Microbiological Medias

Author: Laiba B. (IG: @yourhealthguide_)

SCG ID: 1806201905

Microbiological media or culture medium or growth medium is a liquid or gel designed to support the growth of microorganisms. There are different types of media suitable for growing different types of cells. Here, we will discuss microbiological media used for growing microbes, such as bacteria or yeast.

Origin:

Their origins go back to the 19th century when bacteria were grown on media made from ingredients that were readily available, mainly materials from abattoirs or hospitals, e.g. Blood, beef carcasses and offal.

Constituents:

Many modern media have similar basic chemical and biochemical constituents, that provide protein-derived nutrients in the form of peptides/amino acids for nitrogen and carbohydrates for energy. An important constituent of many microbiological growth media is hydrolyzed protein known as peptone but, in addition, they may contain other ingredients such as sugars, yeast or malt extract, salt, trace elements, vitamins and chemical buffers to maintain a stable ph value.

Complexity:

Over the years, microbiological growth media have become increasingly complex and many are now specially adapted for specific organisms; for example, they may contain milk protein hydrolysates instead of meat peptones. Vegetable (soy) derived peptones are also used.

Purpose:

Such media also contain a range of other constituents for specific purposes. For example, They may contain specific, growth enhancing ingredients that encourage the target organism to grow while the growth of other organisms that may be present in the sample is restricted because they cannot utilize the added ingredients (elective media). 

They may contain ingredients that change color or appearance as a result of a particular microbial metabolic process that the target organism causes a distinctive reaction (diagnostic media).

They may contain inhibitors that prevent the growth of organisms that compete with the target organism (selective media).

1. Diluents:

Many microbiological tests are designed to provide quantitative results, i.e. they are designed to allow the enumeration (count) of a particular microorganism in a test sample. To do this requires accurate dilution of the test sample which, in turn, requires the use of a suitable diluent.

Function:

Ideally, a diluent should protect microorganisms, especially those that may have been damaged or stressed by the food processing operations or the intrinsic or extrinsic properties of the food so that they remain viable but neither multiply nor die.

Method:

To achieve protection, a solution of physiological saline (salt) that is isotonic with the microorganisms suspended in it is used, i.e. the solution has similar osmotic pressure to cytoplasm of an average food borne microorganism.

Examples:

Today, perhaps, the most widely used diluent formulation is known as Maximum (or Maximal) Recovery Diluent (MRD) (or peptone-saline diluent. This contains 0.85% saline and 0.1% peptone.

1. Liquid Growth Media:

Liquid growth media, called broths, are usually used to grow microorganisms so that their numbers are amplified to a level that is useful as an inoculum for other forms of test, e.g. antibody based assay.

Function:

For many decades, broth media provided the main means for conducting batteries of tests to determine the biochemical profile of microorganisms for their identification and are still used for carrying out most probable tests.

Method:

They can be made elective or selective and also diagnostic. Broths are usually dispensed into tubes or bottles of varying capacity between about 5ml and 1 liter.

Uses:

Broth are often used for the primary cultures of organisms from foods, e.g. in detection tests for Salmonella or Listeria.

2. Gel (Solid) Growth Media:

Agar can be added to liquid media to make them into a firm gel. Agar is a complex galactan (polymer of galactose) obtained from some seaweed and is usually added to media at a concentration of around 1-2%. Sometimes lower concentrations are used to form a sloppy gel known as semi solid agar, which is particularly useful when studying motile organisms that can swim through the viscous matrix.

Function:

Agar is usually sold as a powder and has the unusual property that it only dissolves in water when heated to above 84 C but, upon cooling, it remains liquid until the temperature falls to less than about 42 C, when a firm gel is formed.

Method:

Organisms can be suspended in cooled molten agar which is allowed to set and then is incubated until visible growth has occurred, as in pour plates commonly used for counting microorganisms, e.g. total colony counts (TCC).

Example:

If a gelling agent, such as gelatin is used, many more microorganisms can, and do, break down the gelatin by hydrolysis, and the gelatin slowly liquefies as the organism grow. This characteristic is usually used as one of a range of diagnostic tests for the identification of some organisms, e.g. Clostridium species and Enterobacteriaceae.

3. General Purpose Growth Media:

General purpose growth media are used to grow as wide a range of microorganisms as possible from the particular test sample under examination, and so do not contain selective agents.

Purpose:

Different general purpose media have been developed to grow different microbial groups such as bacterial aerobes and anaerobes, yeasts and molds.

Uses:

Probably the most widely used application of general purpose growth media is for making general bacterial colony counts. These counts are known as total colony counts (TCC) but also as total viable counts (TVC), or aerobic plate counts (APC), and other names. Such counts are used to estimate the overall numbers of microorganisms present in a test sample.

Example:

A more correct reporting of a total colony count carried out by the pour plate technique using plate count agar (PCA) incubated at 30 C in aerobic conditions is total aerobic mesophilic colony count. However, the microbiological shorthand of TVC or APC or TCC is still widely used and acceptable.

4. Enrichment Media:

Enrichment media are usually broths and are used to increase the numbers and/or proportion of a target organism from a test sample in the presence of competitive microflora.

Elective Media:

Elective media contain a specific nutrient that the target organism can utilize but many others cannot so that the target organism grows while other microbial growth is discouraged.

Example:

Ethanol is an elective agent used for growing acetic acid bacteria and lactose is an elective agent for growing spoilage yeasts from dairy environments.

Selective Media:

Selective media contains substances that selectively inhibit the growth of many competitor microorganisms present in the test material but allow the target organism to grow.

Example:

Many bacteria inhibited by sodium biselenite but most Salmonellae are able to grow on the presence of this chemical, so sodium biselenite forms the basis of an important selective enrichment medium for Salmonella spp.

5. Pre-enrichment Media:

Microbiological methods may include an initial resuscitation step to allow the target organisms to recover before being exposed to the stress of a selective agent. This is done by inoculating the test sample into a resuscitation medium. Because this medium is used prior to the selective enrichment stage, it is often referred to as a pre-enrichment medium or, more correctly, as a non-selective enrichment medium.

Uses:

Some different pre-enrichment medium formulations are required when isolating Salmonella from specific types of food.

Example:

Perhaps the best known resuscitation medium is buffered peptone water (BPW), a dilute general purpose broth used widely in the isolation of Salmonella from many foods.

6. Diagnostic Media:

Diagnostic media are formulated so that specific products of the metabolism of particular chemicals can be detected easily, usually visually, and used to differentiate readily between cultures of different organisms and aid in their identification.

Uses:

A widely used test is that of acid production from sugars e.g., glucose, lactose, sucrose and ph indicators are used to detect acid production by means of a color change produced as the ph decreases. Such media may be broths, e.g. MacConkey broth used to detect coliforms, but are frequently agars e.g., violet red bile glucose agar (VRBGA) used to detect Enterobacteriaceae. Many of the diagnostic media in use today were developed in the first half of the 20th century and are still very effectively used in food microbiology, but new formulations are continually being developed.

Example:

MUG (4-methylumbelliferyl-β-D glucuronide) is a diagnostic reagent that can be added to media for the detection of E. coli. Another innovation in the formulation of microbiological media is the development of chromogenic agars.