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• Anaerobic Systems
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• Culture Media, Supplements and Raw Materials
• • Are there any guidelines for preparation and reconstitution of culture media?
  • Are there any guidelines for sterilising culture media?
  • Can I prepare culture media in a microwave?
  • Can I store selective supplements after reconstitution?
  • How can I re-melt solid media?
  • How long can I store culture media after preparation?
  • How should I dispose of used culture media?
  • How should I test the pH of culture media?
  • My culture medium does not look right - what has gone wrong?
  • My culture medium is not performing correctly - what has gone wrong?
  • Should I boil media before autoclaving?
  • What are the main components of culture media?
  • What exactly is agar?
  • What grade of water should I use to prepare culture media?
  • What is the shelf life of dehydrated culture media after the pot has been opened?
  • What quality control testing should I carry out on culture media?
  • Which blood product should I choose to make blood agar?
• Diagnostic Reagents
• Dipslides
• General
• Toxin Detection

OXOID AGARS

Agar is a complex mixture of polysaccharides extracted from species of the red algae known as agarophytes (Gelidium, Gracilaria, Pterocladia, Acanthopeltis and Ahnfeltia species). It is a sulphuric acid ester of a linear galactan, soluble in hot water but insoluble in cold water. A 1.5% w/v aqueous solution should set at 32-39°C and not melt below 85°C.

There are two dominating polysaccharides in agar which particularly affect its performance in culture media.

1 A virtually neutral polymer, agarose - (1-4) linked 3,6-anhydro-a-L-galactose alternating with (1-3) linked ß-D- galactose.

2 A charged polymer, agaropectin, having the same repeating unit as agarose but with some of the 3,6-anhydro-L-galactose residues replaced with L-galactose sulphate residues, together with partial replacement of the D-galactose residues with pyruvic acid acetal 4,6-0-(1-carboxyethylidene)-D-galactose.

Agarose is the component responsible for the high-strength gelling properties of agar, whereas agaropectin provides the viscous properties. The proportion of agarose to agaropectin in agar varies according to the algae of origin but it can be as high as 75% agarose to 25% agaropectin.

The characteristic property of agar to form high-strength gels which are reversible with a hysteresis cycle over a range of 40°C is due to three equatorial hydrogen atoms on the 3,6-anhydro-L-galactose residues which constrain the molecule to form a helix with a threefold screw axis. It is the interaction of these helices which causes gel formation.

Agar is hydrolysed with heat at acid pH values because the 3,6-anhydro-a-L-galactoside linkage is very susceptible to acid cleavage.

Agar is manufactured in many parts of the world, although it is essential to locate the industry near suitable beds of algae and have efficient low-cost methods of harvesting the weed. It requires 100 tons of fresh water to produce one ton of dried agar, therefore the quality of the local water will influence the quality of the processed agar.

The presence of `free' metal ions of Ca, Mg and Fe in agar which can react with phosphate salts in culture media to form insoluble precipitates or hazes is undesirable. Equally undesirable is the presence of chelating compounds which can bind these cations and make them unavailable to the organisms. Lowering the phosphate level of the culture medium to overcome its interaction with the metals usually results in poor growth-promoting properties. Compatibility tests between agar and the various culture media formulae are essential.

The agars used in such tests vary as follows:

1 Bacteriological agar
Clear, colourless products in which the mineral/metal components may be reduced making them satisfactory for most formulae.

2 Processed bacteriological agar
Clear, colourless products in which the mineral/metal components have been reduced to low levels, making them compatible with all formulations.

A further advantage of chemical processing to reduce divalent cations is that it overcomes the antagonism of certain agars to amino-glycoside antimicrobials and tetracycline. It also considerably improves the diffusion of antimicrobials in the disc-diffusion assay.

3 Technical grade agar
Less clear and colourless products in which the higher mineral/metal components may have advantages in certain low-phosphate formulations.

All such agars must be free from toxicity to micro-organisms and free from impurities such as non-agar gums, nitrogenous compounds, insoluble salts, free sugar compounds, dead micro-organisms and live thermophilic organisms.

The process of agar production has been fully described by Whistler ¹, Chapman ² and Bridson & Brecker ³ further details on the properties and testing of bacteriological agar can be found in Bridson ⁴.

References
1 Whistler R. L. (1973) Industrial Gums, 2nd Edn., Academic Press, New York, pp. 29--48.
2 Chapman V. J. (1970) Seaweeds and their Uses. 2nd Edn., Methuen & Co. London. pp. 151--193.
3 Bridson E. Y. and Brecker A. (1970) Methods in Microbiology. Vol. 3A, Academic Press, London, pp. 257--266.
4 Bridson E. Y. (1978) Natural and Synthetic Culture Media for Bacteria. In: Handbook series in nutrition and food. Section G. Vol III. Ohio. CRC Press. 91--281.