野口英世著

Journal of Experimental Medicine

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P L E O M O R P H I S M AND PLEOBIOSIS OF BACILLUS
BIFIDUS COMMUNIS.*BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of the Rockefeller Institute for Medical Research.
New York.)
PLATES X-XIII.

Notwithstanding the extensive studies of Tissier, 1 Moro, 2 Rodella, a Cahn, 4 Passini, 5 Jacobson, 6 Herter and Kendall, 7 and a few
others, s the relation between B. bifidus communis (Tissier, 19oo )and certain other Gram-positive organisms of the feces, such as
B. acidophilus (Moro, 19oo), Kopfchenbacillus, B. tuberculiformis
intestinalis (Jacobson, 1908 ) and B. infantilis (Kendall, 19o9) remains still unsolved. At present it is difficult to say whether they
are entirely different organisms, or whether they are different formsof one and the same organism.
It is a recognized fact that the
purity of a culture of B. bifidus cannot be guaranteed absolutely,as it often produces forms which differ in many respects from theoriginal strain and which resemble, not infrequently, B. acidophilus,
B. tuberculiformis intestinalis, or the Ko.pfchenbacillus.
Moroabandoned his effort to identify the Kopfchenbacillus with B. bifidus* Received for publication December 3o, 19o9.
Tissier, Compt. rend. Soe. de biol., 1899, li, 943; Recherches sur la flore
in~testinale des nourrissons, Paris, 19oo; Ann. de l'Inst. Pasteur, 19o5, xix, lO9.
"-Moro, ]ahrb. f. Kinderh., ~9oo, Iii, 38; Wiener klin. Vgoch., I9oo, xiii, 114;
fahrb, f. Kinderh., 19o5, lxi, 687.
3 Rodella, Cent. f. Bakt, I Abt., Orig., 19Ol, xxix, 717; Zeit. f. Hyg., 19o2,
xxxix, 2Ol; Cent. f. Bakt, I Abt., Orig., 19o3, xxxiv, 14.
Cahn, Ccnt. f. Balet, I Abt., Orig., 19Ol, xxx, 721.
Passini, ]ahrb. f. Kinderh., 19o3, 1vii, 87.
6 Jacobson, Ann. de l'Inst. Pasteur, 19o8, xxi, 3o3.
*Herter and Kendall, four. of Biol. Chem., 19o8, v, 289; Kendall, four. of
Biol. Chem., 19o9, v, 419.
Finkelstein, Deutsche reed. Woeh., I9oO, xxvi, 263; Cipollina, Cent. f. Bakt,
I Abt., Orig., 19o2, xxxii, 576; Weiss, Cent. f. Bakt, I Abt., Orig., 19o4, xxxvi, 13.

182

Hideyo Noguchi.

183

because of his inability to produce a bifurcating strain from theformer, while Jacobson met a similar failure in his attempt toreverse B. tuberculiformis inteslinalis into B. bifidus. In spite ofnumerous attempts, as yet no one has succeeded in tracing thesource of B. bifidus outside of the intestinal tract.
In the course of my study of anaerobic bacterial flora of theintestinal tract of healthy and diseased children, my attention was
directed to B. bifidus.
The results I obtained from my present
study on B. bifidus convinced me that B. bifidus communis of Tissier
is an anaerobic phase of life of an aerobic sporogenous organism
belonging to the subtiloid group and closely resembling, especiallymorphologically and biologically, B mesentericus fuscus. By certain cultural methods, I was able to induce sporulation and adaptation of the aerobic life of B. bifidus and then lead the aerobized
B. bifidus back to the anaerobic bifurcating phase. In the aerobic
phase of B. bifidus, no bifiurcation has been observed, and it seems
almost incredible that it should be related to anaerobic B. bifidus atall. With adaptation of aerobiosis and anaerobiosis the entire setsof morphological and biological characteristics undergo profoundalterations. There are intermediate phases between these two extremes, which give the bacillus characteristics of semi-anaerobiosis
and extreme morphological variabilities.
The following experiments were made. Eight pure colonies of
B. bifidus, two of which consisted of a somewhat more delicate
type of bacillus, and one pure colony of a bacillus resembling a typeof B. acidophilus were isolated from the fresh stool of a healthy,
breast-fed girl, aged two months, by means of glucose-agar plating
in an anaerobic apparatus constructed on the same principle as thatof Schattenfroh and Grassberger.
The purity of these colonies
was ascertained first by microscopical examination and then byculture.
For the latter purpose, I found it advisable to use highlayer agar containing 1. 5 per cent. lactose or glucose, because withthis bifurcation was more general and uniform than with any otherliquid media for which an anaerobic apparatus is required. Even
with the use of fresh tissue in bouillon or glucose bouillon, B. bifidus
was seen to bifurcate less constantly than in high layer sugar agar,

184

Pleomorphism of Bacillus bifidus communis.

and to produce a pleomorphic condition 9 which makes it difficult
to determine the purity of an organism. The bacillus which appeared like B. acidophiIus in colony turned out in high layer glucose
agar to be a regular bifurcating strain. On the other hand, a typicalbifidus strain often grew out of colonies with some bifurcating andnon-branching acidophilus types, giving the appearance of mixed
colonies.
SPORULATION OF B. BIEIDUS COMMUNIS.

Inoculations with these nine strains were made into solid andliquid media. The organisms were inoculated in the melted state,or by stab, into the high layer agar containing lactose or glucose(1. 5 per cent.) and were cultivated at 37 ° C. ; the inoculations into
liquid media (litmus milk, beer wort bouillon, glucose bouillon, andespecially Hiss's serum water, containing different sugars) were
cultivated usually for seven days before examination in an anaerobic
apparatus at 37 ° C.
The agar cultures were examined from time to time and in mostcases I found bifurcation within twenty-four hours.
As thebacilli grew older, the number of bifurcations and the length of the
branches increased, and at the same time lost their ability to stain
with Gram's method (Figs. I, 2, 3, 4, 5).
The lactose- and glucose-serum-water were suitable media forB. bifidus and the sugars were fermented, without gas production,
up to coagulation of the serum (acid production). Microscopicalexamination showed the presence of pleomgrphic, non-branchingforms (diplobacilli with tapering ends, spindles, short rods, beaded
forms, coccobacilli, and a few bifuracted specimens). Vesicularforms were also seen occasionally. Besides these, I observed occasional free or attached oval spores (Figs. 6, 7, 8, 9).
By cultivating in succession pure strains of B. bifidus in sugar
containing agar by my modification of the Marino plat@ ° I was
There are in such cultures usually non-branching forms, including delicate
bacilli with tapering ends, club-shaped, beaded, or tad-pole forms. Bifurcation
is rare. In beer wort bouillon, delicate bifurcation may sometimes be produced.
1oMarino, Ann. de l'Inst. Pasteur, 19o8, xxi, IOO5. I made two narrow breaks
on opposite sides of a small Petri dish. I then placed it, with the opening downwards, in a larger Petri dish so that the liquid agar could flow from the large
dish into the smaller one.

Hideyo Noguchi.

185

able to obtain, at intervals of two or three weeks, colonies in which
a few straight Gram-positive bacilli were seen side by side with
Gram-negative, bifurcated forms apparently of a degeneratingstage. A few spores were often seen in such cultures (Figs. IO,
II,

I2, I3).

The spore-forming organisms of these old cultures of B. bifidus
were isolated by the usual methods. The cultures were heated to
IOO° C. for five minutes and plated out with modified Marino
plates or diluted in high layer agar containing lactose and glucose.
From each strain I obtained two distinct strains of bacilli, which
differed in their sensitiveness to oxygen, and also slightly in theirmorphological and cultural characteristics. One of the two wasstrictly aerobic, while the other was a facultative anaerobe. The
first produced felted, dry colonies and contained a spore-forming,
Gram-positive, motile bacillus, while the second formed rather
moist, greyish-white colonies and the bacilli were somewhat more
slender than those of the first. I found, however, that the second
variety becomes gradually strictly aerobic and like the first variety
after a few successive aerobic cultivations (Figs. 14, 15, 16, 17).
In the following protocols, I have given detailed descriptions ofthese two varieties of bacilli which have sprung from apparently
pure cultures of B. bifidus under certain circumstances. The morphology of these two strains will be given in TaMe I which follows
the experiments on reversion.
CULTURAL
Aerobic Phase.

A g a r plate
plain agar.

CHARACTERISTICS.

(Ist Strain.)Semi-Aerobic Phase.

(2d Strain.)

24 hours at 37 ° C.-~The colonies 24 hours at 37 ° C.--The colonies
are very small, irregularly
are round or oval, irregularlyround or oval, and rather
contoured, elevated, fin e l yopaque. Diameter less than I
granular, faintly grayish-brown,ram. U n d e r low power magdimly edged, opaque, some connification: edge fairly well detaining thick centers. Deep
fined with curled filaments
colonies roundish or lenticular.
projecting from the entire cirByreflected light, grayishcumference, some as long as thewhite and shiny. Diameter ofdiameter of the colony. These
single colonies about I mm.
curled filaments are irregularly 4 days at 37 ° C.--A felted edge
interwoven, but sparse. S t r u t is formed around the colonies

MEDICAL LIBI AR

MONTEFIORE HOSPI
iAL

186

Pleomorphism...........

T . . . . . . . . . .

JofBacillusbifidus

Aerobic Phase. (xst Strain.)ture: yellowish-brown, granular appearance due to the interwoven
curvedfilaments
without distinctive nucleus.
By reflected light the colony
looks grayish-white, dry, raised,and of irregular surface.
72 hours at 37 ° C.--The colonies
are about 6 ram. in diameter,and show typical felted appearance.

communis.

Semi-Aerobic Phase.

(2d Strain.)

without changing smoothnessof the latter.

Glucoseagar.

24 hours at 37 ° C.--The size of 24 hours at 37 ° C . - - M u c h thecolonies is somewhat larger
same as colonies on plain agar,
than that of plain agar colonies.
but there is mucin production
Irregularly round or oval.
and often the colonies have a
Opaque, yellowish-brown andfelted edge of long filaments
darker than in plain. Edge ircomparable with the aerobic
regularly serrated, fairly wellphase colonies.
defined, and projecting from it 4 days at 37 ° C.--Growth more
many uneven, almost transvigorous but no other essential
parent, finely granular, wartchange.
like outgrowths. Outer contour is surrounded by a lamellated marginal zone.
This
peripheral zone is caused by
mucilaginous secretion of thecolonies. By reflected lightthe colonies look grayish, pulvinated, with dried edge andmoist center, which give apearl-like appearance. Corrugated structure.
72 hours at 37 ° C . - - M a n y colonies sent out typical felted
filaments.

Agar slab
plain agar.

24 hours at 37 ° C.--Filamentous
growth which is better nearer
the surface.
48 h o u r s . - - S l i g h t
outgrowthalong the stab near the surface.
7 days.--Growth covered the surface with thick membrane,
3o days.--Browned the media.

24 hours at 37 ° C.--Filiform
growth along entire s t a b .
T h e r e is a slight surface
growth out of the puncture.
48 hours.--Slight surface growth.
7 days.--No further change.
3o days.--Browned the media
slightly.

Hideyo Nog~chi.
Aerobic Phase.

(lSt Strain.)

187

Semi-Aerobic Phase.

( 2d Strain )Glucoseagar.

24 hours at 37 ° C.--Thin, uni- 24 hours at 37 ° C.--Similar toform, smooth, filiform growth,
the above.
Surface outgrowth is abun- 30 days.--Slightly browned thedant.
media.
48 hours.--Little outgrowth near
the surface. Thick, mucoid,grayish membrane covered thesurface.
30 days.--Browned the media.

Meltedagar-inoculation.
Plain agar.

24 hours at 37 ° C::--Growth ob- 24 hours at 37 ° C.--Smal] punetitained only on the surface,form colonies throughout thenone in the deeper layer.
entire agar column. No gas
formation. Surface is covered
After a few days the colony bewith thin layer of colony.
came thick, grayish, wrinkled, 3o days.--Apparently no change,
tough.
except browning the media.
3o days.--Some root-like colonies grown into the upper layerof the agar. Browned the
agar.
Similar to the above, but more 24 hours at 37 ° C.--Somewhat
mucoid in character,better growth in the layer
30 days.--Browned the agar.
nearer the air.
30 days.--Apparently no change,
except browning the media.

Glucoseagar.

Agar stroke
plain agar.

24 hours at 37 ° C.--Abundant, 24 hours at 37 ° C.--Moist, graybroad, spreading, rather flat,
ish-white, smooth-edged, butslightly more elevated in thequite zigzag near the conmiddle, and firmly adherent,
densed water, where it spreads.
rugose,opaque,felt-edged
Condensed water is turbid,colony. Grayish-white, in genwithout a film. Medium uneral dry, but beset with minutechanged.
glistening droplets. Not larger 4 days at 37 ° C.--The edge ofthan I ram. By transmitted
the colony assumed a zigzag
light, the margin and center
appearance.
distinctly defined, the margin
being dense and opaque--about
4 ram.; the center is lessopaque and is finely wrinkled.
Condensed water is slightly
turbid and has thin, imperfect,
grayish-white film.
Mediumunchanged.

188

Pleomorphism of Bacilhts bifi&~s comm,~ds.
Aerobic Phase.

(ist Strain.)Semi-Aerobic Phase.

(2d Strai¢~.)72 hours at 37 ° C.--Growth ad~vanced.
Glucoseagar.

Litmusmilk.

24 hours at 37 ° C.--Spread over 24 hours at 37 ° C.--Spreading,grayish white, undefined edge
all surface. Raised by the acwith numerous
outgrowths
cumulation of mucoid droplets
which are almost transparent.
proiected from it. There istendency to confluence. T h e
Entire surface is covered withcolony is rather flat and very
mucus drops of varying sizes.
slightly elevated. The conMedium unchanged. Colonies
densed water is c l o u d y . Medifficult to scrape from agar.
dium somewhat turbid.
72 hours at 37 ° C.--Began topresent more felted appearance 4 days at 37 ° C.--Slowly growand to show a tendency to geting, but no felted appearance
developed. T h e colonies do
dry.
not adhere to the agar firmly,
but can easily be removed withloop.
The consistence issticky.
24 hours at 37 ° C.--Color turned 24 hours at 37 ° C.--Reddened on
the top, and deeper layer dered, and a heavy yellowishcolorized.
white deposit is formed on thebottom.
48 h o u r s . - - M o r e yellowish.
7 days.--Coagulated.
30 days.--Coagulation seems to
72 hours.--Coagulated.
have dissolved somewhat. The
30 days.--Mueh of the coagureaction remains acid.
lated easei~ dissolved. Reaction is slightly acid.
24. hours at 37 ° C . - - W h i t i s h gray, shiny, elevated, wavy
edged, no depression of themedium.
48 h o u r s . - - N o definite liquefaction.
4 days.--No further change.
30 days. - - Somewhat
sunkenalong the colony, indicating afeeble power of proteolysis.

Loeffler'sserum.

24 hours at 37 ° C.--Grayishwhite, rugose, spreading colony.
T h e substance beneath thecolony appears to be depressed.
There are mucoid droplets on
the colony.
48 hours.--Liquefaction alreadyset in and advancing.
4 days.--Not completely liquefied.
30 days.--Lower portion of themedium almost entirely dissolved. Brownish.

Gelatin.

24 hours at 37 ° C.--Minute floe- Much the s.ame as the aerobic
r_uli in clear medium. On thephase, but the liquefaction issurface are floating grayishdecidedly slower.

Hideyo Noguchi.
Aerobic Phase.

(ist Strain.)

189

Semi-Aerobic Phase.

(2d Strain.)white, dull, irregularly con- At 20 ° C.--Liquefaetion is slight
toured pieces of colonies.
a f t e r 30 days.
72 hours at 37 ° C.--Scum is Growth is also poorer.
formed.
7 days.--Liquefaction apparent.
A t 2o ° C . ~ V e r y limited amountof liquefaction of gelatin along
the stab puncture in 2 weeks.
3o days.--Moderate liquefaction.
Slight surface g r o w t h .
In the stab there are many
lateral projections of fine
growthnear
thesurface.
Liquefaction is about I ram.
deep and 6 ram. in diameter.
Plainbouillon.

24 hours at 37 ° C.--Slight tur- 24 hours at 37 ° C.--Slightly
bidity with strong surface
turbid, with slight whitish
membrane, which sinks when
sediment. No surface meretorn, with the exception ofbrane.
narrow annul~e around the sur- 72 h o u r s . - - N o further change,
face on the tube-wall. On
except more turbidity andvigorous shaking it breaks up
deposit.
into a fine flocculence.
48 h o u r s . - - N o new scum.
72 h o u r s . - - A new, firm, wrinkled,whitish-gray membrane overthe surface.

Glucosebouillon.

24 hours at 37 ° C.--More turbidand finely granular sedimentthan in plain bouillon. On
shaking, the sediment diffuses.
No surface membrane except
the annula.
48 hours.--Distinctly acid.
72 h o u r s . - - F i r m surface membrane formed.

Potato.

24 hours at 37 ° C.--Grayish- 24 hours at 37 ° C.--Growth on
white (faintly b r o w n i s h a t the
potato invisible, with the fluid
margin of the c o l o n y ) ,very turbid.
wrinkled, thick, scaly, dull, 4 days.--No
visible surface
spreading. T h e colony is adgrowth.
herent to potato, but is quite 3o days.--No further change.

Similar to the above. Someacid production. No scum.

Pleomorphism of Bacillus bifidus communis.

190
l

]
!

Aerobic Phase.

(ist Strain.)
.

r

]
| .

Semi-Aerobic. . . .

Phase.

(~d S t r a i n . )moist and brittle (suggestiveof colonies B. tuberculosis onveal bouillon-gIycerin agar
slant).
48 hours.--Color intensified andcolony thicker and spreading.
72 hours.--Distinctly brownis'hgray, somewhat reddish inplaces. Thickened and heavily
wrinkled. Scum on the fluid.
4 days.--Brownish and dirty hue.
30 days.--Dark, brownish, grayhue.

The descriptions given of the aerobic strain of the two organisms
agree closely with those of B. mesentericus fuscus.
REVERSION OF AEROBIC PHASE OF B. BIFIDUS COMMUNIS INTO
ANAEROBIC PHASE.

In the foregoing pages I described "the springing out " of twovarieties of spore-forming, non-branching aerobes from certain
cultures of B. bifidus. This finding might, of course, be considered
as a gross contamination with the brown potato bacillus, had I notbeen able to produce all typical vegetative forms characteristic inevery detail of B. bifidus communis from the spore material heated
to IOO° C. for five minutes of these two strains. However, bygradual training of the organisms to anaerobic life, I was able toaccomplish complete reversion of the aerobic phase of this bacillus
into the anaerobic. I cultivated the bacilli first semi-aerobically,
abruptly diminishing the quantity of oxygen. After three or four
successive cultivations, I obtained anaerobiosis in which condition
the organism had grown well. Glucose bouillon and Hiss's serum
water containing different carbohydrates, especially lactose, dextrin,
inulin, saceharose and amygdalin, were found to be very suitable
for reversing the biological phase. Anaerobic condition was produced usually by hydrogen gas, although nitrogen, carbon" dioxideand methan were equally suitable for the production of typicalbifurcating forms.
The degree of reversibility of the strictly

Hideyo Noguchi.

191

aerobic variety is found to be inferior to that of the semi-aerobic
strain, and more generations are required before complete reversion
is attained.
During the reversing processes, I observed that the first step ofreversion of the aerobic spore-material towards anaerobiosis ischaracterized by the appearance of streptococcal or young staphylococcal forms with, occasionally, minute forms of bifurcating types.
In the next stage the numbers of coeeobaeillary forms graduallydiminish and more typical bifurcating bacilli of regular size andshape, with many short primitive types of bifidus, appear. In thisstage, the developmental steps from coceal forms to regular bifidus
are clearly seen (Figs. I8, I9). Stellated arrangements of young
bifurcating rods are often seen.
A further step towards anaerobization shows the tendency toform more bifurcated bacilli of regular size, and in the stage following this phase, we obtained B. bifidus communis in the sense
used by Tissier (Figs. 2o, 21, 22).
The fermentative faculty of B. bifidus and its aerobic varieties
were tested upon different kinds of sugars and glucosides, but theresults were extremely inconstant. In one series of experiments
they split almost every sugar employed with acid production and,on other occasions, they did not attack any of them. I found,however, that in the strictly aerobic phase of the bacillus it is rare
to get coagulation of serum or milk, while in semi-aerobic andstrictly anaerobic phases, acid-production seems to cause coagulation of these proteids in several days. This difference may be due
to simultaneous proteolysis in the aerobic phase. Table I describes
summarily the morphology of the four different phases of B. bifidus.
CONCLUSIONS.

From the foregoing experiments, the conclusion may be drawnthat B. bifidus communis of Tissier has an aerobic phase, in which
it resembles B. mesentericus fuscus. Numerous intermediate phases
can occur between these two extremes; and their morphological andbiological variabilities demand the utmost attention in order tointerpret more intelligently the various phases of a given organism,
constantly found in the stools of sucklings, and to avoid the artificial
creation of two or more organisms from a single microbic type.

192

Plcom.orphism of Bacillus bifidus communis.

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194

Pleomorphism of Bacillus bifidus communis.

The frequent occurrence of B. mesentericus in the stools of sucklings has been described by Tissier, Moro, and many others. That
B. mesentericus is one of the most wide spread saprophytes and isconstantly found on the surface of our skin, is a well establishedfact, Scheurlen 11 was once led to consider this bacillus as the causeof carcinoma, because he isolated it from the cancers of mamma(interior of the tumors).
But Rosentha112 found the same organism in the breasts of healthy persons.
I, therefore, consider that the one source of B. bifidus communisin the stools of breast-fed infants is the breast of the lactating
mother.
EXPLANATION OF PLATES.
PLATE X.
FIG. I. Non-branching forms of B. bifidus communis. From a young culture in high layer glucose agar.
FIG. 2. The beginning of bifurcation of B. bifidus. (I8-hour-old glucose
agar stab culture.)
FIG. 3. Pleomorphic state of young B. bifidus culture, showing the tridents,
Y-forms, wedges, geniculations, non-branching individuals, etc. From a young,lactose agar high layer culture (48 hours old).
FIG. 4. Multiple bifurcations of B. bifidus. From an old, high lactose agar
culture.
FIG. 5. Multiple bifurcations of B. bifidus showing abnormally large individual with enormously long projections. From an old, high glucose agar culture.
Fro. 6. Sporulation of B. bifidus. A spore-bearing short bacillus with two
individuals is seen on the left. Straight, curved, or geniculated forms are alsoseen, but very few bifurcated forms. This shows the stage of springing out of
aerobic individuals from an old culture of B. bifidus. From an old, inulin-serum
water culture.
PLATE XI.
FIe. 7. Non-branching phase of B. bifidus. Diplobacilli forms, candle-flame
forms, banded and striated forms and many other forms are seen. From 7-day
old lactose serum water culture.
FIG. 8. Pleomorphic feature of B. bifidus. From a young saccharose serum
water culture.
FIG. 9. Sporulation of B. bifidus. Besides Gram-negative bifurcated forms,
there are several Gram-positive straight bacilli which represent the semi-aerobic
phase of B. bifidus. From an old culture in modified Marino plate.
FIGS. IO and II. Sporulation of B. bifidus. From a I4-day-old lactose agar
plate after modified Marino method.
n Scheurlen, Berliner klin. Woch., 1887, xxiv, 935.
~2Rosenthal, Zeit. f. Hyg., 1889, v, 161.

T H E J O U R N A L OF E X P E R I M E N T A L

M E D I C I N E VOL. XII.

FIG. I.

FIG. 2.

FIG. 3'

YIG. 4-

FIG. 5'

F1G. 6.

P L A T E X.

T H E J O U R N A L OF E X P E R I M E N T A L MEDICINE VOL. XII,

FIG. 7.

FIG. S.

FIG. 9.

FIG. IO.

FIG. I I .

FIG. I2.

PLATE Xl.

THE JOURNAL OF EXPERIMENTAL

M E D I C I N E VOL. Xll.

FIG. 13 .

FIG. I4.

FIG. I 5.

FIG. I6.

FIG. 17.

FIG. [8.

P L A T E Xll.

T H E J O U R N A L OF E X P E R I M E N T A L

M E D I C I N E VOL. XlI.

FIG. 19.

FIG. 20.

FIG. 2 [ .

F]G. 22.

P L A T E XIII.

H i d e y o Noguchi.
FIG. 12. Transition of bifurcating forms to straight type.
Marino plates.
PLATE XII.

195
From old modified

FI~. 13. Transition of bifurcating forms to straight type. From old modified Marino plates.
Fro. 14. Immediate semi-aerobic generation of B. bifidus just sprung fromthe anaerobic phase and showing transitory forms from non-branching anaerobic
to straight sporulating phase. From a 24-hour colony in the deep layer of lactose
agar near the anaerobic sphere.
FIG. 15. Semi-aerobic phase of B. bifidus. From a 2-day-old surface colony
oi1 glucose agar plate.
FIG. 16. Sporulation of semi-aerobic phase of B. bifidus. From a 3-day-old
agar plate colony grown aerobically.
FIG. 17. Reversion of aerobiosis into anaerobiosis. Coecic stage with a few
minute, bifurcated forms. From a semi-anaerobic cultivation of aerobic phasein glucose bouillon for 6 days at 37° C.
FIG. 18. Reversion. A step nearer anaerobiosis than the foregoing coccic
stage. Extremely pleomorphie.
PLATE XIII.
FIG. 19. Reversion almost completed. From the third generation in reversing cultivation. Six days in glucose bouillon.
FIG. 20. Reversion completed. From dextrin serum water culture. Anaerobically cultivated.
FIG. 21. Reversion completed. Still numerous irregular types.
FIG. 22. Reversion completed, Very regular B. bifidus forms. From alactose bouillon culture. Third anaerobic generation of the aerobic strain inglucose bouillon for 6 days at 37° C.