This document provides information on culture media and methods used to culture bacteria. It discusses the requirements bacteria have for growth and how laboratory culture media aims to provide a captive environment for bacteria to grow. Various types of culture media are described, including solid, liquid and semi-solid media made with ingredients like agar. Special media like enriched, selective, differential and indicator media are also outlined. Common biochemical tests performed on cultured bacteria like TSI, oxidase, indole and citrate are briefly explained. The document provides an overview of basic microbiology laboratory techniques for culturing and identifying bacteria.

1. CULTURE MEDIA &CULTURE MEDIA &
CULTURE METHODSCULTURE METHODS

2. Culture Media

3. - 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

4. •Bacteria have to be grown (cultured) forBacteria have to be grown (cultured) for
them to be identified.them to be identified.
•By appropriate procedures they have toBy appropriate procedures they have to
be grown separately (isolated) on culturebe 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 PasteurThe 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.

5. ColonyColony – macroscopically visible– macroscopically visible
collection of millions of bacteriacollection of millions of bacteria
originating from a single bacterialoriginating 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 24oo
CC

6. 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 theNot affected by the growth of the
bacteria.bacteria.
•Melts at 98Melts at 98oo
C & sets at 42C & sets at 42oo
CC
•2% agar is employed in solid2% agar is employed in solid
mediummedium

7. •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

8. 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)

10. Aseptically pouring
agar plates

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

13. • All labeling is done on the bottom of the agar plate
1. Initials
2. Date (mm/dd/yy)
3. Code # or letter

14. Environmental sampling
Surface samples are normally taken using sterile swabs

15. 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)

16. Applying oral sample to surface of agar.

17. •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 )

18. Agar plates are stored upside down to
prevent condensation.

19. •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.

20. Typical environmental sampling results

21. 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

22. 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

23. Solid mediaSolid media – contains 2% agar– contains 2% agar
•Colony morphology, pigmentation, hemolysis canColony 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 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

25. Simple media / basal mediaSimple media / basal media
-- Eg: Nutrient Broth, NutrientEg: 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

26. 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 chemicalMedia prepared from pure chemical
substances and its exact composition issubstances 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

27. Enriched mediaEnriched media
•Substances like blood, serum,Substances like blood, serum,
egg are added to the basalegg are added to the basal
medium.medium.
•Used to grow bacteria that areUsed to grow bacteria that are
exacting in their nutritionalexacting in their nutritional
needs.needs.
•Eg: Blood agar, Chocolate agarEg: Blood agar, Chocolate agar

28. Blood agar Chocolate agar

29. • 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.

30. 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 toinhibitory substances to
suppress the unwantedsuppress the unwanted
organism.organism.
•Eg:Eg:
•Selenite F BrothSelenite F Broth – for the isolation– for the isolation
of Salmonella, Shigellaof Salmonella, Shigella
• Alkaline Peptone WaterAlkaline Peptone Water – for– for
Vibrio choleraeVibrio cholerae

31. 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 bacteriafor gram negative bacteria
•Thiosulphate Citrate Bile Salt Sucrose AgarThiosulphate 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 (-)

32. TCBSMac Conkey’s medium

33. Potassium Tellurite media LJ media

34. Indicator mediaIndicator media
•These media contain an indicatorThese media contain an indicator
which changes its colour when awhich 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

36. Urease mediumUrease medium

37. Differential mediaDifferential media
•A media which has substances incorporated inA 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
•TTaurocholateaurocholate
•Distinguish between lactose fermenters & nonDistinguish between lactose fermenters & non
lactose fermenters.lactose fermenters.

38. •Lactose fermenters –Lactose fermenters – PinkPink coloniescolonies
•Non lactose fermenters – colourless coloniesNon lactose fermenters – colourless colonies

39. 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.

41. left: no lactose fermentation
right: 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

42. Sugar mediaSugar media
•Media containing any fermentableMedia containing any fermentable
substance.substance.
•Eg: glucose, arabinose, lactose, starchEg: glucose, arabinose, lactose, starch
etc.etc.
•Media consists of 1% of the sugar inMedia 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 thefor the detection of gas by the
bacteria.bacteria.

44. Transport mediaTransport media
•Media used for transportingMedia used for transporting
the samples.the 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– non nutrient
soft agar gel containing asoft agar gel containing a
reducing agentreducing agent
•Buffered glycerol salineBuffered glycerol saline – enteric– enteric
bacillibacilli

45. 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.

46. BIOCHEMICAL TEST & REACTIONSBIOCHEMICAL TEST & REACTIONS
•They provide additional informationThey provide additional information
for the identification of thefor 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

47. OXIDASE TESTOXIDASE TEST
•Detects the presence of an enzymeDetects 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 thePositive test is indicated by the
development of adevelopment of a purplepurple colour.colour.
•Oxidase positive – Pseudomonas, Vibrio,Oxidase positive – Pseudomonas, Vibrio,
NeisseriaeNeisseriae
•Oxidase negative – Salmonella, ShigellaOxidase negative – Salmonella, Shigella

49. 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,properties of a bacterium – sugar fermentation,
gas production and Hgas production and H22S production.S production.
•In addition to peptone, yeast extract & agar, itIn 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 H22SS
production.production.
•Phenol red is the indicator.Phenol red is the indicator.
•It is an orange red medium with a slant and aIt is an orange red medium with a slant and a
butt.butt.
•pH of the medium – 7.4pH of the medium – 7.4

50. TSI REACTIONSTSI REACTIONS::
Yellow – AcidYellow – Acid
Pink - AlkalinePink - Alkaline
•Yellow slantYellow slant // Yellow buttYellow butt (A/A) – Lactose(A/A) – Lactose
fermenters.fermenters.
•Pink slantPink slant // Yellow buttYellow butt (K/A) – Non lactose(K/A) – Non lactose
fermenters.fermenters.
•Pink slantPink slant // no colour changeno colour change (K/K) – Non fermenters(K/K) – Non fermenters
•Black colour – H– H22S production.S production.
•Gas bubbles or crack in the medium – gasGas bubbles or crack in the medium – gas
production.production.
•LF – E.coli, KlebsiellaLF – E.coli, Klebsiella
•NLF – Salmonella, ShigellaNLF – Salmonella, Shigella

52. INDOLE TESTINDOLE TEST
•Used to detect indole production by theUsed to detect indole production by the
organism.organism.
•They produce indole from tryptophan presentThey produce indole from tryptophan present
in peptone water.in peptone water.
•After overnight incubation, a few drops ofAfter 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 ringring
• NegativeNegative indole test -indole test - yellowyellow ringring
•Indole positive – E.coliIndole positive – E.coli
•Indole negative – Klebsiella, Salmonella.Indole negative – Klebsiella, Salmonella.

54. CITRATE UTILIZATIONCITRATE UTILIZATION
•Done in Simmon’s Citrate medium.Done in Simmon’s Citrate medium.
•To detect the ability of certain bacteria toTo 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 bromothymolContains Sodium citrate and bromothymol
blue as the indicator.blue as the indicator.
•If citrate is utilized, alkali is produced whichIf citrate is utilized, alkali is produced which
turns the medium to blue.turns the medium to blue.
• Citrate positive –Citrate positive – blueblue colourcolour
• Citrate negative –Citrate negative – greengreen colourcolour
•Positive – KlebsiellaPositive – Klebsiella
•Negative – E.coliNegative – E.coli

56. UREASE TESTUREASE TEST
•Done in Christensen’s urease medium.Done in Christensen’s urease medium.
•This test is used to detect organisms thatThis test is used to detect organisms that
produce urease.produce urease.
•Urease produced by the organisms splitUrease produced by the organisms split
urea into ammonia and COurea into ammonia 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

58. CULTURE METHODSCULTURE METHODS
•Culture methods employed depend onCulture 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 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.

59. 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

60. STREAK CULTURESTREAK CULTURE
•Used for the isolation of bacteria in pure cultureUsed 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 ontoOne 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 theThe inoculum is then distributed thinly over the
plate by streaking it with a loop in a series ofplate 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 obtainedOn incubation, separated colonies are obtained
over the last series of streaks.over the last series of streaks.

63. LAWN CULTURELAWN CULTURE
•Provides a uniform surface growth of theProvides 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 floodingLawn cultures are prepared by flooding
the surface of the plate with a liquidthe surface of the plate with a liquid
suspension of the bacterium.suspension of the bacterium.

64. Antibiotic sensitivity testing

65. 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 growthProvide a pure growth
of bacterium for slideof bacterium for slide
agglutination andagglutination and
other diagnostic tests.other diagnostic tests.

66. 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 gelatinDemonstration of gelatin
liquefaction.liquefaction.
•Oxygen requirements of theOxygen requirements of the
bacterium under study.bacterium under study.
•Maintenance of stoke cultures.Maintenance of stoke cultures.

67. Gelatin liquefaction Oxidation – Fermentation
medium

68. 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.

69. LIQUID CULTURESLIQUID CULTURES
•Liquid cultures are inoculated byLiquid cultures are inoculated by
touching with a charged loop or bytouching with a charged loop or by
adding the inoculum with pipettes oradding 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.

70. Blood culture bottles

71. ANAEROBIC CULTURE METHODSANAEROBIC CULTURE METHODS
•Anaerobic bacteria differ in theirAnaerobic bacteria differ in their
requirement and sensitivity to oxygen.requirement and sensitivity to oxygen.
•Cl.tetani is a strict anaerobe – grows atCl.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

72. Production of vacuum:Production of vacuum:
•Incubate the cultures in a vacuumIncubate the cultures in a vacuum
desiccator.desiccator.
Displacement of oxygen with otherDisplacement of oxygen with other
gasesgases
•Displacement of oxygen withDisplacement of oxygen with
hydrogen, nitrogen, helium or COhydrogen, nitrogen, helium or CO22..
•Eg: Candle jarEg: Candle jar

74. 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 metalConsists 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 toThe 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.

76. Working:Working:
• Inoculated plates are placed inside the jar and the lidInoculated plates are placed inside the jar and the lid
clamped air tight.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 hydrogen withAct as a catalyst for the combination of hydrogen with
residual oxygen.residual oxygen.

77. 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

79. Biological methodBiological method
•Absorption of oxygen by incubationAbsorption of oxygen by incubation
with aerobic bacteria, germinatingwith 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

80. THANK YOUTHANK YOU