Experiments in microbial genetics / edited by R.C. Clowes and W. Hayes.

Date:: [1968]

Licence: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

Credit: Experiments in microbial genetics / edited by R.C. Clowes and W. Hayes. Source: Wellcome Collection.

20/260 page 6

$Experiments in Microbial Genetics Standard dilutions may be achieved as follows ; — indicates o.i ml into 0.9 ml Dilutions are made in buffer (Appendix A14) unless otherwise stated. 6. Assay of bacterial cells or phage particles (a) 'Total' bacterial cells Çtotal counf) The number of bacteria (living or dead), present in suspensions with cell densities greater than lo'^/ml can be counted by the use of a counting chamber (Helber or Petroff-Hauser ; see p. 238) viewed by dark-field or phase-contrast microscopy. All the cells in the culture, whether viable or not, are counted by this technique. (b) Colony-forming bacteria {^viable count') A liquid culture is diluted so that its cell density is about i to 5 X lo^/ml. Samples of o.i ml are then assayed: (i) By spreading. A o. i ml drop is placed by pipette on an agar plate and spread over the surface using a bent glass rod (a i inch right-angled bend on a 3mm diam. glass rod) which is pre-sterilized by flaming after im¬ mersion in alcohol; after 18 hr incubation, each viable cell will produce a colony. (ii) By overlay. The 0.1 ml sample is added to 2.5 ml of soft agar (Ap¬ pendix An) in a small tube, held molten at 46° in a waterbath. The whole contents of the tube are then poured on an agar plate, which is rocked to distribute as a thin layer over the entire surface. Thin disc-like colonies are produced within the soft agar overlay which may be counted after 24 hr incubation. (c) Standard assay for bacteriophage The most widely used method for assaying phage stocks is the agar- layer method. A small volume (0.05-0.5 ml) of a diluted phage suspen¬ sion is pipetted into a small tube, containing 2.5 ml of molten 'soft' agar$

Experiments in microbial genetics / edited by R.C. Clowes and W. Hayes.

Licence and re-use

Contents