34 General Bacteriology. 
Inasmuch as the first plate is invariably too thickly seeded to be of much service, 
this gelatin tube is often replaced by a water blank, which is treated exactly as the gela- 
tin tube No. 1, but is not of course ‘‘plated’’ but simply serves to dilute the material. 
Rererences. A. 124; H.57; L. & K. 88; M. & W.108; M.& R.61; MeF. 140; 
N.171; P. 224; 8S. 72. 
SPECIAL DIRECTIONS. 
a. Make three gelatin plate cultures, as directed above, and inoculate with B. sub- 
tilis, introducing a minute portion of agar culture (XIII) into tube No. 1, two loops of 
No. 1 into No. 2, and three of No. 2 into No. 3. Label, and when the gelatin has solidi- 
fied, place plates in cool chamber (XV). 
b. Also make a “‘blank’’ plate from an uninoculated gelatin tube, observing all pre- 
cautions to prevent contamination. This will serve as a control or check on your other 
plates. If any colonies develop on this it indicates carelessness. 
EXERCISE XXVIII. AGAR PLATE CULTURES. 
GENERAL DIRECTIONS. These are made in the same way as the gelatin plates ex- 
cept that the high melting point (96° C.) of agar makes it necessary to use boiling water 
to melt it. Inasmuch as the vitality of vegetative bacteria is destroyed at a temperature 
. much above 42° C., it must be cooled down before inoculating, but as agar solidifies at 
39-40° C. it must not, therefore, be cooled below that point. It is best to keep the melted 
agar at about 42° C. for 10 minutes before it is inoculated. For this purpose a water- 
so bath should be so arranged that the temperature can be controlled 
by means of a thermo-regulator. A cheap and yet satisfactory 
arrangement is represented in Fig. 11. Inoculate, make dilu- 
tions and pour as in case of gelatin, except that before the agar 
is poured, it is well to slightly warm the Petri dishes by placing 
them in the incubator at 38° C. for a few minutes, other- 
wise the agar may solidify in lumps in the plate. In cooling, 
agar shrinks somewhat, and in doing so water is expressed from 
the solid jelly. In the incubator this condenses on the under 
side of the cover of the Petri dish to such an extent that drops 
run down on to the culture surface thus causing the developing 
Pred, Wher eth, ir codiice superficial colonies to ‘‘run.’’ To obviate this the Petri dishes, 
agar. when placed in the incubator, should be inverted. 
REFERENCES. H. 61; L. & K. 94; M.& R. 66; N. 285; P. 225; P. B.C. 28. 
SpeciaL DirEcTIONS. a. Make three agar plates of B. coli; useone loop of bou- 
illon culture (XIII) for tube No. land proceed as in XXVI. 0b. Place in incubator at 
28° C. inverted. 
EXERCISE XXVIII. ROLL=CULTURES (Esmarch). 
GENERAL DrrEcTIons. These are essentially plate cultures in which the medium 
instead of being poured out into dishes is solidified in a thin, even layer on the inner surface 
: of the test-tubes. This is best accomplished by means of a 
piece of ice placed in a dish on a piece of cloth by which it 
can be kept in the desired position (Fig. 12). A horizontal 
groove is melted in the ice by means of a test-tube filled with 
; hot water. In this groove the test-tubes, inoculated as in case 
ree ee is of plate cultures, are rapidly whirled until the medium is thor- 
oughly set. Both agar and gelatin can be used, although gelatin cannot be used suc- 
