#11: Gelling together: Why agar gel-strength matters (especially for settle plates)

Agar – the jelly-like substance - has been used in culture media since 1882, providing the basis for a medium for the cultivation of bacteria and fungi (the polysaccharides of the agar - agarose and agaropectin - are combined with various nutrients and growth factors to encourage microbial growth).

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Agar is derived from agarophyte seaweeds. The gelation and melting of agar (and reversing of either state) is based on the formation of hydrogen bridges (physical gels). ?Agar has a gelling temperature of 32-36°C and a melting temperature of 85-86°C (what is sometimes referred to as hysteresis, indicating a large differential between agar’s setting and melting temperature). Of the two components, it is agarose, consisting of alternating β-D-galactose and 3,6-anhydro-L-galactose units linked by glycosidic bonds, that mostly determines the gelling properties of agar.

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Across microbiological applications (as well as many applications for other scientific disciplines such as electrophoresis) one of the most important characteristics of agar, across different physical and chemical properties, is gel-strength – the rheological property of the agar.

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Settle plates and data integrity

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This is important for different uses, not least when agar plates are used as settle plates and subjected to the relatively harsh rapid and dry airflows provided by devices seeking to achieve a EU GMP Grade A / ISO 14644 class 5 environment. Where gel strength is weak, the cracking of agar can occur together with a loss in volume. Excessive losses in volume will affect growth promotion and cracking is a data integrity issue, and both phenomena can lead to an underestimation of any microbial carrying particles that might settle onto the agar surface.

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Variables affecting gel-strength

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To make agar to scale, red algae tends to be used and species of ‘seaweed’ like Hypnea musciformis, Gelidiaceae species, and Gracilaria cornea are common sources. Not all source agars are equal in terms of their gel-strength, with H. musciformis having considerably greater strength than G. cornea. Furthermore, within a genus species can differ widely, as one assessment of Gracilaria species demonstrates. However, also influential are the environmental condition of seaweed growth and the physiological factors, as well as the extraction methods deployed.

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Gel strength is not simply the agar in itself but a combination of the agar, water potential and water availability. Here, high gel strength is associated with reduced water availability from the medium to the cultures.

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Different factors account for gel strength, when different culture media are compared (either different media or the same medium prepared by different manufacturers). One variable is with diffusion rate of ions in gels; another is the time of autoclaving; another is chemical differences between agars. In one study, superior agars were found to possess a relatively low salt content. Another researcher established that sulphate units at points in the polysaccharide chains cause kinks in the helical structure responsible for gel formation, leading to agars of lower gel strength.

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Assessing gel-strength

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In terms of what makes a suitable gel-strength, ≥750 g/cm^2 is typically regarded as a suitable target, although many manufacturers aim for >1000 g/cm^2. The method of assessment involves the use of a plunger where a weight is progressively increased so that the breaking point of the gel can be measured. The time that the gel can support the weight for is assessed and the test is conducted at a specified temperature. This represents an essential quality control test that media manufacturers (should) undertake.

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Conclusion

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Gel-strength is not something that the typical laboratory can assess easily, although the effects of weak gels when settle plates are used is noticeable (as indicated by cracking or excessive desiccation lading to volume loss). In terms of resolving this problem, assessing gel-strength and engaging in conversations with media suppliers represents an important first step.

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Note: Readers who wish to conduct settle plate studies to assess desiccation and other factors under unidirectional airflow can access my paper “Settle plate exposure under unidirectional airflow and the effect of weight loss upon microbial growth” here.

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Tim Sandle is the author of the book ‘Pharmaceutical Microbiology: Essentials for Quality Assurance and Quality Control’ published by Elsevier.