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

Date:
[1968], ©1968
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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.

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    Experiments in Microbial Genetics small volume of phage buffer (Appendix A17) and allowed to stand over¬ night. It is finally well dispersed and centrifuged at low speed to remove the last few bacterial cells. (b) Liquid lysates {of temperate phages^ e.g. P22) To a similar log. culture of the host bacteria, aerated at 37° in nutrient broth, phage is added at a multiplicity (phage per bacterium) of about i in 200. Aeration is continued either for 5 hr, or until the culture shows signs of clearing (whichever is earlier), when a titre of to pfu/ml is usually achieved. Further purification and concentration as above, noting that P22 is a large phage and Л much smaller. (There is frequently no correlation between the final titre and the extent of 'clearing'.) (c) Soft-agar lysates [of temperate phages e.g. Pi or Л) To a small tube containing 2.5 ml of molten soft agar, held at 46° in a waterbath, is added o.i ml of a log. culture of host bacteria [any non- Pi-lysogenic K12 strain for Pi, or a C600 strain (EMG 10-Appendix B) for Л] at about 5 X 10^ cells/ml and o.i ml of a lo^ particles/ml prepara¬ tion of the phage. The tube is overlayed on a plate containing 50 ml of T-phage nutrient agar (TNA; Appendix A16) (containing M/ioo Ca++, in the case of phage Pi and M¡100 Mg++ for phage A). After overnight* incubation, the plate shows confluent lysis of the host cells. The soft agar is scraped off into 2 ml phage buffer, shaken vigorously for a few seconds and immediately centrifuged at low speed. The supernatant is decanted, i drop CHCI3 is added and it is recentrifuged at low speed. The preparation will usually contain to phage particles per ml and can be further concentrated by high-speed centrifugation. 8. Replica plating Plates that are to be replicated are surface-spread with a dilution of a bacterial culture that will give 100-200 colonies per plate, avoiding the periphery of the plate. They are then incubated for a shorter than usual period until the well-separated colonies reach a diameter of about i mm. These are the 'master plates'. A suitable block for replica plating is a N0.79 rubber stopper (79 mm smaller diameter), to which a sterile disc of nylon velveteen or other similar materialf about 12 cm in diameter is * With Pi lysates, an incubation time of 8-10 hr is optimal, t 11 cm filter paper may be used, provided colonies are well-separated and few replicas are required. 8