Preparation of bacterial culture media

These slides explain how media preparation in microbiology lab
for bacterial culture, history of culture media, types of culture media based on concentration and ingredients and practical section.

1. Preparation of Culture Media By: Reem Riyadh , BSC

2. Topics History of bacterial culture media Basic requirements and components of culture media. Types of culture media. Tools used in the preparation of media Tips for success media culture preparation

3. c ulture media : Culture media gives artificial environment stimulating natural conditions necessary for growth of bacteria. Can be used to : Enrich the numbers of bacteria. Select for certain bacteria and suppress other. Differentiate among different kinds of bacteria.

4. Major contribution to culture media

5. Basic requirements for culture media Nutrients: Energy source Carbon source Nitrogen source. Mineral Salt- Sulphate, phosphates, chloride & carbonate of K, Mg and Ca Optimal PH (7.2-7.4) Optimal Temperature (35-37C) Adequate oxidation-reduction potential Growth factor : Tryptophan for Salmonella Typhi X &V factor for H. influenza-

6. Components of culture media: Agar-agar (gelling agent) : Isolated from seaweed, is not digested by bacteria. prepared by treating algae with boiling water the extract is filtered, concentrated and then dried. Agar contain long chain polysaccharide. Melt at 95°C and Solidifies at 43°C.

7. Gelatin Agar agar Animal plant Source YES NO Digested by microbes 28 ° C 95 ° C Melt degree

8. 2. Water source for hydrogen and oxygen. 3.Protein source. Peptone (complex mixture of partially digested enzyme protein) Meat extract . 4. Mineral salt – sodium chloride

9. Types of culture media based on agar concentration (consistency)

10. Types of culture media Based on the ingredients Remember : all media must be sterile before use

11. 1. Synthetic or defined medium T hese type of media is prepared from ‘ CHEMICALS ’. Therefore, their exact chemical composition is known. Defined synthetic media are commonly used for research purposes . Further, they are of two types: simple and complex. Eg : peptone water-1% peptone+ 0.5%Nacl in water .

12. 1.1 Simple Synthetic Media Simple synthetic media contain a carbon and energy source, an organic source of nitrogen and various inorganic salts in buffered aqueous solution. 1.2 Complex Synthetic Media This media contains all the ingredients of simple synthetic media. In addition, it also contains amino acids, purines, pyrimidines and many other growth factors depending upon the nature and growth requirements of the organism. the chemical composition of complex media is unknown

13. 2. Routine laboratory media

14. 2.1 Simple or Basal medium Examples: Nutrient agar ,Nutrient broth and peptone water Consist of peptone, meat extract, sodium chloride and water Used to isolate separate colonies for studying. 1.Colony morphology 2.Pigmentation 3.Biochemical identification test

15. 2.2 Enriched media: Enriched media refers to the media which contain the nutrients required to support the growth of a wide variety of organisms, including some fastidious ones. This media are enriched by adding blood , serum or egg. Enriched media are solid media as they are agar-based. The examples of enriched media are blood agar, chocolate agar, Loeffler’s serum, etc.

16. 2.3Enrichment media: Enrichment media refers to the liquid media that inhibits the growth of unwanted bacteria. The examples of enrichment media : Selenite F broth: isolation salmonella and shigella Tetrathionate broth: isolation salmonella, proteus and inhibit coliform alkaline peptone water (APW) :for vibrio cholera

17. 2.2Enriched Media 2.3Enrichment Media Enriched media refers to the media which contain the nutrients required to support the growth of a wide variety of organisms, including some fastidious ones. Enrichment media refers to the liquid media that inhibits the growth of unwanted bacteria. Enriched media allow the growth of a wide variety of microorganisms Enrichment media allow the growth of a particular type of microorganism in the medium. The enriched media facilitate the growth of fastidious microorganisms The enrichment media inhibits the growth of unwanted commensal or contaminating bacteria Enriched media are solid media as they are agar-based Enrichment media are liquid media The examples of enriched media are blood agar, chocolate agar, Loeffler’s serum, etc. The examples of enrichment media are Selenite F broth, tetrathionate broth, alkaline peptone water (APW), etc.

18. 2.4Selective media: Principle:  Differential growth suppression. Selective medium is designed to suppress the growth of some microorganisms while allowing the growth of others. Selective medium is an agar-based (solid) medium so that individual colonies may be isolated.

19. Thayer Martin Agar For Neisseria sp Lowenstein Jensen Medium For M. Tuberculosis

20. Thiosulfate citrate bile salts sucrose(TCBS), Alkaline PH Inhibit of most throat organism except Diphtheria bacilli

21. Sabouraud Dextrose Agar (SDA) PH5.4 isolate fungi

22. 2.5 Differentiate (Indicator) media : These media contain an indicator which changes its colour when bacterium grows in them. E.g.: Blood agar Mac Coney's medium Christensen’s urease medium

23. Mannitol salt agar PH indicator: phenol red 7.5% NaCl Yellow :coagulase (+) S.aureus Pink: coagulase(-) ( epidermidis,saprophticus) inhibit Gram (-) MacConkey agar PH indicator: Neutral Red Crystal violet ,Bile salt Pink: lactose fermenter Yellow :non lactose fermenter Inhibit gram+

24. 2.6Transport media : T hese media are used when the sample will not be cultured soon after collection. Examples : Cary-Blair medium for faeces Amies medium for gonococci Stuart medium. non nutritional medium   which maintains the viability of organisms without significant multiplication.

25. 2.7 Storage media: These media are useful for storing the bacteria for a long period of time . For Example, Dorset egg medium.

26. 2.8 Sugar media: S ugar media contains 1% “sugar ” such as glucose, sucrose, lactose, dextrose, mannitol, etc . in peptone water beside an appropriate indicator

27. The indicator used in sugar media is Andrade’s indicator , A small tube ( Durham’s tube ) is kept inverted in the sugar tube to detect gas production.

28. Based on oxygen requirement: Anaerobic media: These media are used to grow anaerobic organism. Eg: Robertson’s cooked meat medium, Thioglycolate medium .

29. Practical culture media

30. Biological Safety Cabinet Pipette

31. Tips for success for media preparation Storage Selective materials are high quality Check the expired date

32. Problems caused by faulty culture media preparation Reduced growth Atypical colonial morphology Inhibition of target organism Failure to inhibit competing flora Reduced shelf life of prepared medium

33. Thank you Any questions ??