For education

1. CULTURE MEDIA
DR. ANVI

2. CULTURE MEDIA
▰ Liquid or solid substance - contains nutrients to support the growth, and
survival of microorganisms.
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3. Constituents of Culture Media
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Constituent Explanation
Water and
Electrolytes
Sodium chloride
Peptone Mixture of partially digested proteins - obtained from various sources -
heart muscle, casein or fibrin, or soya.
Agar Used for solidifying the culture media
Source: Prepared from - cell wall of seaweeds and available
commercially in powder form
Preparation: Agar powder is dissolved in water and subjected to
sterilization by autoclave. When the temperature of the molten agar
comes down to 45°C, it is poured into the Petri dishes and then allowed
to set for 20 minutes.
Concentration:
Solid agar preparation - 1-2%
Semisolid agar- 0.5%
Solid agar to inhibit Proteus swarming- 6%

4. Constituents of Culture Media
(Cont..)
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Constituent Explanation
Meat extract Highly concentrated meat stock, usually made from
beef.
Yeast extract (Prepared from Baker’s yeast) and malt extract
(contains maltose)
Blood and
serum
Important components of enriched media; provide
extra nutrition to fastidious
bacteria. 5–10% of sheep blood is used. Alternatively,
horse, ox or human blood can also be used.

5. Types of Culture Media
Based on consistency, culture media are grouped into:
▰ Liquid (or broth) media
▰ semisolid media
▰ solid media
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6. Types of Culture Media
Based on the growth detection, culture media are classified as:
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Conventional culture media:
Prepared from nutrients -
aqueous extract of meat,
peptone.
 Simple/basal media
 Enriched media
 Enrichment broth
 Selective media
 Differential media
 Transport media
 Anaerobic media.
Automated culture media:
Mainly available for blood and
sterile body fluid culture.

7. Classification
CONSISTENCY
• SOLID MEDIA
• LIQUID MEDIA
• SEMISOLID
MEDIA
• BIPHASIC MEDIA
OXYGEN
REQUIREMENT
• AEROBIC MEDIA
• ANAEROBIC
MEDIA
NUTRITION
• SIMPLE MEDIA
• COMPLEX
MEDIA
• SYNTHETIC
/DEFINED
MEDIA
Special
Characteristics
• ENRICHED
MEDIA
• ENRICHMENT
MEDIA
• SELECTIVE
MEDIA
• INDICATOR
MEDIA
• DIFFERENTIAL
MEDIA
• BIOCHEMICAL
MEDIA

8. Con Consistency
Decided by Agar concentration :
- Solid media: 2% Agar
- Semisolid media: 0.5% Agar
- Liquid media: Agar not added
- Biphasic media : Solid and a liquid phase in the
same media container (Bottle)

9. Semisolid Media
Mannitol motility
media
Used to
demonstrate
motile organisms –
indicated by a
haziness spreading
away from the
stab line

10. Brain heart infusion
agar
- Both liquid & solid
phase
-Reduces
contamination and
isolate even a low
count of pathogen

11. Simple/Basal Media
Contain minimum ingredients that support the
growth of non-fastidious bacteria.
▰ Peptone water: Contains peptone (1%) + NaCl
(0.5%) + water
▰ Nutrient broth: Peptone water + meat extract
(1%).
▰ Nutrient agar: Nutrient broth + 2% agar
▰ Semisolid medium: Concentration of agar -
reduced to 0.2–0.5 %.
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12. Uses of Basal Media
▰ Testing the non-fastidiousness of bacteria.
▰ Serve as base for the preparation of many other media.
▰ Nutrient broth - used for studying the bacterial growth curve.
▰ Nutrient agar - preferred medium for:
 Performing the biochemical tests, such as oxidase, catalase, etc.
 Study the colony morphology
 Pigment demonstration.
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13. Enriched Media
▰ Basal medium added with additional nutrients - blood, serum or egg
▰ In addition to non-fastidious organisms, support the growth of fastidious
nutritionally exacting bacteria.
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14. Enriched Media (Cont..)
Blood agar –
▰ Prepared by adding 5-10% of sheep blood to the
molten nutrient agar at 450C.
▰ Tests the hemolytic property of the bacteria, which
may be either- i) partial or α (green) hemolysis and
ii) complete or β hemolysis
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15. Enriched Media (Cont..)
Chocolate agar:
▰ Heated blood agar, prepared by adding 5 -10%
of sheep blood to the molten nutrient agar at
70°C.
▰ More nutritious than blood agar - supports
certain highly fastidious bacteria -
Haemophilus influenzae that does not grow
on blood agar.
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16. Enriched Media (Cont..)
Loeffler’s serum slope: Contains serum - used for isolation of
Corynebacterium diphtheriae
Blood culture media: Used for isolating microorganisms from blood - available
as conventional or automated blood culture media.
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17. Enrichment Broth
▰ Liquid media added with inhibitory agents which selectively allow certain
organism to grow and inhibit others.
▰ Important for isolation of pathogens from clinical specimens which also
contain normal flora (e.g. stool and sputum specimen).
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18. Enrichment Broth (Cont..)
Examples :
▰ Tetrathionate broth—Used for Salmonella Typhi
▰ Gram-negative broth—Used for Shigella
▰ Selenite F broth—Used for Shigella
▰ Alkaline peptone water (APW)—Used for Vibrio cholerae.
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19. Selective Media
Solid media containing inhibitory substances - inhibit the normal flora
present in the specimen and allow the pathogens to grow.
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Media Used for isolation of
Lowenstein Jensen (LJ) medium Mycobacterium tuberculosis
Thiosulphate Citrate Bile salt
Sucrose (TCBS)
Vibrio species
DCA (Deoxycholate Citrate Agar) Salmonella and Shigella from stool
XLD (Xylose Lysine Deoxycholate)
agar
Salmonella and Shigella from stool
Potassium tellurite agar (PTA) Corynebacterium diphtheriae

20. Selective Media (Cont..)
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Lowenstein–Jensen
medium
TCBS
agar

21. Lowenstein Jensen (JL) Medium:
Used for the growth of
Mycobacterium tuberculosis
Contains malachite green as an
inhibiting substance
Mycobacterium
tuberculosis
colonies are
described as
rough, tough, buff

22. TCBS Agar
Thiosulphate Citrate
Bile salt Sucrose (TCBS)
agar:
Used for the isolation of
Vibrio cholerae
Contains bile salt as an
inhibiting substance
Large yellow coloured
colonies

23. Potassium tellurite agar:
Used for the isolation of
Corynebacterium diphtheria
Contains potassium tellurite as an
inhibiting substance

24. Selective media for Salmonella typhi :
Wilson Blair bismuth sulfite medium –
jet black colonies with a metallic
sheen
Deoxycholate citrate agar – NLF
colonies with a black centre
Xylose lysine deoxycholate agar – Red
colonies with a black centre
Salmonella Shigella agar – Colourless
colonies with a black centre

25. Selective Media (Cont..)
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DCA (Deoxycholate Citrate Agar) XLD (Xylose Lysine Deoxycholate) agar

26. ▰ WILSON BLAIR
▰ XLD DCA
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27. Transport Media
▰ Used for the transport of the clinical specimens suspected to contain
delicate organism or when delay is expected while transporting the
specimens from the site of collection to the laboratory
▰ Bacteria do not multiply in the transport media - they only remain viable.
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28. Transport Media (Cont..)
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Organism Transport media
Neisseria Amies medium, Stuart’s medium
Vibrio cholerae VR (Venkatraman-Ramakrishnan)
medium
Autoclaved sea water
Cary Blair medium
Shigella, Salmonella Buffered glycerol saline
Cary Blair medium

29. Differential Media
▰ Differentiate between two groups of bacteria - by using an indicator.
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Differential media Features
MacConkey agar  Differential and low selective medium - used for
the isolation of enteric GNB.
 Differentiates organisms into LF (pink colonies,
e.g. Escherichia coli) and NLF or (colorless
colonies, e.g. Shigella).
 Composition- Peptone, lactose, agar, neutral red
(indicator) and taurocholate

30. Differential Media (Cont..)
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Differential media Features
CLED agar (cysteine
lactose electrolyte-
deficient agar)
Capable of differentiating between LF
and NLF.
Used as an alternative to combination
of blood agar and MacConkey agar, for
the processing of urine specimens

31. Differential Media (Cont..)
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MacConkey agar CLED agar

32. Anaerobic Culture Media
▰ Contain reducing substances which take up oxygen and create lower redox
potential - permit the growth of obligate anaerobes, such as Clostridium.
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Media Features
Robertson’s cooked meat
(RCM) broth
 Contains chopped meat particles
(beef heart), which provide
glutathione (a sulfhydryl group
containing reducing substance) and
unsaturated fatty acids.
 Widely used
 Used for maintenance of stock
cultures.

33. Anaerobic Culture Media (Cont..)
Other anaerobic media include:
▰ Thioglycollate broth
▰ Anaerobic blood agar
▰ BHIS agar (Brain-heart infusion agar) with supplements (vitamin K and
hemin)
▰ Neomycin blood agar
▰ Egg yolk agar
▰ Phenyl ethyl agar
▰ Bacteroides bile esculin agar (BBE agar).
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34. Blood Culture Media
▰ Recovery of bacteria from blood is difficult - they are usually present in
lesser quantity and many of the blood pathogens are fastidious.
▰ Therefore, enriched media - used for isolating microorganisms from blood.
▰ Available either as conventional or automated media
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35. Conventional Blood Culture Media
Two types:
▰ Monophasic medium: Contains brain–heart
infusion (BHI) broth.
▰ Biphasic medium: Liquid phase containing BHI
broth and a solid agar slope made up of BHI
agar
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36. Disadvantages of conventional media
Subcultures are made manually - performed less-frequently (once a
day) as it is cumbersome.
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Broth Disadvantages
Monophasic BHI
broth
 Subcultures are made, periodically for 1 week.
 Higher risk of contamination - due to opening of the
cap of the bottle every time when subcultures are
made.
Biphasic BHI
broth
 Subcultures - made just by tilting the bottles so that
the broth runs over the agar slope.
 Lower risk of contamination as it obviates the
opening of the cap of the bottle

37. Automated Blood Culture Techniques
(Cont..)
▰ Specimens: Used for culture of blood, bone marrow and sterile body
fluids.
▰ More sensitive: Higher yield of positive cultures from clinical specimens
▰ Rapid: Less time than conventional methods
▰ Less labor intensive: fully-automated
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38. Automated Systems
BacT/ALERT 3D:
▰ Principle - colorimetric detection of
growth.
▰ When bacteria multiply - produce CO2 -
increases the pH - changes the color of a
blue-green sensor present at the bottom
of the bottle to yellow
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39. Automated Blood Culture Techniques
▰ Continuous automated monitoring: Blood culture bottles - periodically
monitored for the microbial growth - every 10 minutes by the instrument.
▰ Composition: Tryptic soy broth and/or brain heart infusion broth +
polymeric resin beads - adsorb and neutralize the antimicrobials present in
blood specimen.
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40. Automated Systems (Cont..)
BacT/ALERT VIRTUO (bioMerieux):
Advanced form of BacT/ALERT – has
several advantages, such as:
▰ Automatic loading and unloading of
bottles,
▰ Faster detection of growth,
▰ Determine volume of blood present
in bottle
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41. Automated Systems (Cont..)
BACTEC (BD Diagnostics)
▰ Principle - fluorometric detection of growth.
▰ Uses an oxygen-sensitive fluorescent dye present in the medium.
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42. Disadvantages of automated systems
▰ High cost of the instrument and culture bottles,
▰ Inability to observe the colony morphology as liquid medium is used.
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43. THANK YOU