Preparation of Cultures for Testing

An overnight culture of either pol A or its pol Ai derivative in HA-j-T medium is diluted 10,000-fold in HA-l-T medium, and 0.1-ml portions of the cultures are spread (with a glass spreader) onto the surface of the agar plates. 

It is essential that the bacterial strains be grown in this medium and that the test plates also be of the same composition. Failure to assure these conditions may give unreliable results, since in rich media (i.e., rich nutrient agar), the pol A strain appears to have a selective advantage and hence the occasional pol A revertants present in pol Aj cultures will overgrow (see also Rosenkranz etal ). 

An alternate to the procedure described above is to place 0.1 ml of the culture into 2 ml HA+T soft agar (at 45°C), mix this suspension (Vortex mixer), and pour it onto the surface of the agar plates. This procedure provides for more homogeneous distribution of the bacteria and results in a more uniform zone of growth inhibition. This modification is slightly more time-consuming than the one described above. 

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