The first liquid artificial culture medium was created by Louis Pasteur in 1860.

The first liquid artificial culture medium was created by Louis Pasteur in 1860.

1. CULTURE MEDIA

2. DEFINITION-CULTURE MEDIA AND CULTURE The food materials or substance required for the growth of microorganisms in-vitro is called culture media and the growth of organism is known as culture.

3. HISTORY The first liquid artificial culture medium was created by Louis Pasteur in 1860. Pasteur’s first medium consists of sugar(carbon source), ammonium salts (Nitrogen source, synthetic peptide, nucleic acid) yeast ash (growth factor including vitamins,minerals) Robert koch realized the importance of solid media and used potato pieces to grow bacteria.Gelatin was used as solidifying agent. It was on the suggestion of Fannie eilshemius,that agar was used to solidify solid media. Inventor of petridish-Richard petri in 1887.

4. NEED FOR CULTURE MEDIUM To identify the cause infection from the clinical sample. To study the characteristic properties of microorganisms. To prepare bacteriological products like vaccines,antigen,toxoid etc.

5. Basic requirements of culture media Energy source Carbon source Nitrogen source Salts such as sulphate, phosphate, chloride,and carbonates of K,Na,Fe,Ca,Mg and trace elements such as Mn,Mo,Cu Satisfactory Ph(7.2-7.6) Adequate oxidation-reduction potential Growth factors such as tryptophan for Salmonella Typhi, glutathione for Gonococci. Solidifying agents for solid and semi-solid medias.

6. Characteristics of an ideal culture medium It must give satisfactory growth from small inoculum, ideally from a single cell. Rapid growth Easy to prepare Cheap Easily reproducable It should make it possible for all characteristics in which we are interested to be demonstrated.

7. Common ingredients of culture media Water Essential for the existence of living cell. Source of hydrogen and oxygen. Demineralised distilled water is used. Tap water is often suitable for media preparation,if it has low mineral content. Agar Dried mucilaginous substance from Gelidium Species and other algae(sea weed).

8. Main component used universally for the preparation of solid and semi-solid media. Available as long shreds or in powdered form. Contain mainly long chain polysaccharides,small amount of inorganic salt, protein like material and minerals like Ca,Mg,fatty acids. Does not provide nutrition to growing organisms. Melts at 98°c and generally solidifies at 42°c,hence used as solidifying agent.

9. Two types-Japanese agar Newzealand agar (more jellyfying) agar in solid medium:1-2% Concentration of agar in semi-solid medium:0.3-0.5%

10. 3. Peptone Peptone is the water soluble products of protein hydrolysis. Obtained by digestion of lean meat with proteolytic enzymes. All forms of peptone are heat stable. Peptone from animal protein is the source of nitrogen in the medium,the plant protein(soyabean) is a source of carbohydrate.

11. It is a golden granular hygroscopic powder which act as the source of nitrogen,carbon and as a buffer.
It is sticky on exposure to air so stored in closed bottles. Special grades of peptone- neopeptone,proteose peptone,mycological peptone Certain forms of peptone are used for specific purpose eg:proteose peptone is used in media for toxin production and tryptone is used to enrich media with amino acids.

12. A good brand of peptone must have the following qualities: 1. Must have neutral Ph
2. Must be light in colour
3. Must be hygroscopic in nature
4. Must contain large amount of tryptophan for indole production.

13. 4.Meat extract Hot water extract of meat obtained by special procedure,commercially available as ”lab lemco ”. Constituents- gelatin,peptone,albuminoses,amino acids,certain proteoses,salts such as potassium permagnate,NaCl,accessory growth factors such as glutathione,nicotamic acid,riboflavin,carbohydrate Function:Act as source of growth factor and inorganic salts.

14. 5. Yeast extract Extract of yeast cell act as stimulant for bacterial growth in the culture media. Constituents-protein,amino acids, growth factors (vit b), carbohydrates,and inorganic salts like potassium and phosphates. Source of growth factors and excellent stimulator of growth. Used as substitute for meat extract.

15. 6.Electrolytes Sodium Chloride and other electrolytes Function-essential to maintain osmotic pressure 7. Other ingredients Malt extract Consist mainly of Maltose,starch,dextrins and glucose Contains 5% protein and protein breakdown products and a wide range of mineral salts and growth factors. b) Serum Used for enriching culture media and used in certain media

16. c) Blood Human or animal(horse,sheep,) blood is used. Human blood is mostly available,but sheep blood is better as it has more properties. 5-10% defibrinated blood is used. Blood can be refrigerated for 2months.It is better to store in small bottles than in bulk.

17. Preparation of culture media Steps: Weighing and dissolving Adjustment of Ph Sterilization Dispensing of culture media Heat sensitive ingredients should be added after sterilization when medium has cooled to about 50°c.

18. Classification of culture media Based on physical nature or consistency a) Liquid media/ broth b) Semi-solid media c) Solid media d) Biphasic media Based on constituents a) Simple media b) Complex media

19. 3. Based on nature of constituents a) Synthetic/defined medium b) Semi-synthetic medium/semi defined c) Non-synthetic medium/non defined 4. Based on Oxygen requirement a) Aerobic medium b) anaerobic medium

20. All media other than simple media are called complex media.Complex media have special ingredients are called special media. Enriched media Enrichment media Selective media Differential media Indicator media Sugar media Transport media Assay media Storage media

21. Liquid media In liquid media nutrients are dissolved in water and bacterial growth is indicated by turbidity, surface pellicle, flocculent, sediment and colour change. Atleast 10^6 bacteria per ml of broth are needed for turbidity to be detected with the naked eye. Liquid media provide greater sensitivity for isolation of small number of microorganisms. Examples: Peptone water Nutrient broth Glucose broth

22. Uses: - For preparing bulk cultures - For the preparation of antigen or vaccine - For obtaining bacterial growth from blood or water when large volume have to be tested. Disadvantages: - Growth do not exhibit special characteristic appearance and they are of limited use in identifying species. - Isolation is not possible.

23. Semi-solid media Percentage of agar: 0.2-0.5% This media enables motile bacteria to spread. Uses: To detect motility To check oxygen requirement Storage cultures Example: Mannitol motility medium Stuart’s and amies media (Transport media)

24. Solid media Concentration of agar-2% Bacteria produce discrete visible growth on solid media. Uses: To obtain pure growth To study distinctive colony morphology and other characteristic features Examples: Nutrient agar,blood agar, chocolate agar,macConkey agar

25. Biphasic media Media has both liquid and solid phase. Example: Castaneda medium

26. Simple media/basal media Simple chemical composition, contains only basic nutrients required for the growth of ordinary microorganisms. Used as a base to prepare enriched media. Examples: Nutrient broth Peptone water

27. Nutrient broth Simple basal medium Composition: peptone,NaCl, meat extract, distilled water Uses: Routine culture Base to prepare other media To study growth curve Types: Meat infusion broth Meat extract broth Digest broth

28. Nutrient agar Prepared by adding 2% agar to nutrient broth. Uses: routine lab works To study colony characters To detect pigmentation To maintain stock cultures Peptone water Constituents: peptone,NaCl, distilled water Uses: Basal media for sugar fermentation test Used to test indole production For bacterial subculture

29. Complex media Media have added ingredients for special purpose or for bringing out particular characters of organism or for providing special nutrients required for the growth of organisms under study. Examples: Blood agar Chocolate agar macConkey agar

30. Synthetic media Prepared exclusively from pure chemical and the exact composition is known. Used to study metabolic requirement of experimental organisms. Examples: Dubos medium with tween 80

31. Semi synthetic media Media with chemical composition approximately known. Non synthetic media These media are prepared by using components of unknown chemical composition. Examples: beef extract,meat extract

32. Special media Enriched media Are solid media that facilitates the growth of certain fastidious bacteria. Prepared by adding substance like blood,serum and egg to the basal media. Examples: Blood agar Chocolate agar LJ medium Loeffler’s serum slope

33. Blood agar Enriched with whole blood supplement. Prepared by adding 5-10% blood to sterile nutrient agar cooled at 45-50°c Uses: Used for routine diagnostic purpose Culture of fastidious organisms like streptococcus,pneumococci,Heamophilus etc. Used to differentiate bacteria based on their hemolytic activity.

34. Hemolysis Lysis of red blood cells by extracellular enzymes produced by bacteria. Beta hemolysis: complete lysis of RBCs - Exhibit a wide zone (2-4mm) -Eg: Streptococcus pyogenes, Staphylococcus aureus,Listeria monocytogenes

35. Alpha-hemolysis ( α- hemolysis)   - is a partial or “green” hemolysis associated with reduction of red cell hemoglobin. -  Alpha hemolysis is caused by hydrogen peroxide produced by the bacterium, oxidizing hemoglobin to green methemoglobin. -It exhibit incomplete haemolysis with 1-2 mm wide. Persistence of some unhaemolysed RBC’s can be seen microscopically. -Eg: Streptococcus pneumoniae, Streptococcus viridans

36. Gamma hemolysis - No hemolysis around the colonies. - eg: Enterococcus feacalis

37. 2. Chocolate agar/Heated blood agar Prepared by adding 5-10% blood to sterile nutrient agar cooled to 70-80°c or by heating blood agar at 70-80°c During heating RBCs release substances like Hb,X factor,V factor into the medium. Used for the isolation of H.influenza, Gonococci,Meningococci,Moraxella sp.

38. Enrichment media It is a liquid medium in which certain substances are incorporated which enhances the growth of wanted bacteria(pathogenic) and inhibit the growth of unwanted bacteria (non pathogenic). Examples: Tetrathionate broth Enrichment medium for Salmonella Typhi, Salmonella paratyphi. Tetrathionate inhibit coliform and allowing typhoid and paratyphoid bacilli to grow.

39. Selenite-F-broth Inhibit coliform bacilli while permitting salmonella and many shigella to grow. Alkaline peptone water Enrichment medium for vibrio.

40. Selective media A solid media in which certain substances are incorporated to enhance the growth of wanted bacteria and inhibit the growth of unwanted bacteria is called selective media. Inhibitory agents-dyes,bile salts,alcohols,acids and antibiotics Examples: Deoxycholate citrate agar Used for the isolation of salmonella and shigella. Deoxycholate is the inhibitory substance to other bacteria. Heat sensitive medium. Indicator –neutral red

41. LJ medium Used for selective isolation of Mycobacterium sp. Constituents: mineral salt solution, malachite green solution,egg solution Gycerol enhance the growth of Mycobacterium and malachite green inhibit other microorganisms present in sputum. When heated,the egg albumin coagulates,thus provide solid surface for inoculation.

42. 3.Tellurite blood agar(TBA) Used for selective isolation of Corynebacterium diphtheria. Selective agent-potassium tellurite Corynebacterium reduce potassium tellurite to tellurium and thereby produce grey balck coloured colonies.

43. Thiosulphate citrate bile salt sucrose agar(TCBS) Selective medium for isolation of V.cholerae Ph of the medium is alkaline (8.6) Indicator-bromothymol blue Bile salt inhibit gram positive oraganism and alkaline ph is inhibitory to all other gram negative oraganism except Vibirio. Thayer-martin medium selective media for Neisseria gonorrhoea.

44. Xylose Lysine deoxycholate agar (XLD) Selective medium for the isolation Salmonella and shigella sp. Selective agent- sodium deoxycholate (inhibitory to gram positive oraganism) Indicator – Phenol red H2S indicator system-Sodium thiosulphate and ferric ammonium citrate 3sugars – xylose, sucrose and lactose

45. Xylose is rapidly fermented by most Gram-negative enteric bacteria including Salmonella and causes acidification of the medium turning the phenol red indicator to yellow. Shigella spp doesn’t utilize xylose, acidification does not occur, and red colonies are produced. After the xylose supply is exhausted, Salmonella spp decarboxylates lysine, increasing the pH to alkaline condition, and also produces red colonies like Shigella spp.

46. However, Salmonellae also metabolize thiosulfate to produce hydrogen sulfide, which leads to the formation of colonies with black centers and allows them to be differentiated from the similarly colored Shigella colonies. Organisms that ferment lactose, sucrose, and xylose but are lysine decarboxylase negative, cause an acid pH and produce yellow colonies.

47. Red colonies-Shigella sp.,Providencia sp.,Pseudomonas sp.,H2S non producing Salmonella sp. Red colonies with black centre- H2S producing Salmonella sp., S.typhimurium Yellow colonies- Proteus sp.,E.coli,Serratia,Citrobacter

48. Indicator media This media contains an indicator which changes colour when bacteria grows in them. Indicators used: neutral red, phenol red,bromothymol blue, methylene blue. Examples: Wilson and Blair bismuth sulphite medium There is incorporation of sulphite in this medium. Salmonella Typhi, Salmonella paratyphi B can reduce sulphite to sulphide in presence of glucose and colonies have a black metallic sheen.

49. McLeod’s medium(potassium tellurite agar) For Diphtheria bacilli Potassium tellurite in McLeod’s medium reduced to metallic tellurium by diphtheria bacillus to produce black colured colonies. MacConkey agar It indicate lactose fermenting proberty. Lactose fermenters- pink coloured colonies Non-lactose fermenters-colourless colonies

50. Differential media A medium in which certain substances are incorporated, enabling it to bring out differing characters of bacteria and thus helps to distinguish between them. Examples: MacConkey agar Used to differentiate gram negative bacteria based on its ability to ferment lactose present in the medium. Bile salts present in the medium inhibit the growth of gram positive bacteria.

51. Indicator-Neutral red Lactose fermenters- Ferment lactose,produce acid which decreses the ph of the medium and causes the neutral red indicator to give a pink red colour. Eg: E.coli, Klebsiella Non lactose Fermenters-colourless colonies Eg: Salmonella,Shigella, Proteus Late lactose fermenters-some organism ferment lactose late Eg: shigella sonnei

52. The delayed fermenters have the potential to ferment lactose(produce intracellular enzyme necessary to metabolise lactose) but they lack beta galactosidase- permease enzymes.

53. 2. Cystein lactose electrolyte deficient agar (CLED agar) Differential medium for diagnostic urinary bacteriology. It is electrolyte deficient to prevent swarming of proteus sp. Indicator- bromothymol blue Lactose fermenters will lower the ph and change the colour of medium from green to yellow. Advantage over MacConkey agar- it supports the growth of Staphylococci, StreptococcI and candida stain which fails to grow in macConkey agar.

54. 3. Eosin Methylene blue agar ( EMB agar) Differentiate bacteria based on lactose fermenting proberty. Contains two dyes-eosin y and methylene blue (6:1) LF- dark purple coloured colonies NLF- colourless Most of the strains of E. coli colonies have a characteristic green sheen. Rapid fermentation of lactose & production of strong acids, thus a rapid reduction in the pH of the EMB agar the critical factor in the formation of the green metallic sheen observed with E. coli.

55. 4. Blood agar Blood agar is both differential and an enriched one. Ditinguish between hemolytic and non hemolytic bacterium.

56. Sugar media Consist of 0.5-1% sugar in peptone. Sugar may be glucose, lactose, maltose, sucrose,xylose,arabinose, Mannitol etc Ph indicator-to detect acid production Durham’s tube- to detect gas production Hiss serum sugar used for oraganism which are exacting in their growth requirment like Streptococci, Gonococci, Pneumococci.Consist of 3% serum in distilled water.

57. Transport media It is a holding medium designed to preserve the viability of microorganisms in the clinical specimens during their transport to the laboratory. Contains buffers and salts.They lack C,N,and organic growth factors hence donot facilitate microbial multiplication. Examples: Stuart’s transport media- Gonococci Amie’s media- Gonococci

58. Buffered gycerol saline- Enteric bacilli Pikes medium- Streptococcus pyogenes, Pneumococci, Heamophilus influenza in nose,throat swabs Cary-blair medium- V.cholera,Campylobacter VR fluid- V.cholerae APW- V.cholerae

59. Assay media Used for the assay of certain substances like antibiotics, vitamins,amino acids Examples: antibiotic assay- MHA, Nutrient agar Storage media Employed for storage of bacterial cultures. Semi solid nutrient agar is used as storage medium for non fastidious organisms and semi-solid blood agar for fastidious organisms.

60. Anearobic media Media which are used for cultivating anaerobic organisms. It contains reducing agent to absorbs oxygen and create anaerobic environment. Examples: Robertson’s cooked meat broth (RCM) Medium contains minced cooked meat particles and nutrient broth. Meat particles contain unsaturated fatty acids which take up dissolved oxygen and the reaction is catalysed by heamatin in the meat.

61. After inoculation, culture tube is overlayered with liquid paraffin so as to break oxygen contact. Growth is indicated by turbidity. RCM is also used to distinguish proteolytic and saccharolytic activity of anaerobic bacteria. Proteolytic bacteria turn the meat particles black with a foul smelling gas. Eg:C.histolyticum Saccharolytic bacteria turn meat red with a sour smell. Eg:C.perfringenes

62. Neomycin blood agar Commonly used anaerobic medium,used to isolate bacteria grown in RCM. Thiogycolate broth Forms an oxygen gradient from top to bottom thus facilitating the growth of organism of all oxygen requirement. Contain an oxygen indicator resazurin which turn pink if exposed to oxygen.(or methylene blue which turn greenish-blue )

63. Sodium Thiogycolate,thioglycolic acid and L-cystine reduce oxygen to water.

64. 4. Peptone yeast extract agar Bacterioides bile esculin agar Laked kanamycin-vancomycin blood agar Anaerobic phenyl ethyl alcohol agar Cycloserine cefoxitin fructose agar

65. Preservation of bacterial cultures Refrigeration Used for short term storage. Deep freezing It is the process in which a pure culture of microbe is placed in a suspending liquid and quick frozen at temperature ranging from -50°c to -95°c.

66. Lyophyllisation (freeze drying) A s u spension of microbe is quickly frozen at temperature ranging from -54 to -95°c and water is removed by a high vaccum (sublimation). The oraganism can be revived at any time by hydration with a suitable liquid nutrient medium. Cold storage Common method for preserving strains of bacteria. 5. Drying methods A number of Drying methods have been developed.