culture media and culture methods

1. Dr. Amit kumar
amitkumarshahi90@gmail.com

2. • Culture media:
Clinical
specimens eg
pus ,urine
Inoculat
e BACTERI
A Isolation
CULTURE
MEDIA

3. CONSTITUENTS OF CULTURE MEDIA
SODIUM
CHLORIDE AND
OTHERS
PEPTONE
MEAT
EXTRACT

4. • Peptone:
• source of nitrogen
• digested protein
• SOURCE: lean meat or other protein material (heart muscles , casein) and
digested by pepsin.
• Constituents: proteoses , aminoacids , inorganic salts (phosphates , potassium
and magnesium) and nicotinic acid and riboflavin.
• Neopaptone , proteose peptone , mycological peptone

5. • 0.5% sodium chloride : maintain the salt concentration of media
equal to cytoplasm of microorganism

6. • Agar /agar agar :
• solidifying agent (powder form)
• Melt in water after boiling
• Jellifies after cooling
• Seaweeds
• Components : long chain polysaccharides , protein , long chain fatty acids,
inorganic salt (calcium and magnesium)
• Concentration :
• Solid : 1-2%
• Semisolid : 0.5%
• 6% to inhibit proteus swarming

7. • Meat extract :
• Prepared : beef
• Constituent : protein degradation product , inorganic salts ,
carbohydrates and growth factors
• Yeast extract:
• Prepared : washed cells of Baker’s yeast
• Constituent : aminoacids , inorganic salts (potassium and phosphates) and
carbohydrates
• vitamins, carbohydrates, organic nitrogen compounds and salts.

8. • Blood and serum :
• Enriched media
• Fastidious bacteria
• 5-10% sheep blood
• Serum is sterilized after collection

12. I BASED ON CONSISTENCY

14. II) BASED ON INGRADIENTS
Peptone water

15. Peptone:1% peptone + Nacl (0.5%) + D/W
PEPTONE
NB

16. • USE OF BASAL MEDIA :
• preparation of enriched media
• NB (study bacterial growth curve)
• Study colony morphology
• Pigment demonstration
• Semisolid media: motility of bacteria ,
maintaining stock culture
NA +BLOOD
PSEUDOMONAS ON
NA

17. • B) complex media :
• other than basal media
• added with complex ingredients such as yeast extract or casein hydrolyse
• mixture of many chemical species in unknown properties.

18. C

19. D) SPECIAL MEDIA :
Loeffler’s serum slop Blood culture
media
i)

20. 5-10% SRBC + molten NA at 47o c = BA
Type of media : differential media and enriched
media
Blood
agar

21. • Chocolate agar: 5-10% SRBC + molten NA at 70o c = CA
• haemophilus influenza

22. – Loeffler serum slope: isolation of corynebacterium diphtheriae
Beef serum: solidifying agent
– +
– Heart muscle: amino acids
– +
– Peptic digest of animal tissue: amino acids
– +
• Sodium chloride: ions.
– +
– Dextrose: carbohydrate
– +
– Egg: solidifying agent

23. • Blood culture bottle: microorganism from blood (SEPTECIMEA , BACTERIMEA)
sodium polyanetholsulphonate (SPS) :
• inactivate serum factors toxic to organism
beef heart : amino acids
+
calf brain infusion : amino acids
+
disodium phosphate: buffer
+
sodium chloride : maintain salt = cytoplasm
+
glucose
•
• BRUCELLA
BHI AGAR + BHI
BROTH

24. • Ii) Enrichment media
• Liquid media + inhibitory substances
• used isolation of organisms from mixed culture (stool , sputum)
• Tetrathionate browth : salmonella typhae
• Selenite F broth : shigella
• Alkaline peptone water : vibrio cholerae

27. iii)

30. Magnesium Sulfate
Glycerol
L- Asparagine nitrogen and vitamins
Monopotassium
Phosphate
carbon source , favorable to the growth
of the human type tubercle
Prevent contaminants
coagulates
enhance organism growth
and act as buffer
enhance organism
growth and act as buffers

31. phenyl red
Citrate : bromothymol blue
Triple sugar iron : phenyl red
iv)

33. iv)

35. • CLED (Cysteine lactose electrolyte-deficient agar): differentiate LF
and NLF
• Inhibit swarming of proteus
• Specially use for urine specimen

36. v)

37. • Vi) Transport
medium
• Use for transporting of
samples
• Used for delicate
organism
• Eg : stuart’s medium :
Gonococci
• Buffer glycerol saline:
enteric bacilli

38. VII)

40. METHODS OF CULTURE
SPECIMENS CULTURE MEDIA

41. PLATINUM WIRE :
1) Bacteriological loop:
2) Bacteriological straight wire:
stroke
stab

43. Streak culture
Use : to obtained isolated
colonies

44. LAWN CULTURE/CARPET CULTURE
USE :
AST , BACTERIAL
TYPING

45. STROKE CULTURE
USE : URESE TEST
TO OBTAINED PURE
GROWTH

46. STAB CULTURE

47. POUR –PLATE
CULTURE AND SPREAD
METHOD
USE :
TO OBTAINED VIABLE COUNT
OF BACTERIA
1
ml
9ml nb +
1ml bs
9ml nb 9ml nb 9ml nb
1 ml 1 ml 1 ml

48. LIQUID CULTURE
DISADVANTAGE :
DONOT PROVIDE PURE CULTURE FROM MIXED GROWTH
IDENTIFICATION NOT POSSIBLE
EXAMPLE : BLOOD CULTURE
TURBIDITY

50. • Metal or glass jar with metal lid
with a screw (to close airtight)
• Pressure gauge and outlet and
inlet
• Air evacuate through outlet and
hydrogen induce through inlet
• Residual oxygen remove with
hydrogen gas
• sachet containing aluminium
pellets coated with palladium
react with hydrogen and replace
o2 in the form of water(h2o)
Chemical indicator : methylene blue

51. Gaspak system
sachet containing aluminium pellets coated with palladium
HYDROGEN TO FORM H2O (OXYGEN
REMOVED)
OXYGEN REMOVAL
sachet containing
aluminium pellets
coated with
palladium