A culture media is a special medium used in microbiological laboratories to grow different kinds of microorganisms. A growth or a culture medium is composed of different nutrients that are essential for microbial growth. Since there are many types of microorganisms, each having unique properties and requiring specific nutrients for growth, there are many types based on what nutrients they contain and what function they play in the growth of microorganisms. A culture may be solid or liquid. The solid culture media is composed of a brown jelly like substance known as agar. Different nutrients and chemicals are added to it to allow the growth of different microorganisms.

1. CULTURE MEDIA &CULTURE MEDIA &
CULTURE METHODSCULTURE METHODS

2.  Bacteria have to be grown (cultured) for them toBacteria have to be grown (cultured) for them to
be identified.be identified.
 By appropriate procedures they have to beBy appropriate procedures they have to be
grown separately (isolated) on culture mediagrown separately (isolated) on culture media
and obtained as pure for study.and obtained as pure for study.
HistoryHistory
 The original media used by Louis Pasteur – urineThe original media used by Louis Pasteur – urine
or meat brothor meat broth
 Liquid medium – diffuse growthLiquid medium – diffuse growth
 Solid medium – discrete colonies.Solid medium – discrete colonies.

3. ColonyColony – macroscopically visible collection of– macroscopically visible collection of
millions of bacteria originating from a singlemillions of bacteria originating from a single
bacterial cell.bacterial cell.
 Cooked cut potato by Robert Koch – earliestCooked cut potato by Robert Koch – earliest
solid mediumsolid medium
 Gelatin – not satisfactoryGelatin – not satisfactory
- liquefy at 24- liquefy at 24oo
CC

4. AgarAgar
 Fanny HesseFanny 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 98oo
C & sets at 42C & sets at 42oo
CC
 2% agar is employed in solid medium2% agar is employed in solid medium

5. Types of culture mediaTypes of culture media
I.I. Based on their consistencyBased on their consistency
a) solid mediuma) solid medium
b) liquid mediumb) liquid medium
c) semi solid mediumc) semi solid medium
II.II. Based on the constituents/ ingredientsBased on the constituents/ ingredients
a) simple mediuma) simple medium
b) complex mediumb) complex medium
c) synthetic or defined mediumc) synthetic or defined medium
d) Special mediad) Special media

6. 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

7. Solid mediaSolid media – contains 2% agar– contains 2% agar
 Colony morphology, pigmentation, hemolysis can beColony morphology, pigmentation, hemolysis can be
appreciated.appreciated.
 Eg: Nutrient agar, Blood agarEg: Nutrient agar, Blood agar
Liquid mediaLiquid media – no agar.– no agar.
 For inoculum preparation, Blood culture, for theFor 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 mediumSemi solid medium – 0.5% agar.– 0.5% agar.
 Eg: Motility mediumEg: Motility medium

9. Simple media / basal mediaSimple media / basal media
-- Eg: NB, NAEg: NB, NA
- NB consists of peptone, meat extract, NaCl,- NB consists of peptone, meat extract, NaCl,
-- NB + 2% agar = Nutrient agarNB + 2% agar = Nutrient agar

10. Complex mediaComplex media
 Media other than basal media.Media other than basal media.
 They have added ingredients.They have added ingredients.
 Provide special nutrientsProvide special nutrients
Synthetic or defined mediaSynthetic or defined media
 Media prepared from pure chemical substancesMedia prepared from pure chemical substances
and its exact composition is knownand its exact composition is known
 Eg: peptone water – 1% peptone + 0.5% NaCl inEg: peptone water – 1% peptone + 0.5% NaCl in
waterwater

11. Enriched mediaEnriched media
 Substances like blood, serum, egg are added toSubstances like blood, serum, egg are added to
the basal medium.the basal medium.
 Used to grow bacteria that are exacting in theirUsed to grow bacteria that are exacting in their
nutritional needs.nutritional needs.
 Eg: Blood agar, Chocolate agarEg: Blood agar, Chocolate agar

12. Blood agar Chocolate agar

13. Enrichment mediaEnrichment media
 Liquid media used to isolateLiquid media used to isolate
pathogens from a mixed culture.pathogens from a mixed culture.
 Media is incorporated withMedia is incorporated with
inhibitory substances to suppressinhibitory substances to suppress
the unwanted organism.the unwanted organism.
 Eg:Eg:
 Selenite F BrothSelenite F Broth – for the isolation of– for the isolation of
Salmonella, ShigellaSalmonella, Shigella
 Alkaline PeptoneWaterAlkaline PeptoneWater – forVibrio– forVibrio
choleraecholerae

14. Selective mediaSelective media
 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 bacteriafor gram negative bacteria
 TCBSTCBS – forV.cholerae– forV.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 bacilli– Diphtheria bacilli

15. TCBSMac Conkey’s medium

16. Potassium Tellurite media LJ media

17. Indicator mediaIndicator media
 These media contain an indicator which changesThese media contain an indicator which changes
its colour when a bacterium grows in them.its colour when a bacterium grows in them.
 Eg:Eg:
 Blood agarBlood agar
 Mac Conkey’s mediumMac Conkey’s medium
 Christensen’s urease mediumChristensen’s urease medium

19. Urease mediumUrease medium

20. Differential mediaDifferential media
 A media which has substances incorporated in itA media which has substances incorporated in it
enabling it to distinguish between bacteria.enabling it to distinguish between bacteria.
 Eg: Mac Conkey’s mediumEg: Mac Conkey’s medium
 PPeptoneeptone
 LLactoseactose
 AAgargar
 NNeutral redeutral red
 TTaurocholateaurocholate
 Distinguish between lactose fermenters & nonDistinguish between lactose fermenters & non
lactose fermenters.lactose fermenters.

21.  Lactose fermenters –Lactose fermenters – PinkPink coloniescolonies
 Non lactose fermenters – colourless coloniesNon lactose fermenters – colourless colonies

22. Sugar mediaSugar media
 Media containing any fermentable substance.Media containing any fermentable substance.
 Eg: glucose, arabinose, lactose, starch etc.Eg: glucose, arabinose, lactose, starch etc.
 Media consists of 1% of the sugar in peptoneMedia consists of 1% of the sugar in peptone
water.water.
 Contain a small tube (Durham’s tube) for theContain a small tube (Durham’s tube) for the
detection of gas by the bacteria.detection of gas by the bacteria.

24. Transport mediaTransport media
 Media used for transporting theMedia used for transporting the
samples.samples.
 Delicate organisms may notDelicate organisms may not
survive the time taken forsurvive the time taken for
transporting the specimentransporting the specimen
without a transport media.without a transport media.
 Eg:Eg:
 Stuart’s mediumStuart’s medium – non nutrient soft– non nutrient soft
agar gel containing a reducing agentagar gel containing a reducing agent
 Buffered glycerol salineBuffered glycerol saline – enteric– enteric
bacillibacilli

25. 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,ThioglycolateEg: Robertson’s cooked meat medium,Thioglycolate
medium.medium.

26. BIOCHEMICAL TEST & REACTIONSBIOCHEMICAL TEST & REACTIONS
 They provide additional information for theThey provide additional information for the
identification of the bacterium.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

27. OXIDASE TESTOXIDASE TEST
 Detects the presence of an enzyme “oxidase”Detects the presence of an enzyme “oxidase”
produced by certain bacteria which will reduce theproduced by certain bacteria which will reduce the
dye – tetramethyl-p-phenylene diaminedye – tetramethyl-p-phenylene diamine
dihydrochloride.dihydrochloride.
 Positive test is indicated by the development of aPositive test is indicated by the development of a
purplepurple colour.colour.
 Oxidase positive – Pseudomonas,Vibrio, NeisseriaeOxidase positive – Pseudomonas,Vibrio, Neisseriae
 Oxidase negative – Salmonella, ShigellaOxidase negative – Salmonella, Shigella

29. TRIPLE SUGAR IRON AGAR (TSI)TRIPLE SUGAR IRON AGAR (TSI)
 It is a composite media used to study differentIt is a composite media used to study different
properties of a bacterium – sugar fermentation, gasproperties of a bacterium – sugar fermentation, gas
production and Hproduction and H22S production.S production.
 In addition to peptone, yeast extract & agar, it containsIn addition to peptone, yeast extract & agar, it contains
3 sugars – Glucose, Lactose, Sucrose.3 sugars – Glucose, Lactose, Sucrose.
 The Iron salt - Ferric ammonium citrate - indicates HThe Iron salt - Ferric ammonium citrate - indicates H22SS
production.production.
 Phenol red is the pH indicator.Phenol red is the pH 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

30. TSI REACTIONSTSI REACTIONS::
Yellow – AcidYellow – Acid
PinkPink - Alkaline- Alkaline
 Yellow slantYellow slant // Yellow buttYellow butt (A/A) – Lactose fermenters.(A/A) – Lactose fermenters.
 Pink slantPink slant // Yellow buttYellow butt (K/A) – Non lactose fermenters.(K/A) – Non lactose fermenters.
 Pink slantPink slant // no colour changeno colour change (K/K) – Non fermenters(K/K) – Non fermenters
 LF – E.coli, KlebsiellaLF – E.coli, Klebsiella
 NLF – Salmonella, ShigellaNLF – Salmonella, Shigella
 Gas bubbles or crack in the medium – gas production.Gas bubbles or crack in the medium – gas production.
 HH22S production - ProteusS production - Proteus

33. INDOLE TESTINDOLE TEST
 Used to detect indole production by the organism.Used to detect indole production by the organism.
 They produce indole from tryptophan present inThey produce indole from tryptophan present in
peptone water.peptone water.
 After overnight incubation, a few drops of indoleAfter overnight incubation, a few drops of indole
reagent (Kovac’s reagent) is added.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 ringring
 NegativeNegative indole test -indole test - yellowyellow ringring
 Indole positive – E.coliIndole positive – E.coli
 Indole negative – Klebsiella, Salmonella.Indole negative – Klebsiella, Salmonella.

35. CITRATE UTILIZATIONCITRATE UTILIZATION
 Done in Simmon’s Citrate medium.Done in Simmon’s Citrate medium.
 To detect the ability of certain bacteria to utilizeTo detect the ability of certain bacteria to utilize
citrate as the sole source of carbon.citrate as the sole source of carbon.
 Contains Sodium citrate and bromothymol blue as theContains Sodium citrate and bromothymol blue as the
indicator.indicator.
 If citrate is utilized, alkali is produced which turns theIf citrate is utilized, alkali is produced which turns the
medium to blue.medium to blue.
 Citrate positive –Citrate positive – blueblue colourcolour
 Citrate negative –Citrate negative – greengreen colourcolour
 Positive – KlebsiellaPositive – Klebsiella
 Negative – E.coliNegative – E.coli

37. UREASE TESTUREASE TEST
 Done in Christensen’s urease medium.Done in Christensen’s urease medium.
 This test is used to detect organisms that produceThis test is used to detect organisms that produce
urease.urease.
 Urease produced by the organisms split urea intoUrease produced by the organisms split urea into
ammonia and COammonia and CO2.2.
 Urease positive –Urease positive – pinkpink colourcolour
 Urease negative –Urease negative – yellowyellow colourcolour
 Positive – Proteus, KlebsiellaPositive – Proteus, Klebsiella
 Negative – E.coli, SalmonellaNegative – E.coli, Salmonella

39. CULTURE METHODSCULTURE METHODS
 Culture methods employed depend on the purposeCulture 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 ofTo 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.

40. 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

41. STREAK CULTURESTREAK CULTURE
 Used for the isolation of bacteria in pure culture fromUsed for the isolation of bacteria in pure culture from
clinical specimens.clinical specimens.
 Platinum wire or Nichrome wire is used.Platinum wire or Nichrome wire is used.
 One loopful of the specimen is transferred onto theOne loopful of the specimen is transferred onto the
surface of a well dried plate.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 plateThe inoculum is then distributed thinly over the plate
by streaking it with a loop in a series of parallel linesby streaking it with a loop in a series of parallel lines
in different segments of the plate.in different segments of the plate.
 On incubation, separated colonies are obtained overOn incubation, separated colonies are obtained over
the last series of streaks.the last series of streaks.

44. LAWN CULTURELAWN CULTURE
 Provides a uniform surface growth of the bacterium.Provides a uniform surface growth of the 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 the surfaceLawn cultures are prepared by flooding the surface
of the plate with a liquid suspension of theof the plate with a liquid suspension of the
bacterium.bacterium.

45. Antibiotic sensitivity testing

46. STROKE CULTURESTROKE CULTURE
 Stroke culture is made in tubesStroke culture is made in tubes
containing agar slope / slant.containing agar slope / slant.
 UsesUses
 Provide a pure growth ofProvide a pure growth of
bacterium for slidebacterium for slide
agglutination and otheragglutination and other
diagnostic tests.diagnostic tests.

47. 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 liquefaction.Demonstration of gelatin liquefaction.
 Oxygen requirements of the bacteriumOxygen requirements of the bacterium
under study.under study.
 Maintenance of stoke cultures.Maintenance of stoke cultures.

48. Gelatin liquefaction Oxidation – Fermentation
medium

49. POUR PLATE CULTUREPOUR PLATE CULTURE
 Agar medium is melted (15 ml) and cooled to 45Agar medium is melted (15 ml) and cooled to 45oo
C.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 37oo
C, colonies will be distributedC, 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 aGives an estimate of the viable bacterial count in a
suspension.suspension.
 For the quantitative urine cultures.For the quantitative urine cultures.

50. LIQUID CULTURESLIQUID CULTURES
 Liquid cultures are inoculated by touching with aLiquid cultures are inoculated by touching with a
charged loop or by adding the inoculum with pipettescharged loop or by adding the inoculum with pipettes
or syringes.or syringes.
 UsesUses
 Blood cultureBlood culture
 Sterility testsSterility tests
 Continuous culture methodsContinuous culture methods
 DisadvantageDisadvantage
 It does not provide a pure culture from mixedIt does not provide a pure culture from mixed
inoculum.inoculum.

51. Blood culture bottles

52. ANAEROBIC CULTURE METHODSANAEROBIC CULTURE METHODS
 Anaerobic bacteria differ in their requirement andAnaerobic bacteria differ in their requirement and
sensitivity to oxygen.sensitivity to oxygen.
 Cl. tetani is a strict anaerobe – grows at an oxygenCl. tetani is a strict anaerobe – grows at an oxygen
tension < 2 mm Hg.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

53. Production of vacuum:Production of vacuum:
 Incubate the cultures in a vacuum desiccator.Incubate the cultures in a vacuum 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

55. 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 lidConsists of a metal jar or glass jar with a metal lid
which can be clamped air tight.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 electricalThe lid has two terminals – connected to electrical
supply.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.

57. Working:Working:
 Inoculated plates are placed inside the jar and theInoculated plates are placed inside the jar and the
lid clamped air tight.lid clamped air tight.
 The outlet tube is connected to a vacuum pump andThe 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 isThe 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 electricAfter the jar is filled with hydrogen, the electric
terminals are connected to a current supply, so thatterminals 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 hydrogenAct as a catalyst for the combination of hydrogen
with residual oxygen.with residual oxygen.

58. Gaspak
 Commercially available disposable envelope.
 Contains chemicals which generate H2 and CO2
on addition of water.
 Indicator used – reduced methylene blue.
 Colourless – anaerobic
 Blue colour – on exposure to oxygen

60. Biological methodBiological method
 Absorption of oxygen by incubation with aerobicAbsorption of oxygen by incubation with aerobic
bacteria, germinating seeds or choppedbacteria, germinating seeds or chopped
vegetables.vegetables.
Reduction of oxygenReduction of oxygen
 By using reducing agents – 1% glucose, 0.1%By using reducing agents – 1% glucose, 0.1%
ThioglycolateThioglycolate

61. THANK YOUTHANK YOU