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TABLE IV.-TESTS COMPARING ASPARAGINATE-GLYCERIN AGAR WITH PLAIN ASPARAGINATE AGAR AND WITH GELATIN.

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* These are the only two counts on gelatin which are higher than the corresponding counts on asparaginate agar.

†The higher agar count in each test is printed in bold-faced type. These gelatin counts are inexact because of rapid liquefaction.

TABLE V.-COMPARISON OF GELATIN WITH ASPARAGINATE-GLYCERIN AGAR.

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* The higher count in each test is printed in bold-faced type.
† These gelatin counts are inexact because of rapid liquefaction.

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* The higher count in each test is printed in bold-faced type.

† These gelatin counts are inexact because of rapid liqueïaction.

8

†12,000,000

18,000,000

15,000,000

26,000,000

8,000,000

20,000,000

22,000,000

27,500,000

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23,000,000

25,000,000

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10,000,000

12,000,000

PLATING SOIL FOR QUALITATIVE PURPOSES.

Advantages of gelatin.-In making a study of any bacterial flora, it is very important to have a medium that is satisfactory for qualitative as well as quantitative purposes. A perfect medium for qualitative work would be one upon which all the kinds of bacteria present are able to develop, each kind of organism producing a colony easily distinguished from all the other kinds. As a perfect medium is out of the question at present, the best to be hoped for now is one upon which the important soil organisms are able to grow, and on which the colonies of the most common types can be recognized at a glance. If such a medium were available, it would be a fairly easy task to count the number of colonies of each type present, then isolate and study cultures made from the unknown colonies, thus obtaining a satisfactory knowledge of the flora of the soil investigated.

Needless to say, no medium has yet been found that is satisfactory from this point of view. Gelatin has been used most frequently in the present work, because of the ease with which certain common soil forms are recognized by means of their gelatin colonies. Nearly all the spore-forming bacteria common in soil can be recognized on gelatin plates after a little experience. Of the non-spore-formers, certain rapid liquefiers, such as Ps. fluorescens, produce very characteristic gelatin colonies. An even greater advantage of gelatin is the ease with which even the most minute colonies of the Actinomycetes can be distinguished (with a low-power microscope) from the colonies of lower bacteria.

The scheme on page 31 will serve as a guide to help in distinguishing the different kinds of bacteria from their gelatin colonies. It must be remembered, however, that only the typical forms of colonies are included in this classification, and nearly all the organisms in question occur frequently in atypical forms.

The best concentration of gelatin to use for qualitative purposes is less than that recommended for quantitative work. The concentration used should vary according to the temperature available for incubation. If 18° C. is available, 12 per ct. Gold Label gelatin is generally satisfactory, or if that liquefies too rapidly, then 15 per ct.; while if a temperature of 20 or 21° must be used, 18 per ct. Gold Label gelatin proves best. The concentration used should be just great enough to prevent the rapid liquefiers from spreading over the entire plate during a seven-day incubation. If the concentration is less than this the plates may be lost through liquefaction; while if it is greater some of the rapid liquefiers are suppressed entirely which of course gives a false idea as to the relative abundance of the different kinds of bacteria in the soil. U. S. Glue Co. gelatin is not satisfactory for qualitative purposes, because

CLASSIFICATION OF GELATIN COLONIES.

A. 4-day colonies generally over 1 cm. in diameter; liquefaction rapid.
1. Colonies with rhizoid to filamentous edges.
B. mycoides Flügge
(Under certain circumstances, however, B. cereus develops colonies
of this type.)

2. Colonies with smooth edges (macroscopically), with a heavy pellicle that
is granular and generally concentrically ringed. B. cereus Frankland
3. Colonies with entire edges, structureless, sometimes fluorescent but
generally consisting only of a clear liquefied zone, round to amoeboid
in shape, and often covering the entire plate by the seventh day.
Ps. fluorescens (Flügge) Migula

4. Large liquefying colonies other than the above often occur; but generally
belong to the less common spore-formers, or to forms closely related
to Ps. fluorescens.

B. Colonies seldom over 1 cm. in diameter even on seventh day.

1. 7-day colonies generally 2-8 mm. in diameter, consisting of a white
opaque flocculent center usually surrounded by a clear zone of liquefied
gelatin.
B. megatherium De Bary
2. Colonies generally punctiform, and only rarely over 2 mm. in diameter.
Viewed with low-power microscope, they have entire edges.
The non-spore-formers (not including
the Ps. fluorescens group).

3. Colonies punctiform to 3 mm. in diameter. Of tough consistency, so
that the entire colony can be picked out of the medium without losing
its shape. Viewed under low-power microscope, edges are filamentous,
radiate. Colonies are often surrounded by a broad zone of brown
pigment.
Various types of Actinomyces

4. Some small liquefying colonies without characteristic appearance often
occur that are really atypical forms of B. cereus, B. megatherium, Ps.
fluorescens or of some of the less common organisms.

the colonies of Actinomycetes cannot be distinguished on it as readily as upon either of the other two brands. Bacto-gelatin may be used with as good results as Gold Label gelatin; but as it has a higher jellying power, a lower concentration must be employed. The best concentration of Bacto-gelatin for qualitative work proves to be about 10 per ct.

Gelatin, however, is far from perfect for qualitative work. This has become especially apparent since the comparative insignificance of the spore-forming bacteria in soil has been realized. Although the different spore-formers can generally be recognized by their gelatin colonies, the other soil micro-organisms (with the exception of the Ps. fluorescens group) all produce non-characteristic colonies. The Actinomycetes can be distinguished from the lower bacteria, but not from each other; while all the Bacterium forms (together with a few Coccus and Pseudomonas forms) produce punctiform colonies that look exactly alike. These two groups together constitute about 90 per ct. of all the colonies on the plates. There is actually a great variety of Actinomycetes in soil, and quite likely an equal variety of lower bacteria that produce punctiform colonies on gelatin, all of which cannot be distinguished from each other on gelatin plates; so it is plain that gelatin is not entirely satisfactory for qualitative work.

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