Reprinted from the New York Medical Journal 
for March 2, 1895. 
SOME SUGGESTIONS 
IN BACTERIOLOGICAL TECHNIQUE. 
A. P. OI1LMACHER, D., 
CLEVELAND, OHIO. 
The following subjects are here considered: 
I. A simplified method of making a bacteriological au- 
topsy on small animals. 
II. The use of methyl violet in staining the diphtheria 
bacillus. 
III. A note on methylene blue. 
I. 
The object of these modifications in the technique of 
bacteriological autopsies on the small animals used in ex- 
perimental laboratory work is to do away with certain 
troublesome features of the ordinary methods. As prac- 
ticed by the usual methods, a properly conducted bacte- 
riological autopsy is a tedious task, especially in those cases 
in which pieces of the solid organs are required as mate- 
rial for culture experiments. It is necessary to have at 
hand several sets of sterilized cutting instruments-a set 
for each particular organ-or the instruments of a single 
Copyright, 1895, by D. Appleton and Company. 2 
BACTERIOLOGICAL TECHNIQUE. 
set must be heated to a damaging degree in the free flame 
before and after the removal of each piece of tissue. 
The novel feature of the modified method here proposed 
lies in the use of ordinary commercial benzene as the disin- 
fecting agent, both in solution and in ignition. The ben- 
zene is employed as a bath for the instruments used in 
the autopsy, and also to disinfect the surface of the ani- 
mal's body before the final incision. Benzene was chosen 
as the agent for these purposes because of its cheapness, its 
active disinfecting power, and its property of burning off of 
the surface of metallic or other substances without creating 
a great amount of heat. The details of an autopsy per- 
formed with the aid of benzene are as follows: 
Two small artery catch forceps (Pean's forceps), a me- 
dium-sized dissecting forceps, and a pair of medium straight 
dissecting scissors are placed in a suitable beaker glass two 
thirds full of benzene. An extra dish of benzene and a 
medicine dropper are also provided. 
The animal (mouse, rat, guinea-pig, rabbit, etc.) is se- 
cured on its back to the dissecting board by tacks driven 
through the extended fore and hind legs. The skin of the 
ventral surface is incised in the median line from the sym- 
physis pubis well on to the neck; lateral cuts are made on 
both right and left sides at the cephalic and caudal extremi- 
ties of the median incision. The flaps of skin are carefully 
dissected from the underlying tissue throughout the entire 
length of the primary incision so as to thoroughly expose 
the subcutaneous tissues of the thorax and abdomen; then 
the flaps are secured with tacks so as to be well stretched 
out on each side of the body. This primary dissection is 
done with a coarse dissecting forceps and a pair of 
straight scissors, no particular efforts being made to avoid 
contamination of the exposed surfaces. Care must be 
taken, however, in this dissection that only the skin is re- BACTERIOLOGICAL TECHNIQUE. 
3 
moved, and that the abdominal and thoracic walls are not 
penetrated. With the medicine-dropper benzene is now 
applied to the exposed ventral wall of the body. The ben- 
zene is then ignited and allowed to burn until the flames 
spontaneously subside. Should any particular area not ap 
pear properly singed, the benzene may be again applied to 
this spot and set afire. Some care must be taken to keep 
the benzene from running over the flaps and into the hair 
of the animal, for here the fire does not subside so harm- 
lessly as on the exposed moist surfaces. The dissecting 
scissors and forceps are now removed from the dish of 
benzene, and the benzene clinging to them is ignited and 
allowed to burn off. A fold of the abdominal wall in the 
median line is raised with the forceps and cut through with 
the scissors so as to avoid the underlying organs. As soon 
as a moderate cut has been made the edges of the incision 
are grasped with the artery forceps which have been flamed 
on their removal from the benzene; then the incision is 
continued down to the pubes and up through the thoracic 
cavity in the middle line, while the abdominal walls are 
held well up with the artery forceps. The artery forceps 
are now changed so as to grasp the edges of the deep cut 
at the diaphragmatic border, while the diaphragm is cut on 
each side so as to allow the incised ventral walls to be held 
well apart, exposing freely the thoracic and abdominal con- 
tents. If an assistant is available, the artery forceps can 
be intrusted to him; but in the absence of an assistant the 
operator can secure a comfortable exposure of the abdomi- 
nal and thoracic organs by making lateral incisions at the 
ends of the deep incision, when the weight of the artery 
forceps will usually be sufficient to hold the walls well 
apart. The scissors and forceps are now wiped free of 
blood and tissue, replaced in the benzene, then removed 
and the adhering benzene ignited, and the removal of 4 
BACTERIOLOGICAL TECHNIQUE 
pieces of the organs desired for bacteriological study is 
begun. Of course, it will be necessary to sterilize the 
forceps and scissors as each particular organ is dealt with, 
so as not to carry infectious material from one organ to 
another. All that is necessary for sterilization is to wipe 
the instruments, plunge them a moment in benzene, and 
burn off the benzene. It will also be necessary, especially 
in early autopsies, to avoid contamination of the remaining 
organs from the blood which flows from those already cut. 
This can be avoided by following a proper order in attack- 
ing the various organs, depending, of course, on the object 
of the examination. 
From a considerable practical experience with this meth- 
od, the author is ready to conclude that it is simpler, more 
cleanly, more expeditious, and more saving of instruments 
than the usual methods of bacteriological autopsy. The 
proportion of accidental contaminations after this method 
is certainly smaller than I have ever obtained with any other 
procedure, and this occasional contamination is doubtless 
due to air infection and not to imperfect sterilization of 
instruments and body surfaces. Control experiments with 
culture media, made after benzene sterilization of scissors 
and forceps, show the method to be a safe one. Perhaps 
this contains a suggestion for surgeons, especially in emer- 
gencies when ordinary sterilizing appliances are not on hand. 
II. 
A very powerful and brilliant stain for the diphtheria 
bacillus, and one which strikingly shows the irregularities 
of the "typical" form, may be obtained by the use of 
methyl violet. The aniline which I have employed was ob- 
tained from Grubler, and is specified by him as methyl 
violet, 5 B. It is important to obtain this particular brand. 
To make the staining solution a saturated alcoholic BACTERIOLOGICAL TECHNIQUE. 
5 
solution of the dye is added to water in the proportions of 
about one to ten. The exact proportion is not a matter of 
great moment, and a little experimenting will settle this 
question, and enable any one to make the staining solution 
at a moment's notice. This mixture keeps remarkably 
well. 
The technique of staining with this solution differs in 
no essential feature from the common method practiced by 
bacteriologists. A fixed cover-glass preparation from the 
diphtheria culture is stained, with heat, for half a minute, 
then washed with water, dried, and mounted. Or the stain 
may be employed without heating by allowing it to remain 
in contact with the film of the cover-glass preparation for 
one minute, though the results are not quite so good as in 
the heated specimens. In any case the resulting prepara- 
tions are stained with a brilliancy and beauty that quite 
surpass the more commonly employed stains, like Loeffler's 
methylene blue. 
III. 
For some time I have had considerable trouble in mak- 
ing a methylene-blue solution which would stain bacteria in 
a satisfactory manner. The stain was always too faint and 
imperfect to be satisfactory. This was true of the aqueous 
solution, Loeffler's alkaline solution, Gabbet's decolorizing 
solution, and carbolic acid solution, of methylene blue. 
Finally, settling upon the aniline as the source of the trou- 
ble, I tried other brands, and discovered that an aniline 
which is recommended for another purpose was remarkably 
well suited to bacteria staining, while the dye recommended 
for bacteriological use was unsatisfactory. In these experi- 
ments I have confined myself to the use of Griibler's ani- 
lines. The unsatisfactory methylene blue is the brand which 
he recommends for bacteria staining (Methylen Blau fur 
Bacillenfdrbung, Koch), while the blue which gave positive 6 
BACTERIOLOGICAL TECHNIQUE. 
results is the one proposed by Ehrlich for blood work, espe- 
cially for staining intra vitam (Methylen- Blau nach Ehrlich). 
The Ehrlich blue is considerably more expensive than the 
blue after Koch, but its solutions are much more powerful as 
staining agents, so that in the end it is an economy to use 
this brand when methylene blue is called for. As the same 
trouble has occurred in three different lots of aniline ob- 
tained from Griibler in original packages, I am sure the 
failure is not accidental. 
Bacteriological Laboratory, Medical Department, 
University of Wooster.