The Acid-fastness of the Mycobacterium, especially itsCorrelation with the Rapidity of the Development.
By

Saburo DYED/\.*;ffii ~ =~~(Received March, 10, 19;)1)

Introduction.

Observing the minute growth membr-anes just beginning to develop, in
the previous reports'!', some of the Questions which concerne d the morphology
and development process of the mycobacteriu m especially the tubercle bacillus
were elucidated.
The mycobacterium including the tubercle bacillus developed by means of the segmentation and budding.
Those forms found at thebase of the chain and being stained only acid-fast were considered to benothing more than degenerative forms which had already lost the vital substance through budding, while those forms found at the top of the chain andcontaining the easily stainable substance or granules and at the same time
being stained with the Gram and also Heidenhain methods were considered
to be the viable forms of the tubercle bacillus.
Furthermore, the easily
stainable forms of the tubercle bacillus, namely, the mycelar, rod, and budding forms were rather easily observed, utilizing a new staining method called Ziehl-Heidenhain (author's method), within tuberculous tissues at the certain
stage of the infection.
The peculiarity of the staining behavior of this kind of microorganism may
be its acid-fastness.
Concerning the acid-fast staining, there were those
opinions of .EHRLICH (1885), KANAYSI (1929) etc. in the former literatures, andthat of YEGIAN and KURUNG(2) in the recent literatures.
It is, however, notthe subject of this paper to discuss the mechanism of this staining, but toexamine the correlation between the rapidity of the development of this kindof microorganism and its acid-fast staining.
*From the Bacteriological Laborato ry (Chief: S. UYEDA) of the Tuberculosis
Research Institute,

Kyoto TTniversity.

Saburo UYEDA

8Experiment.

1. Three types 0/ mycobacterium.
The appearance 0/ the acid-fastsubstance according to the rapidity 0/ their development.
The human (strain F) and'Fig. 1.

Three kinds of Mycobacterium.

The rapidityof their development and the appearance of theacid-fast substance in them.
M. smegmatis

avian types (strain Chokyo)
and M. smegmatis were ernployed.

avian typehuman type'The proced ure to

make the smears was thesame as that described in
the previous reports.

Adrop of homogeneous suspension of each type of mycobacterium was added toliquid medium of Kirchner
and incubated at 37°e.

Af·ter one or' two weeks incubation the minute growth
membranes juat beginningto develop were taken care.
fully with a platine spatuleand suspended, without stir-(acid-jclst

aring,

ina drop of saline. t'lOIt-dcicl-jcJ.st solution placed previously on
aslide.

The slide wasdried in an incubator and fixed in the flame and then stained with the warm solution ofZiehl and decolorized with 3% hydrochloric acid alcohol for about ten seconds and washedin water.

The result of staining is represented in Fig. 1 as a schema.
In M. smegmatis, the acid-fast substance began to be recognized only within the rodform which arranged at the fifth or sixth link of the chain.
In the avian
type, the acid-fast substance began to appear within the rod form arranging
at the third or: fourth link of the chain and beginning to bud.
In. the human,type, however, the thread form found at the top of the chain was alreadymore or less acid-fast.
It seems as if Fig. 1 shows that the acid-fast substance was produced in the human type earliest of all and a little later in theavian type and latest of aU in M. srnegrnatis.
It may, however, become
soon apparent that such a consideration as stated above was due to a misunderstanding, if one takes into consideration the difference in the rapidity ofthe development of these three types of mycobacterium,

The Acid-fastness of the Mycobacterium.

--------

9-Fig. 1 shows also the difference in the rapidity of the development of thesethree types of mvcobacter ium,
In 1\1. srnegrnatis, the thread form found at
the top of the chain was already followed by five or six rod forms by this

There were also rod forms which had already budded.

period of time.
the avian type,th ree or four rod forms succeeded the thread form,

Inand rodforms found at the base of the chain just began to bud.
In the humanty pe, however, the thread form just finished to produce a rod form bymeans of segmentation of its basal part.

The difference in the rapidity ot

the development of these three types of mvcobacter ium may be shown, therefore, by the inclined line a as seen in Fig. 1, while the rapidity of the appearance of the acid-fast substance in these three types by the line c which runs
parallel to the base line b.
This may indicate that the acid-fast substance appeared always with almost
equal rapidity in each of these three types of mycobacterium independentlyof the difference in the rapidity 01 their development.

. In M. smegmat is,

the acid-fast substance appeared only in rod forms at the base of the chain.
This may not indicate the slowness of the appearance of the acid-fast substance,

but the fastness of the development.

In the human type,on thecontrary, the thread form found at the top of the chain was already acid-fast.
This may r-ot indicate the fastness of the appearance of the acid-fast substancein this type of. mycobacterium, but the slowness of its development.
Fromthese observat ions it may be said,in other words,

that the non-add-fast

forms were easy to observe in the culture of mycobacter iurn developing fast
and that they were difficult to find in the culture of mycobacterium whichdeveloped slowly.

The following observations also supported such an under-

standing as stated above.
a) In 1\1. smegmatis - - also in the avian type - - the non-acid-fast forms
we re easy to find in the minute growth membranes just beginning to develop
on liquid medium of KIRCHNER,

but it became gradually difficult to find week

after week.
b) In M. smegmatis - - also in the avian type - - the non-acid-fast forms
were easy to observe in the minute growth membranes just beginning to de-velop 0:1 liquid medium of KIRCHNER as already shown in Fig 1, but in thecolonies developing in the depth of the same medium - - their development
was remarkably slower than the growth membranes - - the thread form foundat the top of the chain was already stained acid-fast just like the human typein Fig. 1.
c) M. s mcg mat.is cultivated on the surface of liquid medium of KIRCHNER
at 3TC was stained as seen in Fig. 1,the same medium at 25°S'but when one cultivated this strain on

it was stained just like the human strain. as seen

10Saburo UYEDA-- ~ - - - - - - -~._~~~~. . .. ~ ~ - - - - ~ - - - ~ . _ - - - - - - - - -in Fig. 1.
d) It was already described in the previous report that an amount ofeasily stainable forms of the human strain (F) and also those of BeG wasobserved at the stage of their maximal developernent in the lymph nodes ofguinea pigs inoculated subcutaneously.

From these observations, such an understanding as mentioned above is·thought to be reasonable.
Regardless of the type, the tubercle bacillus-mycobacterium in general-- produce the acid-fast substance only within therod form arranging at the base of the chain when its development is fast.
It is, therefore, easy to find the non-acid-fast forms in such cultures.
Onthe contrary, the acid-fast substance is produced already within the thread
form found at the top of the chain, when the tubercle bacillus - - mycobacterium in general-- develops slowly.
The non-acid-fast forms are, therefore, difficult to find in such cultures.
Why was there then such difference
among these three types of mycobacterium as seen in Fig. 1, in spite of thefact that they were all cultivated under the same condition?
This wasprobably because saprophytic M. smegrnatis was considerably promoted todevelop under this condition, while the human strain was not.

2. Theree types 0/ mycobacteium.
The rapidity 0/ their development
and the easily stainable substance within them.
Fig. 2Three kinds of Mycobacterium.

The rapidity ofThe smears were madetheir development and the easily stainable sub-with the minute growthstance in them.

membranes of three types
avian type

M. smegmatisof mycobacterium in thehuman typesame manner as in theabove observation and sta-......

ined with the alkaline sol.

~ution of methylene blue ofo ••o.

'")

:1,Leffler for about one min-'.

••0••• ,ut~

at room temperature.

••••The easily stainable.
substance was demon/'(..... strated in these three/. types of mycobacteriurn as seen in Fig. 2.
_\\,,;....:...---.
-: The forms bearing theeasily stainable substea.sl':r strLl1'lR.hle
ance were called in theprevious reports the easily stainable forms and considered to be the viable
•

1.(.

)

~" --'i!

«"~( t······)

The Acid-fastness 0/ the Mvcobacterinm,

11forms of the tubercle bacillus -_. of mycobacterium in general.

Comparing

Fig. 2 with Fig.!, however, it became soon apparent that there were twokinds of forms bearing the easily stainable substance.
The first forms hadnone of acid-fast substance but only the easily stainable substance, while thesecond form had more or less acid-fast su bstance together with the easily
stainable substance.
It became also apparent from the comparison of Fig. 1 and Fig. 2 that
there was another form bearing almost none of easily stainable substance but
only acid-fast 'substance.
This third form was called the acid-fast form of
mycobacterium arid considered to be the degenerative form which had alreadylost its viability after budding as discussed already in the previous reports.

3.

Different results obtained through the Ziehl-Neelsen and Ziehl-Heidenhain method.
By means of the Ziehl-Neelsen method it was easy to distinguish the firstform bearing only the easily stainable substance from other two forms stained
acid-fast, but it was difficult to distinguish the second form bearing more orless acid-fast substance together with the easily stainable substance from thethird form stained only acid-fast.
The Ziehl-Heidenhain method (author'smethod) rf!entioned in the previous reports was, however, able to distinguish
those forms bearing the easily stainable substance from the third form stained
only acid-fast.
This method could, therefore, distinguish the easily stainable forms, whether they contained some acid-fast substance at the same timeor not, from the form which had only acid-fast substance, while the ZiehlNeelsen method could only distinguish the non-acid-fast forms from other twoforms containing acid-fast substance.

Therefore,

it may be said that theZiehl-Heidenhain method was suitable to distinguish the viable forms of themycobacterium from the degenerative forms.

The Ziehl-Heidenhain method (author's method)
After deing stained previously with carbol-fuchsin slightly warmed for about two minutes and decolorized with S~;; hydrochloric acid alcohol for about ten seconds,
growth membranes attached to the slide were stained as follows:
ion of iron alum overnight (about fifteen hours).

the minute

Treated with 1% solut-

Washed in water.

Stained with 1~;;solution of hematoxylin (1 g of hematoxylin was dissolved in 10 cc of absolute alcohol,
then 90 cc of distilled water was added) for five hours at least.
then 1% solution of iron alum for several minutes.

andDifferentiated in 2% andWashed in water and then dried ..

Saburo UYEDA

124.

In which manner and front what is produced the acid-fast sub-stance?
This may become the next question.
No direct correlation was recognlzed between the production of the the acid-fast substance and the process ofbudding.
The easily stainable substance which was found within the threadform. changed gradually into granule following the maturation and the granule
became larger in the succeeding rod form.
This granule was thought to bea form of the resting stage very likely to the spore of some kinds of fungi.
The acid-fast substance was thought probably to have its origin in those
substances which were left within the thread form or succeeding rod form anddid not take part in the formation of granule and stained faintly with methylene blue of Leffler or with hematoxylin of the Heidenhain method.

n i SCUSSiOD.
There were many former literatures, for example, those of NOCARD andRoux (1887), METCHNIKOFF (1888), DIXON (1889), FISCHEL (1893), CRAIG (1898)
etc. which had discussed the non-acid-fast mycelar, branched or granular for mof the tubercle bacillus found in cultures.
In those discussions, however,
one did not pay much attention to the conditions under which the tubercle
bacillus was promoted to develop.
From the above mentioned observations the following consideration may.
be possible.
Not with rega re d to the type of the mycobacterium, the acidfast substance is produced within the rod forms arranging at the base of thechain, when the development of the mycobacterium is promoted.
It IS, there-.

fore, easy to observe the non-add-fast forms in such cultures.
On thecontr-ary, the acid-fast substance is already produced within the thread form
arranging at the top of the chain, when the mycobacterium develops slowly.
It is, therefore, difficult to find the non-acid-fast forms in such cultures.
There were also several former literatures, for example, those of BAEES
and LEV ADITI (1897), FRIEDRICH (1897) etc. which referred to the non-acid-fast
forms of the tubercle bacillus within living tissues.
This is, above all, important for the tubercle bacillus did not always appear as the acid-fast form
within tuberculous tissues as described in the previous report.
In the previous report, through the Ziehl-Heidenhain method, an amount of the hematoxylin-stained mycelar and branched forms at the tubercle bacillus was observed easily in the smears of the lymph nodes of guinea pigs inoculated sul.cutaneously
It is to be noticed that these hematoxylin-stained forms wereobserved, above all, easily within the lymph nodes in which the tubercle
bacillus was at the stage of his maximal development,

The Acid-fastness 0/ the Mycobacterium.

13----------

These findings may support not only such a consideration as mentioned
above concerning the correlation between the rapidity in the development of
the tubercle bacillus and the acid-fastness, but may also afford some suggestions to re-examine those opinions concerning the histogenesis, immunity etc.
of tuberculosis.

Summary.
From the observations of the minute growth membranes of the tubercle
bacillus and M. smegmatis,

just beginning to develop,

it may be summarized

as follows:
The acid-fast substance was produced only within the rod for ms arranging
at the base of the chain, when the development of the mycobacterium was promoted.
The non-add-fast forms were, therefore, easy to observe in such
cultures.
On the contrary, the acid-fast substance was already produced in
the thread form arranging at the top of the chain, when the mycobacterium
developed slowly.
It was, therefore, difficult to find the non-acid-fast forms
in such cultures.
The non-acid-fast for ms of the human strain were generally difficult to findin the culture.
This may be because the human strain was not speciallypromoted to develop in usual culture media.
It is, however, noticable thatthe non-acid-fast forms of the human strain were easy to observe at the stageof their maximal development in animal t issues.
The author wishes to extend grateful acknowledgments to the Ministry ofEducation for a grant in aid of this work.
References.
1.

UYEDA, S.,

Acta Scholae Medicinalis Universitatis in Kioto, 23: 31 (1951); 29: 43 (1951)

2. YEGIAN, D. and KURUNG, J., Amer. Rev. Tube rc., 56: 36, (1947).