Different Media of Microbial Culture
- Nutrient media for culture must be easily assimilable by microorganisms.
- Therefore, they have to contain a certain amount of organogens (vitally required elements – oxygen, carbon, hydrogen, nitrogen, phosphorus), growth factors (vitamins, some carbohydrates, microelements, amino acids etc.), and necessary concentration of salts.
- Inaddition, they should be sterile, isotonic, maintain optimal buffer properties, viscosity, and proper reduction-oxidation (or redox) potential.
- Notably, the bacteria should be cultured at optimal temperature. The closed chamber, mantaining the required temperature for culture, is known as thermostat.
- LouisPasteur and his collaborators introduced non-protein media for the cultivation of microbes.
- Then R. Koch with co-workers proposed meat broth, peptone, and sodium chloride as essential components for satisfactory microbial growth.
- Asthe result, a meat-peptone broth or MPB (otherwise, beef-peptone broth) and meat-peptone agar (MPA) were successfully introduced into microbiological practice as basic nutrient media. Meat-peptone agar is prepared by adding to the medium 2-4% of agar.
Agar (from Malayan – jelly)
- It is a fibrous matter produced from certain seaweeds that creates a solid gel, when solidifies in water solutions.
- Agar is predominantly composed of polysaccharides (70-75%); also it includes proteins (about 2-3%) together with other nitrogen-containing substances, and 3-4% of ashes. T
- major constituents of agar are the highly polymeric carbohydrates – agarose and agaropectin.
- Agar easily dissolves in hot water and hardens at room temperature.
- It is distributed as colorless powder ready to use.
In order to design an appropriate liquid medium M. Hottinger proposed to use the tryptic breakdown of proteins that contain free amino acids and short polypeptides. Similarly, L. Martin used plant derived proteolytic enzyme papain for the breakdown of proteins.
All the scope of nutrient media is classified into certain groups depending on their laboratory use.
According to composition the nutrient media are divided into ordinary (simple) and complex media.
- Ordinary or simple media usually have single basic component. There are only few examples of such products, e.g. gelatin or coagulated serum.
- Complex media contain many components, being the combination of various compounds.
All complex media are composed on the ground of basic nutrient media supplemented with other nutrients and growth factors. Essential basic nutrient media included into the most of other complex media are meat-peptone broth and meat-peptone agar.
In consistency nutrient media may be solid (meat-peptone agar, coagulated serum, gelatin, etc.), semisolid (the medium contains about 0.5-1% of agar), and liquid (meat-peptone broth, sugar broth, Mueller Hinton broth, etc.).
According to their destination the nutrient media are also divided into several groups.
- Differential media
- For determination of differencies in biochemical activity of bacteria.
- There are media for the determination of fermentation of carbohydrates, for instance, liquid Hiss’ media for identification of enterobacteria.
- Intube version of these media every tube includes MPB, one kind of mono- or disaccharide, the tube (float) for gas accumulation and acid-base indicator dye, for example – fuchsin, decolorized with 1 M NaOH.
- If grown bacteria ferment carbohydrate, the color of indicator is changed due to appearance of aldehydes and acid products of carbohydrate degradation.
- The multi well plate version of similar media includes the broad panel of sugars for bacterial identification.
- This enables parallel mass screening of biochemical activities of many isolated microbial strains, followed by automated colorimetric registration of the results and their computer analysis.
Also there are numerous solid media for the differentiation of bacteria by lactose fermentation (McConkey agar, Endo agar, eosin-methylene blue or EMB medium, etc.); for determination of the proteolytic action of microbes (gelatin, MPB with indicator dyes), media for the determination of hemolytic activity (blood agar); media for evaluation of oxidative and reductive activity of microorganisms and many others.
Selective media create the growth conditions permissive to only definite species of bacteria; and all other microbial species should be suppressed or don’t grow at all. These media include biocide components that inhibit the majority of outside species except the investigated one (for instance, bismuth sulphite agar for Salmonella enterica var. typhi culture, or egg yolk salt agar for S. aureus with 7,5% NaCl).
Nutrient media that contain antibiotics are selective for antibiotic-resistant strains, inhibiting the propagation of strains, susceptible to these antibiotics.
Special media are used for the growth of fastidious bacteria that are hard for culture. Usually they include a plethora of growth factors ensuring the beneficial conditions for microbial reproduction (ascite agar for gonococci, complex hemin-containing media for Hemophilus influenza culture, Mueller Hinton agar with blood for testing antibiotic susceptibility of streptococci, etc.).
Enrichment media are also implicated for culture of the questioned species, which grows more actively and more rapidly than concomitant bacteria. They can be used for primary inoculation of clinical specimen. In a number of cases they include biocide to suppress accompanying bacteria. In this vein, for instance, alkaline peptone broth serves as enrichment and selective medium for cholera vibrio.
Transport media provide the delivery of microbial-containing clinical specimen to the laboratory without damage of microbial cells. They may support the growth of pathogenic bacteria and damp the activity of saprophytes. As an example of such a medium, it can be glycerol-containing phosphate buffered saline.
Synthetic media are composed of chemically-defined synthesized components of known standard structure.
Now all nutrient media as well as their components are manufactured and commercially delivered as dry powders. They are stable, convenient for routine laboratory work and demonstrate high efficacy of microbial isolation.
Evaluation of growth properties (or cultural characteristics) of isolated microorganisms enhances the accuracy of their final identification.
When cultured on solid nutrient media, the microbes produce the colonies of different sizes and shapes.
Microbial colony is the isolated group of microorganisms, grown on solid nutrient media, which are the offsprings of one microbial cell.
Hence, the microbial cells of one colony form the single genetic clone. Clonality of colonies provides successful discrimination and identification of representatives of all microbial taxa present in clinical specimen.
The colonies possess the number of intrinsic descriptive characteristics.
According to their sizes, the colonies can be separated into large (more than 4-5 mm in diameter), intermediate (2-4 mm), small (1-2 mm), and dwarf (less than 1 mm).
The shapes of the colonies are also variable – the basic types are regular or irregular round-shaped, also they can be rosette-shaped, tree-like, star-shaped, etc.
The edges of colonies are characterized as straight or irregular, fibrous, serrated, tasseled and others
According to their plane, the colonies may be flat, convex or pitted, dome-shaped, etc.; depending on colony surface – smooth (S-forms) or rough (R-forms), bumpy or ridged.
The colonies differ in their density, consistency, and color. They may appear as slimy, moist, or dry; transparent or opaque, pigmented colored or colorless.
Inner structure of colony is studied by optical microscopy with low-level magnification.
The growth of bacteria on liquid media displays diffuse turbid suspension, or visible bottom precipitate, or the biofilm, attached to the inner surface of the well or test tube.