Culture media and Cultivation of Bacteria DR.C.P.PRINCE

Purpose of culturing are
Isolation of bacteria ( pure culture)
Diagnosis of infectious diseases
Properties of bacteria i.e. culturing bacteria is the initial step in studying its morphology and its identification.
Maintenance of stock cultures.
Estimate viable counts. Water , air, milk testing
To test for antibiotic sensitivity.
To create antigens for laboratory use.
Vaccine preparation
Sterility testing
Preparation of pharmaceutical products like antibiotics, enzymes, toxins etc
Certain genetic studies and manipulations of the cells also need that bacteria to be cultured in vitro.
Culturing on solid media is another convenient way of separating bacteria in mixture.
An artificial culture media must provide similar environmental and nutritional conditions that exist in the natural habitat of a bacterium.
A culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some growth factors.
PPT prepared by:
DR.C. P. PRINCE
HOD & Associate Professor
Department of Microbiology
Mother Theresa Post Graduate & Research Institute of Health Sciences (Government of Puducherry Institution)

1. Culture Media and Bacterial Culture methods DR.C. P. PRINCE HOD & Associate Professor Department of Microbiology Mother Theresa Post Graduate & Research Institute of Health Sciences (Government of Puducherry Institution)

2. Cultivation/Culturing of Bacteria A microbial culture, is a method of multiplying microorganisms by letting them to reproduce in predetermined culture media under controlled laboratory conditions .( Growing Bacteria in Laboratory) Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both.

3. Purpose of culturing Isolation of bacteria ( pure culture) Diagnosis of infectious diseases Properties of bacteria i.e. culturing bacteria is the initial step in studying its morphology and its identification. Maintenance of stock cultures . Estimate viable counts . Water , air, milk testing To test for antibiotic sensitivity . To create antigens for laboratory use. Vaccine preparation Sterility testing Preparation of pharmaceutical product s like antibiotics, enzymes, toxins etc Certain genetic studies and manipulations of the cells also need that bacteria to be cultured in vitro. Culturing on solid media is another convenient way of separating bacteria in mixture.

4. Culture Media An artificial culture media must provide similar environmental and nutritional conditions that exist in the natural habitat of a bacterium. A culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some growth factors. The pH of the medium must be set accordingly. Uses: *Enrich the number of bacteria. *Select for certain bacteria and suppress others. *Differentiate among different kinds of bacteria.

5. Agar Solidifying agent used for preparation of solid culture medium Agar, a polysaccharide extracted from marine algae, is used to solidify a specific nutrient solution. Unlike other gelling agent, it is not easily degraded by many bacteria. It is not easily destroyed at higher temperatures, and therefore it can be sterilized by heating, the process which also liquefies it. Once solidified, agar medium will remain solid

6. Pure culture In the laboratory bacteria are isolated and grown in pure culture in order to study the functions of a particular specie. A pure culture is a population of cells or growing in the absence of other species or types. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another Medium containing only one type of bacterial growth

7. Classification of Culture Media Bacterial culture media can be classified in at least three ways. 1. Consistency 2. Nutritional component 3. Functional use

8. Classification based on consistency 1. Liquid media. 2. Solid media. 3. Semi solid media.

9. Classification based on Nutritional Components 1. Simple media. 2. Complex media. 3. Synthetic or chemically defined media.

10. Classification based on Functional Use or Application 1. Enriched media. 2. Selective media. 3. Differential media. 4. Transport media. 5. Indicator media. 6. Anaerobic media.

11. BASIC MEDIA These are simple media used to support the growth of microorganisms that do not have special nutritional requirements. They include nutrient broth, peptone water, and nutrient agar.

12. ENRICHED MEDIA Prepared by the addition of substances such as blood, serum or egg to a basic medium. Used for cultivation of fastidious organisms that cannot grow on simple media and need highly nutritive substances for growth. Used for culturing sterile body fluids such as blood or CSF, where the finding of any organisms = infection due to that organism. And also for primary identification of microorganisms e.g. haemolysis on blood e.g. blood agar, chocolate agar, Loeffler’s serum slope

13. Blood agar sterile de- fibrinated sheep or human 5-10%. blood + melted nutrient agar at 55°C Red opaque solid medium. N. agar is sterilized in autoclave at 121°C for 30 min. Blood is added under complete aseptic condition at 45- .55 oC Supports the growth of most delicate organisms e.g - .Streptococcus pyogenes Identifying bacteria according to their haemolytic-action on the red cells

14. Chocolate agar Prepared as blood agar followed by raising the temperature to l00 °C for 2 min to rupture red cells and release nutrients as X and V factors Brown opaque solid medium. Sterilized as blood agar. Used for the isolation of Neisseria meningitides, Haemophilus .influenza and Streptococcus pneumonae

15. Loeffler's serum slope Opaque whitish solid medium. The medium is solidified in hot air inspissator at.. 75°C for 2 hr for 2 successive days Uses : Culture of Corynebacterium diphtheria

16. SELECTIVE MEDIA Solid media that contain substances (e.g. Bile salts or other chemicals, dyes, antibiotics) which inhibit the growth of one organism to allow the growth of another . Used when culturing a specimen from a site having a normal microbial flora to prevent unwanted contaminants overgrowing a pathogen.

17. Lowenstein Jensen medium Selective medium for Mycobacterium tuberculosis Contains beaten eggs +malachite green Green opaque solid medium. Sterilized in hot air inspissator at 75 °C for 2hr for 2 successive days

18. MacLeod's tellurite blood agar(TBA) Blood agar + 0.02-0.04% K tellurite . Red opaque solid medium. . Selective medium Used for isolation of Corynebacterium diphtheriae .

19. Modified Thayer-Martin agar Chocolate agar + vancomycin + colistin + nystatin Brown opaque-solid medium. Selective medium for Used for Isolation of Neisseria from non-sterile specimens.

20. Thiosulphate citrate bile sucrose agar (TCBS ) Alkaline agar + sucrose + thiosulphate + citrate and . bromothymol blue indicator Greenish transparent solid medium. Sterilized in autoclave at 121°C for 30 min. Selective medium Used for isolation of Vibrio cholerae .

21. Deoxycholate citrate agar (DCA) Na deoxycholate and citrate + Agar + lactose + neutral red indicator. Reddish semi transparent solid medium. Selective medium Used for isolation of Shigella and Salmonella.

22. XLD Media Agar + lactose + phenol red indicator + ferric citrate + desoxycholate + xylose + lysine +sucrose + yeast extract Reddish semi transparent solid medium. Selective medium Used for isolation of Shigella and Salmonella

23. ENRICHMENT MEDIA Fluid media that contain substances which favour the growth of wanted organisms on the expense of others. Usually used as a preliminary step for isolation of pathogens before subculturing on solid selective media. Examples are: Selenite broth for isolation of Salmonella and Shigella species from faeces Tetrathionate broth for isolation of Salmonella from faeces Alkaline peptone water for isolation of Vibrio cholerea

24. INDICATOR (DIFFERENTIAL MEDIA) These are media to which dyes or other substances (Indicators()are added to differentiate microorganisms. Indicators change colour when acid is produced following fermentation of a specific carbohydrate e.g. MacConkey's agar medium.

25. MacConkey's agar medium Peptone, agar, lactose, bile salt and neutral red indicator. Reddish transparent solid medium Detects lactose fermenting and non lactose fermenting bacteria

26. IDENTIFICATION MEDIA These include media to which substrates or chemicals are added to help identify bacteria isolated on primary cultures. i.e. organisms identified must be first isolated in pure culture. Organisms are mainly identified by a change in the colour of the medium and or the production of gas. They include peptone water sugars, litmus milk, and gelatin media.

27. TRANSPORT MEDIA Semisolid media that contain ingredients to prevent the overgrowth of commensal & ensure the survival of aerobic and anaerobic pathogens when specimens cannot be cultured immediately. Examples: 1- Cary-Blair medium for preserving enteric pathogens. 2- Stuarts and Amies transport medium for ensuring the viability of gonococci 3- Thioglycollate broth and deep agar for- 3 anaerobic organisms

28. CULTURE MEDIA FOR ANAEROBES Media for anaerobes is the same as media for aerobes except that: 1. They are richer in organic constituents . 2. Contain reducing agents ( cysteine & haemin). 3. Contain a redox indicator . The inoculated media are incubated in anaerobic environment using anaerobic gas pack .

29. Robertson's cooked meat medium Anaerobic enrichment media cooked minced meat to which broth is added Anaerobiosis is achieved through (reducing substances in the meat).e.g. haemin and glutathione Sterilized in autoclave at 121°C-for 30 min

30. Anaerobic GasPak System A method for the exclusion of oxygen from a sealed jar used for incubation of anaerobic cultures in a non-reducing medium .

31. Robertson's cooked meat medium

32. Anaerobic Culture Methods Production of a vacuum Displacement of Oxygen with other gases Absorption of Oxygen by chemical or biological methods By using reducing agents

33. McIntosh & Filde’s Jar

34. Anaerobic GasPak System

35. Displacement of Oxygen

36. Anaerobic Glove Chamber

37. Anaerobic Glove Chamber

38. Nutrient broth

39. L-J medium

40. TCBS

41. XLD Agar

42. MacConkey's agar medium

43. Robertson's cooked meat medium

44. Culture Methods Streak culture Lawn culture Stroke culture Stab culture Pour plate method

45. Streak culture Used for the isolation of bacteria in pure culture from clinical specimens. Platinum wire is used. One loop full of the specimen is transferred onto the surface of a well dried plate. Spread over a small area at the periphery. The inoculum is then distributed thinly over the plate by streaking it with a loop in a series of parallel lines in different segments of the plate. On incubation, separated colonies are obtained over the last series of streaks.

46. The streak-plate method to obtain pure cultures

47. Streak culture

48. Lawn Culture Provides a uniform surface growth of the bacterium. Lawn cultures are prepared by flooding the surface of the plate with a liquid suspension of the bacterium Uses – For bacteriophage typing. – Antibiotic sensitivity testing. – In the preparation of bacterial antigens and vaccines.

49. Lawn Culture

50. Stroke Culture • Stroke culture is made in tubes containing agar slope / slant. Uses: Provides a pure growth of bacterium for slide agglutination and other diagnostic tests.

51. Stroke Culture

52. Stab Culture Prepared by puncturing a suitable medium – gelatin or glucose agar with a long, straight, charged wire. Uses – Demonstration of gelatin liquefaction. – Oxygen requirements of the bacterium under study. – Maintenance of stock cultures.

53. Pour Plate Culture 1 ml of the innoculum is added to the molten agar. Mix well and pour to a sterile Petri dish. Allow it to set. Uses: – Gives an estimate of the viable bacterial count in a suspension. – For the quantitative urine cultures

54. Thanks