Dr.T.V.Rao MD
Major Contribution to Culture
Media
2Dr.T.V.Rao MD
Agar - Agar Frau Hesse
3Dr.T.V.Rao MD
Agar – Agar
Solid medium is made by
adding Agar
Agar is obtained from Sea
weeds New Zealand agar is
more
Agar contain long...
5Dr.T.V.Rao MD
Agar - Agar
Complex polysaccharide
Used as solidifying agent for culture
media in Petri plates, slants, and deeps
Generall...
Media and Culture
•Media: Nutrients (agar, pH indicators, proteins
and carbohydrates) used to grow organisms
outside of th...
Culture media
Used to grow bacteria
Can be used to:
Enrich the numbers of bacteria
Select for certain bacteria and suppres...
Culture and Medium
Culture is the term given to microorganisms that
are cultivated in the lab for the purpose of
identifyi...
Need for Culture Media
It is usually essential to obtain a culture by
growing the organism in an artificial medium.
If mor...
Basic requirements of
culture media
Nutrients
- Energy source
- Carbon source
- Nitrogen source
Mineral salts – Sulphate, ...
Classification of Culture media
Based on the consistency:
Peptone water, Nutrient broth
Semisolid -- Nutrient agar stabs
S...
Pouring the Culture
Plates
13Dr.T.V.Rao MD
Pouring a plate
http://www.biotopics.co.uk/microbes/pourp2.gif
14Dr.T.V.Rao MD
Petri dish with Media
Plate: provide large surface
for isolation and
observation of colonies
Using a sterile loop or a
ste...
Aerobic Media
Simple media
Complex media
May be Synthetic or Defined Medium
- Enriched media
- Differential media
- Enrich...
Aerobic media
Liquid media
- Peptone water(1% peptone +0.5%Nacl +
100 ml water)
- Nutrient broth ( peptone water + 1% meat...
Liquid Medium
Difficult to identify
all types of
organisms
Suitable for
isolation of
bacteria from
Blood culturing
and wat...
Peptone Water
Peptone contain partially digested
proteins
Proteases
Polypeptides
Aminoacids
Inorganic salts
Phosphates
Pot...
Nutrient Agar
Contain 2% agar added
to Nutrient agar
commonly used
Concentration can be
increased to 6% to
prevent swarmin...
Pigment producing
Staphylococci
on Nutrient Agar
21Dr.T.V.Rao MD
Defined Media
Defined media are media composed of
pure ingredients in carefully measured
concentrations dissolved in doubl...
Selective / Differential Media
Selective/differential media are media based
on either of the two categories above
suppleme...
Salmonella Shigella agar
24Dr.T.V.Rao MD
TCBS medium
25Dr.T.V.Rao MD
Complex media
Nutrient agar + 5 to 10% sheep blood
Melt the sterile nutrient agar by steaming, cool, to
450 c
Add the bloo...
Enriched Medium
To culture medium
Blood serum or egg
are added to
medium eg Blood
agar, Chocolate
agar
Egg based medium
27...
Different types of hemolysis
on Blood Agar
28Dr.T.V.Rao MD
Other Enrichments – Chocolate Agar
Several organic
materials are added
to the basic
constituents of the
Medium such as
Blo...
Chocolate agar
30Dr.T.V.Rao MD
Differential Medium Mac
MacConkey agar
Bringing out different
characters of bacteria
their atypical characters
Mac Conkey’...
Lactose fermenting Mucoid
colonies on MacConkey Agar
32Dr.T.V.Rao MD
MacConkey agar
MacConkey agar is
useful medium for
cultivation of coliforms
It contains a bile salt to
inhibit non intesti...
Lactose fermenting and Non
lactose fermenting
34Dr.T.V.Rao MD
Enrichment Medium
If the sample contain more
than one type of bacteria,
undesired bacteria grwoth
can be reduced or
elimin...
Selective media
Serve the same purpose as
Enrichment media but are solid in
consistency
- Wilson & Blair’s medium -
- Lowe...
Selective Medium
Deoxycholate citrate agar
Inhibitory
substances to
solid medium
as in
Deoxycholate
citrate agar
37Dr.T.V....
Deoxycholate citrate Agar
Suitable for isolation of
dysentery bacilli, food
poisoning Salmonella and
S.paratyphi B, and le...
Indicator Medium Wilson-Blair
medium
Indicate by change
of color Sulphite to
sulphide in Wilson-
Blair medium
S.typhi redu...
Carbohydrate media
Peptone water – 100 ml, Desired sugar 1 gm%
and Andrade's indicator – 0.005% soln(1ml)
Dissolve the des...
Carbohydrate media
Peptone water – 100 ml,
Desired sugar 1 gm% and
Andrade's indicator –
0.005% soln(1ml)
Dissolve the des...
Carbohydrate media
Peptone water – 100 ml,
Desired sugar 1 gm% and
Andrade's indicator –
0.005% soln(1ml)
Dissolve the des...
Sugar Medium
Sugars are fermenting substances
Monosaccharide – peptone, arabinose, xylose and hexose's, dextrose
and manno...
Sugar Medium
Sugar medium
contain 1% sugar
Durham’s tube
indicates production
of gas
Hiss Serum sugars
apart from sugar ,
...
Urease Test
45Dr.T.V.Rao MD
Loffler's serum slope
46Dr.T.V.Rao MD
Lowenstein Jensen Medium
47Dr.T.V.Rao MD
Lowenstein-Jensen’s medium
Mineral salt soln - 600ml
Malachite green soln - 20ml
(2gm% in D.water)
Beaten egg - 1000ml
(20...
Transport Medium
Stuart’s medium
contain reducing
agents to prevent
oxidation.
Charcoal to
neutralize certain
bacterial in...
Hiss Serum Sugars
Sugar Medium with Serum enrichment
50Dr.T.V.Rao MD
Anaerobic Medium
Robertson’s cooked
meat medium
Thioglyclolate liquid
medium
51Dr.T.V.Rao MD
Anaerobic Culture Methods
Anaerobic jar
Anaerobic
jar
Figure 6.552Dr.T.V.Rao MD
Sabouraud's Dextrose Agar
Dextrose - 4 gm%
Neopeptone - 1 gm%
Agar - 1.5 gm%
Distilled water - 100 ml
Dissolve the ingredi...
Sabouraud's Dextrose agar
54Dr.T.V.Rao MD
Sterilization of culture media
Media are sterilized in the autoclave at
1210 c for 15’ under 15lbs of Pressure
Heat-labile...
Storage of culture media
Prepared media in individual
screw capped bottles can be
stored for weeks at room temp
Poured pla...
Colonies of Bacteria in Pure
Culture
57Dr.T.V.Rao MD
Complex Medium
Complex media are rich in nutrients, they
contain water soluble extracts of plant or
animal tissue (e.g., e...
Blood culture –
‘Liquid Medium’
59Dr.T.V.Rao MD
Muller Hinton Agar for
Antibiotic Testing
60Dr.T.V.Rao MD
Blood Agar for Antibiotic
Testing
61Dr.T.V.Rao MD
Programme Created by
Dr.T.V.Rao MD for Medical and
Paramedical students in
Microbiology
Email
doctortvrao@gmail.com
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CULTURE MEDIA IN BACTERIOLOGY

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CULTURE MEDIA IN BACTERIOLOGY

  1. 1. Dr.T.V.Rao MD
  2. 2. Major Contribution to Culture Media 2Dr.T.V.Rao MD
  3. 3. Agar - Agar Frau Hesse 3Dr.T.V.Rao MD
  4. 4. Agar – Agar Solid medium is made by adding Agar Agar is obtained from Sea weeds New Zealand agar is more Agar contain long chain poly saccharides.Inoranic salts and protein like substance Melts at 980c and sets at 420c 4Dr.T.V.Rao MD
  5. 5. 5Dr.T.V.Rao MD
  6. 6. Agar - Agar Complex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 98°C Solidifies ~42°C 6Dr.T.V.Rao MD
  7. 7. Media and Culture •Media: Nutrients (agar, pH indicators, proteins and carbohydrates) used to grow organisms outside of their natural habitats •Culture: The propagation of microorganisms using various media 7Dr.T.V.Rao MD
  8. 8. Culture media Used to grow bacteria Can be used to: Enrich the numbers of bacteria Select for certain bacteria and suppress others Differentiate among different kinds of bacteria 8Dr.T.V.Rao MD
  9. 9. 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.
  10. 10. Need for Culture Media It is usually essential to obtain a culture by growing the organism in an artificial medium. If more than one species or type of organism are present each requires to be carefully separated or isolated in pure culture. Several organism need the determination of Antibiotic sensitivity pattern for optimal antibiotic selection 10Dr.T.V.Rao MD
  11. 11. 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 Dr.T.V.Rao MD
  12. 12. Classification of Culture media Based on the consistency: Peptone water, Nutrient broth Semisolid -- Nutrient agar stabs Solid -- Blood agar, Serum agar Liquid -- Based on Oxygen requirement: -- Aerobic medium -- Anaerobic media 12Dr.T.V.Rao MD
  13. 13. Pouring the Culture Plates 13Dr.T.V.Rao MD
  14. 14. Pouring a plate http://www.biotopics.co.uk/microbes/pourp2.gif 14Dr.T.V.Rao MD
  15. 15. Petri dish with Media Plate: provide large surface for isolation and observation of colonies Using a sterile loop or a sterile swab streak your sample on the petri plate Important let your sterilized loop cool before you pick up your sample 15Dr.T.V.Rao MD
  16. 16. 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 16Dr.T.V.Rao MD
  17. 17. 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 necessities 17Dr.T.V.Rao MD
  18. 18. Liquid Medium Difficult to identify all types of organisms Suitable for isolation of bacteria from Blood culturing and water analysis 18Dr.T.V.Rao MD
  19. 19. Peptone Water Peptone contain partially digested proteins Proteases Polypeptides Aminoacids Inorganic salts Phosphates Potassium and Magnesium Riboflavin Meat exract called as Lab lemco 19Dr.T.V.Rao MD
  20. 20. Nutrient Agar Contain 2% agar added to Nutrient agar commonly used Concentration can be increased to 6% to prevent swarming Can be reduced to 0’5% 20Dr.T.V.Rao MD
  21. 21. Pigment producing Staphylococci on Nutrient Agar 21Dr.T.V.Rao MD
  22. 22. Defined Media Defined media are media composed of pure ingredients in carefully measured concentrations dissolved in double distilled water 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 pyrimidine's 22Dr.T.V.Rao MD
  23. 23. Selective / Differential Media Selective/differential media are media based on either of the two categories above supplemented with growth-promoting or growth-inhibiting additives. The additives may be species- or organism-selective (e.g., a specific substrate, or an inhibitor such as Cyclohexamide (artidione) which inhibits all eukaryotic growth and is typically used to prevent fungal growth in mixed cultures). 23Dr.T.V.Rao MD
  24. 24. Salmonella Shigella agar 24Dr.T.V.Rao MD
  25. 25. TCBS medium 25Dr.T.V.Rao MD
  26. 26. Complex media Nutrient agar + 5 to 10% sheep 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 26Dr.T.V.Rao MD
  27. 27. Enriched Medium To culture medium Blood serum or egg are added to medium eg Blood agar, Chocolate agar Egg based medium 27Dr.T.V.Rao MD
  28. 28. Different types of hemolysis on Blood Agar 28Dr.T.V.Rao MD
  29. 29. Other Enrichments – Chocolate Agar Several organic materials are added to the basic constituents of the Medium such as Blood, yeast, yeast extract etc 29Dr.T.V.Rao MD
  30. 30. Chocolate agar 30Dr.T.V.Rao MD
  31. 31. Differential Medium Mac MacConkey agar Bringing out different characters of bacteria their atypical characters Mac Conkey’s medium Contain peptone, Lactose Agar, Neutral red and taurocholate and show growth of Lactose fermenters as pink colored colonies 31Dr.T.V.Rao MD
  32. 32. Lactose fermenting Mucoid colonies on MacConkey Agar 32Dr.T.V.Rao MD
  33. 33. MacConkey agar MacConkey agar is useful medium for cultivation of coliforms It contains a bile salt to inhibit non intestinal bacteria Lactose in combination with Neutral red distinguish the lactose fermenting from the non lactose fermenting Salmonella and Dysentery group 33Dr.T.V.Rao MD
  34. 34. Lactose fermenting and Non lactose fermenting 34Dr.T.V.Rao MD
  35. 35. Enrichment Medium If the sample contain more than one type of bacteria, undesired bacteria grwoth can be reduced or eliminated. The desired organism is facilitated to grow Eg Tetrathionate broth Selenite F broth 35Dr.T.V.Rao MD
  36. 36. Selective media Serve 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 36Dr.T.V.Rao MD
  37. 37. Selective Medium Deoxycholate citrate agar Inhibitory substances to solid medium as in Deoxycholate citrate agar 37Dr.T.V.Rao MD
  38. 38. 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 It should not be autoclaved or remelted When prepared from commercial medium it should be dissolved and sterilized at 1000c for a short period 38Dr.T.V.Rao MD
  39. 39. Indicator Medium 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 39Dr.T.V.Rao MD
  40. 40. Carbohydrate media Peptone water – 100 ml, Desired sugar 1 gm% and Andrade's indicator – 0.005% soln(1ml) Dissolve the desired carbohydrate in peptone water and steam for 30 min or sterilize by filtration. Distribute into sterile test tube containing inverted Durham’s tubes to detect gas production and steam for 30 min  Use: To test the fermenting ability of an organism 40Dr.T.V.Rao MD
  41. 41. Carbohydrate media Peptone water – 100 ml, Desired sugar 1 gm% and Andrade's indicator – 0.005% soln(1ml) Dissolve the desired carbohydrate in peptone water and steam for 30 min or sterilize by filtration. Use: To test the fermenting ability of an organism 41Dr.T.V.Rao MD
  42. 42. Carbohydrate media Peptone water – 100 ml, Desired sugar 1 gm% and Andrade's indicator – 0.005% soln(1ml) Dissolve the desired carbohydrate in peptone water and steam for 30 min or sterilize by filtration. Use: To test the fermenting ability of an organism 42Dr.T.V.Rao MD
  43. 43. Sugar Medium Sugars are fermenting substances Monosaccharide – peptone, arabinose, xylose and hexose's, dextrose and mannose Disaccharides Sucrose and Lactose Polysaccharides – Starch and Inulin Alcohols – Glycerol. Sorbitol Sugar medium contain 1% sugar Durham’s tube indicates production of gas Hiss Serum sugars apart from sugar , serum is added.Dr.T.V.Rao MD
  44. 44. Sugar Medium Sugar medium contain 1% sugar Durham’s tube indicates production of gas Hiss Serum sugars apart from sugar , serum is added. 44Dr.T.V.Rao MD
  45. 45. Urease Test 45Dr.T.V.Rao MD
  46. 46. Loffler's serum slope 46Dr.T.V.Rao MD
  47. 47. Lowenstein Jensen Medium 47Dr.T.V.Rao MD
  48. 48. Lowenstein-Jensen’s medium Mineral salt soln - 600ml Malachite green soln - 20ml (2gm% in D.water) Beaten egg - 1000ml (20-22 eggs) Mix the above Distribute in Mc Cartney bottles Sterilize by Inspissation  Use: To cultivate Mycobacteria 48Dr.T.V.Rao MD
  49. 49. Transport Medium Stuart’s medium contain reducing agents to prevent oxidation. Charcoal to neutralize certain bacterial inhibitors to Gonococci, 49Dr.T.V.Rao MD
  50. 50. Hiss Serum Sugars Sugar Medium with Serum enrichment 50Dr.T.V.Rao MD
  51. 51. Anaerobic Medium Robertson’s cooked meat medium Thioglyclolate liquid medium 51Dr.T.V.Rao MD
  52. 52. Anaerobic Culture Methods Anaerobic jar Anaerobic jar Figure 6.552Dr.T.V.Rao MD
  53. 53. 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 53Dr.T.V.Rao MD
  54. 54. Sabouraud's Dextrose agar 54Dr.T.V.Rao MD
  55. 55. Sterilization of culture media Media are sterilized in the autoclave at 1210 c for 15’ under 15lbs of Pressure Heat-labile substances like serum & sugar solutions must be sterilized by free-steam or filtration Egg containing media –-- Lowenstein- Jensen’s medium, Loffler's serum slope by inspissation Discarded culture plates are to be sterilized by autoclaving prior to washing Dr.T.V.Rao MD
  56. 56. Storage of culture media Prepared media in individual screw capped bottles can be stored for weeks at room temp Poured plates deteriorate quickly and often contaminated, hence cold storage is necessary For smaller labs domestic refrigerators & for larger labs insulated cold room(4-5oc) Deep freeze refrigerators for preservation of sera, antibiotics & amino acids (-10 to - 400c) 56Dr.T.V.Rao MD
  57. 57. Colonies of Bacteria in Pure Culture 57Dr.T.V.Rao MD
  58. 58. Complex Medium Complex media are rich in nutrients, they contain water soluble extracts of plant or animal tissue (e.g., enzymatic ally digested animal proteins such as peptone and tryptone). Usually a sugar, often glucose is added to serve as the main carbon and energy source. The combination of extracts and sugar creates a medium which is rich in minerals and organic nutrients, but since the exact composition is unknown, the medium is called complex 58Dr.T.V.Rao MD
  59. 59. Blood culture – ‘Liquid Medium’ 59Dr.T.V.Rao MD
  60. 60. Muller Hinton Agar for Antibiotic Testing 60Dr.T.V.Rao MD
  61. 61. Blood Agar for Antibiotic Testing 61Dr.T.V.Rao MD
  62. 62. Programme Created by Dr.T.V.Rao MD for Medical and Paramedical students in Microbiology Email doctortvrao@gmail.com
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