1. CULTURE MEDIA USEDCULTURE MEDIA USED
IN MICROBIOLOGYIN MICROBIOLOGY
SHEIKH JONAID NIZAMI
Trainee Technologist
Clinical Laboratory
AGA KHAN UNIVERSITY
HOSPITAL KARACHI

2. Definition, purpose/importance
History of culture media
Classification of culture media
Growth pattern of bacteria

3. Microbiological culture
Method of cultivating microbialMethod of cultivating microbial
organisms by letting them reproduceorganisms by letting them reproduce
in predetermined culture media underin predetermined culture media under
controlled laboratory conditions.controlled laboratory conditions.

4. Bacteria have to be grown (cultured) for themBacteria have to be grown (cultured) for them
to be identified and subsequent clinicalto be identified and subsequent clinical
diagnosis.diagnosis.
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.
Bacteria have to be cultured in order to obtainBacteria have to be cultured in order to obtain
antigens from developing serological assay forantigens from developing serological assay for
vaccines.vaccines.
Certain genetic studies and manipulations ofCertain genetic studies and manipulations of
the cells also need that bacteria be cultured inthe cells also need that bacteria be cultured in
vitro.vitro.

5. Louis PasteurLouis Pasteur used simple broths made upused simple broths made up
of urine or meat extracts.of urine or meat extracts. Robert KochRobert Koch
realized the importance of solid media andrealized the importance of solid media and
usedused potato piecespotato pieces to grow bacteria.to grow bacteria. It wasIt was
on the suggestion of Fannie Eilshemius,on the suggestion of Fannie Eilshemius,
wife of Walther Hesse (who was anwife of Walther Hesse (who was an
assistant to Robert Koch) that agar wasassistant to Robert Koch) that agar was
used to solidify culture media.used to solidify culture media.
History of culture medias

6. Before the use of agar, attempts were made toBefore the use of agar, attempts were made to
use gelatin as solidifying agent.use gelatin as solidifying agent. GelatinGelatin hadhad
some inherent problems….some inherent problems….
It existed as liquid at normalIt existed as liquid at normal
incubating temperatures (35-37incubating temperatures (35-37oo
C)C)
Digested by certain bacteriaDigested by certain bacteria
Continued….

7. AgarAgar
 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

8. During typical bacteria growth (growth cycle)
bacteria cell divide by binary fission and
their mass and number increase in an
exponential manners. Bacterial growth in
culture can be separated into at least four
distinct phases.
Bacterial Growth Curve

9. Bacterial Growth Curve

10. This is period of intense physiologic adjustment
involving the induction of new enzymes and the
synthesis and assembly of ribosome. In lag phase
and during this phase there occur
1. increase in size of cells
2. increase in metabolic rate
3. adaptation to new environment and
necessary enzymes.
1. Lag phase

11. The length of lag phase depend upon
a. Type of bacteria.
b. Better the medium, shorter the lag phase.
c. The phase of culture from which inoculation in
taken
d. Size or volume of inoculum.
e. Environmental factors like temperature.
Continued….

12. 2. Logarithmic (Exponential) phase
In logarithmic phase the bacterial cell
start dividing and their number increase by
geometric progression with time. During this
periods…
a. bacteria have high rate of metabolism
b. bacteria are more sensitive to antibiotics
c. rate of penetration of the medium it
depends on the concentration of material in
the media

13. 3. Stationary phase
In stationary phase after some time a stage
comes when rate of multiplication and death
becomes almost equal it may be due to
a. depletion of nutrient
b. accumulation of toxic products and
sporulation may occur during this stage.

14. 4. Decline or death phase
In decline (death) phase, during this phase
population decreases due to death of cells the
factors responsible are
a. nutritional exhaustion
b. toxic accumulation
c. autolysis enzymes

15. 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

16. Special mediaSpecial media
 Enriched mediaEnriched media
 Enrichment mediaEnrichment media
 Selective mediaSelective media
 Indicator mediaIndicator media
 Differential mediaDifferential media
 Transport mediaTransport media
III.III. Based on Oxygen requirementBased on Oxygen requirement
- Aerobic media- Aerobic media
- Anaerobic media- Anaerobic media

17. 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:Eg: SIMSIM

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

20. 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% NaClEg: peptone water – 1% peptone + 0.5% NaCl
in waterin water

21. 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

22. Blood agar Chocolate agar

23. 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 Vibrio– for Vibrio
choleraecholerae

24. 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

25. TCBSMac Conkey’s medium

26. Potassium Tellurite media LJ media

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

29. Urease mediumUrease medium

30. Differential mediaDifferential media
 A media which has substances incorporatedA media which has substances incorporated
in it enabling it to distinguish betweenin it enabling it to distinguish between
bacteria.bacteria.
 Eg:Eg: Mac Conkey’s mediumMac Conkey’s medium
 Distinguish between lactose fermenters & nonDistinguish between lactose fermenters & non
lactose fermenters.lactose fermenters.

31.  Lactose fermenters –Lactose fermenters – PinkPink coloniescolonies
 Non lactose fermenters – colourless coloniesNon lactose fermenters – colourless colonies

32. 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 reducingagar gel containing a reducing
agentagent
 Buffered glycerol salineBuffered glycerol saline – enteric– enteric
bacillibacilli

33. Anaerobic mediaAnaerobic media
 These media are used to grow anaerobic organisms.These media are used to grow anaerobic organisms.
 Eg:Eg: Robertson’s cooked meat medium, ThioglycolateRobertson’s cooked meat medium, Thioglycolate
medium.medium.