Study on the Cleaning of Tuberculous Cavity
by Artificial Contamination

First Report. Experiments on Bacillus subtilis
By
Shusuke TSUJI and Yasushi NAMIKAWA *(Received April 6, 1951)

Introduction.

1. The significance 0/ the contamination in tuberculous cavity.
It is true that the contamination of cavity exerts sometimes very bad influe-nces upon pulmonary tuberculosis.
But it is too much emphasized in general.
On the contrary, as DR. SATA already proved (l899), the contamination accerelates remarkably the softening and the removing of caseous substances intuberculous cavities and thus the inner walls of cavities can be easily cleaned.
Therefore, if contaminated microorganisms are adequate, it may be -posaible
that the contamination promotes the cleaning of tuberculous cavities without
destruction.
On the other hand, when we examine the contents of tuberculous cavities
in living bodies, contamination is surprisingly very rare.
We assume thatin living bodies tubercle bacilli may hinder the growth of other microorganism.
This may be one reason of the difficulty of the cleaning ot tuberculous cavities.
We expect that the contamination with organisms, which are almost harmless
to living individuals and can grow saprophitically in caseous substances andexcrete antituberculous substances, will be convenient to the healing of tuberculous cavities.
"From the 5th Division (Chief: SHUSUKE TSUJI) of Tuberculosis ResearchInstitute, Kyoto University.

The Cleaning of Tuberculous Cavity

2. Study on Bacillus subtilis group.
As the first step we take up B. subt illis group.
The reasons are as follows 1) Strains, which arc capable of producing antituberculous substances,have been often detected within subtilis group, for examples Subtilin, Subtenolin and others.
2) It is assurred, that B. subsilis has no pathogenicityagainst human body. 3) As this group is vel-y sensitive to penicillin or streptomycin, we can easily remove those bacilli supposing they exert p rthogenicity to human body.

A.

Fundamental experiments

Exoeriment 1.
bacilli.
Method:Selection of strains,which affect antibiotically to tubercle

Slide culture method W2,S used in exper irnents of antagonism ofB. subtilis and tubercle bacilli.
Technical managements were following.
1) Slide glasses, which had been previously cut in half along their longitudinal axes and had been disinfected by dry heating, were put in an aseptic
glass shell and were dried for 30 minutes in an incubator.
2) They were treated for 20 minutes in an bottle filled with 5;?; sulfuric
acid solution of 37'C.
(in order to remove contaminating bacilli in sputa.)3) Sulfuric acid was washed away twice through two bottles containing
sterile distilled water.
4) Then cultivation in ordinary test tubes.
Culture media were KIRCHNER'S media (10 % goat serum involved.)
5) After 24 hours cultivations, cultures were contaminated with a loopfulof test bacilli.
6) After 6 days cultivation of tubercle bacilli slide glasses were taken out
from culture media, and were" washed with 10 % formalin solution.
Thus
culture were fixed and disinfected.
7) Dried in room temperature.
Stained with Ziehl-Neelsen.
Microscopic examination.
We compared these slides with control slides,
which were taken out from culture media after 24 hours cultivation without
adding contaminations, and select slides, in which the growth of tubercle
bacilli were completely inhibited.
Results obtained:
202 strains of subtilis group were isolated and examined.
34 strains were capable of inhibiting tubercle bacilli by antagonisticcultivations.

Experiment 2.

Selection of strains, which were not pathogeneous to animals but were able to live saprophitlcally in animal tissues.
Method:Male mice of about 15g body weights were inoculated intra-

Shusuke TSUJI and Yasushi NAMIKAWA

peritoneally with 0,5 ml of bacillary saline suspensions, which' involved ~~oloopful of 24 hours cultivation of B. subtilis on nutrient agar.
After 20 daysanimals were killed and bloods and peritoneal fluids were examined to detect
living subtilis bacilli by culture methods.
At the same time pathologicalchanges in intraperitoneal organs were observed with naked eyes, and theseorgans were fixed and preserved in formalin solution.
Results obtained:
Experiments were performed with 23 strains, which
had been "elected in the first experiment.
10 strains were selected.
Animals, which were inoculated with these strains, increased their body weights
until slaughter.
From peritoneal fluids of these animals living bacilli weredetected, but not from bloods.
No pathological changes were recognized in
intraperitoneal organs of these animals.
Adding experiments:
After following third experiment we tested again
on 5 selected strains in guinea pigs by the same methods as this chapter andconfirmed that no pathogenicity were recognized.

Experiment 3.

Selection of strains, which are sensitive to penicillin.
Method:One loopful of 4 days culture on nutrient agar were suspendedin 2 ml of distilled water.
One loopful of this suspension were inoculated
into diluted broth, previously mixed with penicillin.
Penicillin contents were1.5, 0.5, 0.1, 0.01 and 0.005 units in 1.0 ml of media.
After 18 hours and 5 days
results were decided.
Results obtained:
Aften 18 hour s cultivations 5 among 10 strains, which
had been selected in the second experiment, were inhibited their growth in 0.05
units penicillin.
After 5 days cultivations every strain grew in 1.0 units
penicillin solutions.

Experiment 4.

Selection of strains, which are capable of growing incontents of tuberculous cavities.
Method:Each 1 ml of content of tuberculous cavity, which was collected
aseptically from a patient and was not contaminated, was poured into disinfected small glass tubes.
Add to each these glass tubes one droplet of highly
diluted emulsion of test bacilli and stir to mix homogeniously,
At once cultivate a loopfulof this emulsion on a nutrient agar.
Then glass tubes wereincubated at 3TC.
After 24 hours incubation one loopful of these emulsions
was smeared on nutrient agar.
It was decided from the numbers of colonies,
whether test bacilli had increased in the contents of cavities after 24 hours
incubations or not.
Results obtained:
Until now we have examined 17 strains, which wereselected by the first experiment.
None of these strains could grow very
good in the contents of cavities.

.17

The Cleaning of T'uberculous Cavity-----------~._~~-~

Discussion and summary:--------------~_._-.._----_.-

From fundamental experiments 1 ... 3, we 'suc-ceeded to select [) str airis of R.· subt ilis,

which were assumed to be useful foe

the artificial contamination of tuberculous cavities.
As for the forth experiment, it was performed just afte r following clinical experiment.
We havelearned that this forth experiment was very important as a fundamental experiment, because the results of this experiment· .... B. subtilis did not easily
grow in the content of cavity······ agreed very good with the results of clinicalexperiment .... B, subtilis, which had been inoculated int the cavities of patients, disappeared relatively early from the sputa of patients.

B. Clinicai experiments
Clinical experiments were performed with a strain S17,
previously selected in the fundamental experiments 1 ... 3.
Method:In 4 patients experiments were perfor rned.

one of 5 strains
All these patients

had very serious pathological changes in both lungs and cavities were giant(larger than 6 ern in diameter) in each patients.
Therefore, every patient
was too far advanced to be treated by collapstherapy.
Sputum Quantities
were almost 50 ml a day.
Tubercle bacilli in sputa were found as Gaffky
3 -- 8.

Temperatures were normal in most cases,

but in a very serious

case temperature was inclined to be above 37°C, and sometimes temperature
exceeded 38°C.

0.5 or 1.0 ml of saline suspensions ot B. subtilis (5 mg or 10mg wet weight of bacilli) were inoculated slowly into cavities by large needles.
Results obtained:

Summary of results,obtained from 7 times inocula-tions in 4 patients were following.
1. Body temperature:
In 3 cases temperature raised soon after inoculation with slight chill or without chill
ture reached to 38

CCor moreOn the first or second day tempera-

but without any treatment it became normal

after one week.
In another case no reaction of temperature was observed.
2. General conditions:Appetite decreased, when temperature raised,
but it became normal soon after removal of fever.

There were no otheruncomfortable reactions than those, mentioned above.
3. Cough and sputum:
There was no effect upon cough, except in one
case,in whom cough became seldom by reason of reduction 01 viscosity ofsputum.
4. Tubercle bacilli in sputum:

In one case the number of tubercle bacilli

increased temporarily and there were clumps of bacilli.

Therefore,

to be as if the cleaning of cavity wall had taken place,but this was a tempo-rary phenomenon.

it seemed

In other cases there were no remakable changes.

5. B. subtilis in sputum:
Although these bacilli were found in sputa
4 days after inoculations, gerierally they disappeared and could r.ot l.e dete-

.18

Shusuke TS['!1 and Yasushi NAMIKAWA

cted also in cultures alr-eady after 7 days.
Discussion and summary :As the clinical experiments .began just early
and treated cases were few and tested .bacillus was ,only one str.ain, it may .betoo .early to reach the conclusion.
But as for only S17 strain it .seerns to be
impossible to grow in human body.
Therefor-e, there should be almost .nopossibility toacquireu .good clinical result.
.On the other hand pretty strong reactions occured in the tceatments withbacilli as saprophitic as these subttliabacilli, when their quanttttes were verylarge.
Morover, ,go04 result may beexpectedwhenbaciUi, which ad~pt
easier to human body than those bacilli used above, are found.
Therefore,
we should be more careful in future experiments.
We desire to continue thepreceeding forth experiment using many other stt:C\ins of s"btilisgr~p andto discover better strains.
On the other .hand, we will soon ~in <thetqndamental experiment using -microonganisrns .other -than aubnilis .bacilli.