Culture media & Quality Control

This slide describes the different types of media used in the field of microbiology for the growth of microorganisms.

1. CULTURE MEDIA HITEN SHANKER NAVANI III B.Sc MICROBIOLOGY

2. CONTENTS DEFINITION HISTORY GENERAL REQUIREMENTS OF MICROBES MEDIA CONSTITUENTS MEDIA INGREDIENTS ENVIRONMENTAL CONDITIONS IN CULTURE MEDIA AGAR CLASSIFICATION OF MEDIA TYPES OF CULTURE MEDIA CULTURE METHODS QUALITY CONTROL

3. DEFINITION The food materials on which microbes are grown artificially in a lab is known as culture medium. Any liquid or solid preparation made specifically for the growth, storage or transport of microbes or other types of cells.

4. HISTORY First person to identify was Lazaro Spallanzani . Later, was developed by Koch Koch, first used a cut, half boiled potato, but later he found out that it was getting eaten away . Later he started using Gelatin but it had a drawback of low melting point and also it was degraded by the enzyme gelatinase. Later, Mrs Hess, suggested the use of agar which she used for jams and jellies.

5. GENERAL REQUIREMENTS Proper nutrients must be available. Oxygen or other gases must be available, as required. Moisture is necessary. The medium must have an appropriate p H . Proper temperature relations must prevail. The medium must be free of interfering bioburden. Contamination must be prevented.

6. GENERAL REQUIREMENTS A satisfactory microbiological culture medium must contain available sources of: Carbon Nitrogen Inorganic phosphate and sulfur Trace metals Water Vitamins

7. MEDIA INGREDIENTS

8. CONSTITUENTS SOURCE Amino-Nitrogen Peptone, protein hydrolysate, infusions and extracts Growth Factors Blood, serum, yeast extract or vitamins, NAD (Nicotinamide adenine dinucleotide) Energy Sources Sugar, alcohols and carbohydrates Buffer Salts Phosphates, acetates and citrates Mineral Salts and Metals Phosphate, sulfate, magnesium, calcium, iron Selective Agents Chemicals, antimicrobials and dyes Indicator Dyes Phenol red, neutral red Gelling agents Agar, gelatin, alginate, silica gel

9. Additional Points Purified water is recommended for use in the preparation of culture media. Chromogens & Fluorogens are substrates incorporated into culture media to allow organism differentiation and identification. When these substrates are degraded by specific enzymes, they release differently colored or fluorescent compounds Antimicrobial Agents are used in media to inhibit the growth of bacteria, yeasts and fungi.

10. ENVIRONMENTAL CONDITIONS IN CULTURE MEDIA 1. ATMOSPHERE Microbes that we grow can be Obligate or Facultative aerobes, Microaerophilic, Obligate or Facultative anaerobes. Anaerobic conditions for growth of microorganisms are obtained in a number of ways: Addition of small amounts of agar to liquid media Displacement of the air by hydrogen or nitrogen Absorption of the oxygen by chemicals Inoculation into the deep layers of solid media or under a layer of oil in liquid media 2. WATER CONDITIONS 3. PROTECTIVE AGENTS AND GROWTH FACTORS

11. AGAR Universally used for preparing solid medium Obtained from seaweed: Gelidium No nutritive value Not affected by the growth of the bacteria. Melts at 98°C & sets at 42°C 2% agar is employed in solid medium & 1% in semisolid media

12. CLASSIFICATION OF MEDIA

13. CULTURE MEDIUM LIQUID MEDIUM Diffused growth No characteristics for identification Difficult to isolate Earliest liquid medium urine or meat brot h used by Louis Pasteur SOLID MEDIUM Distinct colony morphology Characteristics – easy to identify Colony - macroscopically visible collection of millions of bacteria originating from a single bacterial cell Earliest solid medium: Cooked cut potato by Robert Koch Gelatin - not satisfactory - liquefy at 24℃

14. TYPES OF CULTURE MEDIA I. BASED ON CONSISTENCY Solid medium Liquid medium Semi solid medium II. BASED ON CONSTITUENTS Simple medium Complex medium Synthetic or defined medium Special media III . BASED ON OXYGEN REQUIREMENT Aerobic media Anaerobic media

15. SPECIAL MEDIA Enriched media Enrichment media Selective media Indicator media Differential media Sugar media Transport media Anaerobic Media Assay Media

16. TYPES OF MEDIA Solid media – contains 2% agar Colony morphology, pigmentation, hemolysis can be appreciated. Generally prepared as slants, deeps and plates Eg : Nutrient agar, Blood agar Liquid media – no agar. For inoculum preparation, Blood culture, continuous culture and also for bulk production in fermentations. Eg : Nutrient broth, peptone water Semi solid medium – 0.5% agar. Eg : Motility medium – for studying the motility of bacterial cultures

17. SIMPLE MEDIUM / BASAL MEDIUM Most common in routine diagnostic laboratories Eg : Nutrient Broth, Nutrient Agar. Generally for non-fastidious organisms NB consists of peptone, meat extract, NaCl, water NB + 0.5% Glucose = Glucose Broth NB + 2% agar = Nutrient agar Agar conc. Reduced (0.2 - 0.5%) = Semi-solid medium

18. COMPLEX MEDIA Media other than basal media They have added complex ingredients such as yeast extract or casein hydrolysate, which consist of a mixture of many chemical species in unknown proportions Eg. Yeast Extract, Caesin hydrolysate, Corn Steep liquor, etc.

19. SYNTHETHIC OR DEFINED MEDIUM Media prepared from pure chemical substances Exact composition is known Used for special studies, eg. metabolic requirements Eg : peptone water- (1% peptone + 0.5% NaCl in water), Nutrient agar, Trypticase soya agar

20. SPECIAL MEDIA Media which may be solid of liquid Designed to support the growth of specific microorganisms, to isolate them, to transport them, etc. Examples are as follows

21. ENRICHED MEDIA Substances like blood, serum, egg are added to the basal medium. Used to grow bacteria that are exacting in their nutritional needs. Eg : Blood agar for Streptococcal sps ., Chocolate agar is for highly fastidious organisms. Blood agar when heated gets charred with appearance of chocolate releasing the serum proteins from RBCs Chocolate agar is used for Neisseria sps .

22. ENRICHMENT MEDIA Liquid media used to isolate pathogens from a mixed culture. Stimulate growth of desired bacterium Inhibit growth of unwanted bacterium Media is incorporated with inhibitory substances to suppress the unwanted organism - increase in numbers of desired bacteria Eg : Selenite F Broth – for the isolation of Salmonella , Shigella Tetrathionate Broth – inhibit coliforms Alkaline peptone water for Vibrio sps . from stool samples.

23. SELECTIVE MEDIA Inhibitory substances like bile salts, antibiotics, dyes, etc. are added Increase in number of colonies of desired bacterium Eg. SDA - for fungi Eosin Methylene Blue (EMB) agar – For E.coli Mannitol Salt Agar for Staphylococci sps – High pH and mannitol Pseudomonas Cetrimide Agar Thiosulphate Citrate Bile Sucrose (TCBS) agar – Alkaline pH for Vibrio cholera LJ medium for Mycobacterium tuberculosis TCBS

24. INDICATOR MEDIA It contains an indicator which changes its colour when a bacterium grows in them Eg : Wilson- blair medium – Salmonella t yphimurium forms black colonies Mcleod’s medium (potassium tellurite)– Diphtheria Bacilli Christensen’s urease medium MRVP broth Simmon’s Citrate Agar

25. DIFFERENTIAL MEDIA Used to differentiate two types of organisms in the same media This uses biochemical characteristics of microbes Specific nutrients or indicators are added like phenol red, eosin, methylene blue, etc.) Visible changes are observed in the medium that indicates the defining characteristics of microbes. Eg. MacConkey Agar – Lactose fermenting and non lactose fermenting. Neutral red acts as an indicator Blood agar - Differentiates hemolytic and non hemolytic species of Streptococci sps .

26. SUGAR MEDIA Media containing any fermentable substance Eg : glucose, arabinose, lactose, starch etc. Media consists: 1% of the sugar in peptone water + Indicator May contain a small tube (Durham’s tube) for the detection of gas by the bacteria For fastidious organisms Hiss’s serum sugar media is used with 3% serum

27. TRANSPORT MEDIA Media used for transporting the samples. Delicate organisms may not survive the time taken for transporting the specimen without a transport media. Eg : Stuart’s medium – non nutrient soft agar gel containing a reducing agent & charcoal used for Gonnococci Buffered glycerol saline – enteric bacilli Amies medium with charcoal – Campylobacter sps . Venkataraman Ramakrishnan (VR) medium – For faeces transport of cholera suspected patients.

28. ANAEROBIC MEDIA This media is used to grow anaerobic microorgansims Eg. Robertson’s cooked meat broth Thioglycolate Medium THIOGLYCOLATE MEDIUM

29. ASSAY MEDIA Assay media are generally used to test the efficiency of antimicrobial drugs Eg. Muller Hinton Agar (MHA) This media is used because it is non-selective & non-differential It contains Starch which absorbs toxins released from bacteria It is also a loose agar which allows better diffusion of antibiotics

30. CULTURE METHODS

31. ANAEROBIC CULTURE METHODS Production of vacuum – by vacuum desiccators Displacement of oxygen with other gases – Candle Jar Chemical method - Biological method Reduction of medium

32. QUALITY CONTROL OF MEDIA It is a routine check in microbiology laboratory This check insures the proper detection of proper performance which may because of a poor and deteriorated of stock of chemicals, laboratory errors, improper maintenance of pH, etc. The following are to be taken care while preparation of medium

33. QUALITY CONTROL OF MEDIA Quality control must be supervised by senior staff member and deficiencies or contamination revealed during the checks Specific indicators should be used to ensure the correctness of sterilization cycle Sterilized media should be poured only into previously sterilized plates only in laminar air flow Proper maintenance of Laminar air flow should be done before and after the procedure

34. QUALITY CONTROL OF MEDIA Liquid media should be dispersed in tubes and then sterilized Quality control must be seen of the additives like sugar, serum proteins, enzymes, etc. All prepared media should be incubated for 24 hours prior to the inoculation All aseptic techniques should be followed strictly like sterilizing the loop, opening and closing the plates and flasks

35. QUALITY CONTROL OF MEDIA Microbiology labs should be frequently fumigated using potassium permanganate and formaldehye Decontamination of used stuff should be done properly Glasswares should be handled properly The skill of a person plays an important role in quality control

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