ACTA TUB~RCULOS~A JAPONICA
Number 1

June 1951

Volume 1

Two Kinds of Culture Methods for the Early
Detection of the Tubercle Bacilli.

First Report. Cultivation from Sputa.
By

Saburo UYEDA, (jsamu YAMADA and Hiroshi OKADA*fiR fIl .=: llIr,

111 fIli~,(Received MarchJJqJ fIlm10, 1951)

Iuteoduction,
It llU1Y be one of the we ak points of the culture method on egg slants that

more or less long period of time is necessary to see the development of thetubercle bacilli.
Many workers tried, using various methods, to obtain theculture as fast as possible.
Above all the method of BERRY and LOWRY!) reported recently in U. S. A. is thought to be interesting.
It is, however, rather difficult to carry out this method and one is still obliged, even throughthis method, to spend from one week to ten days to wait for the the development of the tubercle bacilli.
KIMURA and MIZUN0 2 ) reported recently in ourcountry a similar method which involves also the same difficulties as theformer.
Two kinds of culture methods which enabled us to observe the tuberclebacilli just beginning to develop were described below.
Through these methods one could detect the tubercle bacilli after only two or three days of incubation.
Exper-iments.

1.

The Slide Culture Method in the il10ist Chamber,""Fro:11 the Bacte riologic al Lnbor ato ry (Chief; S. UYEDA) Tuberculosis ResearchInstitute, Kyoto University.

.2

Saburo UYEDA, Osamu yAMADA and Hiroshi OKADA

Materials and methods.
70 samples of sputa were examined, of which the smears stained with theZiehl-Neelsen showed none of acid-fast bacilli.
About 1 cc of each sputum
was put Into the centrifuge tube and was homogenized with five volumes of
4 % solution of sodium hydroxyde,
After being kept for 20 minutes in anincubator, the tube was centrifuged strongly for 20 minutes.

The super-natant fluid was poured off, and then certain volumes of sterile destilled water
was added to the sediment and shaken strongly.
The tube was centrifugedagain and the supernatant fluid was poured off.
Equal or double amount ofliquid medium of Kirchner was mixed to the washed sediment.
A dr op of the mixed fluid was placed on six sterile slides respectively.
Til) slid i s thus prepared were mounted on the shelf in the moist chamber andincubated.
After 1, 2, 3, 4, and 5 days of incubation respectively, the slides
were taken out from the moist chamber and dried, being kept in an incubator.
The dried slides were fixed, being passed through the flame and stained with
the Ziehl-Neelsen method.
100 microscopic fields of each slide were observed,
In order to compare with the above mentioned method, a loopful of thewashed sediment was inoculated on two egg slants respectively and incubated.

The moist chamber.
The moist chamber is composed of a glass box (10 ern long, 8 ern wide and7 5 ern deep) with a plain glass cover and a metal shelf (8 ern long, 5,5 ern wide
and 6 em high) having five stairs on both sides as shown in Fig. 1.
A small amount of cotton being
spread previously at the bottomof the glass box was moistenedwith 60 to 100 cc ot destilled waterand the metal shelf place on it.
The glass box with the glass coverthus prepared was sterilized invapor.
The slides having a dropof the culture material on themas described above were mounted
on each stair of the metal shelf.
The box was covered and kept in
an incubator.
Much attention
I'. was paid to keep the glass box in a horizontal position so that the culture
mate rial did' not spread over the slide and flow down from it.
" 1F 19."'The motst,.
c h am b ere

Culture Methods for the Early Detection of the Tubercie Eacilii.

Results.
Amoung 70 samples of sputa examined 6 showed bacilli in the smears made with their washed sediments.
Results obtained from remaining 64 samples were as follows.
Table 1. Numbers of sputa showed bacilli when cultivatedin the moist chamber.
Days of incubation
2g45Totalo34

1
Numbers of
positive sputa

g22o9

Numbers ofnegative sputa

30

Table 2. Numbers of colonies found in each 100 microscopic
fields after two days of incubation.

Less than

Numbers of colonies
More thanMore than

1010

More than

2030

Table 3. Comparison of the culture in the moist chamberwith that on egg slants.
Numbers ofsputa

In the moistchamber

Onegg slants

33++27+

13+

Table 4.

Days necessary to detect the bacilli.
- - - - - - ----------Culture
methods
In the moistchamber.

Numbers ofsputa examined

Numbers ofpositive sputa

Days necessary
to detect(average)

64

34

2,264

S620,6iOn egg slants.i

The above mentioned culture method showed almost equal numbers oftubercle bacilli in sputa when compared with those in which the culture on

Saburo UYEDA Osamu yAMADA and Hiroshi OKADA

egg slants gave positive results.
It must be noted, moreover, that the bacilli were detected, in this method, in few numbers of sputa after only one
day of incubation, in large numbers after two days and in, the remaining
few numbers after three days.
In this method, numbers .of days whichwere necessary to detect the bacilli were shortened remarkably: only 2,2 dayson an average were necessary in this method while an average of 20,6 days
were required in the culture method on egg slants.

2. The culture method in the centrifuge tube.
Materials and methods.
89 samples of sputa were examined, of which the smears stained with theZiehl-Neelsen showed none of acid-fast becllli.
About 1 cc of each sputum
was put into three centrifuge tubes respectively and shaken thoroughly with
5 cc of 4.% solution of sodium hydroxyde.
in an incubator, the tubes were centrifugedwith sterile destilled water.

After being kept for 20 minutes
The sediment was washed once

Equal or double amount of liquid medium ofKirchner was mixed with the washed sediment.
After being sealed, the tubes were kept in an incubator.
After 2, 3, and 7 days of incubation respectively, the tubes were centrifuged again and a loopful of the sediment wassmeared on a slide.

The slid e was dried and fixed in the flame and thenstained with th ~ Ziehl-N eelsen method.
were observed.

100 microscopic fields of each smear

Results.
Table 5. Numbers of sputa which showed bacilli when cultivated in thecentrifuge tubes.
Days of incubation
237
Numbers ofposit.ive sputa

376

Totalo43

Numbers ofnegative sputa

Table 6.

Comparison of the culture in the centrifuge
tubes with that on egg slants.

-N~:~::S ~f---I-~:ntnifUgetUb::-- ~g: Slants~-··
37++

43
6g++

'.

-- _".--------..

Culture Methods fo: the Early Detection of the Tubercle Bacilli.
.~~-_._-,--~ .. ~_--._--- -.., ----_ ..

-- - - ~Table 7.

-- - ---,_._--- ------,--------_.~----_._-_.._-_ ..

_----~------~._~----._~._-------.")~---_.., - , , - -[Jays necessary 10 detect the bacilli.

CulturemethodNumbers ofs p uta examined

Numbers ofpositive sputa

In centrifuge
tubes.

8943On egg slants.

8940

Days necessary
to detect(average)

20,1

Tubercle bacllli were demonstrated in more numbers of sputa through theculture method in the centrifuge tubes than through the culture method on egg
slants.

It must be noticed, rnoteover, that the bacilli were detected, throughthe culture method in the centrifuge tubes, in large numbt:rs of sputa after
two days of incubation and in remaining few numbers after three days.
In
this method, days which were necessary to detect the bacilli were as short as
the slide culture method in the moist chamber. Only 2,2 days on an average
were necessary in this method while it required an average of 20,1 days in
the culture on egg slants.
Discussion.
The tubercle bacilli can be detected fr orn sputa through these two kindsof culture methods described above within 2 or 3 days.
This is rernakably
earlier than the ordinary culture method on egg slants and a little earlier than
the slide culture mehod of BERRY and LOWRY.
This is, after all, because
one can observe, in these culture methods described above, the tubercle bacilli
just beginning to develop.
However, one should always pay attention to the contamination of the saprophytic acid-fast bacilli which may come from saliva or other materials.
We did not encounter such difficulties in the above observations,
In orderto make sure, we examined, however. several cultures of the saprophytic acidfast bacilli,
It became certain that the differentiation of such saprophytic
bacilli from tubercle bacilli was probably possible, if one examined thoroughly
the mor pology and staining behavior of the bacilli ar.d at the same time thelargeness of their colonies, namely, the fastness or the development of the bacilli.
After 2 or 3 days of incubation the colonies of the tubercle bacilli wereconsisted of less tan 10 bacilli, generally 2 or 3.
On the contrary, the coloniesof the saprophytic bacilli were consisted generally or more bacilli than theformer.
Within these colonies one could find often nonacid.fast forms together with acid-fast ones.
Such difference as descr ib rd above between tubercle bacilli and saprophytic bacilli concerning the morphology and also the fast-Saburo UYEDA Osamu YAMADA and Hiroshi OKADA

6

ness of the development became more distinct after 5 or 7 days of incubation.
It seemed, therefore, possible to distinguish saprophytic bacilli from tubercle
bacilli, supposing that the culture 'were contaminated by them.

Summary.
By means of those two kinds of culture methods as described above, namely, "the slide culture method in the moist chamber " and ce the culture method in the centrifuge tub~s", the detection of the tubercle bacilli from sputa
showing none of bacilli through the staining method was possible already after
two or three days (2,2 days on an average) of incubation.
The authors wish to extend grateful acknowledgments to the Ministry of Education
for a grant in aid of this work.

References.
1.

BERRY, J. W. and LOWRY, H.,

Arner. Rev. Tuberc., 60: 151, 1949.

2. KIMURA, R. and MIZUNO, D." Acta Scholae Medicinalis Universitatis in Kioto, 27: 31,
1949.