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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    General methods and techniques and about 2 X lo^ bacteria which are sensitive to the phage [indicator bacteria). These indicator bacteria are conveniently added as 3-4 drops from an o/n broth culture using a i ml pipette or, if large numbers of over¬ lays are required, by using the culture in a 'dropping bottle'. The agar is maintained in the molten state by holding the tube in a 46° waterbath. The mixture of phage, indicator bacteria and soft agar is then poured over the surface of a nutrient agar plate (i.e. 'plated'), and the plate is gently rocked to distribute the mixture evenly over the surface as a thin layer (overlay). When the top layer has solidified (10-15 min), the plate is inverted and placed in an incubator at 37° and incubated overnight. The next day, a continuous lawn of bacterial growth will be seen over the surface of the agar, except where a clone of phage particles has lysed the bacteria, and produced a visible, clear area or 'plaque'. It has been shown that a single phage particle will produce a plaque. The number of plaques, therefore, gives a direct assay of the number of phage particles put on the plate. The titre of a phage preparation can then be expressed as a concentration of 'plaque-forming units' (pfu.). In the case of T phages, this is equivalent to the concentration of phage particles. 7. Preparation of phage lysates (a) Liquid lysates {of virulent phages, e.g. ' T' phages) About 20 ml of phage-broth (Appendix A15)* in a bubbler tube is inoculated with 0.2 ml of an o/n broth culture of the bacterial host strain and aerated by bubbling at 37° in a waterbath for about 2| hr, until a concentration of about i X 10® cells/ml is reached. Phage is then added at a concentration of lo^ particles/ml, so that all bacteria are infected.f Aeration is continued for two to three hours when the turbid culture will often 'clear' and a final titre of lo^o to phage particles/ml will usually be achieved. Several drops of chloroform are then added, the lysate is well shaken, allowed to stand for 10 min and then decanted. It is then spun at low speed in a small, angle centrifuge to sediment bacterial debris. For further concentration, the phage may be sedimented as a pellet, after high-speed centrifugation [12,000 g for i hr in the case of large, T-'even' (T2, T4, T6) phages and appropriately longer for the smaller, T-'odd' (Ti, T3, T5, T7) phages], which is then taken up in a * M9 medium + 0.2% casamino acids can be used as an alternative. Problems of frothing associated with bubbling are then much less (see Appendix A), t Alternatively, inoculate 35 X 10' cells/ml culture with a straight needle after 'stabbing' a phage plaque. 7