Identification of new bacteria strain, bacillus funicularius n.sp

Kluyver, A. J.

English

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DISCUSSION OF THE BACILLUS FUNICULARIUS N.SP.s AND FE ^	 *
REMARKS ABOUT THE GALLION ELLA FERRUGINEA EHRENBERG r
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by
A.J. Kluyver and C.B. van Niel
	
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With 8 figures in the text
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(From the Microbiology Laboratory, Technical University in Delft, Holland)
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1. Introduction
Recently, diploma engineer A.P. Neeb isolated and preliminarily investigated
in our laboratory a bacteria strain which, at least in one respect, seems to be
worthy of further investigation.
The bacteria strain in question was found on a culture plate which had been
prepared for the isolation of azotobacter chrooccum according to the classical
Beijerinck's formula and which -- in addition to tap water agar -- contained 2%
glucose, 2% whiting, and 0.1% K2HPO4 . This plate was inoculated with a liquid
culture medium which contained the same nutrients and which had been previously
infected with garden earth and incubated at 300C for two days.
For one reason or another, the expected azotobacter colonies did not develop
on this plate. Instead, we found pure water-bright colonies of mucous,consis-
tence with an average diameter of 4 mm.
It is well known that the above-mentioned culture plates, which are prepared
without the conscious addition of nitrogenous compounds, can be overgrown not
only with true nitrogen-binding organisms, but occasionally also with numerous
other microbes which are able to coyer their nitrogen requirements from the
small quantities of nitrogenous contaminants contained in the agar. Thus, there
would have been no reason to pay much attention to the organisms found on our
plate if the microscopic examination of the colonies had not revealed a remarkable
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It turned out that we were dealing with an entangled fiber complex in which
no natural fiber ends could be found with certainty. The occasionally observed
ends made the impression of fractures which had occurred during sampling. It
was equally strange that we were unable to disclose a cellular structure of the
fibers. The diameter of the fibers was 1.0-1.8p, with an average of 1.4u. The
mucous consistency of the colonies suggested that the fibers were covered with a
4
distinct mucous sheath, and this presupposition was subsequently confirmed by
microscopic examination of an india ink slide (See Figure 1).
Figure 1
An india ink slide of a few fiber parts obtained from a facillus
funicularius colony grown of yeast agar with 2% whiting and 2% glucose.
The.thi:ck mucous sheath surrounding the actual bacteria fiber is clearly
visible.
The rather strange morphological relations stimulated us to a closer investi-
gation of this bacteria strain. For this purpose, we needed, in the first place,
a pure culture of this organism. Because there was a high probability that the
bacteria lacked the ability to bind elementary nitrogen -- and thus the addition
of suitable nitrogen sources would not yield a better growth -- we continued our
cultivation on an agar plate made of the usual yeast decoction to which 2%
2
nglucose and 2% whiting had been added.
The fibrous structure of the original material made the preparation of a good
suspension in sterile water rather difficult. Thus, inoculation of our culture
plate with the suspension and subsequent cultivation at 30 0 C resulted in the
development of only a few colonies. These, how^v^r, grew vigorously and reached
a diameter of 1 cm and more in several days. The colonies were round and rather
flat, and displayed a peculiar structure in so far as the central part of each
colony was surrounded by a circular indentation followed, distally, by a wall-
like elevation. A typical radial striation was observed on the surface of the
colonies. t.'hen the culture dishes were opened, a faint and pleasant cstcr scent
was perceived.
N
Figure 2. Fibers from the same colony as above, photographed in a
dilute potassium iodide solution. Cellular fiber structure is clearly
visible on places which are in focus (the fibers had not been fixed).
Microscopic examination of these colonies showed again a typical fiber complex.
No cellular structure was observed in the fibers, and they were apparently
filled with a homogenous protoplasm from which several fine kernels distinctly
protruded at various places. The fact was clearly notices'.ble t:.at several narts
of the fiber complex were characterized by a more or less distinct spiral coiling
of the fibers around each other. The presence of a thick mucous sheath was
3
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,remonstrated by means of india ink staining.
Since it is possible to demonstrate a cellular structure in tha seemingly
homogenous fibers of the fibrous iron bacteria through treatment with a po_assium
iodide solution, we decided to apply this treatment to our cultures. Suc-
cessful results did not fail to appear: it became clearly apparent that the fibers
were c. .posed of numerous cells which were 4 -6i: long (see Figure 2). The appear-
ance of a deep red-brown color proved the presence o.: abunc:ant glycogen.
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Figure 3. Typical bacteria cable from a culture of bacillus funicularius
grown in yeast decoction with 2% glucose. Formation of cables from
simpler spiral tufts is clearly visible.
Aging changed the microscopic picture of the colonies developed in our cul-
ture medium very greatly. The fiber content soon changed into primarily an ir-
regular, highly refractive, and more or less spherically shaped mass, while the
cell walls usually dissolved and, finally, a typical cell detritus developed.
We did not succeed in determine::; the chemical nature of the above-mentioned in-
elusion bodies. The usual fat reactions failed completely; only 3 small amount
of fat was found on occasional places next to the above-mentioned 'bodies. The
bodies were kept in concentrated sulfuric acid for severai days and, strangely
enough, they did not dissolve. Only a few fragments of a two-month old culture
proved to be capable of growth. This circumstance has to be taken into account
4
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.Figure 4. A second example of a typical
bacteria cable of bacillus funicularius
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Figure 5. A spiral tuft of
bacillus funicularis at high
magnification. It shows great
similarity with Migula's
pictures of gallionella
ferruginea Enrenb.
when an old culture is used for inoculation and continuous cultivation.
In order to gain some insight into the physiology of this bacteria strain,
which is rather unique in its morphological aspects, we decided to investigate
whether the bacteria were able to develop under anaerobic conditions. For this
purpose, a bottle was filled wi g:, a yeast decoction containing 2% glucose, the
medium was inoculated with Neeb's pure culture,.and the bottle was plugged with a
stopper. At the same time, a control culture was kept under aerobic conditions.
It was found that no development, or only a very meager one, took place in the
bottle while prolific growth was observed in the aerobic culture after a few
days. The liquid did not become opaque, but a rather large cotton-like flake
^c
It turned out to be very important to keep the hydrogen ion concentration
low, so that the pH would range between 6 and 7.5. A simple method of doing this
is to sterilize the liquid with chalk and to decant the clear supernatant fluid
after the chalk has settled.
5
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developed in the lower part of the culture madium. Mf3cro scopic cxc;-Minat^.on of
a part of this flake surprised us very much. It turned out that the flare con-
sisted mostly of fibers spirally coiled around each other, and soma of these
coiled fibers were again coiled around each other, with the result that virtual
cables were formed. Figures 3, 4, and S may give some insight into thcae
structures. This strange formation stimulated us to further study.
2. Closer Investigation of the Isolated Bacteria Strain
It seemed important to investigate the behavior of the bacterium in other
nutrient media. When samples of the pure culture, developed on Schrager agar
of the above-mentioned composition, were transferred to strong culture media,
it was repeatedly observed that growth occurred only on plates which obtained
large samples. When this was taken into account, development was observed in
wort agar, pea leaf agar, peptone agar, etc.
In wort agar, large, white -- later somewhat brownish -- colonies developed
which, at first sight, were similar to those of various yeast types. A micro-
scopic picture showed long fibers which were curved in a very irregular manner.
This made them similar to the well-known picture of the lactobacetrium delbricki
culture grown on a Strong substrate.
In pea leaf agar, white and flat colonies appeared, having a diameter of 3 mm.
Slowly, a yellow field of diffusion would form around each colony. Microscopic
examination of a young culture displayed nothing remarkable. However, when older
colonies were studied in the microscope, we could observe in the fibers -- in
addition to the irregular inclusion bodies occuring also in other nutrient media --
certain little bodies strongly resembling end spores.
The peptone agar culture dissipated all our doubts about the ability of the
investigated bacteria strain to form spores. The 24 --hour old colonies were trans-
parent and showed a typical irridescence in transmitted light. Observation of
these colonies under low magnification revealed a typical wavy structure which is
well known for bacillus anthracis. Within three days, the colonies attained a
6
diameter of 3 nun, and they became white and opaque. Their microscopic examina-
tion showed that the fibers were filled with typical spores, as repruo need in
Figure 6. In still older colonies, we could observe only a spore t,-.ass. The
spores were relatively large, approximately 1. 8p long and 1.1p wide.
47	
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Figure 6. Slide of a 5-day old culture grown in peptone agar. Spore
formation is shown. Slide stained with simple methylene blue; spores
remained unstained.
In order to prove that the inclusion bodies were spores, we subjected a sus-
pension of an old colony to pasteurization at 80 0 C for the duration of 10 minutes.
Smears of this suspension upon peptone agar yielded a prolific growth quite
identical to that obtained from smears of a nonpasteurized suspension. When
smears of the pasteurized suspension were made 3a yeast decoction agar containing
2% glucose and 2% chalk, the familiar and large colonies were obtained again.
These findings made it clear that the bacteria strain under in^estigation
9	 must have belonged to the bacillus genus (Lehmann and Neumann). According to
our expectations, the bacillus yielded a Gram-positive reaction, while the pep-
tone gelatin was slightly liquefied. However, motility was absent in all stages
of development.
7
IWith respect to a diagnosis of the species ) it waa not p000-lblu to ldc"ti47Y
the bacterium with any one of the known specieG. That wki.,3	 in 1.ay
case, because the peculiar growth in liquid media -- a ointilar growtli ,pat-tura
was observed in 1% peptone water as in the yeast decoction with 2Z tlucooa --
would have attracted the attention of investigators, and nothing could be
found in the literature about this type of growth. For purposes of bein doubly
sure, we checked whether it was possible to identify the bacterium by mano of
the determination key given for the species of the bacillus genus in B^.lrgay's0	 0
Manual of Determinative Bacteriology. 	 It was not possible to do this we found.
Under these circumstances, it seems justifiable to us to regard the bacterium
as a new type, from the view point of the literature. We should like to su( ,'gest0
for it the name, bacillus funicularius, because this name would express the
most characteristic property of the organism -- namely, the formation of typical
cables in liquid media.
3. The Mode of Formation of Tufts and Cables
in Bacillus Funicularius
The growth of spiral tufts -- which is so characteristic for the bacillus
funicularius -- has been known for a long period of time to be true of 0-allionalla
ferruginea and spirchaeta plicatilis. Migula (l)
 particularly called attention
to the similarity in the growth pattern of these two organisms. A picture of
spirochaeta plicatilis with spiral tufts can be found in Zopf (2) This author
ascribed such growth forms also to a fiber portion of beggiatoa alba (3)
Ber. d.dtsch. botan. Ges., 15, 321, 1897, and Migula, W.: Bacteriv.,
Systems. Jena, 1031, 1897-1900.
(2)Zopf, W. Bacteria. Breslau, 1885. See Figure 4C. Also Magarete Zuelzer
mentioned these tufts in her comprehensive treatise on spirochaeta plicatilis.
Arch. f. Protistenkunde, 24,1, 1912.(3) See Figure 1 by Zopf.
Second Edition, Baltimore, 1926.
8
6
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100
Fechuar (l) raported on a similar formation of tufts in eyanophycee owc .l.l.atoria
Formosa.
In view of the fact that various authors give various explanation; for the
formation of tufts in the above-mentiot.cd organisms, it seemed appropriate: to
perform a closer investigation of the well developed tufts in the easily -,rown
bacillus funicularius. This was especially important because recently Ch+olodny
has expressed revolutionary views about the morphology of oaa of thu best
known of the above-mentioned organisms -- namely, gallionella ferruginea.
Temporarily leaving Cholodny's studies of gallionella embryology out of consid-
eration (they will be thoroughly discussed in the next section), we can estab-
lish the fact that there are primarily two opinions about the mode of tuft
formation in the above-mentioned microorganisms.
During Cholodny's tame, most authors believed that the typical screw--like
coiling of the gallionella ferruginea originated from a contact irritability
which is characteristic for this organism. Stated Migula (loc. cit., page 325);
"It is difficult to decade what is the cause of the peculiar behavior of the
fibers in that they now form screws, now bend only slightly and grow without
coiling around each other. Perhaps we should assume that the abvious tendency
of the gallionella fibers toward a spiral growth is elicited also by contact
stimuli. Even though curvatures can be observed in single fibers, these un-
dulations are always irregular and never spiral-shaped. From the moment two
fibers or fiber ends have touched each other, they never continue to grow in
juxtaposition in straight or winding lines, but they immediately begin to form
coils".
A similar view was expressed by Ellis in his book titled, "Iron Bacteria",
(1) Fechner, R. 7eitschr. f Botanik, 7, 289, 1915.	 See Figure 1,
page 321.
9
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as follows (1) : "A de .ailed investigation oll' thit;
chiefly to t►la want of success that has attendo.4 ufZorL6 to obtain 4-4= I flalal
cultures of Gallionalla. Certain conclusions Pay, aowevov, be arr.1vod at baued
on comparisons with other organisms in which the sa-me pheno ►ie-na are "-pparo-at.
There seems to be little doubt that Gallionella offi;trs anotllur uxa-,.1j)1Q of
contact irritability". On the other hand, special credit io due 'Lo V%.Iahiitr for
having directed our attention to a completely different explanation of tau forma-
tion of spiral tufts in oscillatoria Formosa. For the formation o lf tug is in
the	 (German text ends at this point).
( ' ) Ellis, D. Iron Bacteria. London, 24, 1919.
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Discussion of the bacillus funcicularius n.sp., and a few remarks about the gallionella ferruginea ehrenberg

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