458 HILDA HEMPL HELLER 



dioxide, nitrogen, illuminating gas, nitrogen-hydrogen-carbon-di- 

 oxide and vacuum with varying degrees of moisture, pressure and 

 oxygen present. How can one hope to standardize type colonies 

 under such conditions? And what, may we ask, is the proper mois- 

 ture for the surface of a plate? There is no universal proper mois- 

 ture. Agar moist enough to grow tetanus will allow the spread of 5. 

 sporogenes till the B . sporogenes has increased a million times more 

 than the tetanus has. Some mixtures of organisms allow isolation 

 of their components by surface methods, and some do not. When 

 discouraged with plates that have dried too long, the worker 

 dries them less, and finds to his joy beautiful discrete colonies, 

 some round and some lobed. He must fish them immediately 

 onto plates or into a deep medium or they may die. But let him 

 beware of a pitfall. Let him hold them to the light without 

 a cover and look between the colonies. A slight film of moisture 

 there may represent a spread of growth which contaminates all 

 his colonies. But such a spread may be difficult or impossible 

 of detection. A fragment of coverslip dropped between colonies 

 may show bacilli. I venture to suggest that it is almost impos- 

 sible to determine in an agar slant the non-existence of such a thin 

 spread, and such a thin spreading film is far more likely to occur 

 in the confines of a tube than on a plate. 



Methods of spreading a culture on a surface do not separate the 

 individual organisms from one another so well as does a shaking 

 in liquid agar — in properly made shakes the colonies are beauti- 

 fully distributed. 



Other minor disadvantages of a surface method are that the 

 plates must be incubated immediately after sowing and be fished 

 immediately after opening; they are usually valueless when reincu- 

 bated after opening for inspection because of too much drying, and 

 they require the use of more glassware than do deep-tube methods, 

 and also the use of an anaerobic jar or other anaerobic apparatus. 



The method of Marino should be recommended for organisms 

 which form minute colonies, and for demonstration plates. 

 Marino poured inoculated agar in the upper half of a Petri dish, 

 and covered it directly with the inverted lower half, and covered 

 the whole with a larger Petri dish. This method is convenient but 



