basic and general culture media used in microbiology laboratory.

1. Bacterial
Culture Media
Deepika Jalal

2. Culture and Medium
 Culture is the term given to
microorganisms that are cultivated in
the lab for the purpose of identifying
and studying them.
 Medium is the term given to the
combination of ingredients that will
support the growth and cultivation of
microorganisms by providing all the
essential nutrients required for the
growth (that is, multiplication) in order
to cultivate these microorganisms in
large numbers to study them.

3. Culture Media
Indications/ Need for culture -
 Isolation of bacteria in pure cultures.
 Demonstrate their properties.
 Obtain sufficient growth for preparation of
antigens & for other tests.
 Typing bacterial isolates.
 Antibiotic sensitivity.
 Estimate viable counts.
 Maintain stock cultures.

4. Classification of Culture media
 Based on the consistency:
Liquid -- Peptone water, Nutrient
broth
Semisolid -- Nutrient agar slopes
Solid -- Blood agar, Serum agar
 Based on Oxygen requirement:
-- Aerobic media
-- Anaerobic media

5. Aerobic Media
 Simple media
 Complex media
May be Synthetic or Defined
Medium
- Enriched media
- Differential media
- Enrichment media
- Selective media
Semisynthetic Medium
- Sugar media
- Transport media

6. Aerobic media
 Liquid media
- Peptone water(1% peptone +0.5%Nacl +
100 ml water)
- Nutrient broth ( peptone water + 1% meat extract
 Solid media
- Nutrient agar (nutrient broth + 2% Agar)
 Use: To grow non-fastidious microorganisms
Simple media- consists of only basic components

7. Liquid Medium
 Difficult to identify all
types of organisms
 Suitable for isolation of
bacteria from Blood
culture.

8. Brain heart infusion broth
 Is a highly nutritious general-purpose
growth medium for culturing fastidious
and nonfastidious microorganisms, such
as Streptococci, Pneumococci and
Meningococci
 BHI broth contains sodium chloride
which is used to differentiate
enterococci from nonenterococcal group
D streptococci.

9. Constituents Of Media
 Agar or agar- agar
 Peptone - mixture of partially digested proteins
 Yeast or meat extract
 NaCl
 Melting point (Agar) : 98°C
 Solidifying point (Agar): 42°C
 % of agar : Solid media (2%)
Semisolid media (.05to 1%)

10. Basic requirements of culture media
 Nutrients
- Energy source
- Carbon source
- Nitrogen source
 Mineral salts – Sulphate, phosphates, chlorides &
carbonates of K, Mg & Ca.
 A suitable pH – 7.2 – 7.4
 Accessory growth factors
- Tryptophan for Salmonella typhi
- X & V factors for H. influenzae

11. Agar – Agar
 Agar is obtained from Sea
weeds
 Two types :New Zealand
agar & Japanese agar.
 New Zealand agar(1.2%)
is more jellyfying than
Japanese agar(2%).
 Agar contains long chain
polysaccharides.

12. Culture Media

13. Nutrient Agar
 Contain 2% agar added to
Nutrient broth commonly
used
 Concentration can be
increased to 6% to
prevent swarming
 Used for maintaining
stock cultures
 Sub-cultures
 Demonstration of pigment
production

14. Pigment producing
Staphylococci

15. Complex media
 Nutrient agar + 5 to 10% sheep’s blood
 Melt the sterile nutrient agar by steaming, cool, to 450
c
 Add the blood aseptically with constant shaking
 Mix the blood with molten nutrient agar thoroughly
but gently avoiding froth formation
 Immediately pour in to the Petri dishes or tubes and
allow to set
Enriched media: Blood agar
 Use: To cultivate all the fastidious organisms

16. Enriched Medium
 To culture medium
Blood, serum or egg are
added to medium
 Blood agar
 Chocolate agar
 Loeffler’s serum slope
 L J medium (Egg)

17. Different types of hemolysis
on Blood Agar

18. Other Enrichments – Chocolate Agar
 Several organic
materials are added to
the basic constituents of
the Medium such as
Blood, yeast, yeast
extract etc

19. Chocolate agar

20. Differential Medium
Mac Conkey's agar
 Bringing out different
characters of bacteria &
their atypical characters
 Mac Conkey’s medium
Contains peptone,
Lactose, Agar, Neutral
red and Na taurocholate
and shows growth of
Lactose fermenters as
pink coloured colonies.

21. MacConkey agar
 MacConkey agar is useful
medium for cultivation of
enterobacteriaceae.
 It contains a bile salt to
inhibit non intestinal
bacteria (cocci)
 Lactose in combination with
Neutral red distinguish the
lactose fermenting from the
non lactose fermenting
Salmonella and Dysentery
group

22. Lactose fermenting and
Non lactose fermenting

23. Deoxycholate citrate Agar
 Suitable for isolation of dysentery
bacilli, food poisoning Salmonella
and S.paratyphi B, and less so, but
superior to MacConkey agar for S.
typhi.
 It is a heat sensitive medium hence
should not be autoclaved or
remelted
 When prepared from commercial
medium it should be dissolved and
sterilized at 1000C for a short
period.

24. Enrichment media
 In mixed cultures, the bacterium to be isolated
is often overgrown by the unwanted bacteria
 Usually nonpathogenic or commensal bacteria
tend to overgrow the pathogenic ones
 Add substances which has
 a stimulating effect on the pathogenic
bacteria or
 an inhibitory effect on others
 E.g. Selenite F broth,
Tetrathionate broth

25. Enriched media Enrichment media
Solid Liquid
Inhibitory substance Both stimulatory and inhibitory
substance

26. Selective media
 Serves the same purpose as Enrichment media but are
solid in consistency.
- Wilson & Blair’s medium -
- Lowenstein Jensen’s medium -
 Use: To cultivate Salmonella, Shigella &
Mycobacteria

27. Wilson & Blair medium
 Indicate by change of
color Sulphite to
sulphide in Wilson-Blair
medium
 S.typhi reduces sulphite
to sulphide in the
presence of Glucose

28. Lowenstein-Jensen’s
medium Selective for mycobacteria
 Lowenstein Jensen medium
Hen’s eggs
Mineral salt solution
Asparagine
Malachite green
Glycerol
 Distributed in Mc Cartney bottles
 Sterilized by Inspissation

29. Lowenstein Jensen Medium
Rough, tough and buff-colored colonies suggestive
of Mycobacterium tuberculosis

30. TCBS medium
(Thiosulphate citrate bile salt sucrose
agar) • Used for Vibrio
cholerae
• Yellow coloured
colonies by V. cholerae &
• Green coloured colonies
by V. parahemolyticus.

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

32. Sabouraud's Dextrose Agar
Dextrose - 4 gm%
Neopeptone - 1 gm%
Agar - 1.5 gm%
Distilled water - 100 ml
 Dissolve the ingredients by heating in a water bath,
cool and adjust pH to 5.4
 Autoclave and dispense 20 ml amount in test tubes
 Use: For the cultivation of Fungi

33. Sabouraud's Dextrose agar
commonly used Fungal
Isolation Medium

34. Anaerobic Medium
 Robertson’s cooked
meat medium
 Thioglycollate liquid
medium

35. Robertsons’cooked Meat Medium
 To cultivate the anaerobic bacteria
 Place meat in 1 ounce bottles to the
depth of 2.5 cms and cover it with 15 ml
of broth
 Contains a column of meat particles in
peptone infusion broth
 Identification of
Saccharolytic/proteolytic bacteria
 Autoclaved at 1210 c for 20 mins
 After sterilization, adjust the pH to 7.5

36. Anaerobic Culture Methods
Anaerobic jar
 Anaerobic
jar
Figure 6.5
Control
Pseudomonas ATCC 27853

37. Transport Medium
 Stuart’s medium contain
reducing agents to
prevent oxidation.
 Charcoal to neutralize
certain bacterial
inhibitors to Gonococci.

38. Sugar media
 Consist of 1% of sugar in peptone water along with
an appropriate indicator
 A small tube (Durham’s tube) is kept inverted to
detect gas production
 Sugars tested are
 Lactose (lylac)
 Glucose (green) - Durham’s tube
 Mannitol ( mauve)
 Dulcitol (.5 %) (dull orange)
 Sucrose (sky blue)
 Maltose (no colour)

39. Muller Hinton Agar for
Antibiotic Testing

40. Sterilization of culture media
 Media are sterilized in the autoclave at 1210 c
for 15’ under 15lbs/inch2 of Pressure
 Heat-labile substances like serum & sugar
solutions must be sterilized by free-steam or
filtration
 Egg containing media - Lowenstein-Jensen’s
medium, Loeffler's serum slope by inspissation
 Discarded culture plates are to be sterilized by
autoclaving prior to washing

41. Thank you….