culture media

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.

3. 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 gr owth 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

4. 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

5. 6

6. 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.

7. According to Special Purposes: Simple/Basal media Complex media Enriched media Enrichment media Selective media Indicator/Differential media Sugar media Defined synthetic media Transport Media Storage Media

8. 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

9. NUTRIENT AGAR

10. 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.

11. 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.

12. BLOOD AGAR

13. DIFFERENT TYPES OF HAEMOLYSIS ON BLOOD AGAR

14. CHOCOLATE AGAR

15. 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.

16. 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 .

17. 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

18. 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 .

19. 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

20. BLOOD CULTURE BOTTLE Enrichment Liquid medium.

21. 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.

22. XLD AGAR Red pink black centred colonies salmonella Red pink colonies shigella

23. 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 .

24. 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.

25. LOWENSTEIN JENSEN MEDIUM

26. 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

27. THIOSULPHATE CITRATE BILE SUCROSE AGAR (TCBS) Selective and Differential H igh pH 8.6, salt and sucrose. Used for growth of Vibrios Indicator used is Bromothymol blue.

28. 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

29. MacConkey Agar Used for the cultivation of entericbacteria It contains bile salt i.e. sodium taurocholate , lactose with neutral red and crystal voilet .

30. Lactose fermenting and Non lactose fermenting

31. 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 .

32. WILSON AND BLAIR’S brilliant-green bismuth sulphite Agar (BBSA) Selective and Indicator medium. Salmonella spp

33. 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.

34. FERMENTATION OF SUGAR

35. 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).

36. STAURT’S Transport Medium

37. 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.

38. Muller Hinton Agar for Antibiotic Testing It’s a basic solid media, dispensed in Petridishses. It contains Beef infusion, Casein hydrolysate, Starch, Agar.

39. 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 121 c for 20 min After sterilization, adjust the pH to 7.1. Use t o cultivate the anaerobic bacteria

40. 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

41. CULTURE METHODS

42. 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.

43. Streak culture Stroke Stab Pour plate Liquid culture Special methods for anaerobic cultures Methods to isolate the Bacteria

44. 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.

45. Streak Plate (Quadrant Streak)

46. 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

47. STROKE CULTURE Done in tubes containing agar slopes Used for:- Pure growth of bacterium. For biochemical testing. Other diagnostic tests.

48. 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.

49. Pour Plate technique

50. 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.

51. Anaerobic Bacterial Isolation and Identification Needs specified conditions

52. Desiccator In Desiccator some oxygen is left. Not suitable for fluid culture. Displacement of oxygen is done with Hydrogen,Nitrogen , Helium,Co 2

53. Candle Jar Inoculated plates are kept Burning candle use up all oxygen But a little o 2 is left But presence of Co 2 stimulates the most bacterium 56

54. 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

55. 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.

56. 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).

57. ANAEROBIC CHAMBER