8 PRINCIPLES OF SOIL MICROBIOLOGY 



first sediment suspended in distilled water settles immediately. During these 

 manipulations, which require about 10 minutes, a second sediment is formed 

 in the tube of the centrifuge. About half of the suspension is carefully taken 

 out and placed in another centrifuge tube; on centrifuging, a third sediment is 

 formed. Preparations are then made from each sediment and from the non- 

 centrifuged and centrifuged suspensions. One drop of the various preparations 

 is placed upon a slide covering just 1 sq. cm. ; the preparations are dried in an oven 

 and are rapidly covered with a very dilute agar solution. One per cent warm 

 agar solution is best for the first two sediments and 0.1 per cent cold agar solution 

 for the third sediment. For the suspensions, no fixative is necessary. When the 

 agar is dried, several drops of absolute alcohol are used for fixing and the prepa- 

 ration is stained by means of a solution of an acid dye in 5 per cent phenol solution. 

 Rose bengal may be used, but its action is prolonged, followed by a drop of acetic 

 acid, then washed. Extra erythrosine in 5 per cent phenol solution is superior. 

 The bacterial cells are colored, but not the capsules and mucus; this is especially 

 true of the compact colonies as those of Nitrosomonads and other soil forms 

 which so readily over-color with basic dyes; the colloids are only faintly colored; 

 the agar is readily discolored by the process of washing with cold water. The 

 dye is allowed to act 5 to 15 minutes in the cold or on slight warming, then washed 

 a few seconds in water. 



The preparations from the first sediment are usually free from bacteria, except 

 in soils rich in organic matter, when some of the particles are not removed by 

 three washings. The second preparation shows on examination the same mi- 

 crobes, qualitatively and quantitatively, as the third sediment, where conditions 

 for examination are most favorable. The fourth preparation made from the 

 suspension is usually most instructive. The living cells only take the stain, 

 while the spores stain only very faintly or not at all and can be seen only when 

 present in large numbers. Protozoan cysts are recognized by their intense 

 coloration and can easily be counted. 



Winogradsky suggested to use always for comparison a control soil, 

 which had no addition of fresh organic matter for a considerable period 

 of time. A normal arable soil contains a native or autochtonous flora 

 consisting of short bacteria with rounded ends and of cocci, 1 to 1.5/z in 

 diameter. Often larger forms, 1 to 3/x in diameter, resembling Azoto- 

 bacter are found. They group into rounded colonies consisting of 

 about 100 cells in a compact mass with a common capsule, but occa- 

 sionally with as few as a dozen individuals (PI. I). The field between is 

 completely devoid of microbes. The colonies are situated on the soil 

 colloidal matter. This is the reason why the centrifuged suspension is 

 practically free from colonies which are carried down by the flakes of 

 organic matter. Spore-bearing bacilli, filamentous bacteria, spirals, 

 mycelial filaments, actinomyces, and protozoan cysts are absent 

 or are very rare. The presence of these indicates that the soil is in an 

 active state of fermentation, due to recent addition of organic matter. 



