culturing & isolating

1. Culturing and Isolating
COURSE : B.SC. (MICRO)
SUBJECT : ELEMENTARY
MICROBIOLOGY
UNIT 4.4

2.  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 to beBy appropriate procedures they have to be
grown separately (isolated) on culturegrown 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.

3. ColonyColony – macroscopically visible collection– macroscopically visible collection
of millions of bacteria originating from aof 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 24oo
CC

4. 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 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 canColony morphology, pigmentation, hemolysis can
be appreciated.be 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

8. Simple media / basal mediaSimple 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

9. 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

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

11. Enrichment mediaEnrichment media
 Liquid media used to isolateLiquid media used to isolate
pathogens from a mixedpathogens from a mixed
culture.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– for the
isolation of Salmonella, Shigellaisolation of Salmonella, Shigella
– Alkaline Peptone WaterAlkaline Peptone Water – for– for
Vibrio choleraeVibrio cholerae

12. Selective mediaSelective media
 The inhibitory substance is added to a solidThe inhibitory substance is added to a solid
media.media.
Eg:Eg:
 Mac Conkey’s mediumMac Conkey’s medium for gram negativefor gram negative
bacteriabacteria
 TCBSTCBS – 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

13. Indicator mediaIndicator media
 These media contain an indicator whichThese media contain an indicator which
changes its colour when a bacteriumchanges 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

14. Differential mediaDifferential media
 A media which has substancesA media which has substances
incorporated in it enabling it to distinguishincorporated 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
– TTaurocholateaurocholate
 Distinguish between lactose fermenters &Distinguish between lactose fermenters &
non lactose fermenters.non lactose fermenters.

15. 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) forContain a small tube (Durham’s tube) for
the detection of gas by the bacteria.the detection of gas by the bacteria.

16. 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– non nutrient
soft agar gel containing asoft agar gel containing a
reducing agentreducing agent
– Buffered glycerol salineBuffered glycerol saline – enteric– enteric
bacillibacilli

17. Anaerobic mediaAnaerobic media
 These media are used to grow anaerobicThese media are used to grow anaerobic
organisms.organisms.
 Eg: Robertson’s cooked meat medium,Eg: Robertson’s cooked meat medium,
Thioglycolate medium.Thioglycolate medium.

18. 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 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

19. Isolation & Culturing
Isolation: Colony on media, one kind of
microbe, pure culture: isolation on general
and special “differential media”
General growth media: NA, TSA
Differential: Mac, EMB, SS
These have dyes, salts, inhibiting
agents : see differences on
plates

20. Microbiology – Chapter 3
Isolation: Colony on media, one kind of
microbe, pure culture
1
2

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

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

23. Microbiology – Chapter 3
Differential: Mac, EMB, SS
These have dyes, salts, inhibiting
agents : see differences on plates
4
5

24. Microbiology – Chapter 3
 Blood agar : rich with nutrients, can see a
difference, thus differential; much more
later
6
7

25. 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.

26. 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

27. 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.

28. 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 andIn the preparation of bacterial antigens and
vaccinesvaccines..
 Lawn cultures are prepared by flooding theLawn cultures are prepared by flooding the
surface of the plate with a liquid suspension ofsurface of the plate with a liquid suspension of
the bacterium.the bacterium.

29. Antibiotic sensitivity testing
8

30. STROKE CULTURESTROKE CULTURE
 Stroke culture is made inStroke culture is made in
tubes containing agar slope /tubes containing agar slope /
slant.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.
9

31. STAB CULTURESTAB CULTURE
 Prepared by puncturing a suitable mediumPrepared 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 bacteriumOxygen requirements of the bacterium
under study.under study.
– Maintenance of stoke cultures.Maintenance of stoke cultures.

32. POUR PLATE CULTUREPOUR PLATE CULTURE
 Agar medium is melted (15 ml) and cooled toAgar medium is melted (15 ml) and cooled to
4545oo
C.C.
 1 ml of the inoculum is added to the molten1 ml of the inoculum is added to the molten
agar.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.

33. 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 withcharged 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 mixedIt does not provide a pure culture from mixed
inocula.inocula.

34. Blood culture bottles
10

35. ANAEROBIC CULTURE METHODSANAEROBIC CULTURE METHODS
 Anaerobic bacteria differ in their requirementAnaerobic bacteria differ in their requirement
and sensitivity to oxygen.and sensitivity to oxygen.
 Cl.tetani is a strict anaerobe – grows at anCl.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

36. Production of vacuum:Production of vacuum:
 Incubate the cultures in a vacuumIncubate 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

37. 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.

38. 11

39. Working:Working:
 Inoculated plates are placed inside the jar andInoculated 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 pumpThe 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 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, soterminals 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 hydrogenAct as a catalyst for the combination of hydrogen
with residual oxygen.with residual oxygen.

40. 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

41. 12

42. Biological methodBiological method
 Absorption of oxygen by incubation withAbsorption of oxygen by incubation with
aerobic bacteria, germinating seeds oraerobic 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

43. Reference
 Image (1 to 12)
1. Microbiology by Gerard J. Tortora, Christine L Case, and Berdell R.
Funke
Books:
1. Microbiology VI Edition, M.J. Pelczar, E.C.S. Chan and N.R. Kreig,
Tata McGraw Hill
2. 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.
Funke
4. Microbiology by Prescott, Harley, and Klein's Microbiology Joanne M.
Willey, Linda M

44. THANK YOUTHANK YOU