ene green } in 0.5 per cent. aqueous solutions.
Vesuvin, }

_Negative Stain_ (Burri).--By this method of demonstration the
appearances presented by dark ground illumination (by means of a
paraboloid condenser) are closely simulated, since minute particles,
bacteria, blood or pus cells etc. stand out as brilliantly white or
colourless bodies on a dark grey-brown background.

_Reagent required:_

Any one of the liquid waterproof black drawing inks (Chin-chin, Pelican,
etc.). This is prepared for use as follows:

Measure out and mix:

Liquid black ink, 25 c.c.
Tincture of iodine 1 c.c.

Allow the mixture to stand 24 hours, centrifugalise thoroughly, pipette
off the supernatant liquid to a clean bottle and then add a crystal of
thymol or one drop of formalin as a preservative.

METHOD.--

1. With the sterilised loop deposit one drop of the liquid ink close to
one end of a 3 by 1 slide.

2. With the sterilised loop deposit a drop of the fluid culture (or of
an emulsion from a solid culture) by the side of the drop of ink (Fig.
69, a); mix the two drops thoroughly by the aid of the loop.

3. Sterilise the loop.

4. Hold the slide firmly on the bench with the thumb and forefinger of
the left hand applied to the end nearest the drop of fluid.

5. Take another clean 3 by 1 slide in the right hand and lower its short
end obliquely (at an angle of about 60 deg.) transversely on to the mixed
ink and culture on the first slide, and allow the fluid to spread across
the slide and fill the angle of incidence.

6. Maintaining the original angle, draw the second slide firmly and
evenly along the first toward the end farthest from the left hand (Fig.
69, b).

7. Throw the second slide into a pot of disinfectant; allow the first
slide to dry in the air.

[Illustration: FIG. 69.--Spreading negative film.]

8. Place a drop of immersion oil on the centre of the film, lower the
1/12-inch objective into the oil and examine microscopically without the
intervention of a cover-slip.

(The film of ink may be covered with a long cover-glass and xylol balsam
as a permanent preparation.)

(<b) _Hanging-drop Preparation._--

1. Smear a layer of sterile vaseline on the upper surface of the ring
cell of a hanging-drop slide by means of the glass rod provided with the
vaseline bottle, and place the slide on a piece of filter paper.

2. "Flame" a cover-slip and place it on the filter paper by the s