Advanced Level

Making up nutrient agars

Introduction

The ability to make different culture media for culturing different microorganismsaking is an essential part of any microbiology investigation.

Agar provides a matrix of supporting jelly for dissolved nutrients. Different nutrients are appropriate for culturing different microorganisms.

Apparatus And Chemicals

Add different nutrients to the basic agar depending on the organisms you are planning to culture.

Health & Safety

Read our health and safety guidelines

Refer to CLEAPSS Recipe card 1 for full details of handling agar. The identified hazard relates to inhaling the powder when making up the agar jelly for which the control measure is weighing out the powder in a fume cupboard. The risk of scalds when dealing with hot liquid is reduced by wearing oven gloves.

Technical Notes

1 Basic agar: Mix 1.5 g of agar with 10 cm³ of water into a paste. Slowly add more water with stirring until the volume is 100 cm³. Heat the mixture on a boiling water bath to 95 °C in the required container. This preliminary heating can be omitted if the agar will then be sterilised, unless it is necessary to decant the agar into smaller containers prior to autoclaving.

2 Nutrient agar for bacteria: Mix 2g of ‘Bovril’, 0.5 g of sodium chloride and 1.5 g of agar with 10 cm³ of water into a paste. Slowly add more water with stirring until the volume is 100 cm³. Heat in a boiling water bath to 95 °C in the required container.

3 Malt agar for fungi: Mix 2 g of malt extract with 2 g of agar with 10 cm³ of water into a paste. Slowly add more water with stirring until the volume is 100 cm³. Heat in a boiling water bath to 95 °C in the required container.

4 Starch agar: Make a paste containing 1 g of soluble starch in 10 cm³ of hot water. Add 1.5 g of agar, stir well and slowly add more water with stirring until the volume is 100 cm³. Heat in a boiling water bath to 95 °C in the required container.

5 Starch malt agar for growth of fungi and digestion of starch: Mix 3 g of light malt (crystal or spraymalt) powder (from home-brewing shops), 0.5 g of peptone (to promote growth) in 20 ml of water. Also make a paste containing 1 g of soluble starch in 10 cm³ of hot water. Add these two solutions to 1.5 g of agar with stirring and slowly add more water with stirring until the volume is 100 cm³. Stir before decanting into smaller containers (if required) and sterilising.

6 Mannitol yeast extract agar (MYEA): For 1 litre of mannitol yeast extract agar (MYEA) suspend 10g of agar in 1 litre of water. Heat to dissolve. Add 0.5 g K₂HPO₄ (Hazcard 95C), 0.2 g MgSO₄.7 H₂O (Hazcard 59B), 0.2 g NaCl (Hazcard 47B), 0.2 g CaCl₂.6H₂O (Hazcard 19A), 10 g mannitol and 0.4 g yeast extract. K₂HPO₄, MgSO₄.7 H₂O and NaCl are described as low hazard. CaCl₂.6H₂O is an irritant as a powder, but not in the final medium.

Procedure

CLEAPSS Laboratory Handbook section 15.2.7 has more information on how to manage agar and prepare plates. Or you can buy prepared media in bottles or plates (see Suppliers).

a Calculate the quantity required and prepare just enough agar for the investigation – around 15 cm³ for normal depth in a 90 mm Petri dish. (Any surplus will keep for 6-12 months in tightly-sealed screw-top bottles if sterile.

b Weigh out the agar medium powder (containing the gel and chosen nutrients), add water and sterilise the mixture for the time (and at the temperature) specified by the manufacturer.

c Heat agar and water at 95 °C to dissolve the agar. Always use a water bath to boil agar and never add agar to boiling water.

d Stopper flasks with a well-fitting plug of non-absorbent cotton wool and cover with greaseproof paper or aluminium foil before sterilising by autoclaving.

e After autoclaving, transfer to a water bath to equilibrate at 50 °C. Stack plates after pouring to minimise condensation except in the top plate(s).

f Warm the Petri dishes before pouring to minimise condensation.

g Keep poured plates in a sealed plastic bag until needed to reduce dehydration of the media.

h Divide the agar into individual sterile McCartney bottles if you want the students to pour their own plates (see Standard techniques).

Web Links

www.microbiologyonline.org.uk/teachers/resources
Society for General Microbiology – source of Basic Practical Microbiology, an excellent manual of laboratory techniques and Practical Microbiology for Secondary Schools, a selection of tried and tested practicals using microorganisms.

www.microbiologyonline.org.uk
MiSAC (Microbiology in Schools Advisory Committee) is supported by the Society for General Microbiology (see above) and their websites include more safety information and a link to ask for advice by email.

(Websites accessed July 2010)

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