Bacterial Bacterial CCuullttuurree MMeeddiiaa

basicsbasicsDr.T.V.Rao MDDr.T.V.Rao MD

Major Contribution to Major Contribution to Culture MediaCulture Media

Agar - Agar Agar - Agar Frau Hesse’sFrau Hesse’scontributioncontribution

Agar – AgarAgar – Agar

► Solid medium is made Solid medium is made by adding Agarby adding Agar

► Agar is obtained from Agar is obtained from Sea weeds New Zealand Sea weeds New Zealand agar is more agar is more

► Agar contain long chain Agar contain long chain poly poly saccharides.Inoranic saccharides.Inoranic salts and protein like salts and protein like substancesubstance

► Melts at 98Melts at 9800c and sets c and sets at 42at 4200cc

Agar - AgarAgar - Agar

►Complex polysaccharide Complex polysaccharide ►Used as solidifying agent for culture Used as solidifying agent for culture

media in Petri plates, slants, and deepsmedia in Petri plates, slants, and deeps►Generally not metabolized by microbesGenerally not metabolized by microbes►Liquefies at 98°CLiquefies at 98°C►Solidifies ~42°CSolidifies ~42°C

► Dr.T.V.Rao MD’s ‘e’ learning seriesDr.T.V.Rao MD’s ‘e’ learning series

Media and CultureMedia and Culture•Media: Nutrients (agar, pH indicators, proteins and carbohydrates) used to grow organisms outside of their natural habitats

•Culture: The propagation of microorganisms using various media

Culture mediaCulture media

►Used to grow bacteriaUsed to grow bacteria►Can be used to:Can be used to:

Enrich the numbers of bacteria Enrich the numbers of bacteria Select for certain bacteria and suppress Select for certain bacteria and suppress

othersothers Differentiate among different kinds of Differentiate among different kinds of

bacteriabacteria

Culture and MediumCulture and Medium

► Culture Culture is the term given to is the term given to microorganisms that are cultivated in microorganisms that are cultivated in the lab for the purpose of identifying the lab for the purpose of identifying and studying them.and studying them.

► Medium Medium is the term given to the is the term given to the

combination of ingredients that will combination of ingredients that will support the growth and cultivation of support the growth and cultivation of microorganisms by providing all the microorganisms by providing all the essential nutrients required for the essential nutrients required for the growth (that is, multiplication) in order growth (that is, multiplication) in order to cultivate these microorganisms in to cultivate these microorganisms in large numbers to study them.large numbers to study them.

Specific MediaSpecific Media

►Defined media are media composed of Defined media are media composed of pure ingredients in carefully measured pure ingredients in carefully measured concentrations dissolved in double concentrations dissolved in double distilled water i.e., the exact chemical distilled water i.e., the exact chemical composition of the medium is known. composition of the medium is known. Typically, they contain a simple sugar as Typically, they contain a simple sugar as the carbon and energy source, an the carbon and energy source, an inorganic nitrogen source, various inorganic nitrogen source, various mineral salts and if necessary growth mineral salts and if necessary growth factors (purified amino acids, vitamins, factors (purified amino acids, vitamins, purines and pyrimidines purines and pyrimidines

Need for Culture MediaNeed for Culture Media

► It is usually essential to obtain a culture by It is usually essential to obtain a culture by grwoing the organism in an artificial grwoing the organism in an artificial medium.medium.

► If more than one species or type of organism If more than one species or type of organism are present each requires to be carefully are present each requires to be carefully separated or isolated in pure culture.separated or isolated in pure culture.

► Several organism need the determination of Several organism need the determination of Antibiotic sensitivity pattern for optimal Antibiotic sensitivity pattern for optimal antibiotic selectionantibiotic selection

Basic requirements of Basic requirements of culture mediaculture media

► NutrientsNutrients - Energy source - Energy source - Carbon source - Carbon source - Nitrogen source - Nitrogen source

► Mineral salts – Sulphate, phosphates, Mineral salts – Sulphate, phosphates, chlorides & carbonates of K, Mg & Ca.chlorides & carbonates of K, Mg & Ca.

► A suitable pH – 7.2 – 7.4A suitable pH – 7.2 – 7.4► Accessory growth factorsAccessory growth factors

- Tryptophan for Salmonella typhi - Tryptophan for Salmonella typhi - X & V factors for H. influenzae - X & V factors for H. influenzae

Pouring the Culture Pouring the Culture PlatesPlates

Petri dish with MediaPetri dish with Media

► Plate: provide large Plate: provide large surface for isolation surface for isolation and observation of and observation of coloniescolonies

► Using a sterile loop or a Using a sterile loop or a sterile swab streak sterile swab streak your sample on the your sample on the petri platepetri plate

► Important let your Important let your sterilized loop cool sterilized loop cool before you pick up your before you pick up your samplesample

Classification of Culture Classification of Culture mediamedia

► Based on the consistency:Based on the consistency:

LiquidLiquid -- Peptone water, -- Peptone water, Nutrient broth Nutrient broth SemisolidSemisolid -- Nutrient agar stabs -- Nutrient agar stabs Solid Solid -- Blood agar, Serum agar -- Blood agar, Serum agar

► Based on Oxygen requirement:Based on Oxygen requirement:

-- Aerobic medium-- Aerobic medium -- Anaerobic media -- Anaerobic media

Aerobic MediaAerobic Media

► Simple mediaSimple media► Complex mediaComplex media

May be Synthetic or Defined May be Synthetic or Defined MediumMedium

► - Enriched media- Enriched media - Differential media - Differential media - Enrichment media - Enrichment media - Selective media - Selective media

► Semisyntetic MediumSemisyntetic Medium - Sugar media - Sugar media - Transport media - Transport media

Aerobic mediaAerobic media

► Liquid mediaLiquid media - Peptone water(1% peptone +0.5%Nacl - Peptone water(1% peptone +0.5%Nacl + + 100 ml water) 100 ml water)

- Nutrient broth ( peptone water + 1% - Nutrient broth ( peptone water + 1% meat meat extract extract

► Solid mediaSolid media- Nutrient agar (nutrient broth + 2% - Nutrient agar (nutrient broth + 2%

Agar)Agar)

Use: To grow non-fastidious microorganisms Use: To grow non-fastidious microorganisms

Simple media- consists of only basic necessities

Liquid MediumLiquid Medium

► Difficulat to identify Difficulat to identify all types of all types of organisms organisms

► Suitable for isolation Suitable for isolation of bacteria from of bacteria from Blood culturing and Blood culturing and water analysiswater analysis

Peptone Peptone

► Peptone contain Peptone contain partially digested partially digested proteinsproteins

► Proteases Proteases ► PolypeptidesPolypeptides► AminoacidsAminoacids► Inorganic saltsInorganic salts PhosphatesPhosphates Potassium and Potassium and

MagnesiumMagnesium RiboflavinRiboflavinMeat exract called as Lab Meat exract called as Lab

lemco lemco

Nutrient AgarNutrient Agar

► Contain 2% agar Contain 2% agar added to Nutrient added to Nutrient agar commonly agar commonly usedused

► Concentration can Concentration can be increased to 6% be increased to 6% to prevent to prevent swarmingswarming

► Can be reduced to Can be reduced to 0’5% 0’5%

Pigment producingPigment producing StaphylococciStaphylococci

Complex mediaComplex media

► Nutrient agar + 5 to 10% sheep bloodNutrient agar + 5 to 10% sheep blood► Melt the sterile nutrient agar by steaming, cool, Melt the sterile nutrient agar by steaming, cool,

to 45to 450 0 cc► Add the blood aseptically with constant shakingAdd the blood aseptically with constant shaking► Mix the blood with molten nutrient agar Mix the blood with molten nutrient agar

thoroughly but gently avoiding froth formationthoroughly but gently avoiding froth formation► Immediately pour in to the Petri dishes or tubes Immediately pour in to the Petri dishes or tubes

and allow to setand allow to set

Enriched media: Blood agar

Use: To cultivate all the fastidious organisms

Enriched MediumEnriched Medium

► To culture medium To culture medium Blood serum or egg Blood serum or egg are added to are added to mediummedium

► eg Blood agareg Blood agar► Chocolate agarChocolate agar► Egg Egg

Different types of hemolysis Different types of hemolysis on Blood Agaron Blood Agar

Other Enrichments – Chocolate AgarOther Enrichments – Chocolate Agar

► Several organic Several organic materials are added materials are added to the basic to the basic constituents of the constituents of the Medium such as Medium such as Blood, yeast, yeast Blood, yeast, yeast extract etcextract etc

Chocolate agarChocolate agar

Enrichment MediumEnrichment Medium

► If the sample contain If the sample contain more than one type of more than one type of bacteria, undesired bacteria, undesired bacteria grwoth can be bacteria grwoth can be reduced or eliminated.reduced or eliminated.

► The desired organism is The desired organism is facilitated to growfacilitated to grow

► Eg Tetrathionate brothEg Tetrathionate broth► Selenite F brothSelenite F broth

Selective mediaSelective media

►Serve the same purpose as Serve the same purpose as Enrichment media but are solid in Enrichment media but are solid in consistency consistency

- Wilson & Blair’s medium - - Wilson & Blair’s medium - - Lowenstein Jensen’s medium -- Lowenstein Jensen’s medium -

Use: To cultivate Salmonella, Shigella Use: To cultivate Salmonella, Shigella & & Mycobacteria Mycobacteria

Deoxycholate citrate AgarDeoxycholate citrate Agar

► Suitable for isolation of Suitable for isolation of dysentery bacilli, food dysentery bacilli, food poisoning Salmonella and poisoning Salmonella and S.paratyphi B, and less so, S.paratyphi B, and less so, but superior to MacConkey but superior to MacConkey agar for S. typhi.agar for S. typhi.

► It is a heat sensitive It is a heat sensitive medium It should not be medium It should not be autoclaved or remeltedautoclaved or remelted

► When prepared from When prepared from commercial medium it commercial medium it should be dissolved and should be dissolved and sterilized at 100sterilized at 10000c for a c for a short periodshort period

Indicator Medium Wilson-Indicator Medium Wilson-Blair mediumBlair medium

► Indicate by change Indicate by change of color Sulphite to of color Sulphite to sulphide in Wilson-sulphide in Wilson-Blair mediumBlair medium

► S.typhi reduces S.typhi reduces sulphite to sulphide sulphite to sulphide in the presence of in the presence of GlucoseGlucose

Differential Medium Differential Medium Mac Conkey's agar Mac Conkey's agar

► Bringing out different Bringing out different characters of bacteria characters of bacteria their atypical their atypical characterscharacters

► Mac Conkey’s mediumMac Conkey’s medium

Contain peptone, Lactose Contain peptone, Lactose Agar, Neutral red and Agar, Neutral red and taurocholate and show taurocholate and show grwoth of Lactose grwoth of Lactose fermenters as pink fermenters as pink colored coloniescolored colonies

MacConkey agarMacConkey agar

► MacConkey agar is MacConkey agar is useful medium for useful medium for cultivation of cultivation of enterobacteria enterobacteria

► It contains a bile salt to It contains a bile salt to inhibit non intestinal inhibit non intestinal bacteria bacteria

► Lactose in combination Lactose in combination with Neutral red with Neutral red distinguish the lactose distinguish the lactose fermenting from the non fermenting from the non lactose fermenting lactose fermenting Salmonella and Salmonella and Dysentery groupDysentery group

Lactose fermenting and Non Lactose fermenting and Non lactose fermentinglactose fermenting

Carbohydrate mediaCarbohydrate media

► Peptone water – 100 ml, Desired sugar 1 Peptone water – 100 ml, Desired sugar 1 gm% and Andrade's indicator – 0.005% gm% and Andrade's indicator – 0.005% soln(1ml)soln(1ml)

► Dissolve the desired carbohydrate in Dissolve the desired carbohydrate in peptone water and steam for 30 min or peptone water and steam for 30 min or sterilize by filtration. sterilize by filtration.

► Distribute into sterile test tube containing Distribute into sterile test tube containing inverted Durham’s tubes to detect gas inverted Durham’s tubes to detect gas production and steam for 30 minproduction and steam for 30 min

Use: To test the fermenting ability of an Use: To test the fermenting ability of an organism organism

Carbohydrate mediaCarbohydrate media

► Peptone water – 100 ml, Peptone water – 100 ml, Desired sugar 1 gm% Desired sugar 1 gm% and Andrade's indicator and Andrade's indicator – 0.005% soln(1ml)– 0.005% soln(1ml)

► Dissolve the desired Dissolve the desired carbohydrate in peptone carbohydrate in peptone water and steam for 30 water and steam for 30 min or sterilize by min or sterilize by filtration. filtration.

► Use: To test the Use: To test the fermenting ability of an fermenting ability of an organism organism

Sugar MediumSugar Medium

► Sugars are fermenting substancesSugars are fermenting substances► Monosaccharide – peptone, arabinose,xylose Monosaccharide – peptone, arabinose,xylose

and hexose's, dextrose and mannoseand hexose's, dextrose and mannose► Disaccharides Sucrose and LactoseDisaccharides Sucrose and Lactose► Polysaccharides – Starch and InulinPolysaccharides – Starch and Inulin► Alcohols – Glycerol. SorbitalAlcohols – Glycerol. Sorbital► Sugar medium contain 1% sugar Sugar medium contain 1% sugar ► Durham’s tube indicates production of gasDurham’s tube indicates production of gas► Hiss Serum sugars apart from sugar , serum Hiss Serum sugars apart from sugar , serum

is added.is added.

Sugar MediumSugar Medium

► Sugar medium Sugar medium contain 1% sugar contain 1% sugar

► Durham’s tube Durham’s tube indicates production indicates production of gasof gas

► Hiss Serum sugars Hiss Serum sugars apart from sugar , apart from sugar , serum is added.serum is added.

Urease TestUrease Test

Loeffler’s serum slopeLoeffler’s serum slope

Lowenstein Jensen Lowenstein Jensen MediumMedium

Transport MediumTransport Medium

► Stuart’s medium Stuart’s medium contain reducing contain reducing agents to prevent agents to prevent oxidation.oxidation.

► Charcoal to Charcoal to neutralize certain neutralize certain bacterial inhibitors bacterial inhibitors to Gonococci, to Gonococci,

Hiss Serum SugarsHiss Serum SugarsSugar Medium with Serum enrichmentSugar Medium with Serum enrichment

Anaerobic MediumAnaerobic Medium

► Robertson’s Robertson’s cooked meat cooked meat mediummedium

► Thiglyclolate liquid Thiglyclolate liquid mediummedium

Anaerobic Culture Methods Anaerobic Culture Methods Anaerobic jarAnaerobic jar

► Anaerobic Anaerobic jarjar

Figure 6.5

Sabouraud's Dextrose agar Sabouraud's Dextrose agar commonly used Fungal commonly used Fungal

Isolation MediumIsolation Medium

Sabouraud's Dextrose Sabouraud's Dextrose AgarAgar

DextroseDextrose - 4 gm%- 4 gm%NeopeptoneNeopeptone - 1 gm%- 1 gm%AgarAgar - 1.5 gm% - 1.5 gm% Distilled waterDistilled water - 100 ml- 100 ml

►Dissolve the ingredients by heating in a Dissolve the ingredients by heating in a water bath, cool and adjust pH to 5.4water bath, cool and adjust pH to 5.4

►Autoclave and dispense 20 ml amount Autoclave and dispense 20 ml amount in test tubesin test tubes

Use: For the cultivation of Fungi Use: For the cultivation of Fungi

Robertsons’cooked Meat Robertsons’cooked Meat MediumMedium

► Place meat in 1 ounce Place meat in 1 ounce bottles to the depth of bottles to the depth of 2.5 cms and cover it 2.5 cms and cover it with 15 ml of brothwith 15 ml of broth

► Autoclave at 121Autoclave at 12100 c for c for 20 min20 min

► After sterilization, After sterilization, adjust the pH to 7.5adjust the pH to 7.5

Use: To cultivate the Use: To cultivate the anaerobic bacteriaanaerobic bacteria

Lowenstein Jensen Medium - cultivation Lowenstein Jensen Medium - cultivation of Mycobacterium tuberculosisof Mycobacterium tuberculosis

Lowenstein-Jensen’s mediumLowenstein-Jensen’s medium

►Mineral salt solnMineral salt soln - 600ml- 600mlMalachite green soln - 20mlMalachite green soln - 20ml(2gm% in D.water)(2gm% in D.water)Beaten egg - 1000mlBeaten egg - 1000ml(20-22 eggs)(20-22 eggs)

►Mix the aboveMix the above►Distribute in Mc Cartney bottlesDistribute in Mc Cartney bottles►Sterilize by InspissationSterilize by Inspissation

Use: To cultivate MycobacteriaUse: To cultivate Mycobacteria

Sterilization of culture Sterilization of culture mediamedia

► Media are sterilized in the autoclave at 121Media are sterilized in the autoclave at 12100 c c for 15’ under 15lbs of Pressurefor 15’ under 15lbs of Pressure

► Heat-labile substances like serum & sugar Heat-labile substances like serum & sugar solutions must be sterilized by free-steam or solutions must be sterilized by free-steam or filtrationfiltration

► Egg containing media –-- Lowenstein-Jensen’s Egg containing media –-- Lowenstein-Jensen’s medium, Loeffler's serum slope by medium, Loeffler's serum slope by inspissationinspissation

► Discarded culture plates are to be sterilized Discarded culture plates are to be sterilized by autoclaving prior to washingby autoclaving prior to washing

Colonies of Bacteria on Colonies of Bacteria on Culture platesCulture plates

Salmonella Shigella agarSalmonella Shigella agar

TCBS mediumTCBS medium

Blood culture – ‘Liquid Blood culture – ‘Liquid Medium’Medium’

Carbohydrate mediaCarbohydrate media

► Peptone water – 100 ml, Peptone water – 100 ml, Desired sugar 1 gm% Desired sugar 1 gm% and Andrade's indicator and Andrade's indicator – 0.005% soln(1ml)– 0.005% soln(1ml)

► Dissolve the desired Dissolve the desired carbohydrate in peptone carbohydrate in peptone water and steam for 30 water and steam for 30 min or sterilize by min or sterilize by filtration. filtration.

► Use: To test the Use: To test the fermenting ability of an fermenting ability of an organism organism

Muller Hinton Agar for Muller Hinton Agar for Antibiotic TestingAntibiotic Testing

Antibiotic Testing on Antibiotic Testing on Blood Agar MediumBlood Agar Medium

Storage of culture mediaStorage of culture media► Prepared media in Prepared media in

individual screw capped individual screw capped bottles can be stored for bottles can be stored for weeks at room tempweeks at room temp

► Poured plates deteriorate Poured plates deteriorate quickly and often quickly and often contaminated, hence cold contaminated, hence cold storage is necessarystorage is necessary

► For smaller labs domestic For smaller labs domestic refrigerators & for larger refrigerators & for larger labs insulated cold room(4-labs insulated cold room(4-55ooc)c)

► Deep freeze refrigerators Deep freeze refrigerators for preservation of sera, for preservation of sera, antibiotics & amino acids antibiotics & amino acids (-10 to - 40(-10 to - 4000c)c)

Created for benefit of Created for benefit of Medical and Technical Medical and Technical students in Developing students in Developing

WorldWorld

Dr.T.V.Rao MDDr.T.V.Rao MD

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