Agar is a polysaccharide that is derived from plants, mostly marine algae. Agar is mostly employed as a coagulant in microbiological medium. Agar is added to fluid media at 0.5-2g/L, semi-solid media at 3.5-4g/L, plant tissue media at 5-10g/L, and solid media at 12-15g/L.
Agar powder is a crystalline substance that is white, yellowish, or greyish in appearance and is insoluble in cold water. Agar can be dissolved by boiling or autoclaving (the melting point of most agars is about 90°C) and then frozen after cooling (the freezing point of most agars is around 40°C).
(1) Dissolve the required amount of dry medium in purified water according to the medium's label, heat and boil until the agar is completely dissolved, then distribute into test tubes or conical flasks.
(2) Sterilize the medium according to the instructions. The sterilised medium can be cooked 4-6 times more until it is totally dissolved.
(3) After sterilisation, after the agar medium has cooled to around 50°C, it can be kept warm in a water bath set at 47-50°C for no more than 4 hours.
(4) Add the required addition components, shake well, and pour onto the plate, harden, and prepare for use, or form a slant, pour, and use, etc. before using.
(1) When dissolving dry powder, the water temperature should be below 40°C and no more than 60°C. When agar comes into contact with hot water, its surface absorbs the water quickly and hardens, generating hard lumps that are difficult to disperse and dissolve. When dissolving dry powder, it's best to start with cold water and mix until it's uniformly spread and lump-free before heating.
(2) Before sterilisation, the agar must be heated and boiled to dissolve completely and evenly before being dispensed into tubes or conical vials; otherwise, agar distribution may be uneven and coagulation may vary after dispensing; heating and boiling before sterilisation of agar medium also helps to speed up heat penetration in the autoclaving process, improve the sterilisation effect, and reduce the chance of sterilisation failure.
(3) Once the medium has solidified, it should not be autoclaved again. If the medium is stable, it can be dissolved and reused in a boiling water bath.
(4) Antibiotics or other unstable components should be introduced to the medium after it has been chilled to 50-55°C before use. If these components are added too soon or while the medium is too hot, unstable components will decompose and the medium's impact will be reduced.
(5) When using the solidified agar medium, it should not be heated and dissolved repeatedly. Because repeated heating causes the nutrients in the medium to disintegrate and the gel strength of the agar to deteriorate, the more stable components of the medium are usually allowed to dissolve just once for use.
(6) The solidified medium must not be stored in icing conditions. The agar's internal structure can be damaged by freezing, resulting in water loss, poor coagulation, and agar denaturation.