Culturing and Isolating COURSE : B.SC. (MICRO)SUBJECT : ELEMENTARY

MICROBIOLOGYUNIT 4.4

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 be By appropriate procedures they have to be grown separately (isolated) on culture 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 collection of – macroscopically visible collection of millions of bacteria originating from a millions of bacteria originating from a single bacterial cell.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 bacteria.Not affected by the growth of the 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 can Colony morphology, pigmentation, hemolysis can

be appreciated.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 in Used to grow bacteria that are exacting in

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

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 The inhibitory substance is added to a solid The inhibitory substance is added to a solid

media.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

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

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

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

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) for Contain a small tube (Durham’s tube) for

the detection of gas by the bacteria.the detection of gas by the 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 reducing soft agar gel containing a reducing agentagent

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

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, gas properties of a bacterium – sugar fermentation, gas production and Hproduction 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 butt.It is an orange red medium with a slant and a butt. pH of the medium – 7.4pH of the medium – 7.4

Isolation & CulturingIsolation: Colony on media, one kind of microbe, pure culture: isolation on general and special “differential media”

General growth media: NA, TSADifferential: Mac, EMB, SS

These have dyes, salts, inhibiting agents : see differences on

plates

Microbiology – Chapter 3Isolation: Colony on media, one kind of microbe, pure culture

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Microbiology – Chapter 3Isolation: Colony on media, one kind of microbe, pure culture – Streak Plates

3

Microbiology – Chapter 3Isolation: Colony on media, one kind of microbe, pure culture. Many colonies? Use a needle, pick one, and redo streak plate

Microbiology – Chapter 3Differential: Mac, EMB, SS

These have dyes, salts, inhibiting agents : see differences on plates

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Microbiology – Chapter 3 Blood agar : rich with nutrients, can see a

difference, thus differential; much more later

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

for which they are intended.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 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 of surface of the plate with a liquid suspension of the bacterium.the bacterium.

Antibiotic sensitivity testing8

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.

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

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

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

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

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 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 touching with a Liquid cultures are inoculated by touching with a

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

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

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

inocula.inocula.

Blood culture bottles10

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 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.

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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 pump The outlet tube is connected to a vacuum pump

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

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

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

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

0.1% Thioglycolate0.1% Thioglycolate

Reference Image (1 to 12)1. Microbiology by Gerard J. Tortora, Christine L Case, and Berdell R.

FunkeBooks: 1. Microbiology VI Edition, M.J. Pelczar, E.C.S. Chan and N.R. Kreig,

Tata McGraw Hill2. Brock Biology of Microorganisms (13th Edition) by Michael T. Madigan,

John M. Martinko, David Stahl.3. Microbiology by Gerard J. Tortora, Christine L Case, and Berdell R.

Funke4. Microbiology by Prescott, Harley, and Klein's Microbiology Joanne M.

Willey, Linda M

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