Oxoid - Technical Support

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FAQs

Answers to our more common technical enquiries.

Technical Support - FAQs

FORMULATION OF CULTURE MEDIA

DEVELOPMENT AND MANUFACTURE

The formulation of all Oxoid culture media and the components can be divided into different roles or functions:

1 Nutrients: proteins/peptides/amino-acids.

2 Energy: carbohydrates.

3 Essential metals and minerals: calcium, magnesium, iron, trace metals: phosphates, sulphates etc.

4 Buffering agents: phosphates, acetates etc.

5 Indicators for pH change: phenol red, bromo-cresol purple etc.

6 Selective agents: chemicals, antimicrobial agents.

7 Gelling agent: usually agar.

There is often an overlap of functions of some media components, thus protein hydrolysates will supply amino-nitrogen, energy, some metals/minerals and act as buffering agents. Phosphate buffers are important suppliers of minerals and agar contributes metals.

1 Nutrients
Naegeli is credited with the earliest publications (1880/82) describing the requirements of micro-organisms for a protein component which he called `peptone'.

Later work showed that the group of bacteria, now defined as chemo-organotrophs, required amino-nitrogen compounds as essential growth factors in their culture media.

Meat infusions contain water-soluble fractions of protein (amino-acids and small peptides) along with other water-soluble products such as vitamins, trace metals, minerals and carbohydrates (glycogen). Such infusions or extracts may have been regarded as `peptones' but their amino-nitrogen content was usually too low to sustain the growth of large numbers of bacteria.

It was not until deliberate attempts were made to hydrolyse proteins with acids or enzymes that sufficiently high concentrations of water-soluble protein fractions (peptides) were made available for bacterial growth. Many nutrient media usually contain a mixture of protein hydrolysate (peptone) and meat infusion (meat extract/Lab-Lemco).

The difficulties associated with the production of protein hydrolysates were soon recognised and commercial suppliers of peptones became established by the 1920s. The commercial supply of dried peptone eventually led to complete culture media being produced in the form of dehydrated media.

Although meat was the first and most obvious protein to hydrolyse, other proteins were tried later and some showed specific advantages which ensured their retention in culture media to this day. Casein hydrolysate with its pale colour and high tryptophan content and soya peptone with its high energy carbohydrate content are popular examples of non-meat peptones.

A detailed description of these products is given in "Peptones-Hydrolysates" section.

The nutrient components of culture media are carefully selected to recover the required spectrum of organisms in the sample e.g. coliforms or anaerobes. General purpose media such as blood agar in its various forms will often contain mixtures of peptones to ensure that peptides of sufficient variety are available for the great majority of organisms likely to be present. However, more demanding organisms will require supplemental growth factors to be added and examples of such requirements can be seen in media for Legionella species.

Most of the components used for the nutrition of micro-organisms are undefined and require extensive testing with careful selection to ensure a reasonable degree of uniformity. Would it not be better to use wholly defined peptides and amino-acids to produce a totally defined medium? Whilst such media would improve uniformity, experience has shown that they lack good performance as general purpose media. They would also be very expensive compared with undefined media. The use of totally defined culture media is an understandable goal of most microbiologists but defined media have yet to prove themselves equal in performance to currently used complex mixtures of meat and plant protein hydrolysates.

2 Energy
The most common substance added to culture media as a source of energy to increase the rate of growth of organisms is glucose. Other carbohydrates may be used as required.

Carbohydrates added to media at 5-10 grammes per litre are usually present as biochemical substrates to detect the production of specific enzymes in the identification of organisms. It is usual to add pH indicators to such formulations.

3 Essential Metals and Minerals
The inorganic essential components of culture media are many and can be divided on a semi-quantitative basis:
Typical macro-components (gm/litre): Na, K, Cl , P, S, Ca, Mg, Fe.
Typical micro-components (mgm-microgm/litre): Zn, Mn, Br, B, Cu, Co, Mo, V, Sr, etc.
As previously mentioned, a formulation may not have specific metals and minerals listed in its formulation. In such cases it is assumed that all the factors required are present in the hydrolysates, buffers and agar components.

4 Buffering Agents
It is important that the pH of a culture medium is poised around the optimum necessary for growth of the desired micro-organisms. The use of buffer compounds at specific pK values is especially necessary when fermentable carbohydrates are added as energy sources.

Phosphates, acetates, citrates, zwitterion compounds and specific amino-acids are examples of buffering agents that may be added to culture media.

A side effect of such compounds is their ability to chelate (or bind) divalent cations (Ca ++ and Mg ++). Polyphosphate salts, sometimes present in sodium phosphate, are compounds which can bind essential cations so firmly that they are made inaccessible to the micro-organisms.

The effect of these binding or chelating agents will be seen in diminished growth or failure to grow at all, unless care has been taken to supplement the essential cations in the formulation. Opacity forming in a medium, after heating or on standing at 50°C for several hours, is commonly caused by phosphate interaction with metals. Such phosphate precipitates can very effectively bind Fe and lower the available amount of this essential metal in the medium.

5 Indicator Substances
The addition of coloured indicator substances is a very effective way of detecting fermentation of specific carbohydrates in a culture medium. Such compounds should change colour distinctly and rapidly at critical pH values.

Most of the compounds used e.g. phenol red, bromo-cresol purple, fuchsin, etc., are toxic and it is essential to use low concentrations of pre-screened batches/lots. Known sensitive strains of micro-organisms are used in the screening tests.

6 Selective Agents
Chemicals or antimicrobials are added to culture media to make them selective for certain micro-organisms. The selective agents are chosen and added at specific concentrations to suppress the growth of unwanted organisms in a polymicrobial sample. It is, of course, essential to have established that the selective agents, at the appropriate concentration, will allow uninhibited growth of the desired organisms.

Common chemical selective agents are: bile salts, dye-stuffs, selenite, tetrathionate, tellurite and azide. Antimicrobial agents are commonly used in mixtures when suppressing polymicrobial contaminating flora. Antimicrobials are more specific in their selective action than the chemical agents shown above. However, the critical weighing and heat-lability of most antimicrobials demand special care and post-sterilisation addition.

The wide variety of organisms and their almost infinite ability to adapt to changing conditions makes a truly selective medium unlikely. Selective media can be said to suppress most of the unwanted organisms and allow most of the desired organisms to grow. The final formulation is usually a compromise which achieves the best of these criteria.

7 Gelling Agents
Although gelatin is still used for a few specific media and carrageenans, alginates, silica gel and polyacrylamides are sometimes used as gelling agents, the outstanding gel-forming substance used in culture media is agar.

Hesse, a worker in Robert Koch's laboratory, is credited with its first use in culture media, although Frau Hesse gave him the idea from its use in table-jellies in hot climates.

Its inertness to microbial action, the unique setting and melting temperatures (38°C and 84°C respectively) the high gel strength which allows low concentrations of agar to be used, its clarity and low toxicity have contributed to its wide popularity with microbiologists. Its ability to retain its gel structure at 60°C makes agar of special value to culture media which have to be incubated at this temperature to isolate thermophilic organisms.

Agar is obtained from agarophyte sea-weeds mainly Gelidium, Gracilaria and Pterocladia species. It is extracted as an aqueous solution at greater than 100°C, decolourised, filtered, dried and milled to a powder.

Agar is not an inert gelling agent; it contributes nutrients and/or toxic agents to culture media, depending on the chemical processing carried out by the suppliers.

Microbiological agar is specially processed to yield a low toxicity, high clarity, low mineral and high diffusion gel.

Other Components
There are many other substances added to culture media for specific purposes e.g. growth factors for fastidious organisms, eH-reducing compounds for anaerobic organisms (thioglycollate and cysteine), whole blood to detect haemolytic enzymes and encourage the growth of organisms which are vulnerable to oxidation products.