Making up nutrient agars
The ability to make different culture media for culturing different microorganisms 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 and Technical notes
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 – 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.
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 2 g 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 while 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), and 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 while 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 10 g 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.
7 Nitrogen-free mineral salts agar For 500 cm3, first dissolve 0.05 g FeCl3.6H2O in 500 cm3 distilled water. Add 2 g K2HPO4, 0.25 g MgSO4.7H2O, and 10 g glucose. Dissolve and check pH, adjusting to 8.3 if necessary with 0.1M NaOH (Refer to CLEAPSS Hazcard 91: Irritant at this concentration). Pour into a bottle containing 1 g CaCO3 and 7.5 g agar. Autoclave at 121°C. Mix to disperse the CaCO3 before pouring. You can buy ready-made nitrogen-free mineral salts agar. K2HPO4 (Hazcard 95C), MgSO4.7H2O (Hazcard 59B), glucose (Hazcard 40C), and CaCO3 (Hazcard 19B) are all listed as Low hazard. FeCl3.6H2O is harmful as a solid, but not at the low concentration of the final agar.
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 below).
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. 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 Pouring an agar plate).
Web links
Microbiology teacher 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.
Microbiology online
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 October 2011)