Introduction of culture media
Media –Plural
While medium is singular
Culture media are required to grow the organisms from infected material to identify the causative agent and its basic constituents are-
Water: source of hydrogen and oxygen
Electrolyte: Sodium chloride or other electrolytes
Peptone: It is a complex mixture of partially digested protein. It contains proteoses, amino acids, polypeptides, phosphates, minerals (K, Mg) and accessory growth factors like nicotinic acid and riboflavin.
Meat extract: Available commercially as “Lab-Lamco”
It contains protein degradation products, inorganic salts, carbohydrates and growth factors.
Blood or serum: They are used for enriching culture media. Usually 5-10% defibrinated sheep blood is used. In certain media, serum is used.
Agar: It is long chain polysaccharide and prepared from sea wood (Algae –Geladium Species).
It does not provide any nutrition to the bacteria but acts as a solidifying agent only.
Used in concentration of 2-3%
Melts at 98°C
Solidifies at 42°C
New Zealand agar has twice the capacity jellifying capacity than that of Japanese agar.
Types of culture media
Media are classified in flowing ways:
Based on physical state
- Liquid media
- Semisolid media (Agar, 0.2-0.4% which enables motile bacteria to spread)
- Solid media
On the basis of presence of molecular oxygen and reducing substances in the media
- Aerobic media
- Anaerobic media
Based on nutritional factors
- Simple media
- Complex media
- Synthetic media
- Special media
Special media
- Enriched media
- Enrichment media
- Selective media
- Differential media
- Indicator media
- Transport media
- Sugar media
Simple media
Nutrient broth is an example of simple medium. It contains peptone water and meat extract 1%. When agar is added to nutrient broth, it becomes nutrient agar. This is the simplest and routinely employed medium in the laboratory for diagnostic purposes.
Complex media
All media other than simple media are complex media.
Synthetic media
These are prepared from pure chemicals and the exact composition of the medium is known. These are used for special studied such as metabolic requirements. Dubo’s medium with tween 80 is an example of a synthetic medium.
Special Media
Enriched media
When basal medium is added with some nutrients such as blood, serum or egg. It is called enriched medium. For e.g.
Blood agar-Blood is added to nutrient agar. It may be used for growing a number of bacteria but one specific example is Streptococcus which requires blood for its growth.
Loeffler’s serum slope-Serum is added for enriching the medium. This medium is used for growing Corynebacterium diphtheriae.
Enrichment media
A fluid type of selective medium in which some substances are incorporated that have either a stimulating effect on the bacteria to be grown or inhibits its competitors or both.
This results in an absolute increase in the number of wanted bacteria related to other bacteria. Such medium is called enrichment medium.
Tetrathionate broth-Tetrathionate is added which inhibits coliforms while allows typhoid-paratyphoid bacilli to grow.
Selenite F broth –Selenite has similar action as that of tetrathionate in tetrathionate broth.
Selective media
Selective media contain substances that inhibit all but a few types of bacteria and facilitate the isolation of a particular species. These media are used to isolate a particular bacterium from specimen where mixed bacterial flora is expected.
Selective media are solid in contrast to enrichment media which are liquid. Example of selective media are:
Deoxycholate citrate agar (DCA)-Addition of deoxycholate acts as selective agent for enteric bacilli (Salmonella, Shigella).
Bile salt agar (BSA)-Bile salt is a selective agent. It favors the growth of only Vibrio cholerae whereas inhibits the growth of other intestinal organisms.
Differential media
When a medium contains substances, which help to distinguish differing characteristics of bacteria, it is called differential medium e.g.
MacConkey’s medium, which contains peptone, lactose, agar, sodium taurocholate and neutral red. The lactose fermenters (LF) form pink colored colonies where as non-lactose fermenters (NLF) produce colorless or pale colonies.
Indicator Media
These media contain an indicator which changes color when a bacterium grows in them. Salmonella enterica serotype Typhi grow as black colonies on Wilson and Blair medium containing sulphite.
MacConkey ‘s medium is also an indicator medium. Due to fermentation of lactose, there is acidic pH which forms the pink colonies in the presence of neutral red indicator.
Transport media
These are used in the case of delicate organisms (e.g. gonococci) which may not survive the time taken for transit or may be overgrown by non-pathogenic bacteria (e.g. cholera organisms). They maintain only viability.
Examples of transport media are:
Stuart’s transport medium: It is a non-nutrient soft agar gel containing a reducing agent to prevent oxidation, and charcoal to neutralize bacterial inhibitors. It may be used for organisms such as gonococci.
Buffered glycerol saline transport medium for enteric bacilli.
Sugar media
Sugar media help in identification of bacteria. The term sugar in microbiology denotes any fermentable substance. Glucose, lactose, sucrose and mannitol are routinely employed for fermentation tests.
Anaerobic Media
These are used for cultivation of anaerobic bacteria e.g.
Cooked meat broth (CMB)
Thioglycolate broth
About Nutrient agar
Intended use
Nutrient Agar is used as a general-purpose medium for the cultivation of less fastidious microorganisms, can be enriched with blood or other biological fluids.
Composition
Ingredients Gms / Litre
Peptone 5.000
Sodium chloride 5.000
peptone 1.500
Yeast extract 1.500
Agar 15.000
Final pH (at 25°C) 7.4±0.2
Directions
Suspend 28 grams in 1000 ml distilled water.
Heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs. pressure (121°C) for 15 minutes.
Cool to 45-50°C. If desired, the medium can be enriched with 5-10% blood or other biological fluids.
Mix well and pour into sterile Petri plates.
About blood agar base
Intended Use
Blood Agar Base is recommended as a base to which blood may be added for use in the isolation and cultivation of fastidious pathogenic microorganisms like Neisseria, Streptococci etc.
Composition
Ingredients Gms / Litre
peptone 10.000
Tryptose 10.000
Sodium chloride 5.000
Agar 15.000
Final pH (at 25°C) 7.3±0.2
Directions
Suspend 40.0 grams in 1000 ml distilled water.
Heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs. pressure (121°C) for 15 minutes.
Cool to 45-50°C and aseptically add 5% v/v sterile defibrinated blood.
Mix well and pour into sterile Petri plates.
About Robertson’s Cooked Meat Medium (RCM)
Intended use
Cooked Meat Medium (R.C. Medium) is used for cultivation of aerobes and anaerobes, especially pathogenic Clostridia from clinical, food and water samples. This can also be used as a maintenance medium for stock cultures.
Composition
Ingredients Gms / Litre
peptone 98.000
Proteose peptone 20.000
Dextrose (Glucose) 2.000
Sodium chloride 5.000
Final pH (at 25°C) 7.2±0.2
Directions
Suspend 12.5 grams in 100 ml distilled water (or suspend 1.25 grams in 10 ml distilled water in test tubes).
Mix thoroughly and allow to stand for 15 minutes until all the particles are thoroughly wetted.
Dispense into tubes or flasks as desired.
Sterilize by autoclaving at 15 lbs. pressure (121°C) for 15 minutes.
References
- Topley and Wilson’s microbiology and microbial infection Bacteriology-2-10th Edn.
- Manual of Clinical Microbiology Patrick R. Murray -8th Edn
- Koneman Colour Atlas and Text Book of Diagnostic Microbiology-6th Edn.
- Jawetz Melnick and Adelberg’s Medical Microbiology- 25th Edn.
- Lippincotts –Illustrated- review-Microbiology-3rd Edn.
- Mandell’s Infectious Disease-7th Edn.
- Bergey’s Manual of Systemic Bacteriology – 2nd Edn.
- Medical Microbiology-The Practice of Medical Microbiology Vol-2-12th Edn. –Robert Cruickshanky