The document discusses culture media and methods for microbial cultivation, emphasizing the components necessary for bacterial growth, such as nutrients and solidifying agents. It describes various types of media, including enriched, selective, and differential media, detailing their applications and advantages. Additionally, it outlines culture methods for isolating bacteria and methods for anaerobic bacterial identification.

1. CULTURE MEDIA
&
CULTURE
METHODS
ROMA GOYAL

2.  CULTURE – Is term given to microorganisms that are
cultivated in the lab for the purpose of studying them.
 MEDIUM – Is the term given to the combination of
ingredients that will support the growth & cultivation of
microorganisms outside their natural habitats.

4. Necessary Requirements for Growth of
Bacteria
 Distilled Water
 Nitrogen containing compounds
• Peptone- Golden granular powder
Complex mixture of partially digested protiens by proteolytic
enzymes pepsin, trysin or papain
Peptones, Proteoses, polypeptides, aminoacids, inorganic salts like
phosphates
potassium & magnesium
Accessory growth factors like nicotinic acid & riboflavin
 Energy sources
 Suitable Ph- 7.2 – 7.4
 Solidifying agents:
• Gelatin– Protien
• Agar— Chief component is Long chain Polysaccharide
Melts at 95°c & solidify only when cooled to about 42°c
1- 2% yields a suitable gel eg. Non-nutritive agar

5. CLASSIFICATION
 According to Physical State:
Liquid – Peptone Water, Nutrient
Broth
Semisolid – Nutrient Agar Stabs
Solid – Blood Agar
 According to Oxygen requirement:
Aerobic Medium
Anaerobic Media

6. 6

7. Advantages of Liquid Medium
 For obtaining bacterial growth from blood or water when
large volumes have to be tested.
 For prepairing bulk cultures of antigens or vaccines.
Disadvantages of Liquid Media over Solid
Media
 Bacteria growing in them may not exibit specific
characteristics for their identification.
 Also difficult to isolate different types of bacteria from
mixed populations.

8.  According to Special Purposes:
A. Simple/Basal media
B. Complex media
 Enriched media
 Enrichment media
 Selective media
 Indicator/Differential media
 Sugar media
 Defined synthetic media
 Transport Media
 Storage Media

9. BASAL MEDIA
 Simplest and most common medium in routine diagnostic
laboratories.
 liquid Media
• Meat infusion broth
• Meat extract broth
• Hartley’s digest broth
• Nutrient broth
 Nutrient agar
 Semi-solid agar
 Firm agar
 Peptone water

10. NUTRIENT AGAR

11. ENRICHED MEDIA
 Prepared to meet the nutritional requirements of more
exacting bacteria by the addition of substances such as
blood, serum or egg to a basal medium.
 Used for cultivation of fastidious organisms.
 Examples are:
 Blood agar
 Chocolate agar
 Loefller’s serum slope.

12. BLOOD AGAR
 It’s a solid culture media, consist of agar, peptone and
blood.
 Sheep blood is the commonest and 10% is the most usual
conc.
 It’s an enriched as well as Indicator medium.
 It shows the haemolytic properties. Eg Streptococcus.
 It supports the growth of most aerobic and anaerobic
bacteria and fungi.
 Vit K, cysteine and hemin supplement enhances growth of
anaerobic bacteria.

13. BLOOD AGAR

14. DIFFERENT TYPES OF
HAEMOLYSIS ON BLOOD AGAR

15. CHOCOLATE AGAR

16. CHOCOLATE AGAR
 Solid culture media made by heating a mixture of blood
and nutrient agar.
 Heat ruptures the red cells and librates nutrients i.e.
hemoglobin, hemin/X factor and Nicotinamide adenine
dinucleotide/V factor.
 Useful for isolation of fastidious organisms, Haemophilus
infuenzae, Neisseriae meningitidis, N. gonorrhoeae,
Moraxella spp and Pneumococcus.

17. LOEFFLER’S SERUM SLOPE
• It’s consist of media
containing blood (fresh,
heated or lysed), serum
as solidifying agent.
• Serum has created
difficulties in
immunodiffusion tests
and biochemical tests.
• Useful for
Corynebacterium
diphtheriae.

18. ENRICHMENT MEDIA
 It facilitate the isolation of particular spp from a mixed
inoculum.
 It’s a liquid medium, which favours the multiplication of
a particular spp, either by containing enrichments that
selectively favour it or inhibitory substances that
suppress competitors.
 Examples are:-
Selenite F broth
 Tetrathionate broth
 Alkaline peptone water

19. SELENITE F BROTH
 Consists of Disodium
hydrogen phosphate,
Sodium Hydrogen
Selenite.
 It inhibits coliform
bacilli while permit
dysentry bacilli to grow
i.e. Salmonella typhi and
Shigella sonnei and
Shigella flexneri.

20. TETRATHIONATE BROTH
 Used for Salmonella and Shigella. It inhibits gram
positive organisms and enterobacteriaceae.
 It contains Sodium thiosulphate, Phenol red.
THIOGLYCOLATE BROTH
 Oxidation reduction indicator – methylene blue or
resazurin
 Thioglycolic acid and 0.075% agar

21. BLOOD CULTURE BOTTLE
 Enrichment Liquid medium.

22. SELECTIVE MEDIA
 It’s an solid medium.
 Inhibits the growth of unwanted bacteria but
permits the growth of wanted bacteria in
form of colonies.
 Eg. Deoxycholate Citrat Agar(DCA), Xylose Lysine
Deoxycholate agar, Lowenstein Jensen Medium.

23. XLD AGAR
Red pink black
centred
colonies
salmonella
Red pink
colonies
shigella

24. XYLOSE LYSINE DEOXYCHOLATE AGAR
 Selective or Differential medium
 Esp. used for Shigella and Salmonella spp.
 It contains Phenol red, sodium deoxycholate at conc of 0.1%-
0.25%, instead of 0.5% in DCA.
DEOXYCHOLATE CITRATE AGAR (DCA)
 Selective or Differential medium.
 Used for isolation of dysentery bacilli like salmonella and shigella
and for E.coli too.
 It contains Neutral red, Sodium citrate, Sodium thiosulphate,
Ferric ammonium citrate and Sodium deoxycholate.

25. DEOXYCHOLATE CIRATE AGAR
 It is an heat
sensetive medium,
should not be
autoclaved or
remelted.
 When prepared from
commercial medium it
should be dissolved
and sterilized at 100°c
for a short period.

26. LOWENSTEIN JENSEN MEDIUM

27. THAYER-MARTIN AGAR
 Selective and Enriched medium for the isolation of
Neisseria gonorrhoeae and N. meningitidis.
 It contains chocolate blood agar with antibiotics.
 Antibiotics - Colistin, Vancomycin, Nystatin and
antimicrobial Trimethoprim

28. THIOSULPHATE CITRATE BILE SUCROSE
AGAR (TCBS)
Selective and
Differential
 High pH 8.6, salt and
sucrose.
 Used for growth of
Vibrios
 Indicator used is
Bromothymol blue.

29. DIFFERENTIAL MEDIUM
 These medium contain some substances that is changed
visibly as a result of metabolic activities of particular
organisms.
 These are combinations of enriched media with selective
agents and indicator systems.
 Eg. MacConkey medium, Blood agar, Wilson and Blair’s
medium

30. MacConkey Agar
 Used for the
cultivation of
entericbacteria
 It contains bile salt
i.e. sodium
taurocholate, lactose
with neutral red and
crystal voilet.

31. Lactose fermenting and Non
lactose fermenting

32. CYSTINE LACTOSE ELECTROLYTE
DEFICIENT (CLED) AGAR
 It’s a semi-quantative
method for urine.
 It distinguish LF from NLF
colonies and inhibit Proteus
from swarming.
 Advantage over Mac
Conkey: It supports the
growth of certain Staph,
Strept and Candida that fail
to grow on Mac Conkey.
 It contains Lactose, L-
cystine, Bromothymol blue,
Tryptone.

33. WILSON AND BLAIR’S brilliant-green
bismuth sulphite Agar (BBSA)
 Selective and
Indicator medium.
 Salmonella spp

34. SUGAR MEDIUM
 It is used to determine the ability of an organism to
ferment a specific carbohydrate present in the medium.
 Sugar can be glucose, lactose, sucrose, maltose and
mannitol.
 It contains 1% sugar in peptone water along with
Durham’s tube is kept inverted to detect the gas
production and with Andrade’s indicator.

35. FERMENTATION OF SUGAR

36. TRANSPORT MEDIA
 Used when delay in culture and specimen has to be
transported from a distance and pathognomic bacteria
may not survive or may be over grown by commensals.
 Eg. Stuart’s media (urethral discharge for N. gonococci),
Cary Blair (stool/rectal swab specimens).

37. STAURT’S Transport Medium

38. STORAGE MEDIUM
 Cold storage for the preservation of culture media.
 Temp should be 4-5 c (39°- 41°F), should never be low
to cause freezing.
 Prepared sterilized media in individual screw-capped
bottles (broths and nutrient agars) can be stored at room
temp for weeks but can deteriorate.

39. Muller Hinton Agar for Antibiotic Testing
It’s a basic solid
media, dispensed in
Petridishses.
 It contains Beef
infusion, Casein
hydrolysate, Starch,
Agar.

41. BRAIN-HEART INFUSION WITH COOKED
MEAT PARTICLES (BHI/CMP)
 70ml BHI (broth) in
120ml medical flat
bottles with 2.5cm
layer of CMP
 Autoclave at 1210 c
for 20 min
 After sterilization,
adjust the pH to 7.1.
 Use to cultivate the
anaerobic bacteria

42. Sabouraud's Dextrose agar
Commonly used
Fungal Isolation
 It contains Peptone,
Dextrose, Agar and
Water.
 Adjust pH at 5.4,
Autoclave at 115°c for
20min

43. CULTURE
METHODS

44. Culture methods are done to:
• Isolate bacteria in pure culture from the clinical specimens
and their idintification by various methods.
• Determination of antibiotic sensitivity.
• Prepare antiges for serodiagnosis of infective diseases.
• Maintain stock cultures.

45. • Streak culture
• Stroke
• Stab
• Pour plate
• Liquid culture
• Special methods for
anaerobic cultures
Methods to isolate the Bacteria

46. STREAK CULTURE
• Used majorly to isolate
the bacteria in pure
culture.
• A platinum or nichrome
wire loop of 2-4 mm
internal diameter is
used.
•

47. Streak Plate
(Quadrant Streak)

49. LAWN CULTURE
• Lawn culture are obtained
by flooding the surface of
the surface of the plate with
suspension of the bacterium
• Mainly used for:-
• Antibiotic sensitivity
testing
• Preparation of bacterial
antigens and vaccines.
• Bacteriophage typing

50. STROKE CULTURE
• Done in tubes containing
agar slopes
• Used for:-
• Pure growth of
bacterium.
• For biochemical testing.
• Other diagnostic tests.

51. STAB CULTURE
• It is performed by a straight
wire, charged with culture
material, by puncturing deep
inside the agar.
• It is used to:
• Demonstrate gelatin
liqefaction
• Oxygen requirement of
the bacterium
• To maintain stock culture
for preservation of
bacteria.

52. Pour Plate technique

53. LIQUID CULTURE
• Test tubes are
inoculated by a
charged loop.
• Uses are:
• For blood culture.
• Prefered for specimens
containing antibiotics.
• For biochemical
examination.
• When large fields of
bacteria are required.

54. Anaerobic Bacterial
Isolation and
Identification Needs
specified conditions

55. Desiccator
• In Desiccator some
oxygen is left.
• Not suitable for fluid
culture.
• Displacement of
oxygen is done with
Hydrogen,Nitrogen,
Helium,Co2

56. Candle Jar
• Inoculated plates are
kept
• Burning candle use
up all oxygen
• But a little o2 is left
• But presence of Co2
stimulates the most
bacterium
56

57. Mac Intosh Fildes Anaerobic Jar
• Contain inlet and
outlet.
• Electrical supply
• Inoculated culture
plates
• When electrified
palladinised asbestos
heating acts as catalyst for
combination of hydrogen
with residual oxygen
causes complete
anaerobiasis

58. Gas pack
• A disposable envelop contains
chemicals which generate
hydrogen and carbon dioxide
on addition of water
• Inoculated plates are kept in
jar
• Water is added hydrogen and
carbon dioxide are liberated
• Presence of cold catalyst in the
envelop permits the
combination of Hydrogen and
oxygen to produce anaerobic
environment
• Indicator is methylene blue
• Colorless when anaerobic
environment.

59. Other Reducing agents
• Reducing agents
O.1% Thiglyclolate
0.1% Ascorbic acid
0.05 % cysteine.
• Cooked meat broth
• It is also known
as Robertson’s
cooked meat
broth(RCM).

60. ANAEROBIC CHAMBER