.wrapped with a layer of palladinised asbestos.

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

clamped air tight.clamped air tight.• The outlet tube is connected to a vacuum pump and The outlet tube is connected to a vacuum pump and

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

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

• Act as a catalyst for the combination of hydrogen with Act as a catalyst for the combination of hydrogen with residual oxygen.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% glucose, By using reducing agents – 1% glucose, 0.1% Thioglycolate0.1% Thioglycolate

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