The material used in the work came from abattoir animals, bull calves
and adult bulls raised in the department herd, and sires upon which
clinical observations had been made by various veterinarians in the
field. Semen samples, many of which were sent in, were collected as
often as possible after the method described by Williams (16). The
genital organs were removed with as little chance of contamination as
possible, and taken or sent to the laboratory where the examinations
were made soon after arrival.
All cultures were made by searing the surface carefully, tearing out a
small portion of the tissue with sterile forceps, and placing it upon
the media. In most cases, however, where fluids were present, tubes were
inoculated with the material which had been drawn off with a sterile
pipette. As stated by Carpenter (9), in his work on the female genital
tract, the organisms usually live in the depths of the tissue. The media
used principally were glucose glycerin agar (glucose 1 per cent,
glycerin 3 per cent); plain agar, both with a pH value of 7.4, and
Loeffler’s blood serum. Small amounts of sterile blood serum or
defibrinated blood were added to most of the agar slants to insure
better growths of streptococci when present. All tubes, to which the
serum had been added, were incubated for forty-eight hours before
inoculation to insure absolute sterility.
After inoculation, the agar tubes were sealed with sealing wax to give a
partial oxygen tension which was quite necessary in isolating the
streptococci. The growth of other organisms was by no means hindered by
the procedure, for one tube from each organ was often left unsealed.
Incubation was at 37° C, and the routine method of examining the tubes
was identical with the method of Carpenter (9).
Whenever possible, a sample of blood was obtained from the animal either
before, or at the time of slaughter, for agglutination with _Bact.
abortum_ antigen. Extracts from the seminal vesicles, testes, and
epididymes were injected into the guinea pigs and examined at the end of
four to six weeks for the presence of _Bact. abortum_.
Sections of all organs were fixed as soon as possible in either Zenker’s
or Helly’s fluid. Hematoxylin and eosin were used as routine tissue
stains. Eosin and methylene blue, and Mallory’s connective tissue stain
were, however, frequently utilized for special staining reactions.
The motility of the spermatozoa is best observed about half an hour
after ejaculation, when the thick tenacious clot has started to liquefy.
A drop of the fluid is placed upon a warmed slide, preferably one with a
slight depression in it, and observation made with high or low powered
objectives. The semen may be examined whole, or diluted with
physiological saline solution. In the latter case, the sperms have a
greater opportunity for freedom of motion in the absence of the thick
viscid coagulate. A small vial of saline solution may be carried in
one’s pocket where it will be kept warm, and a drop of this placed upon
the glass slide. If the clot of semen is merely touched to this drop on
the slide, plenty of spermatozoa will be deposited for an examination.
This method is very satisfactory for the observation of motility, but
needless to say, the undiluted semen must be used for the determination
of the number of sperms present. If necessary, the specimen may be
covered with a cover glass and the oil immersion objective used. While a
warmed slide is quite sufficient to enable one to detect the presence of
motility, the field soon cools and the sperms gradually become less
motile. If possible, it is best to use a small electrically heated stage
warmer, which keeps the field at a constant body temperature, so that
the duration of the motion may be observed for hours if warmed
physiological saline solution is added as the fluid evaporates.
Stained preparations are best made with _thin_ smears on the glass
slides. This is conveniently done by placing a drop of the semen on a
slide and smearing it over the surface with the edge of another slide. A
fairly thin and even field is thus obtained. A still better method is to
first dilute the semen with physiological saline solution, so as to
obtain fewer sperms in the field. After drying the preparation in air,
fixation may be produced by drawing the slide through a gas flame
several times, by immersion in equal parts of alcohol and ether, or even
by the use of tissue fixers such as Helly’s or Zenker’s fluids. For
ordinary staining, heat fixation is the quickest, and at the same time
is quite satisfactory. After the slide is cooled, or washed, to remove
the fixing solutions, it should be placed for a few minutes in a
_freshly_ prepared solution (1 per cent) of chlorazene, as recommended
by Williams, to remove the mucus and proteid material which otherwise
blur the field. Other authors (38) have recommended diluting the semen
with about twenty volumes of a 0.12 per cent sodium carbonate solution
in 0.8 per cent sodium chloride. From this liquid the cells should be
centrifuged for several minutes, removed with a pipette, and smeared on
the slide. Following this, the slide should be thoroughly washed,
preferably for ten minutes in running water, after which it is ready for
staining. Numerous methods have been used for this procedure, but the
sperms are more or less erratic in their reactions to the dyes, and one
must be very careful to use the same method in all samples, in order to
obtain uniform results. For quick staining, to bring out gross
abnormalities of structure, and number of sperms present, one may use
Gram’s stain, or a light stain with any of the aniline dyes, such as
fuchsin. To bring out the finer structure, particularly of the head,
more careful technic must be employed.
Carnett and others (38) recommend the following: “The method of staining
by iron-hematoxylin, particularly when supplemented by a cytoplasm
stain, has proved, on the whole, the most satisfactory, and possesses
the additional advantage of being absolutely permanent, a quality that
few anilines can boast. The method consisted of treating the fixed
object—and here the fixing agent was heat—with a two per cent solution
of iron-alum for from two to four hours. The excess of iron-alum was
then _completely_ removed by pure water, and the object treated with a
solution of hematoxylin (one per cent aqueous) for twelve hours or
longer. The cells by this time were perfectly black. However, a 1 per
cent solution of iron-alum removed the stain from the cytoplasm, leaving
the chromatin of the head, the centrosome, and the axial filament a
brilliant blue-black. Care must be taken that the preparation is not
over-decolorized. After decolorization a saturated aqueous solution of
eosin was added for from one to three minutes. This stained the
protoplasmic envelope pink, and, unless the envelope is overstained, the
view of the inner structures is not impaired in the least.”
Williams (17) recommends using two staining solutions, one of alcoholic
eosin and fuchsin, the other a diluted methylene blue. The results
obtained are, however, more or less erratic, due to the unstable
character of the former stain, and the ease with which one may over or
under stain. Many beautiful specimens may, nevertheless, be obtained by
this method. I have frequently used a fairly quick method, though one
not satisfactory in all cases, which consists of staining for five or
six minutes in a saturated aqueous solution of fuchsin, washing in
water, and counterstaining for a few seconds in a strong solution of
methylene blue. A quite satisfactory method is to stain from two to five
minutes in a saturated aqueous solution of methyl green, with the
application of gentle heat. The heat may be applied by warming the slide
over a gas flame as it steams, or by placing the jar containing the
stain in a hot water bath. The slide is then washed thoroughly and
counterstained for five minutes in a strong aqueous solution of eosin.
This is a fairly reliable method, and many excellent preparations may be
obtained by its use. The nucleus is stained green, the anterior part of
the head and all of the tail pink. So far, I have found this a very
reliable stain for routine work.