Add 0.2ml of sterile saline  to a screw-cap tube

Use a sterile loop/spade to  emulsify two loopfuls/spadefuls of a 4-week old culture in the saline

Add 2ml of the NaNo₃ substrate

Shake well and incubate  upright in a 37EC  water bath for 3 hours and remove

Add in the following order:

1 drop diluted HCI (shake tube well)
2 drops 0.2% sulfanilamide
2 drops 01.% N-napthtylethylene-diamine

Examine immediately for a  pink to red colour and compare to colour standard