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

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