NUMBERS OF MICROORGANISMS 19 



already acid in reaction, or a medium to which enough lactic acid is 

 added to bring the reaction to pH 4.0. Because of the high acidity, 

 25 gm. of agar are required per liter. For quantitative purposes, 

 however, a synthetic medium is always to be preferred : 32 



Glucose 10 grams MgS0 4 -7H 2 0.5 gram 



Peptone 5 grams Distilled water 1000 cc. 



KH2PO4 1 gram 



The ingredients are dissolved by boiling and enough normal acid (H2SO4 or 

 H3PO4) is added to bring the reaction to pH 3.6 to 3.8. This will require from 

 6 to 7 cc. of normal acid per liter of medium. Twenty-five to 30 grams of agar 

 ara added and dissolved by boiling; the medium is then filtered, tubed and ster- 

 ilized as usual. The final reaction of the medium should be pH 4.0. 



For soils to which an excess Of organic matter has recently been added, the 

 dilution used for the determination of fungi is usually from one hundredth to 

 one-tenth of the dilution used for the determination of numbers of bacteria. 



All the necessary glassware and water blanks should be prepared 

 and sterilized before the soil samples are taken. Three or four 

 pipettes and the same number of water blanks are required for every 

 soil sample. Ninety-nine or 90 cc. portions of tap water, depending 

 on the fact whether 1-cc. or 10-cc. pipettes are employed for making 

 the dilutions, are placed in the flasks which are then plugged with 

 cotton. The glassware (including the Petri dishes) is sterilized in 

 the hot air sterilizer for two hours at 160°C, while the water blanks 

 are sterilized in the autoclave, at 15 pounds pressure for 15 or 20 

 minutes. The sampling bottles may be sterilized either with the 

 other glassware or with the water blanks. 



Sampling of soil. It has been shown both by cultural studies 18 and 

 by the use of the microscope 33 that a given soil will contain at a given 

 time, in a given soil layer, a uniform bacterial flora. Samples taken 

 under the same experimental conditions will, therefore, be the same bac- 

 teriologically, while any change in soil type and treatment will result 

 in a change in bacterial relations. When comparing different soils, 

 samples taken from the same depth should be compared. The varia- 

 bility of a soil itself, even as to physical and chemical factors, is so 

 great, however, that it is not surprising that such a sensitive indicator as 

 bacterial numbers and activities should also vary. To meet the factor 

 of variation, the common practice has been to use composite samples, 



32 Waksman, 8. A. A method for counting the number of fungi in the soil. 

 Jour. Pact. 7: 339-341. 1922. 



33 Winogradsky, 1925 (p. 7). 



