Marine Biological Laboratory Library
Woods Hole, Mass.
Presented by
Academic Press
JulT P, 1960
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ATLAS OF
Bacterial Flagellation
ATLAS OF
Bacterial
Flagellation
EINAR LEIFSON
Stritch School of Medicine
of Loyola University
Chicago, Illinois
ACADEMIC PRESS lyiFJ I960
, New York and London
Copyright (g), 1960, by Academic Press Inc.
ALL RIGHTS RESERVED
NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM,
BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS,
WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.
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Lihrarij of Congress Catalog Card Number 59-15755
printed in the united states of AMERICA
Preface
The main purpose of this Atlas is to present a unified exposition
showing the shape and arrangement of the flagella on representative
strains of all available species of bacteria. The illustrations are all
in the form of photomicrographs of stained preparations personally
prepared by the author. In addition to the normal shapes and
arrangements, the observed variations and mutations of flagellar
shapes and arrangements are illustrated. Measurements of flagellar
wavelengths and amplitudes were made on all cultures studied and
these are recorded in the Atlas. With each genus is included the
source of the cultures studied and a discussion of their authenticity
and identity.
To enhance the practical value of the Atlas for bacteriologists,
several chapters are included dealing with the various factors which
influence bacterial motility and flagellation, technical details on the
staining of flagella, and various methods by which flagellar varia-
tions and mutations may be studied.
Flagella are undoubtedly the locomotor organs of bacteria. Their
composition is much the same as that of contractile tissue in general,
such as muscle tissue. They are extremely thin, averaging only 20-30
millimicrons in diameter. The helical shape is most characteristic
and most efficient for locomotion. The flagella originate from be-
neath the cell wall and perhaps from definite structures or kineto-
plasts. How they operate to move the bacteria is not known, nor
is the nature of the activating stimuli understood. Being locomotor
organs, the shape of the flagella and their arrangement on the
soma are determined by the genetic constitution of the bacteria.
Flagellation is a major basis for bacterial identification and
classification. Because of technical difficulties experienced by many
bacteriologists, the nature of the flagellation has been omitted from
the original descriptions of many bacterial species. In some in-
stances the flagellation has been incorrectly described. The Atlas
should do much to rectify this situation and place morphology in
its proper and preeminent place in bacterial taxonomy.
To the many bacteriologists throughout the world who have so
generously contributed cultures the author expresses his sincere
thanks. ^ _
EiNAB Leifson
Wheaton, Illinois
November, 1959
^Jt . ... ^\ 0
Contents V^^JlSJ^^A
Preface T^TTTTTT . ... v
1. Technical Problems Related to Motility and Flagellation 1
2. Shape and Arrangement of Flagella 8
3. Variation and Mutation of Flagellar Shape and Arrangement .... 14
4. Bacterial Evolution 18
5. N itrosoinonas 20
6. Nitrohacter 22
7. Hydro genomonas 22
8. Thiobacillus 22
9. Pseudomonas 25
10. Methanomonas 34
11. Protaminobacter 34
12. Xanthomonas 36
13. Mijcoplana 40
14. Lophomonas 42
15. Acctomonas 44
16. Acetobacter 46
17. Zijmomonas 46
18. Aeromonas 48
19. Vibrio 52
20. Desulfovibrio 56
21. Cellvibrio 56
22. Succinovibrio 58
23. Lachnospira 58
24. Spirilhim 60
25. Azotobacter 62
26. Azotomonas 66
27. Rhizobium 68
28. Agrobacteriiim 74
29. Chromobacterium 76
30. Sarcina 78
31. Streptococcus 80
32. Lactobacillus 82
33. Conjnebacterium 82
34. Arthrobacter 86
35. Listeria 86
36. Alcaligenes 90
37. Achromobacter 92
38. Flavobacterium 94
39. Cellidomonas 96
40. Escherichia and Paracolon Group 98
41. Aerobacter 100
42. Envinia 102
i
78^oT.
43. Serratia 106
44. Proteus 108
45. Salmonella 112
46. Pasteurella 116
47. Noguchia • 118
48. Photobacterium 120
49. Bacillus 124
50. Clostridium 131
51. Caulohacter 140
52. Chromaiium 144
53. Rhodopseudomonas 144
54. Rhodospirillum 146
55. Nocardia 146
56. Borrelia 148
57. Treponema 150
58. Bartonella 152
59. Selenomonas 154
60. Caryophanon 156
61. DuPage River Organism 158
62. Appendix " 161
Index 165
1. Technical Problems Related to Motility and
Flagellation
The Bacterial Culture
The synthesis of flagella and their activity does not always
correlate with the other physiological activities of the bacteria.
The environment which is best for growth and metabolism is not
always best for flagellation and motility. Substances which have
very minor effects on somatic growth and metabolism may com-
pletely inhibit flagellation or flagellar activity.
As a general rule flagellation is best in cultures incubated at
relatively low temperatures such as 20° C. With some mesophilic
bacteria little difference may be found in the flagellation observed
at 20 and 37° C. With some genera, such as Listeria, the flagella-
tion at 20° C. is good while that at 37° C. is very poor, and at
38° C. flagella are absent. In rare instances flagellation is better
at higher temperatures than at lower temperatures. The only ex-
ample the author can cite from personal experience is a culture of
Salmonella in which both the flagellation and motility were dis-
tinctly better in cultures incubated at 37° C. as compared to 20° C.
In many instances the apparently adverse effect of the higher tem-
peratures of incubation is not due to the temperature as such, but
rather to the growth phase in which the bacteria are examined.
Overnight incubation at 37° C. of bacteria such as those of the
enteric group, finds the bacteria long past the logarithmic phase
and well into the death phase, while after the same length of
incubation at 20° C. the bacteria are in a much more active state.
After growth has ceased, the flagella may deteriorate more rapidly
than the soma.
The optimum length of incubation for best flagellation seems
directly related to the growth rate. In general it appears that the
best flagellation is observed during the logarithmic and maximum
stationary phases of growth. In one genus of bacteria, Aeromoruis,
the nature of the flagellation may change during the growth cycle.
Strains of this genus may show numerous lateral flagella in young
♦ 1
cultures but only polar flagella in older cultures. This phenomenon
has not been observed in other bacterial genera.
The chemical composition of the medium may greatly influence
the flagellation. Of greatest importance perhaps is the pH. A low
pH often has a distinctly deleterious effect on flagella. Ferment-
able carbohydrates should be omitted as much as possible and
only added to the medium if necessary for growth. Phosphates
appear to favor flagellation and the author routinely adds 0.1%
potassium phosphate to his media. As a rule liquid media give
better flagellation than soHd media but sometimes the reverse
seems to be true. For technical reasons, related to the staining
procedure, the liquid medium used should be perfectly clear prior
to inoculation. Any agar in the medium interferes with staining
of the flagella. Satisfactory flagella stains may be made from
thioglycollate cultures but better slides are obtained if the agar is
omitted.
Bacterial Motility
It is a good rule to examine the culture by moist preparation
prior to staining. A motile culture should always show presence
of flagella; if not, the staining technique is faulty. An apparently
nonmotile culture may show presence of flagella for several reasons.
The flagella may be in the "paralyzed" state; changes may have
occurred in the medium, such as low pH, which have damaged
the flagella; or the flagella may simply have ceased to function
from various causes. Bacteria with flagella of the curly type are
sometimes very poorly motile, and those with straight flagella are
comparatively nonmotile. Another reason for making a moist
preparation is that the nature of the motion indicates the nature
of the flagellation. A single polar flagellum moves the soma rapidly
and linearly without much, if any, wiggle. Peritrichous flagella
move the bacteria with a characteristic wiggle. With a little ex-
perience polar and peritrichous flagellation are recognized with
considerable accuracy. In a detailed study of the flagellation of
the genus Chromobocterium, moist preparations of several strains
showed linear motion characteristic of polar flagellation. However,
the flagella stains showed only occasional lateral flagella which
could not account for the motion observed. By modifying the stain
2
the polar flagella were finally visualized but would have been
missed otherwise. Reliance cannot always be placed on spreading
growth in semisolid or motility agar. Polar flagellated bacteria
spread much less than the peritrichous flagellated, and if the
flagellation is poor and polar, the culture may appear entirely
nonmotile in the semisolid agar.
The simplest technique for making a satisfactory moist prepara-
tion is to place a loopful of culture on a slide and observe directly
with low and high dry objectives. With proper adjustment of the
condenser most bacteria may be seen. Light through a ground
glass is better than light through a blue glass. The author uses a
20 X objective. For small bacteria, and poorly motile bacteria, a
disk with an opaque center sHpped into the condenser is very effec-
tive. Using the 10 or 20 X objective and the condenser all the way
up, a very nice dark field may thus be obtained and even the
slightest motion of the smallest bacteria detected. With low mag-
nification the observer is less likely to mistake Brownian motion,
and motion of convection currents, for vital motion.
Staining of Flagella
Bacterial Suspension
If the bacteria are growing on a solid surface a Hght suspension
is made in distilled water, taking care that only bacterial growth
and none of tlie agar is carried into the suspension. For routine
diagnostic purposes staining may be made directly from this sus-
pension. Better preparations may be obtained by washing the
bacteria. If the bacteria are pathogenic, formalin should be added
to the suspension to a concentration of from 5 to 10%. The author
routinely adds formalin to every suspension. Washing is accom-
plished as described for broth cultures.
If the bacteria are in broth culture, add 5-10% formahn, dilute
with distilled water, mix and centrifuge; pour off supernatant and,
wliile tube is still inverted, rinse lip of tube with distilled water to
remove any supernatant which clings; now add 1-2 ml. of distilled
water and shake to resuspend bacteria; dilute with distilled water,
mix and recentrifuge; pour off supernatant as before, rinse lip of
tube; suspend bacteria in 1-2 ml. of water and dilute to light sus-
pension. If the formalin is very acid it seems preferable to neu-
tralize it with sodium hydroxide. The final suspension should show
a barely visible turbidity.
To show the presence of pH-sensitive flagella the bacterial cul-
ture is divided into two tubes. Ten per cent dibasic potassium
phosphate is added to one tube to a concentration of 17c, and
10% monobasic potassium phosphate added to the other to a like
concentration. After mixing, formalin is added and the culture
washed as before. The flagella on some kinds of bacteria assume
the curly shape in the acid phosphate and the normal shape in the
alkaline phosphate.
The author has encountered only one group of bacteria which
is injured by distilled water, namely the red halophiles. These
bacteria completely dissolve in distilled water. They may be
washed in 20% sodium chloride solution but very successful
flagella stains of these bacteria were not obtained.
Preparation of Bacterial Smear
Clean and grease-free sHdes are essential for good stains. In
emergencies powdered cleansers such as Bon Ami may be used.
The author uses concentrated sulfuric acid saturated with potas-
sium dichromate as cleaning solution. A strong solution of the
dichromate is first made in a relatively small quantity of water
and the sulfuric acid poured into this solution. If the cleaning so-
lution is kept at room temperature the slides may require several
days to a week before they are clean. Greater efficiency is obtained
with hot solution, e.g., 70° to 80° C. In this temperature range
most slides are satisfactorily cleaned overnight. A glass rack for
holding the slides in the cleaning solution saves time and trouble.
When the slides have been cleaned they must be thoroughly
washed, first in tap water and then in distilled water, to remove
every trace of the acid. After washing they are dried by being
placed upright against a clean surface, such as a large beaker
placed on a paper towel. The dry slides are stored in a clean slide
box. The fingers must never touch any part of the slide to be used
for staining. It is a good practice to indicate on the storage box
which end of the slide has been handled and use the other end for
the staining. Just before use the slide is heated in the colorless
flame of a Bunsen burner (the side to be used against the flame)
and then laid on a piece of paper, to prevent cracking, until cool.
4
If the burner lias a pilot light this must be turned off or the flame
will be smoky. A smoky or yellow flame ruins the slide. On pro-
longed storage the shdes may become greasy and must be re-
cleaned. In the atmosphere of a large industrial city this may
occur in a few weeks.
Sulfuric acid cleaning solution, if hot, gives off appreciable
amounts of sulfur oxides which may ruin the flagella stains. Re-
move all hot cleaning solution from the vicinity of the staining
place, preferably to another room.
Draw a hne with a wax pencil transversely across the middle of
the slide. Be sure the pencil line reaches both edges. A heaping,
medium size loopful of the prepared suspension is placed on the
distal end of the cool or shghtly warm slide; the slide is tilted to
cause the liquid to run down to the wax line. If the liquid does
not run down readily the slide is not clean and results may not be
good. Two smears, side by side, are readily made on each slide.
The smear is allowed to dry in air and not fixed in any manner.
It is now ready to be stained.
Preparation of Flagella Stain
The stain formula given below has proven satisfactory for the
visualization of the flagella of all bacteria with a few exceptions
such as the polar flagella of some strains of Chromobacterium.
The latter may be visualized by doubling the concentration of
tannic acid in the stain, i.e., instead of using a stock solution of
3% tannic acid use a stock solution of 6% tannic acid. With the
higher concentration of tannic acid the staining time is longer,
close to double. The normal formula of each stock solution is as
follows:
Basic fuchsin in 95% ethyl alcohol, 1.27o; tannic acid in distilled
water, 3.0%; sodium chloride in distilled water, 1.5%.
The basic fuchsin may be purchased certified for flagella stain-
ing. It should either be pure pararosaniline acetate or a mixture
of pararosaniline hydrochloride and pararosaniline acetate, but not
over % parts of the hydrochloride. Basic fuchsin must have an
odor of acetic acid to be satisfactory. Allow about 1 day to insure
complete solution of the fuchsin.
The tannic acid should preferably have a light yellow color.
To prevent molds from growing in the tannic acid solution addi-
tion of phenol to a concentration of about ^^ooo is effective. The
tannic acid and the sodium chloride solutions may be mixed with
equal parts of each or prepared as one solution with 1.5% tannic
acid and 0.757c sodium chloride. The stock solutions should be
kept in the refrigerator.
To prepare the stain, mix together equal parts of the three stock
solutions, or add 2 parts of the tannic acid-salt solution to 1 part
of the dye solution. Keep the stain bottle tightly stoppered. The
stain is ready for use immediately. A precipitate develops in the
bottle on storage which should not be disturbed when the stain
is used. The stain solution undergoes a gradual change during
storage, requiring a longer staining time; the change is faster at
higher temperatures. At room temperature the stain solution is
satisfactory for about 1 week, in the refrigerator for 1-2 months,
and in the deep freeze indefinitely. If the stain is frozen, care
must be taken to mix thoroughly after thawing, since the alcohol
has separated from the water. The author keeps his stain solution
in the refrigerator and discards it when the staining time exceeds
that of the freshly prepared stain by more than about 5 minutes.
Application of the Stain
For application of the stain solution the slides are most con-
veniently placed on a board or rack. The author uses a board,
painted or stained black, about 3 inches wide and 20 inches long
with very short legs slightly higher in front than in back. This
gives a slight tilt to the board and the slides, and the staining solu-
tion is slightly deeper at the distal end of the slide than in the mid-
dle. With a Pasteur pipette, fitted with a rubber bulb and marked
at the 1-ml. level, 1 ml. of the stain is taken from the top of the
solution and is quickly applied to part of the slide holding the
smear. The stain solution must not spread beyond the wax line
or run off the slide.
Staining Time
The time required for staining the flagella varies normally be-
tween 5 and 15 minutes. A short staining time is required with
freshly prepared stain, warm stain, high room temperature, strong
air currents, thin stain layer, and pure pararosaniline acetate dye.
A longer staining time is required with old stain solutions, cold
stain, cold room, little air circulation, deep stain layer, and high
proportion of pararosaniline hydrochloride in the dye. When the
alcohol has evaporated to concentration of 20-25% a colloidal
precipitate forms which settles on the flagella making them thicker
and colored red. Freshly prepared stain usually has a variable
amount of coarse precipitate. On storage in the refrigerator this
precipitate settles and the supernatant used for staining is clear.
Do not disturb this precipitate when removing stain from the bot-
tle. By careful observation of the stain on the slide the formation
of the colloidal precipitate may be observed by the change from
a clear solution to an opaque and rust colored solution. With the
slide on a black background a strong beam of light readily shows
the formation of this precipitate. As soon as the precipitate has
formed the staining is completed and the slides are washed imme-
diately.
Another method is to prepare one or two extra slides. When
the staining time appears to be about up the trial slide is washed.
If the smear is not macroscopically visible the time is too short.
After about 2 more minutes wash off the second slide and observe
the smear. With a little experience one trial slide is usually suffi-
cient and will serve as a guide to the staining time of a dozen or
more slides stained at the same time. When many slides are
stained at one time they should be placed on the board about 1
inch apart to allow the alcohol to evaporate at somewhat the
same rate from the middle and end slides. When the staining time
is up the slide is placed directly under the faucet or a stream of
water. Do not allow any of the stain to run off the slide before it
is placed under the faucet. After washing, the slide is allowed to
drain dry or carefully blotted.
Cotinterstaining
The soma of some species of bacteria characteristically stain
very faintly or not at all. With such organisms a counterstain may
be used for better visualization of the soma. A satisfactory coun-
terstain is the usual methylene blue stain diluted 5-10 times with
water and slightly alkalinized with sodium hydroxide, sodium bi-
carbonate, or sodium borate. Application of this stain for a minute
or so usually stains the soma blue while the flagella remain red.
2. Shape and Arrangement of Flagella
Flagellar Shape
The most common shape of bacterial flagella is a fairly uniform
helix with a pitch characteristic of the species. Since the flagella
are very thin the helix is flattened when they dry on the slide and
they appear to be wavy. The distance from one wave crest to the
next, which is termed the wavelength, may be slightly different
from the pitch of the original helix, but measurements made on
stained flagella and on flagella in moist preparation (dark field)
have shown little difference. The amplitude of the waves is com-
parable to the diameter of the original helix. The exact relation-
ship of these two is still somewhat uncertain but the amplitude
seems definitely greater than the diameter of the helix. With most
bacteria the flagellar shape is quite uniform and constant, but
with a few bacteria the flagellar shape is quite irregular.
Measurement of flagellar wavelength and amplitude is most
conveniently and accurately done by means of a filar micrometer.
A fixed scale micrometer is less accurate but gives good mean
values where many flagella are measured. Since some flagella are
bound to become damaged and distorted when they dry on the
slide, such flagella should not be measured. Figure 1 illustrates
the way in which wavelength and amplitude are defined.
Flagellar Arrangement
The old terminology describing the arrangement of the flagella
on the bacterial soma is rather inadequate. The following ter-
minology will be used throughout this Atlas:
Polar, (a) Monotrichous: Predominantly a single flagellum at
one or both poles. The base of the flagellum usually parallel to the
long axis of the soma, (h) Multitrichous:^ Predominantly two or
1 Since this classification is based primarily on flagellar arrangement the
term lophotrichous is not included. Where this term is used in the Atlas
it refers to polar multitrichous flagellation with flagella of relatively long
wavelength and typically having less than one complete wave.
8
NORMAL
amplitude
Fig. 1. Illustration of the way that wavelength and amplitude are defined.
From E. Leifson, S. R. Carhart, and M. Fulton,). Bacterial. 69, 73-82 (1955).
more flagella at one or both poles. The base of the flagella usually
parallel to the long axis of the soma.
Subpolar, (a) Monotrichous: Predominantly a single flagellum
near the pole with the base of the flagellum usually at a right angle
to the long axis of the soma, (b) Multitrichous: Several flagella
near the pole with the base of the flagella usually at a right angle
to the long axis of the soma.
Lateral, (a) Monotrichous: A single flagellum predominantly
from the middle half of the soma, (b) Multitrichous: Several
flagella as a tuft predominantly from the middle half of the soma.
Peritrichous: Flagella seemingly haphazardly arranged on the
soma, either single or multiple.
Mixed: Two or more flagella of distinctly different appearance
in different locations.
The subpolar arrangement is rare. The monotrichous type has
been seen mainly in Rhizobiutn, the multitrichous type only in
Treponema. The lateral arrangement is also rare. The lateral
monotrichous arrangement may be seen in Lachnospira and the
lateral multitrichous arrangement in Selenomonas. The mixed
arrangement is typical of Chromobacteriiim and young cultures of
Aeromonns. It has also been observed on several occasions, in
cultures physiologically related to Pseiidomoruis, and in unstable
mutants of various bacteria. ( See Figs. 2 and 3. )
Relation of Shape and Arrangement to Motility
In a liquid medium bacteria with polar flagella as a rule move
more rapidly than bacteria with peritrichous flagella. The move-
ment of the polar flagellated bacteria is linear and smooth, while
that of the peritrichous flagellated bacteria is wiggly and more er-
ratic. The shape of the flagella may greatly affect locomotor effi-
ciency and will be discussed in the next section in connection with
flagellar variation and mutation. In media with more soHdity than
ordinary broth the peritrichous flagellated bacteria apparently
move faster than the polar flagellated bacteria. This is readily
demonstrated by inoculating the center of one semisolid plate with
a peritrichous organism such as Salmonella, and similarly inocu-
late another semisolid plate with a polar organism such as Pseitdo-
FiG. 2. a, b, c, d, e, f, g. Examples of polar monotrichous flagellation, illus-
trating variations of wavelength.
h. This illustrates the formation of two variants, one with a normal flagel-
lum and one with an undulant flagellum.
i. A normal and a straight flagellum at the same pole.
j, k, 1, m, n, o. Various types of polar multitrichous and lophotrichous
flagellation. The coiled flagella shown in o are quite rare.
p. Subpolar monotrichous flagellation.
q. Subpolar multitrichous flagellation.
r. Lateral monotrichous flagellation.
s. Lateral multitrichous flagellation.
t, u. In t is shown a stalked organism with a flagellum at the end of the
stalk. In u is shown a rosette of stalked bacteria with polor monotrichous
flagellation.
g,k,q. From E. Lcifson, /. Bacterial. 62, 377-389 (1951). j. From E. Leif-
son, Antonie van Leeuwenhoek, J. Microbiol. Serol. 20, 102-110 (1954).
m, o. From E. Leifson, and R. Hugh, /. Bacteriol. 65, 263-271 ( 1953). p. From
E. Lcifson, and E. W. Erdman, Antonie van Leeutoenhoek, J. Microbiol. Serol.
24, 97-110 (1958).
10
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11
monas. The Salmonella growth will spread faster and more widely
than the Pseudomonas growth. In fact, semisolid agar stabs of
polar flagellated organisms may show so little spreading that they
appear nonmotile.
Fig. 3. a, b, c, d, e, f . The variety of peritrichous flagella which have
been observed and are named in order, normal, curly, small amplitude, coiled,
semicoiled, and straight.
g, h. Two examples of double curvature. Other types, not illustrated, may
be seen in Proteus.
i, j. Examples of peritrichously flagellated bacteria with flagella of difi^erent
wavelength on the same individual.
k, 1, rii. Mixed polar-peritrichous flagellation.
n. The hooked flagellum is the normal one for this organism and is sub-
polar in origin. The exact origin of the other flagella is undetermined.
o. Mixed lophotrichous-peritrichous flagellation. This is an unstable mutant.
p. The nature and function of the spine-like structures is unknown. The
polar flagellum is unusually long but otherwise normal.
a, b, e, h, i. From E. Leifson, S. R. Carhart, and M. Fulton, /. Bacteriol.
69, 73-82 (1955). c. From E. Leifson, and M. I. Palen, /. Bacteriol. 70, 233-
240 (1955). k. From E. Leifson, /. Bacteriol. 71, 399-400 (1956). 1. From E.
Leifson, and R. Hugh, /. Bacteriol. 65, 263-271 (1953). n. From E. Leifson,
and L. W. Erdman, Antonie van Leeuwenhoek, J. Microbiol. Serol. 24, 97-110
(1958). o. From T. P. Galarneault, and E. Leifson, Can. J. Microbiol. 2, 102-
110 (1956).
12
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13
3. Variation and Mutation of Flagellar Shape,
Arrangement, and Function
Variation of Shape
Flagella may show several shape variations which are encoun-
tered with variable frequency in a variety of genera. The shape
most usual for the flagella of a genus is designated as normal. This
shape is usually that of a helix with a wavelength-ampHtude ratio
ranging from 4:1 to 3:1.
The most commonly encountered shape variant is the curly.
This shape variant has been observed in most genera of peritri-
chous flagellated bacteria but infrequently in polar flagellated
bacteria. In the enteric and related groups of bacteria, the curly
flagella have a wavelength close to i/^ that of the normal. The
ratio of wavelength to amplitude approximates 3:1 while in the
normal for these bacteria this ratio approximates 4:1. In other
groups of bacteria such as Bacillus and Clostridium the wavelength
of the curly flagella is about ^ that of the normal flagella. In the
subpolar types of Rhizobium the curly flagella have a wavelength
about y^ that of the normal. In one strain of Sarcina urea three
wavelengths were observed having a relative ratio of 3:2:1. The
curly flagella appear shorter and stiffer than the normal. From a
careful study of the two types of flagella in Proteus the actual
lengths of the normal and the curly flagella when stretched out
straight appeared to be about the same. In some strains of some
genera of bacteria the change from normal to curly and vice versa
may be induced by a change of the pH of the suspending medium.
Strains of Proteus, Azotobacter, Erwinia, and Bacillus have shown
this phenomenon. At pH 6 and below, the flagella are curly while
at pH 7 and above they are normal. In these genera, as well as in
others which do not show this pH sensitivity, both normal and
curly flagella may appear on the same soma. In some genera or
species the curly flagella appear to be stable genetic mutants. Curly
flagella are less efficient locomotor organs than normal flagella. A
pure curly strain of Salmonella Wichita, for example, showed prac-
14
tically no spreading in semisolid agar and only wiggling and spin-
ning motion in liquid media.
Next to the curly the most common variant is the coiled type.
The flagella of one genus of bacteria, Scrmtia, are mainly coiled.
The coiled shape may be the flattened appearance of a helix with
a very large amplitude and short wavelength. This shape is fairly
frequent in many genera of both peritrichous and polar bacteria
but it is rare to find a culture which shows only coiled flagella.
Aside from Serrafio, cultures showing only coiled flagella have been
found in Aeromonos, Listeria (true mutant), Escherichia, and
Erwinia. Locomotor efBciency of coiled flagella is fair but less
than that of normal flagella.
Other shape variations less frequent than those mentioned are
straight, small amplitude, and undulant. Straight flagella are oc-
casionally seen among the normal flagella of many bacteria. Pure
variants or mutants with straight flagella are rare. Stable variants
with straight flagella have been isolated from several cultures of
Listeria. One strain of Arthrobacter studied had mainly straight
flagella. Organisms with straight flagella are either nonmotile or
show only a nonprogressive spin or wiggle. Both types have been
observed in Listeria.
Organisms with small amplitude flagella have been observed
in Listeria, Sarcina, Rhizobium, and occasionally in other genera.
Pure variants with this type of flagella have been isolated from
Listeria strains. The motility of these variants was poor and prac-
tically nonprogressive, like organisms with straight flagella.
The undulant type of flagella was observed on one strain of
Thiobacillus thiopariis, on several strains of halophilic Pseiido-
motias species, and on several strains of Aeromonus. This is the
type of flagella found on algae, such as Chlaniydotnonas, on pro-
tozoa, and apparently on flagellated plant gametes. With the single
exception of a peritrichous organism seen in water all undulant
flagella seen have been polar.
Variation of Flagellar Arrangement
Variation of flagellar arrangement is relatively rare but has
been observed in several genera. The variation is always from
polar flagellation or subpolar flagellation to peritrichous flagella-
15
tion, never the reverse. This seems to indicate to the author an
evolutionary trend in bacteria from polar flagellation to peritrichous
flagellation. Typical strains of the Aeromonas genus show pre-
dominantly polar monotrichous flagellation in cultures which have
attained a relatively dense population. In very young or very Hght
cultures several strains showed peritrichous flagellation in addition
to the polar. The lateral flagella usually have a shorter wave-
length than the polar flagellum. Although the polar flagellum may
be of the normal or undulant type the lateral flagella are alike. One
widely distributed strain, indistinguishable from Aeromonas phys-
iologically, has coiled peritrichous flagella and appears to be a
stable mutant. A well authenticated mutation has been observed
in a strain of Lophomonos. Lophomonas has polar multitrichous
flagella of long wavelength like the spirilla. In one culture typical
of the genus, lateral flagella of relatively short wavelength were
observed in addition to the typical polar flagella. Individuals with
only peritrichous flagella were also observed. The latter were
isolated in pure culture and have remained unchanged over a
period of years. This culture is indistinguishable from typical
Alcaligenes species with curly flagella. In several strains of Rhizo-
biiim with a single subpolar flagellum, occasional individuals with
one or more subpolar or lateral flagella of very short wavelength
have been observed. Most often the normal subpolar flagellum
and the curly flagella are found in the same individual, but occa-
sionally organisms are seen with only the curly flagella. Pure
variants of Rhizobiwn with only the curly flagella have not been
isolated.
Variation in Motility
The change in motility associated with change of flagellar shape
has been discussed. Yet to be mentioned is the complete absence
of motility observed in some strains with otherwise normal flagella.
This phenomenon has been observed in Salmonella and Listeria.
These "paralyzed" mutants have shown fair stability.
16
Detection of Flagellar Variants and Mutants
Flagellar variants of bacteria which show differences in motility
may be isolated by plating in semisolid agar. The most convenient
technique is to streak plates of solid agar with the culture and a
thin layer (about 7 ml. for a 15 cm. plate) of semisolid agar (0.3-
0.5% agar) poured on top. Sometimes better results are gotten by
making proper dilutions of the culture and inoculating the melted
semisolid agar before it is poured on the solid agar. Plates made
by either of these two methods can be turned over and otherwise
handled like ordinary solid agar plates. Colonies of bacteria with
normal peritrichous flagella tend to spread most widely. Colonies
of bacteria with peritrichous flagella of any other shape tend to
spread less widely, if at all. Colonies of polar flagellated bacteria
with normal flagella spread less widely than those of peritrichous
flagellated bacteria with normal flagella.
Detection of spontaneous mutants with greater motility than
the parent strain is best accomplished by making a heavy streak
across the middle of a soHd agar plate followed by a thin layer of
semisolid agar. Mutants with greater motility than the parent strain
show up after a variable length of incubation (up to 20 days) as
outshoots from the main streak.
17
4. Bacterial Evolution with Respect to Flagellation
The available evidence indicates that bacterial evolution is from
polar monotrichous organisms to peritrichous organisms and,
finally, to atrichous organisms. Polar flagellation is most efficient
for locomotion through a liquid medium and such bacteria are
best adapted to an aquatic habitation. All strictly autotrophic
bacteria, and most water types, both fresh water and marine, have
shown only polar flagellation, if any. Current ideas on the evolu-
tion of the earth are that in the early stages the surface was com-
pletely covered with water. In this environment the polar flagel-
lated bacteria were evolved. Peritrichous flagellated bacteria are
more efficient in locomotion through a medium denser than water
and, as more and more land appeared, the peritrichous types
evolved in the soil. With the appearance of animals and plants
some bacteria became parasitic with an environment in which
flagella served no useful purpose and were only a hindrance in that
• they required food and energy to be produced and to function.
Under these conditions the bacteria evolved into atrichous types.
Soil, and even water, rich in bacterial food could also render flagella
superfluous.
Flagellar variations and mutations which have been observed
in the laboratory invariably have been changes from polar flagel-
lation to peritrichous flagellation, never the reverse, and from peri-
trichous to atrichous, rarely the reverse. An unequivocal instance
is a polar flagellated strain of Lophomonas which spontaneously
mutated to a peritrichous type. Strains of Aeromonas produce
peritrichous cells in young cultures, and indirect evidence indicates
that one Aeromonas culture produced a stable peritrichous mu-
tant. There are some indications that the subpolar flagellated
Rhizobium species of soy bean, lima bean, lupine, etc. are evolving
into peritrichous types. The Rhizobium species from pea, garden
bean, alfalfa, clover, etc. have peritrichous flagella, indicating per-
haps a longer period of association with plants and rich soil.
The evidence for the peritrichous to the atrichous type of
lution is very suggestive. The mutation of laboratory cultures f
peritrichous to atrichous is common experience. A majority of tl
bacteria strictly parasitic and pathogenic for animals are atrichous.
18
evo-
rom
le
The flagellated pathogenic types are mainly intestinal where con-
ditions favorable to peritrichous flagellation may exist to some
extent. The plant pathogens, however, are generally flagellated,
both polar and peritrichous. This may indicate that much of their
existence is in water and soil. Instances of mutations from non-
flagellated to flagellated types appear to be very rare and have
never(?) been observed in genera other than those which he-
some species which normally are flagellated.
ave
19
5. Nitrosomonas
A culture labeled Nitrosomonas europaea was received from
Dr. Martin Alexander of Cornell University. Dr. Alexander stated
that the culture was not pure and that attempts at purification had
not been successful. Another culture with the same label was ob-
tained from the American Type Culture Collection (ATCC).
Flagellar Characteristics
Stains were made directly from the broth culture furnished by
Dr. Alexander. Two types of flagellated bacteria were seen. Most
numerous was a small rod with polar monotrichous flagellation.
The wavelength of the flagellum was exceptionally short averaging
0.93 micron. A much smaller number of rod shaped organisms had
polar monotrichous flagella of much greater wavelength, averaging
2.3 microns, or about 2i/^ times the other one. The soma of the
organism with the long wavelength flagellum did not take the
flagella stain and was practically invisible. The author did not
plate or attempt to grow the Alexander culture in the proper syn-
thetic medium. However a transfer was made into peptone-yeast
extract broth, and growth appeared. This growth had a pinkish-
brown color, water insoluble; flagella stain showed polar mono-
trichous flagella of 2.2 micron wavelength. Morphologically the
organism was similar to the one with the long wavelength seen
in the original culture. The real N. europaea would thus seem to
be the type with the short wavelength flagella illustrated in Fig. 4a.
The ATCC culture of N. europaea, 12248 was stained directly
from the original suspension. The predominant flagellation was
polar monotrichous, with a smaller proportion of polar multitri-
chous individuals. The flagellar wavelength was rather variable
ranging from 1.3 to 1.6 microns. On transfer to nutrient broth
good growth was obtained of organisms with the same flagellation
found in the original suspension. No growth was obtained in media
free from organic matter. This organism apparently is not auto-
trophic and not typical of Nitrosomonas.
20
\ ^' /-■ ^
Fig. 4. a. Nitrosomonas europaea, Alexander strain. Polar monotrichou.s
flagellation. Note the very short wavelength.
b. Nitrosomonas (?) sp., Alexander strain. Polar monotrichous flagellation.
Note the relatively long wavelength. This organism is not autotrophic and
probably not a Nitrosomonas sp.
c, d. N. europaea (?), ATCC 12248. The organisms shown in c and d
are typical examples of the organisms in the culture studied. Since the
culture was not strictly autotrophic the organisms shown are probably not
Nitrosomonas.
21
6. Nitrobacter
Only one culture labeled 'Nitrobacter was obtained, namely
'Nitrobacter agilis, ATCC 12812 (Fig. 5). The original liquid cul-
ture did not show motility, but staining showed a fair proportion
of the bacteria with Hagella. The organisms were very small, often
coccoid, with a single flagellum which was not polar. The flagella-
tion is perhaps best described as lateral monotrichous. Further
study of this organism should be made to be sure it is not peri-
trichous.
7. Hydrogenoftionas
Two cultures labeled Hijdrogenomoruis were studied: Hydro-
genomonas pantotropha NRRL, B-935 and Hijdrogenomonas facilis,
ATCC 11228 (Fig. 6). The first of these was nonflagellated. H.
facilis was well flagellated with polar monotrichous flagella of
average wavelength of 1.8 microns. The wavelength was quite
variable ranging from 1.3 to 2.2 microns.
8. Thiobacillus
Two species of the genus Thiobacillus were studied, namely,
Thiobacillus thiooxidans and Thiobacillus thioparus (Fig. 7). A
strain of each species was received from Dr. Robert Starkey of
Rutgers University, and a strain of each species from Dr. J. D.
Stout of the Ministry of Agriculture in New Zealand. The Starkey
strains were isolated from soil in the United States. The Stout
strain of T. thiooxidans was isolated from the water of a hot spring
in New Zealand and the T. thioparus strain from New Zealand
soil. The four strains appeared to be strict autotrophs but further
identification was not made by the author.
Flagellar Characteristics
The two T. thiooxidans cultures were very similar, with normal
polar monotrichous flagella. The Starkey culture of T. thioparus
appeared nonmotile and flagella could not be demonstrated. The
Stout culture of T. thioparus showed undulant polar monotrichous
flagella, often at both ends. No distinct variants were observed in
any of the cultures.
The wavelength of T. thiooxidans averaged 1.63 microns with
amplitude of 0.55 micron. The wavelength of T. thioparus could
not be measured accurately but was about 4.0 microns.
22
Fig. 5. a. Nitrobactcr agilis, ATCC 12812.
The organism illustrated shows the typical flagel-
lation of the individuals in the culture studied.
The flagellation may be labeled lateral mono-
trichous.
J
Fig. 6. a. Hydrogenomonas facilis, ATCC 11228.
Polar monotrichous flagellation.
)'
,' ■ / e^
Fig. 7. a. Thiobacillus thiooxidans showing a typical normal polar mono-
trichous flagellum. b and c. Thiobacillus thioparus showing undulant polar
monotrichous flagella.
23
9. Pseiidomonas
The genus Pseiidomonas has a large number of species many of
which are inadequately described and appear to be unobtainable
from any source. By definition, all motile strains of the genus
must have polar flagella, either polar monotrichous or polar multi-
trichous. Physiologically the most typical members of the genus
oxidize, but do not ferment, carbohydrates. Polar monotrichous or
multitrichous heterotrophic bacteria which ferment carbohydrates
with acid formation are better classified as Vibrio or Aeromoiias.
Still included in the genus are polar flagellated bacteria which
have no effect on carbohydrates. These are often mistakenly la-
beled Alcaligenes. The genus Lophomonas has been suggested for
a polar multitrichous or lophotrichous type which does not attack
carbohydrates. The type species, Fseudomonas aeruginosa, pro-
duces a water soluble greenish pigment, but many otherwise tvpical
species do not produce the pigment. For practical reasons the
genus will be discussed under three headings: (1) The ordinary
pseudomonads of fresh water, soil, and animal body; (2) the plant
pathogens; (3) the halophilic types.
Cultures
More than one hundred strains of the ordinary Pseiidomonas
from fresh water, soil, and the animal body were studied. These
were obtained from a variety of sources over a period of several
years. Among the major sources were Dr. W. D. Haynes of the
United States Department of Agriculture (U.S.D.A. ); Dr. Mac-
Donald Fulton, Stritch School of Medicine; WilHam Keller of
Philadelphia; the IlHnois State Health Laboratory, Chicago. All
of these cultures were identified physiologically and culturally as
well as morphologically. One culture, isolated from a five oyster,
showed mixed flagellation with a normal polar flagellum and one or
(more rarely) several lateral flagella of shorter wavelength. This
culture was physiologically typical of Pseiidomonas.
The majority of the plant pathogens, some twenty species,
were obtained from Dr. Mortimer P. Starr of the University of
California.
The halophihc types (fifteen strains) were mainly from R. A.
MacLeod of the Fisheries Research Board of Canada, Vancouver,
British Columbia. Aside from being halophilic these organisms
were typical Pseiidomonas species.
25
Flagellar Characteristics
The ordinary pseudomonads show two types of flagellation,
polar monotrichous and polar multitrichous (Fig. 8). Both of
these types are quite ubiquitous and often isolated from the hu-
man skin, throat, intestines, etc. Both types may produce a greenish
pigment but as a rule the monotrichous types produce the most
pigment. Pigment production appears to be limited to the carbo-
hydrate oxidizers. The nonoxidizers such as Fseudomonas dimi-
nuta, Fseudomonas stutzeri, etc. are nonpigmented. The most
Fig. 8. The ordinary pseudomonads.
a, b, c. Fseudomonas aeruginosa, a. The typical flagellation of a young
culture, b. Direct stain of the peritoneal fluid of an infected guinea pig. c. A
dividing organism.
d. P. fuorescens. Normal polar monotrichous flagellation.
e. P. diminuta. Note the extremely short wavelength.
f. P. nigrifaciens. This organism is larger than most pseudomonads and
the flagellum has an extraordinary long wavelength.
g. h. Pseudomonas sp. g. Either a polar monotrichous or a polar multi-
trichous organism, h. A typical polar multitrichous type.
i. P. sijnxantha, NCIB 8178. Polar multitrichous or lophotrichous flagella-
tion.
j. P. arsenooxijdans, NCIB 8685. Polar multitrichous flagella with coiling
tendency.
k. Pseudomonas sp. Polar multitrichous flagella of long wavelength. This
culture was received from Dr. S. F. Snieszko as Aeromonas, U-21. Its history
shows it originally came from R. R. Rucker of the Western Disease Laboratory,
Seattle, as No. 28.
1. P. cuneatus, comb, nov., ATCC 6972. Typical polar lophotrichous flagel-
lation. This culture is labeled Vibrio cuneatus in culture collections. Physio-
logically and morphologically it is very much like the phytopathogenic pseudo-
monads.
m. P.pseudomallei (Malleomijces pseudomallei) . Polar multritrichous
flagellation. Organism received from Dr. S. Gowan, British Type Culture Col-
lection.
n. P. chlororaphis, NCIB 8672. Typical polar monotrichous flagellation.
o. Pseudomonas sp., H-163. Short polar monotrichous flagellum. Culture
isolated from sputum lay Dr. Rudolph Hugh.
p. P.saccharophila, ATCC 9114. Typical polar monotrichous flagellation.
q. P. hookeri, comb, nov., ATCC 9128. This organism in culture collections
is labeled Alcaligenes hookeri. It does not attack carbohydrates but the
morphology places it in the Pseudomonas genus.
r. P.jaecalis var. radicans, comb, nov., ATCC 4741. This organism is
labeled Alcaligenes faecalis var. radicans in culture collections. It does not
-attack carbohydrates but has the typical flagellation of Pseudomonas.
a,c,h. From E. Leifson, /. Bactcriol. 62, 377-389 (1951).
26
k
0
J'
/
P .
>
X
i-
n . ^ .
r
common monotrichoiis types have a predominantly single flagellum
at one or both poles, rarely two flagella at one pole. The flagellar
wavelength (Table I) is remarkably uniform averaging from 1.7
to 1.8 microns. The common multitrichous types usually have from
two to five flagella at one pole, less often at both poles. The wave-
length of these flagella is distinctly greater than that of the mono-
trichous types, averaging from 2 to 2.5 microns. Flagella of longer
wavelengths are rare and usually limited to strains of the plant
pathogens. The extremes of wavelengths are illustrated by P.
diminuta, wavelength 0.7 micron, and Fseudomonas nigrifaciens.
Fig. 9. s. Pseudomonas sp., Fulton 3984. Culture isolated from human
pleural fluid at autopsy. Typical polar multitrichous flagellation. The chain of
bacilli show where and when the new flagella develop on the daughter cells.
In this organism the new flagella develop only after cell division is complete
and on the distal pole.
t. Pseudomonas sp. Organism isolated from the water of the DuPage
River. Capsulated organisms of this type are not infrequent in water.
u. Pseudomonas sp. The organism pictured is an example of mixed flagella-
tion. Note the difl^erence in wavelength of the polar and the lateral flagella.
The organism was isolated from a live oyster.
Fig. 10. The phytopathogenic pseudomonads.
a. Pseudomonas angulata, Starr PA 12. Polar multitrichous or lophotrichous
flagella.
b. P. cattleyae, Starr PC 107. Polar multitrichous flagella.
c. P.savastanoi, Starr PS 111. Polar lophotrichous flagella.
d. P. glycinea, NCIB 8613. Polar lophotrichous flagella.
e. f. P. washingtoniae, Starr PW 2. Polar multitrichous flagellation. In e
the two upper flagella have distinctly different wavelengths, the shorter meas-
uring 2.02 microns and the longer 2.57 microns. Many individuals in this
culture were mototrichous as illustrated in f.
g. Pseudomonas sp., Starr YCLS. Polar multitrichous flagella. This organ-
ism was reported by Dr. Starr to be pectolytic. It was physiologically typical
of the genus, and produced a reddish-purple water soluble pigment.
h. P. marginata, Starr PM 15. Polar multitrichous flagella. The soma is
stained rather lightly.
i. P. rihis, Starr PR 5. Polar multitrichous flagella of imusually long wave-
length. Coiled flagella were frequent.
j. Pseudomonas sp.. Smith Cabbage 2B. Polar lophotrichous flagella. This
organism was isolated by M. A. Smith, U.S.D.A., from diseased cabbage.
k. P. savastanoi var. fraxini, Starr PS 19. Polar lophotrichous flagellation.
The flagella are short with a tendency to coil.
1. P. polycolor, Starr PP 2. Polar monotrichous flagella. The phytopatho-
genicity of this organism seems doubtful. Morphologically it is not typical of
the phytopathogenic pseudomonads.
28
^ .4
■ '^0C3^
^
Figure
Q
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<
j
c
d
g .
i
k
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Figure 10.
29
wavelength 3.5 microns. Mixed flagellation with lateral flagella of
shorter wavelength than the polar flagellum was observed in a
culture isolated from a live oyster ( Fig. 9 ) .
The plant pathogens are remarkably uniform in their flagella-
tion which is polar multitrichous, often lophotrichous (Fig. 10).
The only exception studied is Pseiidomonas pohjcolor which, ac-
cording to personal communication from Dr. M. P. Starr, and
Bergey's Manual, is a questionable plant pathogen. The only
flagellar variant observed was in Pseudomoiias washingtoiiiae with
flagella of two distinctly different wavelengths.
All of the halophiles studied were monotrichous. Some showed
the typical flagellation of ordinary pseudomonads, others had
flagella of the undulant type. This type of flagella is rather rare
and has been observed mainly in what may be considered as
typical water bacteria, particularly marine. In two of the cultures
studied (B-13 and B-28) some individuals had normal flagella,
others had undulant flagella. One individual was found with a
normal flagellum at one pole and an undulant flagellum at the
other pole (Fig. 11a).
30
Fig. 11. The halophilic pseudomonads.
a, b. Pseudomonas sp., MacLeod MB-13. The great majority of the in-
dividuals in this culture showed polar monotrichous flagella with the undulant
shape, and a few with the normal shape. In a is shown an individual, probably
about to divide, with a normal flagellum at the upper pole and an undulant
flagellum at the lower pole. The undulant flagellar type was seen in several
strains of halophilic pseudomonads.
c. Fseudomonas sp., MacLeod B-28. Polar monotrichous flagellation of
normal type.
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10. Methanomonas
One culture labeled Pseudomonas methanica was received
from J. W. Foster of the University of Texas ( Fig. 12 ) . The organ-
isms were stained directly from the original slant. Subcultures on
peptone media did not grow. The organism was fairly well flagel-
lated with a single polar flagellum of normal curvature. No var-
iants were observed. The average flagellar wavelength was 1.77
microns with an average amplitude of 0.51 micron.
1 1 . Protaminohacter
One strain each of Protaminohacter ruber (NRRL,B-1048) and
Protaminohacter alboflavus (NRRL,B-1051) were received from
Dr. W. B. Haynes of the Northern Regional Research Laboratory
(NRRL), U.S.D.A., Peoria, Illinois. The culture of P. ruher pro-
duced reddish colonies on agar, did not acidify any carbohydrate
media tested, and was motile (Fig. 13). P. alhofiavus produced
a deep yellow pigment on agar, showed no effect on any carbo-
hydrate tested, and was nonmotile. Both cultures appeared to be
typical.
Flagellar Characteristics
P. alhofiavus was atrichous. P. ruher in moist preparation
showed a few individuals with rapid linear motion characteristic of
polar monotrichous bacteria. Flagella stain showed a few indi-
viduals with a single polar flagellum. The bacterial soma did not
take the flagella stain to any extent and counterstain was used.
The flagellar curvature was uniform, with an average wavelength
of 2.01 microns and average amplitude of 0.54 micron. Flagellar
variations were not observed.
34
Fig. 12. a. Mcthanomoiuis mcthanica (Pseudomonas
methanica) . Polar monotrichous flacjellation.
/
Fig. 13. a. Protaminohacter ruber, NRRL, B-1048.
Typical organism showing polar monotrichous flagella-
tion. With most individuals on the slide the soma did
not take the flagella stain, unlike the one photographed.
^
35
1 2 . Xanthomonas
The genus Xanthomonas is quite well defined. Typical species
are characterized by their polar monotrichous flagellation, the
yellow water-insoluble pigmentation, and rather feeble oxidative
metabolism of glucose and some other carbohydrates. Nonflagel-
lated strains are common. The phytopathogenic types are best
known, but nonpathogenic types also exist.
Cultures
Thirty-nine cultures were studied of which thirty-eight were
phytopathogenic types received from Dr. M. P. Starr of the Uni-
versity of California. Of these thirty-eight cultures twenty-one
were typical with polar monotrichous flagellation. The rest were
either nonflagellated (seventeen strains) or atypical in other re-
spects. Only one nonpathogenic culture was studied, namely,
Xanthomonas arsenooxydans, received from the National Collec-
tion of Industrial Bacteria (NCIB) as 8688.
Fig. 14. a. Xanthomonas campestris, XC-16. Polar monotrichous flagella.
b. X. campestris, XC-16. This picture is included to show the very rare
occurrence in Xanthomonas of two flagella at the same pole.
c. X. amaranthicola, XA-12()R. Polar monotrichous flagella.
d. X. amaranthicola, XA-120R. This picture may be interpreted in two
ways, either as representing one organism with two different polar flagella, or
as two organisms, each with a different polar flagellum. The wavelength of the
shorter flagellum is exactly one half that of the longer flagellum.
e. X. papavericola, XP-161. Polar monotrichous flagella.
f. X. manihotis, XM-12. Rather long polar flagellimi but otherwise typical.
g. X.vignicola, XV-118. Long polar flagellum with irregular wavelengths
and amplitudes. This was common in some strains.
h. X.ricinicola,XR-lOl. Typical polar flagellum.
i. X. rubrilineans, XR-2. Polar monotrichous flagellation.
j. X. zinniae, XZ-101. Typical polar flagellum.
k. X. zinniae, XZ-101. This illustrates a variant with a flagellum of longer
than average wavelength.
1. X.tardicrescens, ST-1. The average wavelength of this species is signifi-
cantly shorter than that of the more typical Xanthomonas species.
m. X. arsenooxijdnns, NCIB 8688. Polar monotrichous flagella. Note the
rather long wavelength and coiling tendency of the flagella.
36
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37
Flagellar Characteristics
All typical strains showed polar monotrichous flagellation.
Atrichous strains are common and the strains which are flagellated
are often very poorly so. In some species the flagella are of uni-
form curvature while in others the curvature is very irregular. In
two species were found individuals with flagella of two distinctly
different wavelengths (Figs. 14d, j, k). The one culture of X.
arsenooxijdans studied was well flagellated with flagella of rather
long wavelength and frequently coiled (Table II).
One culture labeled Bacterium tardicrescens was typical of
Xanthomvnas species except for failure to oxidize glucose. The
flagellar wavelength was significantly less than that of all the other
Xanthomonas species. In spite of these variations this organism
appears sufficiently typical to be classified as Xanthomonas tardi-
crescens comb. nov. X. rubrilineans did not oxidize glucose, was
nonpigmented but morphologically typical. The strain of Xantho-
morms heticola studied was peritrichously flagellated, nonpig-
mented, and oxidized glucose and sucrose. This organism could
be classified in the genus Agrohacterium.
38
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39
1 3 . Mycoplana
A culture of each of the two species of Mycoplana, Mycoplana
dimorpha and Mycoplana bullata (Fig. 15) were received from
Dr. Wilham B. Haynes of the Northern Regional Research Labora-
tory in Peoria, Illinois. The history of these cultures show that
they were received by the Peoria Laboratory directly from Dr.
Thornton and presumably are authentic. Physiologically both cul-
tures showed some differences from the original description as
recorded in the Bergey Manual. Neither strain reduced nitrate to
either nitrite or nitrogen gas. Both strains oxidized glucose, di-
morpha with slight acidity and bullata with considerable acidity.
Flagellar Characteristics
M. dimorpha was rather poorly flagellated with most often one
flagellum per organism, fairly often two flagella, and occasionally
three or four flagella per organism. The flagellar arrangement was
peritrichous. No flagellar variants were seen. The average wave-
length was 1.57 microns with amphtude of 0.56 micron. M. bul-
lata was very motile and well flagellated with polar monotrichous
flagella. Only one type of flagella was seen. The flagellar wave-
length was very short averaging only 0.91 micron.
ToxoNOMic Comments
The writer cannot see much, if any, justification for the con-
tinued existence of the genus Mi/coplaiia. M. bullata fits very well
into the genus Pseudomonas. Its somewhat unusual flagellar curva-
ture and, perhaps, physiology would justify retention of the specific
epithet and the organism could well be named Pseudomonas bul-
lata comb. nov. M. dimorpha could well fit into the genus Achro-
mobacter. As a matter of fact the characteristics given for
Achromobacter cycloclastes in Bergey's Manual agree more closely
with the characteristics of the strain of M. dimorpha studied than
with the characteristics given for M. dimorpha. The suggestion is
made that M. dimorpha be considered a synonym of Achromo-
bacter cycloclastes and that the genus Mycoplana be dropped.
40
b ^- •; V
Fig. 15. a. Mijcoplana dimorpha (Achromobacter cycloclastes) , NRRL B-
1091. Flagella with peritrichous arrangement and normal shape.
h,c. M.buUota {Pseudomoms hullata), NRRL B-1090. Polar mono-
trichous flageUation. Flagellar wavelength exceptionally short.
41
14, Lophofttonas
In the genus Lophomonas are classified a group of ubiquitous
bacteria found in soil, water, and occasionally isolated from human
and animal material such as feces, blood, etc. They are charac-
terized by their distinctive flagellation and failure to metabolize
carbohydrates. Thirty-seven strains have been studied in the au-
thor's laboratory. All were sufficiently alike to be placed in a
single species, Lophomonas faecalis (Fig. 16).
Flagellar Characteristics
All strains of Lophomonas by definition are polar multitrichous
or lophotrichous. The flagella tend to be relatively short with very
long wavelength such that it is rare to find a flagellum with a
complete wave. This type of flagellation is also typical of spirilla
but rare in other bacteria.
Several years ago a culture was received from the American
Type Culture Collection labeled Vibrio percohns, 8461. When this
culture was stained both polar and peritrichous individuals were
observed on the slides. Some individuals showed both polar and
lateral flagella of different wavelength. By plating, the original
culture could be separated into pure variants with polar and
peritrichous flagella, respectively. The mixed type could not be
isolated and is an unstable transitional type. The two flagellar
variants were physiologically identical but differed slightly in
flagellar antigenicity. The polar variant continued to produce
peritrichous forms but the peritrichous variant appears to be stable.
In other words, the mutation is always from the polar type to the
peritrichous type and never the reverse. This type of mutation
appears to be the first of its kind ever described and substantiated
by photomicrographs of the mutants and their intermediates. The
mutation raises a serious problem in bacterial taxonomy and sug-
gests that a closer relationship exists between polar and nonpolar
flagellates than our present taxonomy indicates. The peritrichous
mutant is a typical Alcaligenes sp. with curly flagella. None of the
other thirty-six strains of Lophomonas studied have shown any
evidence of a similar, or any, morphological mutation.
The wavelength of the polar flagella is somewhat indefinite
since a complete wave is rarely found and only half waves can
be measured. The wavelengths and amplitudes of the polar flagella
of the various strains studied did not show significant differences.
The mean wavelength of the polar flagella for the species was
42
v..
# I
^
:V.- »
i^^
9
^
L
Fig. 16. a, b, c. Lopho7nonas faecalis. Typical polar lophotrichous flagella-
tion. In c is shown the filamentous form.
d, e, f, g, h, i. L. faecalis {Vibrio percolans), ATCC 8461. In d is shown
the polar lophotrichous type which mutates through intermediates e, f, and g to
the peritriclious flagellated types h and i. Note the difference in wavelengtli
of the polar and the lateral flagella.
b. From E. Leifson, /. Bacteriol. 62, 377-389 (1951). d, f. From E. Leif-
son, and R. Hugh, /. Bacterid. 65, 263-271 (1953). a,c, e. From T. P. Galar-
neault, and E. Leifson, Can. J. Microbiol. 2, 102-110 (1956).
3.10 microns and the amplitude 1.08 microns. The mean wave-
length of the peritrichous mutant was 1.05 microns with amplitude
of 0.42 micron. The wavelength of the polar flagella is thus
almost exactly three times that of the lateral flagella.
15. Acetofttonas
The bacteria which are used for the commercial production
of vinegar may be classified into two groups which are distinctly
different both physiologically and morphologically. For these
groups the generic names Acetomonas and Acetobacter have been
suggested. In the genus Acetobacter are placed the organisms
which oxidize acetic acid ( and some other acids ) to carbon dioxide
and water, and have peritrichous flagella, if any. In the genus
Acetomon^as are placed the organisms wliich do not oxidize acetic
acid and which have polar flagella, if any (Fig. 17).
Ten cultures representing the various named species of Aceto-
monas were studied. Five of these were received from Prof.
Frateur of Louvain University in Belgium, and five from W. B.
Haynes of the U.S.D.A. in Peoria, Illinois. All were physiologically
and culturally typical of the genus. Nine of the cultures were
motile and one was nonmotile.
Flagellar Characteristics
The motility and flagellation of Acetomonas is generally much
better than that of Acetobacter. All strains showed polar multi-
trichous flagellation with flagella of uniform shape and quite short
wavelength. The number of flagella per individual varied among
the strains with some strains showing many monotrichous indi-
viduals, others only a few. The flagellar wavelengths of all the
strains averaged 1.4 microns with a range of 1.2 to 1.5 microns.
This wavelength is unusually short for polar multitrichous bacteria
and strikingly different from the polar multitrichous pseudomonads.
44
b.
Fig. 17. a. Acetomonas suboxydans var. roseum, F-16. Polar miilti-
trichous flagellation. The soma below the flagella is stained rather faintly.
b. A. suboxydans, B-72. Two organisms, one with a tuft of five typical
polar flagella and the other with a single short flagellum.
c. A. melanogena, F-8. A typical tuft of polar flagella extending up from
the faintly stained soma.
d. A. melanogena, B-58. A typical tuft of five polar flagella with a more
faintly stained soma.
a, b, c, d. From E. Leifson, Antonie van Leetnvenhoek, ]. Microbiol. Serol.
20, 102-110 (1954).
45
1 6. Acetobacter
Nine cultures of Acetobacter were received from W. B. Haynes
of the U.S.D.A. in Peoria, Illinois; nine cultures from Prof. Frateur
of Louvain University in Belgium; one culture from C. B, van
Niel, University of California; one culture from ATCC; and
one culture from J. L. Shimwell, British Vinegars Ltd. Of these
twenty-one cultures, six were motile and flagellated. All were
typical of the genus.
Flagellar Characteristics
Acetobacter strains are often nonmotile and those which show
motility are usually poorly flagellated even under the most favor-
able growth conditions. A fairly satisfactory medium is glucose-
peptone-yeast extract broth at pH 6, incubated at 20° C. On
centrifugation the organisms tend to clump and few individual
bacteria are likely to be found on the slides. The flagella, how-
ever, stain readily.
The six motile cultures in the collection showed peritrichous
flagellation. The shape of the flagella varied considerably among
the different strains. In some the flagella were mainly of the
coiled type with long but very irregular wavelength of 3-4 microns.
In other cultures the flagella were more uniform in shape with
wavelengths for example of 2.36 microns for Acetobacter aceti,
F-4; 2.21 microns for Acetobacter orleanense, B-55; and 1.82 mi-
crons for Acetobacter aceti, Shimwell. The Shimwell culture was
somewhat different from the others in being much better flagel-
lated, showed less clumping, and having flagella of shorter wave-
length and smaller ampHtude than the others (Fig. 18).
1 7. Xyntotnonas
Through the courtesy of J. L. Shimwell one culture of Zijmo-
monas (SaccJuiromonas) anaerobia was received from Bristol Uni-
versity in England. A culture of Fseudomonas {Zijmomonas) lind-
neri obtained from Dr. Haynes was nonflagellated. Another species
of this genus, Zijmomonus mobile, was not obtained. These bac-
teria are often referred to in industry as cider sickness organisms.
Flagellar Characteristics
The one culture of Zijmomonas anaerobia from Bristol Univer-
sity showed organisms with a tuft of polar flagella of wavelength
averaging 2.5 microns (Fig. 19). No variants were observed.
46
5?^
^^^^^
Fig. 18. a. Acctohacter rancens, F-5. A clump of two organisms with
lightly stained soma. Peritrichous flagella of normal curvature.
b. A. aceti, B-1036. A single individual, rarely found on slides of this
genus, showing peritrichous flagellation. The flagellar curvature is normal.
c. A. aceti, Shimwell. This strain was better flagellated than the other
strains studied and the organisms showed less tendency to clump. Many in-
dividuals showed from five to seven flagella. The flagellar arrangement is
typically peritrichous.
a, b. From E. Leifson, Antonie van Leeuwenhoek, J. Microbiol. Serol. 20,
102-110 (1954).
Fig. 19. a. Zymomonas anaerobia var. ponia-
ceae. The picture shows the typical flagellation of
this organism. The soma of the organism shown
is somewhat larger than the average.
r
47
1 8. Aerontonas
Tlie genus Aeromoims has fairly recently been created for a
group of organisms formerly classified as Pseudomonos. These or-
ganisms differ from the type species of Pseudomonos, Pseiido-
monas aeruginosa, by their fermentative metabolism of carbohy-
drates, absence of pigmentation, and a tendency to produce lateral
flagella in addition to the polar flagellum in young cultures. In
this group are found bacteria which are pathogenic for frogs
{Pseudomonas hydrophila, Proteus hydrophila, etc.), for fish, and
other cold blooded animals.
Cultures
Ten cultures have been studied. Six of these were isolated in
the United States and supplied by Dr. S. F. Snieszko of the U.S.
Fish and Wildlife Service. They were isolated mainly from dis-
eased fish. Three of the cultures originated from Holland and
were isolated from water. One culture, Aeromonas formicans, was
received from Dr. H. Pivnich, University of Nebraska. All the
cultures were physiologically typical of the genus. One culture
was peritrichously flagellated (Kluyver strain L-418) when re-
ceived and has remained unchanged over a period of several years.
From personal communication this culture apparently was polar
flagellated when first isolated. It has the typical physiology of
Aeromonas and appears to be a stable variant (?). The coiled
shape of the flagella is also found in the polar flagella of other
strains as may be observed from the illustrations.
Flagellar Characteristics
In cultures which were incubated past the logarithmic phase
of growth, i.e. overnight at temperatures between 20° and 37° C,
all strains of Aeromonas studied showed mainly polar monotrichous
flagellation. Some strains produced only the normal type of polar
flagellum while in the other strains several types of polar flagella
were produced: undulant, normal, and coiled. The undulant type
has a wavelength averaging 3.5 microns and the normal type a
48
wavelength averaging 1.7 microns. The ratio of the two wave-
lengths is thus almost exactly 2:1. By plating in semisolid agar,
colonies showing much and little spreading were found. When
these were fished the small spreading colonies showed bacteria
mainly with the undulant type of polar llagellum while the bac-
teria from the larger spreaders had mainly the normal type of
polar flagellum. However the cultures from the fished colonies
were not pure for one or the other type of flagella, and variation
from the one type to the other must take place at a high rate.
Individuals with both types of flagella are occasionally seen, even
at the same pole, as illustrated in Fig. 20d. The undulant flagel-
lum is apparently less efficient for locomotion than the normal
flagellum. Individuals with coiled polar flagella were occasionally
observed. In one strain (Kluyver L-417) the coiled flagella were
very common and often multiple as illustrated in Figs. 20h and i.
The peritrichous variant with coiled flagella may have originated
from this type with multiple coiled polar flagella. However, a
mutation from polar to peritrichous was not observed in the au-
thor's laboratory.
The most unique feature of Aeromonas flagellation is the forma-
tion of lateral flagella in very young cultures. This phenomenon,
to a variable extent, has been observed in all but one of the strains
studied. In one strain (Kluyver L-417) the lateral flagella had the
same wavelength as the polar flagellum. With the other strains
the lateral flagella had a definitely shorter wavelength than the
polar flagellum. In all the strains studied the lateral flagella
showed the same wavelength of 1.5 microns irrespective of the
wavelength of the polar flagellum. Individuals with undulant, nor-
mal, or coiled polar flagella produced normal lateral flagella of
the same wavelength. Coiled or undulant lateral flagella were
never observed in the young cultures.
49
Fig. 20. a, b, c, d, e, f . Aeromonas sp., Snieszko U-6. This typical culture
of Aeromonas was isolated from diseased fish. Old cultures showed a mixture
of polar flagellated individuals, some with the normal type of flagella and some
with the undulant type, as illustrated in a and b. One individual was found
with a normal and an undulant flagellum at the same pole (d). In young
cultures lateral flagella were found, both on individuals with a normal polar
flagellum and with an undulant polar flagellum, as illustrated in e and f. Note
the similarity of the lateral flagella in the two types.
g, h, i, j. Aeromonas hydrophila, ATCC 7965. In old cultures were found in-
dividuals with a normal single polar flagellum, illustrated in g, and individuals
with a polar tuft of coiled flagella, illustrated in h. In young cultures individuals
with lateral flagella of short wavelength were found as illustrated in i and j.
k, 1. A. liquefaciens, Kluyver L-417. Old cultures of this strain showed
normal single polar flagella only, while young cultures showed many individuals
with peritrichous flagellation. In this strain the polar and lateral flagella were
of the same wavelength.
m. A. formicans, Pivnick. Normal polar monotrichous flagellation. Both
old and young cultures showed only this type of flagellation. This organism
may not be a pathogen for cold bloocled animals.
n. Aeromonas sp., Snieszko, U-23. The polar flagellum illustrated is unusual
in that the wavelength gets progressively longer from the soma out. This type
of flagellum was seen frequently in several strains of Aeromonas but is very
rare in other bacterial genera.
o. Aeromonas (?) sp. This culture was received as A. liquefaciens, Kluyver
L-418. It is physiologically typical of Aeromonas and may be a morphological
mutant but definite evidence to this effect is lacking. All individuals in the
culture showed the coiled peritrichous flagellation. Morphologically and physio-
logically this organism is very similar to a nonpigmented Serratia.
g, h, i, j, k, 1, o. From E. Leifson, and R. Hugh, /. Bacteriol. 65, 263-271
(19.53).
50
a b
c Ad.
r: ^ i
<0
m •. ♦.
19. Vibrio
The genus Vibrio is composed of bacteria which typically have
a slight somatic cm-vature and a single polar flagellum (Fig. 21).
The somatic curvature is an unreliable characteristic which has,
perhaps, little taxonomic significance. Carbohydrates are fermented
with acid production but no gas. Most strains are very proteolytic,
actively liquefying gelatin and coagulated serum. Bergey's Manual,
6th ed., recognizes twenty-two species. Several of these species
are definitely out of place in the genus, such as Vibrio percolans
(Lophomonas) and Vibrio cimeatus (Pseiidomonas). Several other
species obtained from culture collections had peritrichous flagella
and therefore are not Vibrio species. These latter, of course, may
have been contaminants which had replaced the original vibrios.
In this category may, perhaps, be Vibrio jejuni, ATCC 11734, which
had peritrichous flagella.
Fig. 21. a. Vibrio cholerae, Freter 144. Typical specimen showing polar
monotrichous flagellation.
b. V. proteus. This picture is of an old stock strain labeled V. finklur-prior.
c. Vibrio { Pseud omonas) rubicundus, Haynes B-782. This organism is
more properly classified as Pseiidomonas sp. since it did not attack carbo-
hydrates.
d. V. tyrogeniis, Haynes B-1033. The soma of this organism often re-
sembled a spirilkim with several curves. A very few individuals had multiple
flagella at one pole.
e. f. Vibrio sp., MacLeod MB-26. This is a halophilic vibrio from the
Pacific Ocean. The organisms with the straight soma were more numerous
than those with a curved soma.
g, h. V. fetus, Hansen. Most of the individuals in the culture studied
showed polar monotrichous flagellation illustrated in g. A fair number showed
polar multitrichous flagellation illustrated in h. The resemblance to spirilla is
striking.
i, j. V. fetus, Rylf 28099. In i is shown an organism with several curves like
a spirillum. In j is shown the rounded types or "microcysts" so typical of
spirilla.
k, 1. V. coli, Di Liello 505. The organism illustrated in 1 is in the process
of microcyst formation.
m. V. coli, Di Liello 498. The bipolar flagellation was common in the
V. coli cultures as it was in the V. fetus cultures.
n. Vibrio (?) jejuni, ATCC 11734. This is obviously not a Vibrio sp. In
the culture studied the soma was generally curved as illustrated. The peri-
trichous flagellation is unmistakable. Cultures other than this could not be
obtained.
52
a
e
b
f
c d -
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53
Cultures
Included in the study were several strains of V. choleroe from
the University of Chicago through Dr. Rolf Freter; one strain of
Vibrio fetus from P. Arne Hansen of the University of Maryland
and four strains from J. F. Ryff of the Wyoming State Veterinary
Laboratory; two strains of Vibrio coli from Leo R. Di Liello in
Maryland; one strain of Vibrio tyrogenus from W. B. Haynes of
the Northern Regional Research Laboratory, U.S.D.A.; and Vibrio
proteus from stock. Vibrio rubicundus, received from W. B.
Haynes, was a polar monotrichous fairly straight rod which did
not ferment glucose and, if authentic, is better classified in the
genus Pseudomonas. Several strains of halophilic vibrios, both
luminescent and nonluminescent types, were studied. The non-
luminescent strains came from R. A. MacLeod, Fisheries Research
Board of Canada, Vancouver. The luminescent strains came from
R. Spencer of Humber Laboratory, Hull, England. The latter
strains are discussed under Photobacterium although the author
does not wholeheartedly favor the existence of the genus Photo-
bacterium.
Flagellar Characteristics
All typical flagellated vibrios appear to have polar monotrichous
flagellation. Multiple flagella at one pole and bipolar flagella are
rare except in V. fetus. Multiple polar flagella and bipolar flagella
are common in V. fetus. The soma of the latter organism often
has several curves like a spirillum. In some cultures the rounded
forms (microcysts) were numerous, which is also characteristic of
spirilla. The polar flagella, when multiple, frequently are short
with few curves of large amplitude, which gives a rather typical
spirillum picture. Physiologically V. fetus has few characteristics
of a typical vibrio and more closely resembles the spirilla. The
original classification of this organism as a SpiriUum has much in
its favor. Morphologically the two cultures of V. coli resembled
those of V. fetus rather closely. Microcysts were also observed in
V. coli but not multiple polar flagella.
Morphologically the halophilic vibrios do not differ significantly
from the nonhalophilic types. In artificial culture the soma is
usually a short or oval rod, rarely curved. Identification as vibrios
is based on the polar monotrichous flagellation, the fermentative
54
action on carbohydrates, and other physiological characteristics.
All strains studied liquefied gelatin. In most instances the flagella
had normal curvature. The flagellar wavelengths of the species
studied are given in Table III.
TABLE III
Mean Flagellar Wavelengths of Vibrio Species
Wavelength
Species Strain ( microns )
2.43
2.01
2.08
1.87
2.06
2.02
1.85
1.95
V. cholerae
F-144
V. proteus
—
V. fetus
Hansen
V. fetus
RyfiF
V. ttjrogenus
NRRL, B-1033
V. rubicundus
NRRL, B-782
V. coli
Di Liello
Vibrio sp. ( halophilic )
MacLeod
55
2 0 . Desulfovihrio
The several species of Desidfovibrio listed in Bergey's Manual,
6th ed., are stated to be morphologically indistinguishable. A
culture was received from Dr. C. E. ZoBell of the Scripps Oceano-
graphic Institute in La Jolla, California. According to Dr. ZoBell
the culture was not pure and this was verified by staining. The
organism pictured was most typical for a vibrio and it is repro-
duced with this questionable identification (Fig. 22).
Flagellar Characteristics
Assuming the organism referred to above to be a Desidfovibrio
species, the flagellation is polar monotrichous. The flagellar wave-
length is quite long, averaging 3.0 microns. Since a pure culture
was not available for study, nothing can be said about variations.
21. Cellvibrio
Bergey's Manual, 6th ed., lists four species of Cellvibrio which
are differentiated on the bases of growth on glucose and starch
agar and degree of pigmentation. Several cultures were obtained
for study. One culture was obtained from W. B. Haynes of
the Northern Regional Research Laboratory, U.S.D.A., Peoria,
Illinois. This culture (B-668) was simply labeled Cellvibrio sp.
Four cultures were obtained from Dr. H. W. Reuszer of Purdue
University. Two were labeled Cellvibrio vulgaris, strain 6, and
strain 122. Two were labeled Cellvibrio fulviis, strain 18, and
strain 102. All five of these cultures grew well on glucose agar
slants, producing at first a yellow water-insoluble pigment which
later turned brown. In glucose-yeast extract broth growth was
fair with the formation of a brown pellicle. In dextrose semisolid
agar all cultures produced a very slight acidity under aerobic con-
ditions but no acidity under anaerobic conditions. Physiologically
and culturally the five cultures appeared to be more or less iden-
tical.
Flagellar Characteristics
All four of the Reuszer strains showed the same two types of
flagellation, differing only in the relative proportions of the two
types. C. fulviis, Reuszer strain 18, showed mainly small curved
rods with single polar flagella of relatively long wavelength as
illustrated in Fig. 23b. Also present in lesser numbers was a small
straight rod with a single polar flagellum of relatively short wave-
56
Fig. 22. a. Desulfovihrio sp. (?), ZoBell strain
249. Typical polar monotrichous flagellation with
long wavelength and large amplitude.
1-.
y i ■;• )
f A
J
Fig. 23. a. Cellvibrio vulgaris, Reuszer 122. The soma is straight with
rounded ends and with a single polar flagellum of relatively short wavelength.
b. C fulvus, Reuszer 18. The soma is typically vibrio shaped with a single
polar flagellum of relatively long wavelength.
c. Cellvibriv sp., Haynes B-668. Single polar flagellum with the long
wavelength. In this individual the soma is only very slightly curved.
d. C. vulgaris, Reuszer 122. Polar monotrichous flagella with the longer
wavelength.
e. C. fulvus, Reuszer 102. This shows the same type of organism illustrated
in a. In addition to the individuals with the single polar flagellum the rather
unusual situation pictured was quite common. With most bacteria in which
the new flagella develop before cell division is completed, bipolar or amphi-
trichous flagellation is produced. In other words, the distal ends of the daugh-
ter cells usually carry the flagella. With the organism pictured it appears as
if the region of cell division develops the new flagella.
f. C. fulvus, Reuszer 102. Polar monotrichous flagellation of same type
shown in b.
57
length as illustrated in Fig. 23a. In C. fiilvus, Reuszer strain 102,
the individuals with the short flagellar wavelength were most
abundant but otherwise the two strains were morphologically
alike. Both strains of C. vulgaris showed the same two types of
individuals with the long wavelength type most abundant in
Reuszer strain 122. The average flagellar wavelength of the two
types was 2.06 microns and 0.84 micron, respectively.
According to letter communication from Dr. Reuszer, he also
had observed two somatic types of individuals in his Cellvibrio
cultures. By plating and fishing single colonies he had not suc-
ceeded in obtaining stable pure cultures of each somatic tv^pe.
The author also plated the cultures and fished a number of typical
yellow-brown colonies. The fresh isolates invariably showed only
individuals with the short wavelength flagella. Transfers from
these isolates showed only individuals with the short wavelength
flagella. The long wavelength type was not observed in pure cul-
ture and its relationship to the short wavelength type remains un-
determined.
22. Succinovibrio
One culture of Succinovibrio dextrinosolvens C-85 was obtained
from Dr. M. Bryant of the U.S.D.A., Beltsville, Maryland (Fig. 24).
This culture was isolated from cow rumen fluid. It was strictlv
anaerobic.
Flagellar Characteristics
The one culture studied showed a large rod shaped organism
with somatic curvature like a vibrio. A few individuals were
flagellated with a single polar flagellum of rather long wave-
length, averaging 3.2 microns. No variations were observed.
2 3 . Lachnospira
A culture of Lachnospira multiparus, D-38, was obtained from
Dr. M. Bryant of the U.S.D.A., Beltsville, Maryland (Fig. 25).
Flagellar Characteristics
The flagellation of this organism is very unusual. The individual
organisms appear as slightly curved rods with a single flagellum
originating from the side, usually near the center. This type of
flagellation is designated as lateral monotrichous and is quite rare.
The one culture studied labeled Nitrobacter agilis also seemed to
have this type of flagellation. The flagellar wavelength is rather
long, averaging 3.0 microns.
58
\
Fig. 24. a. Succinovibrio dextrinosolvcns, Bry-
ant, C-85. Polar monotrichous flagellation. The
flagella stained with some difficulty and rather
lightly.
y
a
V- V
Fig. 25. a. Lachnospira multiparus, Bryant D-38. This shows the organ-
ism in the filamentous form. The polar location of the flagellum at the end
of the filament is not significant since individual organisms did not show
polar flagellation.
b, c. L. multiparus, Bryant D-38. In c is a single individual showing lateral
monotrichous flagellation. In b is shown two individuals, one of which has a
single lateral flagellum.
59
24. Spirillum
A representative collection of twenty-six cultures of Spirillum
were studied morphologically. One culture of SpiriUum vir-
ginianum was received from C. B. van Niel, Hopkins Marine
Station; one culture of SpiriUum serpens came from the Midwest
Culture Service; and all the others from Marion Williams, Uni-
versity of Southern IlHnois. The Williams collection of twenty-
four cultures was about evenly divided into fresh water and
marine forms. The marine forms were cultured in media with
Fig. 26. a. Spirillum serpens, Williams. A typical large fresh water spiril-
lum with a tuft of flagella at both ends. Note the relatively short flagella with
few curves. Organisms with flagella only at one pole do occur but not as
commonly as the amphitrichous types.
b. Spirillum sp., Williams LA-1. A fresh water spirillum with a large
number of flagella. The tufts of lateral flagella presumably originate at points
of somatic cleavage.
c. Spirillum sp., Williams Sb-10. A typical, medium sized fresh water
spirillum.
d. S. itersonii, Giesberger strain. A relatively small fresh water spirilkmi.
e. f. S.virginianum, Hopkins Marine Station 0.1.1. This spirillum tends
to be short and only slightly curved. Some individuals may even be perfectly
straight like that pictured in e. The flagellation is polar lophotrichous and
typical of spirilla.
g. Spirillum sp., Williams Sb-9. A typical fresh water spirillum.
h, i, j. Spirillum sp., Williams 2E-6. This is a typical marine spirillum. In
h is shown the normal or vegetative form, and in i and j, the "microcyst" form.
The microcysts are usually slightly oval and may have flagella at one or both
poles. The flagella apparently are not disturbed when the vegetative form
rounds up or condenses to form the microcyst.
k. Spirillum sp. This organism was not isolated in culture but stained
directly from the intestinal contents of a dog. The soma is short and twisted
into a spiral. The flagellation is polar monotrichous.
1. S. pohjmorphum, Williams. This species was the only one of the twenty-
six cultures studied with polar monotrichous flagellation. The culture grew
poorly and very slowly on the media used. The organism is rather small and
only a few individuals were flagellated. All flagellated indi\iduals had the
same type of polar monotrichous flagella.
m. S. linum, Williams. This is a typical marine spirillum of average size.
The organism to the right shows a flagellum with several curves and relatively
short wavelength, along with the more normal flagella of spirilla. This type
of flagellum appears to be extremely rare in spirilla and was seen only in this
particidar culture. It may be considered as equivalent to the curly type of
flagella in other bacteria.
e,f. From T. P. Galarncault, and E. Leifson, Can. J. Microbiol. 2, 102-110
(1956). k. From E. Leifson, }. Bactcriol. 62, 377-389 (1951).
60
i^A:tj:
» . . *
r
m^ f^
i. ^ • ^ 4
m
61
3% sodium chloride, the others in ordinary media made with soya
peptone. Unfortunately a culture of Spirillum minus was not ob-
tained.
Flagellar Characteristics
All spirilla are polar flagellated, usually polar multitrichous or
lophotrichous. Only one named species showed polar mono-
trichous flagella, namely Spirillum polijmorphum. A polar mono-
trichous type was also observed in a smear from the dog intestine
and is illustrated in Fig. 26k. The typical flagella of spirilla have
a very long wavelength (over 3 microns) and usually less than
one complete wave. Only one culture, Spirillum linum, showed
an occasional flagellum with short wavelength (about 1.5 microns)
and several waves (Fig. 26m). This flagellum may be con-
sidered as the curly variant although the wavelength is consider-
ably greater than the curly flagella of most bacteria. In the marine
forms the oval "microcysts" were much in evidence. These showed
the same flagellation as the normal organisms, as illustrated in
Figs. 26h, i, j. Although it is usual to find flagella at both ends of
a spirillum, organisms with flagella at only one end were present
in every culture. In spite of all the statements in the literature
about the unusual nature of the flagella on spirilla the author
finds them no different fundamentally from the flagella of other
bacteria. The genus Lophomotms has the same type of flagellation
as Spirillum, as have also a few Pseudomonas types, particularly
among the plant pathogens. Most bacteria, however, have flagella
with more curves.
2 5. Azotobacter
Complete agreement on the taxonomy of Azotobacter appar-
ently has not been reached. Bergey's Manual lists three species
and does not include Azotobacter vinelandii. Azotobacter macro-
cytogenes is also not included. Eight strains labeled Azotobacter
chroococcum were received; one from William D. Haynes of the
Northern Regional Research Laboratory of the U.S.D.A., Peoria,
Illinois, and seven strains from Perry Wilson of the University of
Wisconsin. Four of the Wilson strains showed good motility and
were typical in every respect including a light brownish pigmenta-
tion. The other strains were nonmotile or so poorlv motile as to
62
be unsatisfactory for flagellar studies. Two strains each of Azoto-
hacter agilis and A. vinckindii were received from Dr. Wilson.
The two strains of A. agilis showed good motihty, while one strain
of A. vinekindii was motile but the other not. Azotobacter in-
dicum 8597 was received from the National Collection of Industrial
Bacteria in England and also A. nmcrocijtogenes 8700. A. indicum
9037, 9038, 9039, and 9540 were received from ATCC. Of the
indicum strains onlv ATCC 9038 and 9039 were flagellated. (See
Fig. 27.)
Flagellar Characteristics
A. agilis was actively motile and showed good flagellation.
The flagella were peritrichously arranged and either normal, coiled,
or both coiled and normal on the same individual, A. vinelandii,
strain Wilson O, was actively motile with peritrichously arranged
flagella. Only normal flagella were observed. The four strains of
A. chroococcum which were motile showed peritrichously arranged
flagella. The best stains were obtained from mannitol agar slants
without peptone. When the four strains were stained from sus-
pensions made alkaline with dibasic potassium phosphate, prior to
the addition of formalin, all showed normal flagella only. However,
when stained from suspensions made shghtly acid with monobasic
potassium phosphate, strains E-2 and E-3 again showed only normal
flagella while strains E-7 and E-8 showed curly flagella only. This
change of flagellar curvature by change of pH is not common and it
is curious to find this difterence in strains of A. chroococcum which,
superficially at least, appear to be alike. A. indicum, ATCC strains
9038 and 9039, were slightly motile and some individuals showed
peritrichous flagellation. The flagella showed a strong polar tend-
ency, often appearing as a tuft of polar flagella. All the observed
flagella of these strains had a short wavelength comparable to the
curly type of A. chroococcum. Change of pH did not affect the
wavelength of these flagella.
A. macrocijtogenes, NCIB 8700, showed polar flagellation. The
flagella were most frequently single, but occasionally multiple, and
this culture must be regarded as polar multitrichous. This or-
ganism should, therefore, not be classified in the genus Azoto-
bacter. If it is a nonsymbiotic nitrogen fixing organism it could
be classified in the genus Azofomonas.
63
Fig. 27. a. Azotobacter chroococcum, Wilson E-7. Peritrichous flagella
with normal curvature. These organisms are from a suspension to which
K^HPO^ had been added prior to the formalin. The average normal wave-
length of this strain was 2.70 microns.
b. A. chroococcum, Wilson E-7. Peritrichous flagella with curly curvature.
These organisms are from a suspension to which KH-.PO^ had been added
prior to the formalin. The average curly wavelength was 1.20 microns.
c. A. vinelandii, Wilson O. Peritrichous flagella of normal curvature. Aver-
age normal wavelength was 2.71 microns.
d. A. agilis, Wilson 4-4. Peritrichous flagella typical in this culture with
partly normal and partly coiled curvature. Average wavelength about 3.2
microns.
e. A. irulicum, ATCC 9039. Peritrichous flagella of short wavelength com-
parable to the curly type of A. chroococcum. Note the polar tendency of the
flagella. The average flagellar wavelength was 1.0 micron.
f. g. A. (Azotomonas) macrocijtogenes, NCIB 8700. Polar multitrichous
and polar monotrichous flagella. The soma of this organism is much like that of
the typical Azotobacter strains but the flagella are distinctly different both as
to arrangement and shape. Average wavelength was 1.7 microns.
64
"i/^^
I
y
t
^
i
65
26, Azotoftionas
The genus Azotoinonas has only one species, Azotomonas in-
solita (Fig. 28). The organism is described as polar flagellated
with one to three flagella, ferments a variety of carbohydrates, in-
cluding lactose, with acid and gas. In spite of the lactose fermenta-
tion "no change" is recorded for milk which seems improbable
unless the organism fails to grow in milk. Gas production from
carbohydrates by polar flagellated bacteria is so unusual as to cast
doubt on the correctness of the description.
One strain of A. insolita was obtained from the National Col-
lection of Industrial Bacteria in England. This organism showed
peritrichous flagellation. It must be stated, however, that in a
somewhat sparsely flagellated organism many individuals will be
found with polarly located flagella. This was also the case with
the strain of A. insolita studied, but lateral flagella were too fre-
quent to cause any doubt that it is peritrichous flagellated. The
strain studied was rather poorly flagellated with up to four flagella
per individual. The wavelength was rather short, averaging 1.4
microns. If this culture is an authentic A. insolita strain then the
genus Azotomonas must be redefined. Morphologically the or-
ganism resembles rather closely some species of Rhizobium but
does not resemble the common species of Azotobacter.
66
Fig. 28. a. Azotomonas insolita, NCIB 8627.
Peritrichous flagella of rather short wavelength. Many
individuals on the shde showed single flagella often
at the pole, as well as several flagella at the pole.
However, the peritrichous nature of the flagellation
seems unquestionable. The flagellation is very simi-
lar to some species of Rhizobium.
\k
67
27. KhizobiuTti
The genus Rhizohium is composed of a group of bacteria (Fig.
29) which are able to invade the root tissues of specific leguminous
plants with the formation of characteristic nodules. The bacteria
Fig. 29. a. From Vigna sinensis, 3I6nlO. Subpolar nionotrichous flagella.
b. From Phaseolus lunatus, 3I6dlO. Subpolar monotrichous flagellation.
c. From Phaseolus aureus, 3I6h7. Subpolar monotrichous flagellation. The
organism pictured appears to be made up of three cells with the flagellum
originating from the middle of the right end cell.
d. From Phaseolus angularis, 3I6fl. Subpolar monotrichous flagellation.
e. From Phaseolus aconitifolius, 3I6gl. Subpolar monotrichous flagellation.
f. From Albizzia fulibrissen, lBoa2. Subpolar monotrichous flagellation.
g. h, i, j. From Glycina hispida, 3Ilb59. g. The basic subpolar mono-
trichous flagellation of this strain, h. A curly flagellum in addition to the
normal flagellum. i. Four long curly flagella in addition to the normal flagel-
lum. j. Two curly flagella but no normal flagellum.
k. From Ulex europaeus, 3C3al. Subpolar monotrichous flagellation.
1. From Lupinus sp., 3C2k5. Subpolar nionotrichous flagellation.
m, n, o. From Enjthrina itidica, 3I2bl. m. A subpolar flagellum of some-
what peculiar shape, n. Two curly flagella in addition to the normal flagellum
which is short and hooked in this strain, o. Two curly flagella only.
p, q. From Pisum arvense. Peritrichous flagellation. The peculiar flagella
shown in q were common in this strain. Compare it with the illustration of
Agrobacteritim rhizogenes.
r. From Trifolium. dubitim, 3D 1x3. Peritrichous flagellation.
s. From Phaseolus vulgaris, 3I6clO(a). Peritrichous flagellation.
t. From Phaseolus vulgaris, 3I6cl4. A curly and a straight flagellum. A
most unusual variant.
u. From Medicago sativa, 3Doa30. Peritrichous flagellation.
V. From Melilotus alba, 3Dohl3. Peritrichous flagellation. Note the straight
proximal parts of the flagella like those in q above.
w. From Lotus amcricanus, 3Eobl. Peritrichous flagellation.
X. From StropJwstylus paucifora, 3I6ml. Peritrichous flagellation.
y. From Robinia pseudoacacia, 3F4b7. Peritrichous flagellation.
z. From Caragana arborescens, 3F6g2. Peritrichous flagellation.
aa. From Acacia linifolia, lAocl. Peritrichous flagellation. This organism
shows an unusually large number of flagella for a rhizobium.
bb. From Wisteria frutescens, 3F33cl. Peritrichous flagella with strong
polar tendency.
cc. From Lupinus densifiorus, 3C2nl. Peritrichous flagellation. All three
of the strains studied of this origin showed peritrichous flagellation.
dd. From Phaseolus lunatus, 3I6d23. Very nice peritrichous flagella.
This is one of the two strains from Phaseolus lunatus which showed peri-
trichous flagella. The other nine strains studied showed typical subpolar
flagella.
a-d. From E. Leifson, and L. W. Erdman, Antonie van Leeuwenhoek, J
Microbiol. Serol. 24, 97-110 (1958).
68
b
c
d
•.->-.•■-
A.-.
g.
1
h
i
1/
J .
• 1
y-
k
1
1,
rn '.
n . . , 0
• • •
r
s
t
1 • .
, V
z
aa
1/
bb
cc
dd..:
69
within the nodules fix atmospheric nitrogen and thus furnish ni-
trates for the growth of the plant. Because of its economic im-
portance the genus has been studied in great detail. Morpholog-
ically the genus may be divided into two subgroups, mainly on
the basis of flagellation, which correlates fairly well with physio-
logical and cultural characteristics. The "phytopathogenicity" of
the organisms is very specific and the genus may be separated
into a number of so-called inoculation groups, each group infect-
ing a specific genus or group of genera of legumes. The bacteria
in each inoculation group generally have similar morphology and
cultural characteristics as shown in Table IV.
The flagellation of eighty-two strains from thirty-seven plant
species was studied. All of these were furnished by Dr. L. W.
Erdman of the U.S.D.A., Beltsville, Maryland.
Flagellar Characteristics
Most strains of Rhizobium are rather poorly flagellated. Var-
ious media and cultural conditions were tried to improve the
flagellation but with rather limited success. Best results were
usually obtained with a peptone-mannitol broth incubated at 20° C.
In a few instances an agar medium of same composition gave as
good or better results. With a few cultures stains could be made
after 1-2 days incubation but most cultures required 3 or more
days to produce sufficient growth for staining. The flagella stained
readily and none of the cultures were completely devoid of flagel-
lated individuals.
Rhizobium strains show two main types of flagellation, a non-
polar or peritrichous type and a subpolar monotrichous type.
The subpolar monotrichous flagellum usually emerges quite close
to the somatic pole and at a right angle to the long axis of
the soma. This is quite distinct from the situation in polar flagel-
lation where the flagellum emerges in a direction parallel to the
long axis of the soma. The subpolar rhizobia are almost invariably
monotrichous. Two flagella at the same pole are rarely observed
and one flagellum at each pole has never been observed. Of all
the strains of rhizobia studied the best flagellation is generally
found among the subpolar types.
The other main type of flagellation may be called peritrichous.
Most of the strains studied with this type of flagellation were
poorly flagellated with most of the flagellated individuals having
only one flagellum, rarely several. The flagella, whether single or
70
multiple, showed a tendency to emerge at or near the somatic
poles. This same tendency may be observed in other genera of
poorly flagellated peritrichous bacteria and is not characteristic
of rhizobia alone. A "monotrichous" individual may thus have a
polar, a subpolar, or a lateral flagellum. By observing several in-
dividuals, one with a lateral flagellum or one with several flagella
will usually be found and thus establish the flagellation as peri-
trichous. Another difference between the two types of flagellation
is the flagellar wavelengths. The mean wavelengths of the normal
subpolar flagella ranges from 1.9 microns to 2.2 microns, and of
the peritrichous flagella from 1.3 microns to 1.6 microns.
Flagellar Variations
The peritrichous cultures showed no definite variations. The
most striking variation was observed in the subpolar group. Strain
3Ilb59 of the soybean group showed many individuals with one
or more flagella with very short wavelength (curly) in addition
to the normal flagellum. Strain 3I2bl (from Erijfhrina indica)
showed the same type of variants. In a few instances an organism
was found with the curly flagella only. In most cases, however,
the normal long wavelength flagellum was present. The curly
flagella appeared to originate at the same locus as the normal
flagellum. The wavelength of the curly flagella was very uniform,
averaging 0.75 micron or about 1/3 that of the normal subpolar
flagella.
71
o
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bJO M
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D O
+++++++
+
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+++T+++
+ + + +
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q I q in c o] | q oi o | cd Tf co
oi P (>i r-I oi oi C5 oi oi r>i c ^' ^ _;
o o o o c c o o c o c T; 'C C C c c c c c ';:: "C n
cA i«t/ic«CUtA;c/;cA-y5t«cnD-(0-iDH0-icnc« «Djm O-iD-iD.
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72
3
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Oj o
1; o
+ + + ++ t
+ + + ++ T
+ + + ++ J
^' O CO o in o
1 ic lo CO CO ^
++ ++ +i
rt<,— l.-HCI-tOi.-St-
lO CO ^^ 10 10 Ol 1 CD
1
3 3 3 3 3 3
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ous
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ous
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lOUS 1.
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TJ "o '0 "o JH " "o
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Clover Trifolitwi dubium 1 peritr
Trifolium repens 1 peritr
Trifolium ambiguum 1 peritr
Pea Visum arvense 2 peritr
Visum sativum 1 peritr
Bean Vhaseolus vulgaris 13 peritr
Lotus corniculatus 1 peritr
L(jfu5- americanus 1 peritr
Lof«.s" uliginosus 1 ?
Caragana arborescens 2 peritr
Rohinia pseudoacacia 2 peritr
Wwfen'a speciosa 1 subpo
Wisierio frutescens 1 peritr
Amorpha fruticosa 1 peritr
73
28. Agrobacterium
The genus Agrobacterium in Bergey's Manual is grouped
together with Rhizobium and Chromobacterium in the family
Rhizobiaceae. The more typical species of Agrobacterium such as
Agrobacterium tumefaciens, Agrobacterium rhizo genes, and Agro-
bacterium radiobacter are very similar, both physiologically and
morphologically, to the peritrichously flagellated species of Rhizo-
bium. Chromobacterium, however, is so radically different that the
author sees little justification for grouping it in the same family as
Agrobacterium and Rhizobium.
Cultures
Sixteen cultures of Agrobacterium were studied over a period
of several years. Eight strains of various species came from Morti-
mer P. Starr of the University of California, two strains from Joel
Hildebrant of the University of Wisconsin, and the others from
various sources. All the cultures were carefully checked physio-
logically. Typical species of Agrobacterium oxidize, but do not
ferment, carbohydrates. The oxidation of sucrose by these bacteria
seems particularly noteworthy. With the exception of Agrobac-
terium. gypsophilae, Starr TG-101, all cultures were physiologically
typical of the genus. This culture of A. gypsophilae was a poorly
flagellated, peritrichous rod which fermented carbohydrates. It
could be a species of Erwinia but not Agrobacterium.
Flagellar Characteristics
The cultures studied were rather poorly flagellated with the
majority of the individuals without flagella. One or two flagella
per flagellated individual was most common with a few individuals
having three to four flagella but rarely more. The arrangement of
the flagella was peritrichous. In common with other poorly flagel-
lated peritrichous rods the flagella show a strong tendency to
originate at or near the somatic poles. The general appearance of
the flagella of A, tumefaciens, A. radiobacter, and A. rhizogenes
was very similar, and no variants were observed. The average
wavelength of A. tumefaciens was 1.45 microns, of A. radiobacter
1.49 microns, and of A. rhizogenes 1.47 microns. These wave-
74
y '*=^ V >j^
e ■ . ,f . , g .
Fig. 30. a. Agrobacterium tumefaciens, Hildebrand strain. Peritrichous
flagella of rather short wavelength. The organisms pictured are unusually well
flagellated for Agrobacterium, species.
b. A. tumefaciens, Starr TT-116. Peritrichous flagella of short wavelength
as in a.
c. A. rhizogenes, NCIB 8196. Peritrichous flagellation very similar to that
of A. tumefaciens. The flagella show a tendency to originate at or near the
somatic pole, with the proximal part frequently straight as illustrated. This
picture closely resembles some taken of the peritrichously flagellated rhizobia.
d. A.radiobacter, Starr TR-1. Peritrichous flagellation similar to that of
A. tumefaciens.
e. A. pseudotsugae, Starr TP-102. Peritrichous flagella of relatively long
wavelength and very small amplitude quite different from A. tumefaciens.
f. A. pseudotsugae, Starr TP-3. This strain was very poorly flagellated.
Most of the flagella were very short with indefinite curvature.
g. Agrobacterium sp., Keller 72. This is one of several strains isolated from
water and nothing is known about its phytopathogenicity. The flagellation is
peritrichous with rather short flagella of distinctly greater wavelength than
that of A. tumefaciens.
lengths correspond closely with those of the peritrichously flagel-
lated rhizobia. Agrobacterium pseudotsugae had an entirely dif-
ferent type of flagella with much greater wavelength ( 2.4 microns )
and unusually small ampHtude. A culture isolated from water and
physiologically typical of Agrobacterium also had flagella of dis-
tinctly different type from A. tumefaciens. The phytopathogenicity
of this water strain is unknown. Two strains of Agrobacterium
rubi studied did not show either motiHty or flagella. ( See Fig. 30. )
75
29. Chromobacteriutn
The genus Chromohacterium has two characteristics b\' which
it may be identified and differentiated from all other bacterial
genera: the water insoluble purple pigment and the mixed polar
and peritrichous flagellation. Physiologically the genus is hetero-
geneous, including psychrophiles and mesophiles, carbohydrate
fermenters and nonfermenters. Opinions differ regarding the tax-
onomy of the genus but the author recognizes three species:
Chromohacterium viokiceum, Chromohacterium manilue, and
Chromohacterium laurentium (Fig. 31). Twenty-eight selected
cultures were studied, including several strains of each species.
Flagellar Characteristics
A polar, predominantly single, flagellum could be demonstrated
in all strains. With some strains, particularly of C. manilae, the
polar flagellum could not be stained with the standard flagella
stain. Fairly satisfactory staining was obtained by the modified
flagella stain with double the normal concentration of tannic acid.
In addition to the polar flagellum, all but four strains showed a
variable number of lateral flagella. The lateral flagella always
stained readily with the standard stain. They diflered from the
polar flagella by having a much shorter wavelength. By plating
the cultures in semisolid agar and fishing from the periphery of
the most spreading colonies, the number of lateral flagella could
be increased. The four strains which showed only polar flagella,
however, did not show lateral flagella by this technique. The
wavelength and amphtude of ten polar and of ten lateral flagella
on as many individuals were measured. In Table V are recorded
the mean values of these measurements.
Fig. 31. a, b, c, d, e. Chromohacterium manilae. Note the weakly stained
polar flagellum, compared to the lateral flagella, in b, c and d. These polar
flagella did not stain with the usual stain formula.
f. C. violaceum. A polar and a lateral flagellum of different wavelength.
g. C. laurentium. Mixed polar and peritrichous flagellation,
a-g. From E. Lcifson, /. Bacteriol. 71, 393-400 ( 1956).
76
a.
#'-sV.
r ':'^'
h.
4
Flagellar
Table V
Characterlstics of Chromobacterium Species
Strains
Polar
flagella
Species
Wave-
length
( microns )
SD«
Amplitude
( microns )
SD
C. manilae
C. laurentiiim
C. violaceiim
Genus mean
16
6
6
2.21
2.07
2.23
2.19&
0.17
0.13
0.24
0.18
0.55
0.52
0.56
0.54
0.07
0.07
0.07
0.07
Strains
Lateral flagella
Species
Wave-
length
( microns )
SD
Amplitude
( microns )
SD
C. manilae
C. laurentium
C. violaceum
Genus mean
16
6
6
1.31
1.35
1.26
1.31''
0.11
0.07
0.11
0.10
0.46
0.43
0.44
0.45
0.07
0.06
0.06
0.06
'* SD = standard deviation.
WL polar flagella
WL lateral flagella
2.19
1.31
77
1.67,
3 0. Sarcina
Two species of flagellated Sarcina are listed in Bergey's Manual.
Sarcina citrea is described as producing a yellow to orange pig-
ment, in the form of single individuals, pairs, and packets, and with
a single flagellum per individual. A culture of this species could
not be obtained. Sarcina ureae (Sporosarcina ureae) is a fairly
common organism and three strains from different sources were
studied. Namely: Sporosarcina ureae from C. B. van Niel, Hop-
kins Marine Station; Sarcina ureae from Bruce Stocker, London,
England; and Sarcina ureae from Rudolph Hugh, George Wash-
ington University. All were morphologically typical and motile
(Fig. 32).
Flagellar Characteristics
With an organism which characteristically occurs in packets of
eight, and multiples of eight, the number of flagella per individual
is difficult to determine. With packets which appear to consist
of eight individuals the maximum number of flagella found was
nine. A lesser number was more common. From this we may
conclude that each individual coccus generally has only one flagel-
lum. It is also very difficult to determine if a flagellum has a polar
location, if one may use this term with Sarcina. No conclusion has
been reached on this point and no opinion is ventured.
The flagella of S. ureae tend to be exceptionally long and the
normal flagella have a greater wavelength than the great majority
of normal flagella of rod shaped bacteria. Two of the strains
studied showed normal flagella only. The third strain (Hugh)
showed several shape variants: normal, curly, small amplitude, and
one with a wavelength intermediate between normal and curly.
This latter type is labeled subnormal and was only found in asso-
ciation with curly flagella on a packet. A few flagella were also
found which were partly subnormal and partly curly. Attempts
at isolation of pure cultures of the various flagellar types were
not made.
The wavelength of the normal flagella of the three strains
studied averaged 3.19 microns. The wavelength of the curly
flagella averaged 1.4 microns. The few measurements possible
on the subnormal wavelength averaged 2.3 microns. The small
amplitude flagella had wavelengths averaging about 2 microns.
78
a I
Fig. 32. a. Sarcina (Sporosarcina) iireae, L.E.1.1. Hopkins Marine Sta-
tion. A packet of what appears to be eight cocci with seven normal flagella.
This culture showed only normal flagella.
b. Sarcina ureae, Hugh. At the upper left is shown a packet of presumably
eight cocci with eight normal flagella and one young flagellum without definite
curvature. At the lower right is a packet of presumably eight cocci with seven
long flagella, one medium-short and one very short. These flagella are of the
small amplitude type.
c. S. ureae, Hugh. Note the lower flagellum of the packet with subnormal
wavelength in the proximal portion and curly wavelength in the distal portion.
In the upper left of the picture are the ends of normal flagella from other
individuals.
d. S. ureae, Hugh. Illustrated are apparently two and possibly more packets
of eight cocci each. The long flagella on the right are of the curly type. The
relatively shorter flagellum in the upper right corner has the subnormal wave-
length.
79
3 1 . Streptococcus
The incidence of flagellated streptococci may not be as rare
as it is commonly considered to be. Bacteriologists rarely examine
a culture of cocci for motility, assuming it to be nonmotile. All
flagellated streptococci studied to date fall in Lancefield group
D, or the enterococcus group.
Three strains of motile streptococci were studied, one from Dr.
O. Felsenfeld, Hektoen Institute, Chicago; and two from Dr. Hans
Graudal, Statens Serum Institut, Copenhagen, Denmark.
Flagellar Characteristics
All three strains studied showed good motility and were well
flagellated (see Fig. 33). The Felsenfeld strain showed a few
fairly long chains while the Graudal strains showed mainly diplo-
cocci and rarely chains of as many as four individuals. In the
Felsenfeld strain the individual organisms showed mainly one
flagellum, occasionally two. The flagella appeared to originate
most frequently at the point of division of two cells which may
indicate a polar origin. The Graudal strains were definitely multi-
trichous with up to five flagella on a single cell. In these strains
the flagella were definitely of polar origin in most instances. If
one should characterize the flagellation in the usual terms the
streptococci studied should probably be labeled polar multitri-
chous. If this is correct these streptococci are the only gram-posi-
tive bacteria with polar flagella ever encountered by the author.
The shape of the flagella in the three strains studied was mainly
normal with unusually long wavelength, similar to Sarcina ureae.
The normal wavelength of the three strains averaged 3.2 microns.
In the Graudal strains were found a number of flagella which were
partly curly. The wavelength of the curly waves averaged 1.2
microns. There was also found a rare flagelhim with a wavelength
about 2.4 microns which correspond to the subnormal type seen
more distinctly in Sarcina ureae. The small amplitude shape seen
in Sarcina was not found, nor were any other shapes found. In
general, the shapes of the flagella of streptococci and Sarcina are
quite similar and both types of cocci have normal flagella of
distinctly longer wavelength than the great majority of rod shaped
bacteria.
80
^v-^^
Fig. 33. a. Streptococcus sp., Type D, Felsenfeld. A chain of cocci in
various stages of division. The exact origin of the flagella on the soma is not
clear in tliis picture. The flagellar curvature is normal.
b. Streptococcus sp., Type O, Graudal. Two cocci showing distinctly polar
multitrichous flagellation with flagella of normal curvature.
c. Streptococcus sp.. Type D, Graudal. The pair of cocci on the right
shows one flagellum in which the pro.ximal portion is curly and the distal
portion normal. This was very rare.
81
3 2 . Lactobacillus
Motility in the genus Lactobacillus appears to be very rare
and in Bergey's Manual all the species listed are described as
nonmotile. A strain labeled Lactobacillus plantarum was received
from Dr. P. Arne Hansen of the University of Maryland ( Fig. 34 ) .
Flagellar Characteristics
The culture studied was motile and fairly well flagellated with
peritrichous flagella. No variants were seen. The curvature of the
flagella was very uniform with an average wavelength of 2.26
microns and amplitude of 0.56 micron. Whatever phylogenetic
relationship there may be between lactobacilli and streptococci to
justify placing them in the same family is not apparent in the
flagellation.
3 5 . Corynebacterium
Reports of motility in the genus Corynebacterium appears
limited to the phytopathogenic group and one cellulolytic or-
ganism variously labeled CeJlulomonas fimi or Corynebacterium
fimi. No systematic study was made of any other corynebacteria.
One strain of Corynebacterium fimi was received from Dr. H. W.
Reuszer of Purdue University. The following phytopathogens
were furnished by Dr. Mortimer P. Starr of the University of
California: Corynebacterium tritici (CT-102), Corynebacterium
michiganense (CM-6), Corynebacterium poinsettiae (CP-1 and
CP-42), Corynebacterium flaccumfaciens (CF-18 and CF-8). The
phytopathogens studied were typical of the genus morphologically
and physiologically. Three motile strains of Corynebacterium
citreum-mobile were received from Dr. Werner Kohler in Ger-
many. The three strains were culturally and morphologically iden-
tical. They produced a dark yellow pigment and grew readily on
simple peptone media.
Flagellar Characteristics
C. tritici (CT-102) and C. michiganense (CM-6) were non-
motile and flagella could not be demonstrated. Strain CP-1 of C.
poinsettiae was nonmotile and nonflagellated, but strain CP-42
82
Fig. 34. a. Lactobacillus
plantarum. Hansen strain. The
flagella are peritrichous.
is/ -.v
V
83
showed fair motility and about 1% or less of the individuals with
flagella. The flagella were usually quite long and in no instance
could more than one flagellum be found on one organism. The
arrangement of the flagella was nonpolar ( peritrichous ) . In all
nonpolar or peritrichously flagellated bacteria there is a much
greater proportion of the flagella located at or near the poles of
the individuals than one would expect from chance. When dealing
with bacteria which have many flagella per individual this is not
so obvious as with bacteria having only one or two flagella per
individual. An experienced observer may thus mistake nonpolar
for polar flagellation. A polar flagellum usually emerges from the
cell in line with the long axis of the soma of the bacterium while
a nonpolar flagellum usually emerges at right angles to the soma.
In C. poinsettiae CP-42 the flagellated individuals were mono-
trichous with most flagella located at or near the poles but the
majority of the flagella emerged at right angles to the soma, and
in a fair number the flagella emerged from the middle of the
soma. C. fiacciimfaciens, strains CF-8 and CF-18, were both mo-
tile and showed nonpolar (peritrichous) flagellation very similar
to C. poinsettiae. These strains were mainly monotrichous but
occasionally two flagella were found on one individual. C. fimi,
Reuszer strain, was motile though very poorly flagellated. The
flagella were nonpolar (peritrichous) in arrangement. As with
the phytopathogens the majority of the cells were monotrichous
but with this organism a fair number of cells showed two flagella
and a rare individual had three flagella. The Koehler strains
showed the same flagellar arrangement as the phytopathogens. In
these strains individuals with two flagella were fairly common.
The flagella of C. fimi and the Koehler strains showed a much
greater wavelength than the flagella of the phytopathogens, as
indicated in Table VI and also obvious from the illustrations ( Fig.
35). Of some significance perhaps is the difference in wave-
lengths of the two strains of C. flaccumfaciens. These differences
are statistically significant. Strain CF-18 of C. fiacciimfaciens
produced acid in inulin and raffinose while strain CF-8 did not.
84
\ \ v^- / i'
• b c d e *•
> "^^ .^ •/
Fig. 35. a, b, c, d, e. Corynebacteriiim fiaccumfaciens, Starr CF-18. These
figures illustrate the typical flagellation of C. flaccumfaciens. More than one
flagellum per individual is extremely rare. The flagellum usually originates at
or near the somatic pole but should not be confused with polar flagellation.
f. C. poinsettiae, Starr CP-42. Most individuals in this culture showed only
one flagellum, if any. The flagellar arrangement was definitely peritrichous
and similar to that of C. flaccumfaciens.
g, h. Corynebacteriiim (Celliilomonas) fimi, Reuszer 133. The flagella are
peritrichously arranged and of rather long wavelength.
i. C. citreum-mobile, Koehler. The majority of the flagellated individuals in
this culture had only one flagellum. The arrangement was peritrichous like the
phytopathogens. Note the long wavelength similar to C. fimi.
TABLE VI
Mean Wavelengths of Corynebacteriiim, Species
Wavelength
Species
( Microns )
C. poinsettiae, CP-42
2.09
C. flaccumfaciens, CF-8
2.17
C. flaccumfaciens, CF-18
1.74
C.fimi, Reuszer 133
3.0
C. citreum-mobile, Koehler
3.16
85
34. Arthrobacter
One culture labeled Arthrobacter citreus was received from
Dr. L. E. Sacks of U.S.D.A., Albany, California (Fig. 36). The
culture grew well on simple media with a lemon yellow, water
insoluble pigment. The organism was gram positive, very pleo-
morphic and showed some motility in moist preparation. The
motility was nonprogressive and consisted mainly of spinning and
wiggling.
Flagellar Characteristics
The one culture of A. citreus examined showed mainh' straight
flagella (or very small amplitude flagella) with nonpolar or peri-
trichous arrangement. Most individuals showed only one flagel-
lum. One organism only was seen with a normal flagellum. How-
ever, reports in the literature show Arthrobacter simplex with both
normal (wavelength about 2 microns) and curly (wavelength
about 1 micron ) flagella so the strain of A. citreus studied may not
have had the most typical flagellation of the genus.
3 5 . Listeria
All strains of Listeria are classified into the one species Listeria
monocytogenes. The organism is peritrichously flagellated, and
well flagellated if cultured at low temperatures such as 20° C, but
very poorly flagellated if cultured at 37° C. At 38° C. flagella are
not produced. In cultures at 37° C. a single flagellum may be
found on a small proportion of the organisms which led to the
early reports that the organism was polar monotrichous.
Of all the bacteria the author has encountered Listeria has
shown the greatest genetic instability, or mutability, in regard to
flagellar shape and function (see Fig. 37). This apparent mutabil-
ity may partly be due to the fact that a large number of strains
(eighty-one) were studied, since a large proportion (85%) of
the strains did not show any variants. The cultures were all old
stock strains and this in itself appears to contribute to genetic
instability. All of the cultures included in this study were received
from Dr. A. M. Griffin of George Washington University.
Flagellar Characteristics
The flagella of Listeria showed four distinct shapes: normal,
small ampfitude, straight, and coiled. Variants with each type of
flagella were isolated in pure culture. Curiously enough the var-
86
>
Fig. 36. a. Arthrohacter citreus, Sacks. This figure shows the most com-
mon flagellation of the culture studied. The flagella were straight or with a
slight curvature (small amplitude), and usually single. The arrangement was
peritrichous.
b. A. citreus, Sacks. This was the only organism seen on the slide with a
normal flagellum.
87
iant flagellar shape most often encountered in other bacteria,
namely the curly shape, was not observed in Listerki, nor could
the curly shape be induced by lowering the pH. Filamentous
variants could readily be obtained and these invariably retained
the flagellar shape of the parent nonfilamentous type. The normal
flagellar shape was by far the most common and was present in
all of the eighty motile strains studied.
The flagella of Listeria also showed variations in function. One
of the original cultures had normal flagella but was entirely non-
motile. From two other cultures were isolated pure strains of
normal flagellated nonmotile individuals. Organisms with the
coiled flagellar shape showed fair motiHty but those with small
ampHtude and straight flagella showed very poor motility at best
and no progressive motion at all. Two variants with straight
flagella were obtained, one of which was entirely nonmotile while
the other showed only a nonprogressive wiggling and spinning
motion.
On the basis of flagellar shape and function, seven varieties of
individuals were obtained in pure culture: nonflagellated; normal
flagella, motile and nonmotile; straight flagella, motile (very slight)
and nonmotile; coiled flagella, fair motiHty; small amplitude
flagella, slight motility. By plating in semisolid agar a variety of
flagellar mutations were observed but no new types found. The
rate of such mutations were calculated to be in the neighborhood
of 10~^ to 10~^ per cell division.
The normal wavelength and amplitude of different strains
showed only minor differences. From 10 measurements on each of
eight strains was obtained an average wavelength of 2.01 microns
with a standard deviation (S.D. ) of 0.1 micron, and an average
amplitude of 0.48 micron with an S.D. of 0.06 micron. Based on 20
measurements the wavelength of the small amplitude flagella was
1.53 microns with amplitude 0.25 micron. For the coiled flagella
the wavelength was 2.18 microns and the ampHtude 0.76 micron.
Fig. 37. a. Listeria monocytogenes. Normal flagella, peritrichous arrange-
ment.
b. L. monocytogenes. Small amplitude flagella.
c. L. monocytogenes. Straight flagella.
(1. L. monocytogenes. Coiled flagella.
e. L. monocytogenes. Normal flagella. Filamentous soma.
f. L. monocijto genes. Straight flagella. Filamentous soma.
g. L. monocytogenes. Coiled flagella. Filamentous soma.
a-e. From E. Leifson, and M. I.'Palen, /. Bacteriol. 70, 233-240 ( 1955).
88
3 6, Alcaligenes
The genus Alcaligenes is composed of peritrichously flagellated
bacteria and related nonflagellated types which do not attack
carbohydrates. Many types of polar flagellated bacteria have
cultural and physiological characteristics very similar to Alcali-
genes species. These polar flagellated bacteria are often mistaken
for Alcalipenes and are found in culture collections as Alcaligenes
species of one kind or another. It is impossible to identify an or-
ganism as Alcaligenes without determining the nature of the flagel-
lation. Although Alcaligenes species are ubiquitous in nature thev
are not as common as most bacteriologists believe. The author has
studied a fair number of strains of Alcaligenes obtained from a
variety of sources over a period of several years. Most of the
species Hsted in Bergey's Manual are unidentifiable and should be
discarded. The author includes Alcaligenes (Brucella, Bordetella)
bronchisepticus in the genus because of its similar morphological
and physiological characteristics.
Flagellar Characteristics
In some strains the flagella are quite numerous and well-formed
but in most strains the flagellation is only fair or poor (Table VII).
The most common flagellar shape is the normal and the only
definite other shape found was the curly. All strains of Alcaligenes
bronchisepticus studied, twenty in number, had normal flagella
only, and so did the two strains studied of Alcaligenes denitrificans.
One strain of Alcaligenes faecalis had only curly flagella and one
strain had some individuals with normal flagella and some with
curly flagella but not both types of flagella on the same individual.
A peritrichously flagellated mutant of Lophomonas faecalis had
only curly flagella (Fig. 38).
90
^^
i
.^
f^r: ^
Fig. 38. a. Alcaligenes jaecalis, H-222. Two unusually well-flagellated
organisms showing normal flagella peritrichously arranged.
b. A. jaecalis, H-136. Peritrichous flagella showing some coiling tendency.
c. A. denitrificans, H-12. Normal flagella, peritrichously arranged.
d. A. bronchisepticus, H-184. Peritrichous flagella of normal curvature.
e. A. jaecalis, H-247. This is a mutant of Lophomonas jaecalis. Curly,
peritrichous flagella.
c. From E. Leifson, and R. Hugh, /. Gen. Microbiol. 11, 512-513 (1954).
e. From E. Leifson, and R. Hugh, /. Bacterial. 65, 263-271 (1953).
(See p. 93 jor Table VII.)
91
3 7. Achrofnobacter
The three genera of the family Achromobacteriaceae appear to
be rather closely related and a definite distinction cannot always
be made between them. Peritrichoiisly flagellated gram-negative
rods are found in the soil which produce just the faintest trace of
acid in carbohydrate media and the differentiation of these from
Alcaligenes species is difficult. Organisms of the same physiological
nature are also found in the soil which produce a cream colored
or faint yellow pigment. These are on the borderline betsveen
Flavohacterium and Achromobacter and differentiation is difficult.
A few cultures of Achromobacter were studied. They were ob-
tained from various sources and some were old stock strains labeled
Alcaligenes. Much can be said in favor of combining Alcaligenes
and Achromobacter into one genus.
Flagellar Characteristics
All of the more typical cultures studied had peritrichous
flagella with normal curvature (Fig. 39). The flagellation was
generally fair to poor. The average wavelength of the strains
studied was 2.39 microns.
92
Fig. 39. a. Achronwhacter sp., H-
137. Typical normal peritrichous flagella.
The flagellar shape in most strains
studied was quite irregular.
n.^. ^^^
TABLE VII
Mean Flagellar Wavelengths of Alcalwenes Species
Wavelength
Strain
( microns )
Species
Normal
Curly
A. bronchisepticus
H-46
2.70
H-47
2.76
H-48
2.68
H-49
2.85
H-50
2.76
H-51
2.87
H-52
2.73
H-140
2.86
H-171
2.86
H-180
3.04
H-181
2.65
H-182
2.58
H-183
2.62
H-184
2.50
H-225
2.82
H-227
2.89
H-232
2.96
Species
mean
2.78
A. denitrificans
H-12
2.74
—
H-13
2.64
—
Species
mean
2.69
A. faecalis
H-135
2.82
—
H-138
2.24
1.07
H-222
2.58
—
H-223
—
1.29
H-247
—
1.03
Species
mean
2.55
1.13
Genus
mean
2.67
1.13
93
3 8. Flavobacterium
The genus Flavobacterium is characterized by a yellow water-
insoluble pigment and peritrichous flagellation, if any. Some strain
have a weak oxidative action on carbohydrates, others have none.
Many unidentified cultures were studied as well as several named
species.
Flagellar Characteristics
The only well flagellated culture studied was an unnamed but
typical species received from Dr. J. D. Stout of New Zealand.
This organism was rather filamentous with numerous curly peri-
trichous flagella as illustrated in Fig. 40f. Five named cultures
were received from Dr. Owen D. Weeks of the University of
Idaho. Only two of these were flagellated: Flavobacterium ma-
rinotypiciim, Zobell (F-6), showed poor flagellation with only one
nonpolar flagellum per flagellated organism as illustrated in Fig.
40a, The single flagellum had an average wavelength of 2.5 mi-
crons but many were coiled, Flavobacterium suaveolens, ATCC
(F-23), showed fair flagellation with one or two flagella per or-
ganism as illustrated in Fig. 40b. The majority of the flagella had
an average wavelength of 1.85 microns. Two flagellated cultures
typical of the genus were received from Dr. Oleg Lysenko of
Yugoslavia: Flavobacterium sp., BmEl, was isolated from Bombex
mori and was peritrichously flagellated as illustrated in Fig. 40d,
Both normal flagella, with an average wavelength of 2.9 microns,
and curly flagella, with an average wavelength of 1.4 microns,
were found on the same and on separate organisms, Flavobac-
terium sp,, Ac21, was isolated from Aponja crataegi. The flagella-
tion was fair with normal peritrichous flagella of average wave-
length of 2.95 microns as illustrated in Figs, 40c, e.
94
060
-/~no
Fig. 40. a. Flavohacterium marinotijpicimi, Zobell (F-6). A nonpolar
flagellum of rather long wavelength. Coiled flagella were common in this
culture.
b. F.suaveolens, ATCC (F-23). Peritrichous flagella of normal but
somewhat short wavelength.
c. e. Flavohacterium sp., Lysenko Ac21. Peritrichous flagella of normal
shape and rather long wavelength.
d. Flavobacterium sp., Lysenko BmEl. Peritrichous flagella of normal
shape.
f. Flavohacterium sp., Stout K-8. Peritrichous flagellation. This culture
showed only the curly type of flagella. Most individuals were filamentous.
95
39. Cellulomonas
Cellulomonas is the generic name commonly used for a group
of gram-negative, simple rods which decompose cellulose. The
generic name is unfortunate since these bacteria show peritrichous
flagellation.
Six strains labeled Cellulomonas were studied. Five of these
were supplied by Dr. H. W. Reuszer of Purdue University, and
one {Cellulomonas hiazotea) was supplied by William C. Haynes
of the Northern Regional Research Laboratory, U.S.D.A., Peoria,
Illinois. Two cultures failed to show either motility or flagella: C.
hiazotea, NRRL B-401, and Cellulomonas cellasea, Reuszer 124.
One strain was labeled Cellulomonas fima, Reuszer 133, and is
discussed under Conjnebacterium. The other three strains showed
good to fair flagellation and motility.
Flagellar Characteristics
Cellulomonas rossica, Reuszer 128, grew well on simple media,
produced colorless growth, and was very motile. Flagellation was
good with many normal peritrichous flagella per individual. No
variants were observed. Cellulomonas hihula grew well on simple
media with a yellow pigmentation. Flagellation was good but
each individual seldom showed more than one flagellum with
peritrichous arrangement. The curvature of the flagella was pure
curly except for a rare flagellum with the proximal end of very
long wavelength (approximately 2.8 microns) and the distal end
curly. Cellulomonas perlurida showed moderate growth with spots
of yellow on agar media. Flagellation was fair, with most in-
dividuals having only one flagellum and a few with two or more.
Most individuals showed pure curly peritrichous flagella. A few
individuals showed normal peritrichous flagella. Also in this strain
were observed flagella with proximal end normal and distal end
curly. One organism was observed with a long normal flagellum
and a short curly flagellum. Morphologically C. hihula and C.
perlurida were similar, while Cellulomonas rossiea was quite dif-
ferent. The normal flagella of C. rossica had an average wave-
length of 2.1 microns. Curly flagella were not observed in C. ros-
sica. The curly flagella of C. hihula and C. perlurida averaged
1.06 and 1.05 microns, respectively. The normal flagella of C
96
':^
^
^ V'
Fig. 41. a. CeUiilomonas rossica, Reuszer. Normal, peritrichous flagella.
b, c. C. hibula, Reuszer. A single curly flagellum was most common but
individuals were also seen with several curly flagella. The arrangement is
peritrichous.
d. C. perlurida, Reuszer. This shows a flagellum with the proximal end of
normal curvature and the distal end curly. The wavelength ratio of the normal
and curly waves is about 3:1. Normal wavelength, 2.9 microns, curly wave-
length, 1.05 microns.
e. C. perlurida, Reuszer. Long curly flagella, peritrichously arranged.
f. C. perlurida, Reuszer. A normal and a curly flagellum on same indi-
vidual.
perlurida had an average wavelength of 2.9 microns and those of
C. hibulo 2.8 microns. ( See Fig. 41. ) Morphologically C. per-
lurida and C. bibuki appear to be identical and could well be con-
sidered a species of the genus Flnvobacterium.
97
40. Escherichia
Several dozen strains of Escherichia have been studied. Most
of these were from the collection of Dr. MacDonald Fulton and
included all the common physiological types of Escherichia coli,
Escherichia freundii, and the slow lactose fermenting paracolons.
Flagellar Characteristics
All strains showed peritrichous arrangement of the flagella
(Fig. 42). The flagellation of the colon bacilh is quite variable
with atrichous and poorly flagellated strains very common. With
most motile cultures the flagellation can be greatly improved by
culture in semisolid agar and fishing from the periphery of the
spreading growth: Most strains studied showed normal flagella
only. A few strains showed both normal and curly flagellated in-
dividuals. Individuals with both normal and curly flagella were
extremely rare and in no instance could the normal curvature be
changed to the curly by lowering the pH. In this respect the
coliforms are like Salmonella but unlike Proteus. Some strains may
show a considerable proportion of coiled flagella.
The mean wavelength of the normal flagella of fifteen strains
of Escherichia was 2.74 microns. Only one strain with curly
flagella was found and the wavelength was 1.15 microns. In Ta-
ble VIII are given the mean flagellar wavelengths of various
genera in the enteric group.
98
6^ 1^
a.
Fig. 42. a. Escherichia coli, F-3412. Peritrichous flagella of normal shape.
The flagellation of Escherichia strains tends to be rather poor. Coiled and ir-
regularly curved flagella are common. Strains with curly flagella are occasion-
ally encountered.
b. Paracolon type (Bethesda), F-364. This shows a normal flagellum ex-
tending out from the somatic pole and four nicely coiled flagella. The organ-
isms of the paracolon group have much the same type of flagellation as the
typical Escherichia.
c. Paracolon type ( Arizona ) . Peritrichous flagella of normal curvature.
d. e. E. freundii, F-360. In d is illustrated the normal peritrichous flagella.
In e is shown a filamentous form with curly flagella. Curly flagella, of course,
are also found on nonfilamentous individuals.
(See p. 101 for Table VIII.)
99
41. Aerobacter
All the flagellated strains of Aerobacter are classified in the one
species, Aerobacter cloacae (Fig. 43). Eight strains of A. cloacae
were received from Dr. Sverre Dick Henrikson in Norway, and
one strain from Dr. Perry Wilson of the University of Wisconsin.
All strains were physiologically typical and motile.
Flagellar Characteristics
The flagellation of A. cloacae is very similar to that of Escheri-
chia and the paracolon group. Normal and coiled flagella were
most common, with two strains showing a few individuals with
curly flagella. Change in pH did not change the flagellar curva-
ture. The average normal wavelength for the ten strains was 2.77
microns, with a range of 2.59 to 2.90 microns. The curly flagellar
wavelengths averaged 1.17 microns. These wavelengths are not
significantly difterent from those of Escherichia and the paracolon
group as shown in Table VIII.
100
S^f^: ■ ^
Fig. 43. a. Aerohacter cloacae, F-3942. Normal peritrichous llagella.
b. A. cloacae, F-3784. Normal and coiled flagella.
c. A. cloacae, F-3943. This individual shows two partly curly flagella, one
coiled flagellum and one normal flagellum. Curly flagella were seldom seen
in this species.
TABLE VIII
Flagellar Wavelengths of the Enteric Group
Range of wavelength
Number of
strains
( microns )
Group
Normal
Curly
Escherichia
15
2.59-3.02
Paracolon
31
2.58-2.88
0.87-1.20
Aerohacter
9
2.59-2.90
Erwiniao
33
2.31-3.02
1.06-1.35
Salmonella^
39
2.30-2.71
1.12-1.15
Proteus
60
2.13-2.39
1.06-1.16
Mean wavelength
Number
strains
of
( microns )
Group
Normal Curly
Escherichia
15
2.74 1.15
( 1 strain)
Paracolon
31
2.68 1.08
( 6 strains)
Aerohacter
9
2.77 1.17
( 2 strains)
Erwiniao-
33
2.72 1.20
( 9 strains)
Salmonella^
39
2.51 1.13
( 3 strains)
Proteus
60
2.26 1.10
(40 strains)
" The mean nonnal wavelengths of the coHform group and Erwinia are
not significantly different.
^ The mean nonnal wavelengths of Salmonella differ significantly from
the colifomi group Erwinia and Proteus. The mean curly wavelengths of all
groups do not differ significantly.
101
42, Erivinia
The genus Erwinki is composed of a group of phytopathogenic
bacteria closely related to the coliform group. Should any organism
of this genus be isolated in the ordinary public health or clinical
laboratory it would certainly be considered a type of coliform.
Cultures
A total of fifty-four cultures representing fifteen named species
were studied physiologically and morphologically. The great ma-
jority of these cultures were received from Dr. Mortimer P. Starr
of the University of California. A few cultures were from N. A.
Smith, U.S.D.A., and a few from various sources. All cultures
grew readily at 20° C. in simple peptone media with the exception
of Erwinia tracheiphila. Four strains of this latter organism were
studied. All grew slowly and relatively poorly, and were non-
motile and nonflagellated. Many of the other strains studied were
Fig. 44. a. Erwinia amylovora, EA-145. A short filament with normal
peritrichous flagella.
b. E. amylovora, EA-11. A rather unusual situation in this species with
normal and curly flagella on the same organism.
c. E. amylovora, EA-128. A short filament with coiled flagella mainly.
d. E. salicis, ES-4. This culture showed only normal flagella.
e. Erwinia sp., Walnut, WC-1. An exceptionally well flagellated culture
showing normal flagella only.
f. E. lathryi, EL-102. Beautifully flagellated culture with normal flagella
only.
g. E. carotovora, EC-153. Rather short normal flagella typical of this strain,
h. E. carotovora, EC-109. This organism shows one curly flagellum and
several normal ones. This culture showed a mixture of individuals with only
normal and only curly flagella. The organism pictured was one of a very few
with both types of flagella.
i. E. chrysanthemi, EC-176. This species showed mainly coiled flagella.
The organism pictured shows mixed coiled and curly flagella which was
common in this strain.
j. E. aroideae, EA-148. A pure curly individual. This strain also showed
individuals with normal flagella and mixed normal and curly flagella.
k. E. solanisapra, ES-3. This strain showed normal flagella only which did
not change to curly by acid suspension.
1. E. solanisapra, ES-101. This culture showed only curly flagella. It
was labeled nonmotile by the donor. Moist preparation showed nonprogressive
wiggling and turning motion.
102
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nonmotile and nonflagellated but in only one species, Erwinia
phytophthora (in addition to E. tracheiphila) were no motile
strains found.
Flagellar Characteristics
All of the motile strains studied showed peritrichous flagella-
tion. From the standpoint of flagellation the genus is very hetero-
geneous. Some species have flagella resembling those of the
coliform group, others resemble Proteus. The most common flagel-
lar shape was the normal with several species showing only this
shape. The curly shape was fairly common. One strain of Er-
winia solanisapra, ES-101, was completely curly, while another
strain of the same species, ES-3, was completely normal. It is
interesting to note that the donor of the culture described E.
solanisapra, ES-101, as being nonmotile. Careful observation of
this culture in moist preparation did show some motility but only
a nonprogressive wiggling and turning motion. Erwinia aroideae,
EA-8, was mainly curly. Two species, Erwinia atroseptica, EA-
112, and Erwinia nimipressuralis, EN-1, showed only normal
flagella in alkaline suspension and mainly curly flagella in slightly
acid suspension. The coiled shape was found to a variable extent
in several species. One strain of Erwinia amijlovora showed a
great deal of coiling. Three strains of Erwinia chnjsanfhemi had
mainly coiled flagella with some curly and normal flagella mixed
in. Mixed curly and normal, curly and coiled, normal and coiled
were found in several strains. ( See Fig. 44. )
Table IX gives a summary of the morphological characteristics
of the strains studied. The organisms are grouped according to
the pathology they produce: dry necrosis, soft rot, and non-pecto-
lytic.
104
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105
43, Serratia
Species of the genus Serratia generally produce a characteristic
red pigment. Colorless variants are common and these are difficult
to identify with certainty. The genus has been separated into
several species but the differences between these species is not
great and bacteriologists in general tend to label any red pig-
mented gram negative rod with the proper physiological charac-
teristics as Serratia marcescens.
Eight strains labeled S. marcescens were studied. Three of
these came from Dr. MacDonald Fulton and were isolated from
clinical material. The other five strains were of diverse origin
and came from the National Collection of Industrial Bacteria in
England. All these strains were pigmented and physiologically
typical of the genus. From the American Type Culture Collection
were obtained five cultures which appeared typical of the genus:
Serratia kiliensis 992, Serratia plijmiithica 183, and Serratia indica
4002 were well flagellated; S. indica 4003 and Serratia urinae 11111
were nonflagellated. From W. B. Haynes of NRRL were obtained
two cultures: Serratia anolium B-1700 was well flagellated and
physiologically typical except for lack of pigmentation; S. indica
B-341 was nonflagellated.
Flagellar Characteristics
Three of the fifteen cultures studied were nonflagellated which
would indicate that nonflagellated variants of Serratia are fairly
common. With one exception the flagellated strains showed more
or less identical flagellation with coiled peritrichous flagella. From
an occasional uncoiled flagellum a mean wavelength of 4.5 microns
was obtained. In S. marcescens, NCIB 2302, was seen a flagellum
which was curly in its proximal part with a coil at the end. This
organism is illustrated in Fig. 45a. The curly waves measured 1.1
microns. The culture of S. indica, NCIB 4002, showed two types
of individuals, one with the typical coiled flagella and one with
normal peritrichous flagella. On plating a culture was obtained
with organisms having normal flagella only. This culture was
physiologically typical of the genus. The wavelength of the
normal flagella averaged 2.3 microns.
106
*. fi j^.
d»
Fig. 45. a. Serratia marcescens, NCIB 3202. Typical coiled peritrichous
flagella with one flagellum curly in its proximal part, ending in a coil.
b. S. anolium, NRRL B-1700. Typical coiled peritrichous flagella.
c, d. S.indica, NCIB 4002. This culture was a mixture of organisms with
the coiled flagella typical of the genus, illustrated in c, and normal flagella,
illustrated in d. By plating, a pure culture with normal flagella was obtained.
e. S. plymuthica, ATCG 183. A short filament with the typical coiled peri-
trichous flagella.
f. S. kiliensis, ATCC 992. A short filament with typical coiled peritrichous
flagella.
g. S. marcescens, NCIB 2302. The filamentous form of this organism with
typical coiled peritrichous flagella.
a-g. From M. Fulton, C. Forney, and E. Leifson, Can. J. Microbiol. 5,
269-275 (1959).
107
44. Proteus
The genus Proteus is fairly well defined physiologically. Four
species are commonly recognized: Proteus mirahilis, Proteus vul-
garis, Proteus morganii, and Proteus rettgeri. The last named of
these has some characteristics relating it to the SalmoneUa. A
group of bacteria commonly referred to as the "Providence group"
probably should be classified as Proteus. From the large collec-
tion of Dr. MacDonald Fulton were selected seventy-five strains
for study. These were evenly divided among the four species and
the Providence group.
Flagellar Characteristics
All the strains studied showed peritrichous flagellation. The
number of flagella varied greatly from strain to strain and has little
taxonomic significance. The cultures of P. mirahilis and P. vulgaris
which showed the "swarming" phenomenon on agar generally
showed the greatest density of flagella. P. rettgeri strains usually
showed the fewest flagella.
Normal and curly flagella were observed in all strains studied
with the exception of P. rettgeri. In this species only one of the
twelve strains studied showed an occasional curly flagellum. As
may be seen in the illustrations ( Fig. 46 ) , an individual organism
Fig. 46. a, b. Proteus mirahilis, Fulton 52. In a is illustrated the typical
normal peritrichous flagella of Proteus. In b is shown the corresponding curly
flagella on the same strain. The organism in a was stained from a slightly
alkaline suspension while that in b from a slightly acid suspension, both
from the same culture.
c. P. vulgaris. Mainly coiled flagella with two normal flagella at the top
of the soma.
d. Proteus sp., Providence type. Semicoiled flagella. These were quite
rare in the Proteus group and have not been definitely observed in other kinds
of bacteria.
e. P. morganii. Normal and curly flagella on the same individual.
f. P. mirahilis. Many curly flagella and one normal flagellum on the same
individual. These mixed types of flagellation were seen mainly in slides pre-
pared from su.spensions at pH 6.5 to 7.5.
g. h, i, j. P. morganii. These figures show various double curvature arrange-
ments: proximal end curly, distal normal; proximal normal, distal curly; ends
normal, center curly; alternating normal and curly.
k. P. mirahilis, Fulton 52. A filamentous form with mainly normal flagella.
A curly flagellum may be seen on the right end.
a-j. From E. Leifson, S. R. Carhart, and M. Fulton, /. Bactcriol. 69, 73-82
(1955).
108
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may show only normal flagella, only curly flagella, or mixed curly
and normal. Occasionally a flagellum may be part normal and
part curly in various arrangements. Most Proteus flagella, except
those of P. rettgeri, assume the normal shape in media above pH
7.5 and the curly shape in media below pH 6.5. At a pH between
6.5 and 7.5 the flagellation tends to be mixed normal and curly.
This pH sensitivity is not unique for Proteus but is also found to
a limited extent in Erwinia, Azotohacter, and Bacillus.
In addition to the normal and curly shapes, coiled flagella
were observed to a variable extent in all species, absent in some
strains but common in others. In the Providence group, and in
some strains of P. morganii, a very few organisms showed the
semicoiled shape. The semicoiled flagella are very characteristic
having an exceptionally large amplitude in relation to the wave-
length.
Flagellar Measurements
The wavelengths and amphtudes of Proteus flagella were
measured to a great extent. In Table X are summarized the mean
wavelengths and amphtudes of the normal and curly flagella of
the sixty best flagellated strains. Studies were also made of the
effect of variation in culture medium, age of culture, etc. These
studies showed remarkably little variation in wavelength and
amplitude in different media and at different ages of culture. In
summarizing the statistical data it may be stated: statistically sig-
nificant variations are found in the mean strain wavelengths in-
dicating that a species in Proteus is not morphologicalh' homo-
geneous. The mean wavelengths of normal flagella of P. mirabilis,
P. vulgaris, and P. morganii do not differ significantlv but do
differ significantly from the mean wavelengths of P. morganii
(trehalose -(-), P. rettgeri, and the Providence group. The mean
spiral unit lengths
S.U.L. := VWL^ + AmpV
were not significantly different except for P. rettgeri which was
greater. Again P. rettgeri appears different from the others. The
curly flagella of all strains were of quite uniform wavelength, being
slightly greater for the Providence group. P. rettgeri showed so
few curly flagella that it could not be compared with the others.
110
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45. Salmonella
With the exception of a few well recognized types such as
Salmonella gallinarum and Salmonella pullorum, species or types
of Salmonella are generally motile and flagellated. The flagellation
of a large number of strains of a large variety of types have been
studied over a period of several years. Most of the strains studied
were supplied by Dr. MacDonald Fulton of the Stritch School of
Medicine, Loyola University, Chicago, Illinois. Several others
came from the Illinois State Health Laboratory, Chicago, and a
few from diverse sources.
Flagellar Characteristics
All types of flagellated Salmonella appear to have the flagella
peritrichously arranged. By far the most common shape of the
flagella is normal with wavelengths varying between 2.4 and 2.7
microns ( see Table XI ) . Curly variants are encountered occa-
sionally and appear to be genetic mutants of the normal. In some
types such as Salmonella wichita the curly variant appears very
stable, while in other types such as Salmonella fijphimuriitm it
appears much less so. Lowering the pH of a suspension does not
cause a change from normal to curly as with Proteus strains.
Other shapes such as coiled and straight have only been observed
in odd flagella among otherwise normal types. By careful observa-
tion of a large number of organisms a few strains of S. typhi-
murium have shown a rare curly flagellum among the normal
flagella and also a rare flagellum partly curly and partly normal
(Fig. 47c). One strain of S. typhimiirium, supplied by the Illinois
State Health Laboratory, showed normal flagella but no motion.
The paralyzed flagella of this strain were perfectly normal anti-
genically and developed both antigenic phases (i: 1,2,3). The
change of antigenic phase in the diphasic types of Salmonella
does not appear to be associated with any significant change of
flagellar wavelength. In Fig. 48 is shown an interesting filamentous
form of Salmonella typhimiirium.
112
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Fig. 47. a, b, c. Salmonella tijphimurium, Friewer. In a is shown the
normal and in b the curly type of flagella. The curly flagella of S. typhimiirium
seem to have somewhat limited stabihty and pure curly cultures have shown
a high rate of dissociation to the normal type. In c is shown a rare phenom-
enon in Salmonella: a flagellum with the proximal part curly and the distal part
normal.
d. S. typhosa, Watson. Normal peritrichous flagella. Variants with curly
flagella are also found in this species.
e, f. S. Wichita, Fulton 3216. In e is shown the normal variant and in f the
curly variant. These variants have remained stable for years as laboratory
broth cultures. The curly variant is only feebly motile with only wiggling and
turning movements.
g. S. Virginia, Fulton 189. Normal peritrichous flagella on a faintly stained
soma.
h. S. enteritidis, Fulton. Normal peritrichous flagella.
i. S. derby, Fulton. Normal peritrichous flagella.
j, k. S.anatum, Fulton. Typical normal (j) and curly (k) variants of this
species.
1. S. arizona, Fulton. Normal peritrichous flagella.
114
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115
46. Pasteurella
Pasteurella pseudotuberculosis appears to be the only species
in the Pasteurella genus which is flagellated (Fig. 49). One strain
was received from the University of California School of Medi-
cine, San Francisco. Flagellation of this strain at temperatures
between 20° and 30° C. was fair and the organism showed peri-
trichous flagella of long and very irregular wavelength, averaging
3.2 microns, with an amplitude about 0.8 micron.
Fig. 48. m. S. tijphimurium, Fulton. A filamentous form with normal peri-
trichous flagella.
116
Fig. 49. a. Pasteurella pseudotuberculosis,
Peritrichous flagella of very irregular wave-
length and amplitude.
47. Noguchia
Three species of the genus Noguchia are listed in Bergey's
Manual, 6th ed., Noguchia granulosis and Noguchia simiae are
described as having polar flagella and Noguchia cuniculi as hav-
ing peritrichous flagella. One culture labeled N. granulosis was
obtained from Dr. W. E. Clapper of the University of New Mex-
ico. This culture was isolated from the human eye (Fig. 50).
The organism was a medium large rod with normal peritrichous
flagella with average wavelength of 3.3 microns. This morphology
oranu-
does not correspond with the original description of A',
losis. From ATCC was obtained N. granulosis 11479. This or-
ganism grew well on infusion agar with a yellow water insoluble
pigment. Moist preparation showed fair motiUty and staining
showed peritrichous flagellation. The organisms clumped so badly
however that good stains were impossible to get. The wavelength
of the flagella was normal and quite long averaging 3.4 microns.
An occasional curly and part curly part normal flagellum was seen.
In general the ATCC culture and that from Clapper were mor-
phologically much alike. Neither corresponds with the original
description of N. granulosis. The yellow pigmentation of the
ATCC culture fits it nicely into the genus Flavobacterium.
118
JE^
Fig. 50. a. Noguchia granulosis. Clapper. Normal peritriclious flagella.
This picture does not correspond with the original description of the organ-
ism which stated the flagellation as polar.
b, c, d. N. granulosis, ATCC 11479. In b and c are illustrated the normal
peritrichous flagellation of this organism. The organisms clumped so badly that
few separate individuals could be found. In c is shown a curly flagellum
which was quite rare.
119
48. Photobacterium
The light producing or luminescent bacteria do not form a
homogeneous group either physiologically or morphologically. To
group them together into one genus, Photobacterium, may not be
in the best interest of a sound taxonomy. Luminescence, however,
is such a striking phenomenon that one is apt to regard it as of
fundamental taxonomic importance even though it is not a particu-
larly stable characteristic.
Five cultures of the group were received from H. Spencer of
the Humber Laboratory, England; namely Photobacterium sepiae,
P. albensis, P. harveiji, P. phosphoreum, and P. splendidum. P.
fischeri was received from M. J. Cormier, Oak Ridge, Tennessee.
P. phosphor escens, strains L-342 and L-1761, were received from
Kluyver's laboratory in Holland through R. S. Breed (see Fig.
51). All the cultures were halophilic and rather psychrophilic.
With 3% sodium chloride added to the proper medium and in-
cubated at 20° C, good growth was obtained. With the excep-
tion of P. sepiae, all produced some light in one medium or an-
other.
Fig. 51. a. Photobacterium fischeri, Cormier. Polar multitrichous flagella.
The flagella are short with few curves, usually less than one complete wave,
and long wavelength. Resembles Spirillum.
b. P. fischeri, Cormier. Same slide as in a. This picture shows the "micro-
cyst" form which is common in this strain. The flagellation is the same as in
the long, or normal, form.
c. P. phosphor escens, Kluyver L-342. All individuals in this culture had a
spherical soma with a more or less well-defined capsule. Polar monotrichous
flagella.
d and e. P. phosphoreum, Spencer. All individuals in this culture were
spherical with a single flagellum. In e is shown a dividing form with the
typical location of the flagellum, indicating polarity.
f. P. albensis, Spencer. In addition to the curved rod illustrated, this
culture also showed straight rods and spiral forms. The straight and curved rods
showed polar monotrichous flagellation. The spiral forms occasionally had
tufts of polar flagella.
g, h. P. splendidum, Spencer. Both spiral and spherical individuals were
present in this culture. In g is shown a slightly spiral form and in h, a spherical
form. The flagellation is polar monotrichous.
i. P. sepiae, Spencer. Straight rods with polar monotrichous flagella.
j. P. harveiji, Spencer. Straight rods with single polar flagella.
120
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121
Taxonomy
All strains of light producing bacteria appear to be carbohy-
drate fermenters and polar flagellated. They could thus be classi-
fied either in the genus Vibrio or Aeromonas. On the bases of
somatic morphology and sensitivity to a vibrio static substance
(2,4-diamino-6,7-diisopropylpteridine), Spencer would classify P.
sepioe and P. horveyi in the Aeromonas genus and the others in
the Vibrio genus. As recorded in Table XII the flagellar wave-
lengths of P. horveyi and P. sepiue are much alike and distinctly
different from the others, which lends support to the Spencer
classification. The flagellation of P. fischeri, however, is different
from any typical Vibrio species and more like spirilla.
Flagellar Characteristics
P. fischeri showed polar multitrichous flagellation. The flagella
were short, with few curves, usually less than one complete wave,
and of very long wavelength. Some individuals were short spiral
forms, others practically spherical. The somatic morphology and
flagellation resemble the marine spirilla in which the spherical
form, or microcyst, is quite usual. P. phosphoreum, both the
Spencer strain and the two Kluyver strains, showed a spherical
soma and a single flagellum of somewhat longer than the average
wavelength of most polar monotrichous bacteria. By observing
the location of the flagellum in dividing forms it was evident that
the flagellum had a polar location. P. albensis showed a variety
of somatic types — small straight rods, small curved rods, and long
spiral filaments. On the small forms the flagellation was polar
monotrichous with about SO^o of the organisms being amphi-
trichous. The long spiral forms frequently showed tufts of flagella
characteristic of spirilla. P. harveyi was a large straight rod with
polar monotrichous flagella. The size of the soma and the curva-
ture of the flagella showed great variation. P. sepiae showed only
straight rods with polar monotrichous flagella. The flagellar wave-
length was rather variable and may be of two types. P. splendi-
diim showed a very pleomorphic soma with spherical forms,
straight and slightly curved rods, and definitely spiral forms.
The flagellation was polar monotrichous without the flagellar tufts
seen in P. albensis. None of the cultures examined showed any
very definite flagellar variants.
122
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49. Bacillus
Thirty-eight cultures, representing twenty-one species, of the
genus Bacillus were studied morphologically. The identity of the
cultures was accepted as labeled by the donors. The majority of
the cultures were from the R. N. Smith Collection and were re-
ceived from William B. Haynes of the Northern Regional Research
Laboratory, Peoria, Illinois. Several cultures were from Kenneth
Burdon, University of Texas. With the exception of Bacillus
pasteurii, which required addition of 1% urea to the medium, all
cultures grew readily in simple peptone broth. Only one thermo-
phile. Bacillus stearothermophilus, was studied and this was in-
cubated at 55° C.
Fig. 52. a. Bacillus subtilis, NRRL B-642. Typical normal and coiled
flagella.
b. B. subtilis, NRRL B-642. This is a somewhat unusual organism for this
species, showing two normal and two curly flagella.
c. B. pumilus, Burdon Ba 7(5). Normal and coiled flagella.
d. B. megaterium, NRRL B-349. Normal flagella. This strain also showed
some coiled flagella.
e. B.cereus, Burden Ba 2(7). Normal flagella. Flagella of two distinctly
different wavelengths were observed in this culture. The shorter wavelength
flagella had a wavelength about 1.6 microns which is greater than tvpical curly
flagella.
f. g. B. macerans, NRS 1093. In f is shown the normal and in g the curly
variant of this strain.
h. B. macerans, NRRL B-171. This strain had a unique flagellation. The
flagella are short, stiff^, and with a very short wavelength — compare with g.
i. B. macerans, NRRL B-172. Still a difi^erent picture of B. macerans show-
ing lightly stained soma with normal flagella on one individual and curly
flagella on the other. The normal flagella have a shorter wa\elength than
those of strain 1093 shown in f.
j. B. pohjmyxa, NRRL B-173. Normal flagella of somewhat irregular
curvature.
124
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125
Flagellar Characteristics
Some of the cultures were well flagellated, others very poorly
flagellated (see Figs. 52 and 53). The flagellar arrangement was
peritrichous in all cultures. Three types of flagellar curvature
were observed: normal, curly, and coiled. A few species showed
normal flagella only. Some strains of Bacillus macerans and of
B. pasteurii showed only curly flagella, other strains only normal
flagella, and still other strains a mixture with some individuals
with curly flagella, some with normal flagella, but not both kinds
of flagella on the same individual. In several other species normal
and curly flagella were found on the same individual. Also in
several species the same individual might show normal and coiled
flagella, and even normal, curly, and coiled. Bacillus subtilis and
Bacillus pumilus showed the greatest tendency to produce coiled
flagella. One strain of B. macerans was quite unique with onlv
short, stiff curly flagella with shorter wavelength than the curly
Fig. 53. k. Bacillus circulans, NRRL B-378. Normal flagella of rather short
wavelength.
1. B. aZuet, NRS-811. Normal flagella.
m. B. foreuts, NRS-1138. Normal flagella.
n. B. sphaericus, NRS-348. Normal flagella. This strain also showed some
coiling.
o. B. pasteurii, NRS-673. Normal flagella.
p. B. pasteurii, NRS-674. Curly flagella.
q. Bacillus sp., A-J. This picture is from a culture labeled bacillus A-J, re-
ceived from Dr. Eleanor Alexander-Jackson. It is claimed by the donor to
cause human cancer and was isolated from human cancerous tissue. The
flagellar morphology is very similar to B. cereus with flagella of two distinct
wavelengths.
r. B.stearothermophilus, NRRL B-1172. Normal flagella. This was the
only thermophile studied.
s. B. lentus, NRRL B-396. Normal and coiled flagella.
t. B.coagulans, NRRL B-1167. Normal flagella. "
126
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127
flagella of the other strains and species. Considerable variation
was found in the wavelengths of the normal flagella of the differ-
ent species (see Table XIII). The shortest normal wavelengths
were found in Bacillus circulans and the longest in Bacillus mega-
therium.
Bacillus sphaericus showed pH sensitive flagella. When stained
from a 1% dibasic potassium phosphate suspension the flagella
were all of normal curvature, but from a monobasic potassium
phosphate suspension they were mainly curly.
Bacillus cereus and the Alexander-Jackson (A-J) strain showed
mainly normal flagella but mixed with the normal were some
flagella with shorter wavelength but not sufficiently short to be
labeled curly. The wavelength of these flagella averaged 1.6
microns in B. cereus and 1.5 microns in the A-J strain. The differ-
ence between these wavelengths and wavelengths of true curlv
flagella (1.1-1.25 microns) is readily apparent by direct observa-
tion.
The flagellar wavelengths may be of considerable value in the
speciation of the genus Bacillus. However, to have any great
significance, many more strains must be studied than the author
has done to date.
128
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129
5 0. Clostridiutn
Thirty-one species of Clostridium were studied with two or
more strains of each species. The cultures were suppHed by Dr.
L. S. McCking of Indiana University. In agreement with the
Hterature the only definitely nonflagellated and nonmotile species
was Clostridium perfringens, of which five strains were studied.
The single strain of Clostridium nigrificans studied did not show
an)' flagella but the growth was not entirely satisfactory and much
significance should not be attached to this finding.
Flagellar Characteristics
All the motile strains studied showed peritrichous flagellation.
With a few exceptions the flagella stained readily. Some species of
Clostridium grow with difficulty in media without particulate mat-
ter and had to be cultured in thiogly collate medium (O.l^r agar),
corn mash, etc., which made washing not entirely satisfactory.
The flagella of three species stained with more difficulty than
those of the other species. These were Clostridium felsineum, C.
acetohutijlicum, and C. roseum. C. acetobutijlicum and C. roseum
are very similar morphologically. Strain McClung 638 of C. fel-
sineum showed normal flagella only but strain 639 showed flagella
much like C. acetohutijlicum with a mixture of flagella of short
and long wavelength. This species requires further study but
morphologically it appears very similar to C. acetohutijlicum and
C. roseum. The flagellation of Clostridium tetani deserves special
mention. Two strains were studied and, while one was better
flagellated than the other, both showed the same type of flagella
which were rather stiff with relatively short wavelength and large
amplitude. The same type of flagella, with longer wavelength,
was also found in the strains of Clostridium noviji studied. This
flagellation, if similar in all strains, is so distinctive as to be diag-
nostic.
In some species of Clostridium only normal flagella were ob-
served, in other species the flagellation was predominantly normal
with an occasional curly flagellum mixed in with the normal.
Several species showed both normal and curly flagellated indi-
viduals. Coiled flagella were particularly prominent in the aceto-
131
butylicum group. Straight flagella were observed only in Clos-
tridium aerofoetidum, McClung 1148, which showed a few
individuals with straight flagella. Several flagellar variants were
observed in Clostridium tetanomorphum as illustrated (Figs. 54, 55,
and 56).
Fig. 54. a. Clostridium difficile, McClung 871. The three strains studied
showed an identical picture. Note the exceptionally short wavelength, shorter
than that of curly flagella on most bacteria.
b. C. tetani, McClung 148. Two strains studied showed practically an
identical picture. The slides showed only normal flagella with the shortest
normal wavelength of all Clostridia studied, excepting C. difficile which may
have been curly variants. The flagella of C. tetani appear to be very stiff
with relatively large amplitude. They are very characteristic and practically
diagnostic.
c. C. capitovale, McClung 1237. The two strains stvidied were identical
and showed normal flagella only. The flagellar wavelength is relatively short
and amplitude relatively great. Flagella give impression of being rather
delicate.
d. C. noviji, McClung 151. Normal peritrichous flagella of rather large
amplitude. The flagellation resembles that of C. tetani except for greater wave-
length. A few curly types seen.
e. C. botulinum, McClung 662. Normal flagella. Two of the tliree strains
studied were atrichous. The strain pictured showed a few partly curly
flagella.
f. C. septicum, McClung 1019. This is a medium length filament, many
were several times this length, and characteristic of C. septicum. The flagella
pictured are of normal type with rather short wavelength and small amplitude.
In organisms other than that pictured curly flagella were interspersed among
the normal.
g. C.felsineum, McClung 638. The organism pictured is typical of strain
638, with normal flagella which stained readily. However, strain 639 showed
more a picture resembling C. acetobutylicum and the flagella were difficult to
stain. Further study is necessary to definitely elucidate the flagellar character-
istics of this species.
h. C.chauvoei (feseri), McClung 1436. Normal flagella. No variants ob-
served.
i. C. centrosporogenes, McClung 136. Normal flagella. With the exception
of a single curly flagellum all flagella seen in this culture were normal.
j. C. hifennentans, McClung 435. Only normal flagella observed on both
strains studied.
k. C. lentoputrescens, McClung 998. Nornial flagella. The one strain
studied showed excellent flagellation with all flagella of normal curvature.
132
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X
p
The flagellation of Clostridium difficile was most unusual if not
unique for the genus Clostridium. Three strains of this species
were studied (McClung 870, 871, 1253) and all three were iden-
tical. The wavelength of the flagella is shorter than that of curlv
flagella in general and sufficiently distinctive to be diagnostic.
Flagella of more normal wavelength were not found in this species
(see Table XIV).
Fig. 55. 1, m, n. Clostridium aerofoetidum, McClung 1148. In addition to
indix'iduals with pure normal and pure curly flagella a few individuals were
found with straight flagella. This is the only Clostridium species in which
straight flagella have been observed.
o. C. histolxjticum, McClung 1292. Two strains were studied both of which
were well flagellated with predominantly normal flagella. A few part normal
and part curly flagella were seen but no entirely curly flagella nor curly in-
dividuals.
p. C.pamhotulimtm, McClung 489. Typical normal flagella of this species.
Twenty-one strains were studied and only in two was a very rare curly or
partly curly flagellum seen. Most strains were well flagellated.
cj. C. carnis, McClung 1249. Two strains were studied. Both showed good
flagellation with flagella predominantly normal with a few curly flagella on
some individuals along with the normal types.
r. C. thermosaccharolyticum, McClung 919-A. The flagellation of this
strain was very poor and the organism pictured was the best that could be
found. The flagella had normal curvature.
s. C.sphenoides, McClung 1183. The flagellation of this organism is very
similar to that of C. hutyricum. The flagella on the one strain studied were all
normal but seemed to be of two kinds. On those individuals with relati\el\
long flagella the wavelength averaged 2.67 microns, and on those with the
relatively shorter flagella the wavelength averaged 3.12 microns. The possil^le
presence of two variants must be determined by further study.
t. C. cochlearium, McClung 257. This culture showed practically pure
normal flagella. A rare curly flagellum was seen with small amplitude.
u. C. hutyricum, McClung 629. The flagella are peritrichous and of nor-
mal, rather long, wavelength. The flagella tend to be rather short.
V. C. butylicum, McClung 1670. Normal flagella of somewhat irregular
curvature. About 207o of the individuals showed curly flagella and the rest
normal flagella.
134
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Fig. 56. w. Clostridium sp. 3679, McClung 174. Three strains of this
organism were studied. With the exception of a few partly curly and partly
normal flagella the flagellation was pure normal. This organism has highly
heat-resistant spores and is used in the canning industry as a check on steriliza-
tion.
X, y. C. sporogenes, McClung 175. In .\ are shown the typical normal
flagella and in y the curly flagella. Of seven strains studied three were
pure curly types. In C. sporogenes the curly variant apparently is a stable
type.
z, aa, bb, cc. C. tetanomorphiim, McClung 2038. The culture stained was
fairly far advanced into the spore stage and a younger culture perhaps would
show more flagella per individual. Pictured are several variants; normal, curly,
part curly and part normal, curly and normal on the same individual, and
double curvature.
dd. C. tertium, McClung 1272. Two strains were studied and both were
well flagellated with predominantly normal flagella and some coiling tendency.
ee. C aurantibiityricum, McClung 2038. The organism pictured has four
or five normal and two curly flagella. The culture was about half normal
and half curly with a few individuals with mixed flagella like the one pictured.
ff. C. pasteurianum, McClung 308. Normal flagella with long wavelength.
No variants seen.
gg. C. beijerinckii, McClung 1673. Normal but rather short flagella of long
wavelength.
hh. C. roseum, McClung 653. This organism was well flagellated but the
flagella stained with considerable difficulty and always rather lightly. The
flagella were mainly of the curly type but interspersed among the curly flagella
were often flagella of very long wavelength and usually also coiled flagella.
The resemblance to C. acetobutylicum is so striking that a close relationship is
suggested. Best stains were obtained by doubling the tannic acid concentration.
ii. C. acetohuttjlicum, McClung 633. The two strains of this organism
studied showed beautiful flagellation with a flagellar density equal to or sur-
passing such organisms as Proteus. The great majority of the flagella were of
the curly type, usually interspersed with flagella of very long wavelength and
often some coiled types. The flagella stained with some difficulty. The re-
semblance to C. roseum is striking.
136
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139
5 1 . Caulobacter
In Bergey's Manual is listed only one species of Caulobacter,
Caulobacter vibrioides. This organism is one of the most common
bacteria in natural waters. It is present in the distilled water of
the author's laboratory at all times. When bacterial cultures are
washed for flagella staining a few caulobacter may invariably be
found on the slides and may be mistaken for a variant of the cul-
FiG. 57. a. Caulobacter vibrioides. A typical in(li\'idiuil organism showing
a single polar flagellum of very short wavelength.
b. C. vibrioides. This pictnre shows an individual with a stalk and a flagel-
hun at the end of the stalk.
c, d, e. C. vibrioides. Here are shown various degrees of rosette formation.
The individuals appear to be attached to each other. Flagella develop on the
daughter cells when division is about complete.
f. C. vitjrioides attached by a stalk to a staphylococcus. The Caulobacter
Hagellum is still attached to the end of the stalk.
g, h. C. vibrioides attached to an unidentified water organism with a single
polar flagellum. Note the difference in wavelength of the flagella of the un-
identified organism and Caulobacter.
i. C vibrioides attached to Salmonella wichita. There are several salmon-
ella in the clump and the flagella with the longer wavelength are salmonella
flagella. The smaller organisms around the periphery are Caulobacter and
two of these at the bottom of the picture show flagella.
j. C vibrioides attached to Sareina ttreae. The rounded curves of several
sarcinae may be seen along the upper right edge of the dense mass. Several
sarcinae flagella of long wavelength emerge from the upper part of the clump
and one from the lower part. Many caulobacter flagella are evident.
k. C. vibrioides. The three pictures show the morphology of caulobacter
in slightly alkaline media of pH 7.5 to 8.0; pH 8.0 was the upper limit for
growth. In the somewhat alkaline medium caulobacter grew very poorly and
produced few stalks and few flagella. Note the strongly curved soma. The
organism shown in the upper right was very exceptional in having both a stalk
and a flagellum.
1. C. vibrioides. In media with osmotic pressure equivalent to about 1%
sodium chloride and at a pH not lower than 6.5, caulobacter grows slowly and
in the form of long and somewhat curved filaments. The filament may have a
single polar flagellum as illustrated, one or more lateral flagella or, most fre-
quently, no flagella.
m. C. vibrioides from a mixed culture with Bacillus pumilis. This is a
rather unusual involution form.
n. C. vibrioides rosette from which has grown out a long filament. This
picture is from a normal culture in which filaments are extremely rare. Note
the flagellum at the end of the filament.
140
^■ryMt-
f
g
h
/
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ture under study. Other types of small polar monotrichous bacteria
may also be found in the distilled water and consequently on the
flagella slides. If confusion is likely, the water used for washing
the bacteria must be freshly distilled or freshly boiled. In the
author's experience the Caulobacter in distilled water usually have
no flagella but do have a long stalk which makes them readily
recognized. If a washed suspension of bacteria is allowed to stand
for several days at room temperature a goodly number of caulo-
bacter is usually found in the suspension, where they may or may
not be attached by stalks to the washed bacteria (see Figs. 57 and
58).
Flagellar Characteristics
C. vibrioides in the free living state is a small rod which may
be straight, or curved Bke a vibrio. The flagellation is polar mono-
trichous. The flagellar wavelength is very short, averaging 0.95
micron with an amplitude averaging 0.4 micron.
In all the cultures studied, for causes unknown at present, the
individual bacteria soon begin to grow stalks. The stalk develops
on the flagellated end of the organism and the flagellum persists
for a limited time on the end of the stalk, even when the stalk is
attached to some particulate matter, such as other bacteria. When
the stalked bacteria start to divide a new flagellum develops from
the end distal to the stalk. Many individuals may become attached
to a single particle of matter or to each other with the formation
of small and large rosettes. New flagella develop on the distal
ends of the daughter cells and the rosettes are soon bristling with
flagella.
Fig. 58. o. This picture is from a distilled water suspension of Bacillus
megaterium and caulobacter. The caulobacter is attached by a long stalk
to the upper right end of the bacillus. Note the short caulobacter flagellum
at the base of the stalk.
p. An involution form of caulobacter with two polar flagella and appar-
ently attached to a Listeria organism with straight flagella. This is from a
mixed culture of the two organisms. Multiple polar flagella on caulobacter
were very rare.
q. Caulobacter attached to staphylococci and to each other. From the
masses of staphylococci and caulobacter emerge long filaments of caulobacter.
Note two flagella on the filament at the right. This picture is from a mixed
culture of caulobacter and staphylococci.
142
H-.^\
143
52. ChromatiuTn
One culture of Chromatium strain D was received from Dr.
D. D, Hendley of the University of Chicago (Fig. 59). The orig-
inal broth culture was very motile and was stained directly with-
out subculture. The growth had a distinct red color and the
massed bacteria were red.
Flagellar Characteristics
Flagellation was excellent and the individual bacteria showed
long polar monotrichous flagella. The flagella were very uniform
in shape with an average wavelength of 2.05 microns and ampli-
tude of 0.47 micron.
5 3 . Khodopseudomonas
The Bergey's Manual lists four species of Khodopseudomonas.
Three of these species were obtained for study: Rhodopseudo-
monos palustris ATH 2.1.1 from Hopkins Marine Station; R. palus-
tris, NCIB 8252; Rhodopseudonwiuis gelatinosa, NCIB 8290; and
Rhodopseiidomonas spheroides, NCIB 8253, from the National
Collection of Industrial Bacteria in England. All the cultures grew
readily on agar slants producing a reddish growth, and all were
motile. No attempt at species identification was made.
Flagellar Characteristics
The flagellation of the four cultures was monotrichous. In R.
gelatinosa, NCIB 8290, the flagellum was definitely polar in origin.
In the other three cultures the flagellum often originated subpolar
and occasionally actually lateral. R. palustris, Hopkins Marine
Station strain, showed mainly subpolar, coiled monotrichous
flagella which appeared quite different from those of R. palustris,
NCIB 8252. This latter strain also had some individuals with a
subpolar flagellum but the wavelength was only half that of the
former strain. R. spheroides, NCIB 8253, showed a goodly num-
ber of subpolar flagella of long wavelength, or coiled flagella, and
was very similar to the Hopkins Marine Station of R. palustris. In
this strain the flagellum was often lateral. ( See Fig. 60. )
144
Fig. 59. a. Clironiatium sp., Hendley-Gaffroi
Strain D. Polar monotrithoiis flagella.
i
; / S'-^ -'
Fig. 60. a. Rhodopseudomonas palustris, NCIB 8252. Polar monotrichous
flagella. Some individuals of this strain had a subpolar flagellum. Mean fla-
gellar wavelength 1.58 microns.
b. R. palustris, Hopkins Marine Station 2.1.1. This culture was very poorly
flagellated. Most flagellated individuals showed subpolar monotrichous coiled
flagella. The soma stained very lightly. Flagellar wavelength from 3 to 4
microns.
c. R. gelatinosa, NCIB 8290. Polar monotrichous flagella of normal curva-
ture. This strain showed only polar flagella. Mean flagellar wavelength 1.63
microns.
d,e. R.spheroides, NCIB 8253. Note the long wavelength of the flagella
and the lateral origin in d. Individuals with subpolar and lateral flagella were
as frequent in this strain as those with polar flagella. Flagellar wavelength
was 3 to 4 microns.
145
54. Rhodospirillum
Two species of Rhodospirillum are listed in Bergey's Manual,
Rhodospirillum rubrum and Rhodospirillum fulvum. Several strains
of R. rubrum were obtained for study from several sources. R.
fulvum was not obtained.
Flagellar Characteristics
R. rubrujn is a typical spirillum with a tuft of polar flagella at
one or both ends. The flagella rarely have more than one curve
as in most spirilla (Fig. 61).
5 5. Nocardia
The great majority of Nocardia organisms encountered in na-
ture appear to be nonmotile and presumably nonflagellated. A cul-
ture labeled Nocardia, Orskov, was received from Mortimer P.
Starr of the University of Cahfornia (Fig. 62). This strain origi-
nated from Dr. J. Orskov in Denmark. The culture showed mainly
relatively small pleomorphic rods with a few short filaments. Mo-
tility was fair.
Flagellar Characteristics
The majority of individuals showed only one flagellum or none.
A fair number showed two flagella and a few showed many flagella.
The flagella were very irregular as to length and curvature. Some
were almost straight, others coiled, and some showed uneven
waves. The arrangement of the flagella was distinctly nonpolar or
peritrichous. The wavelength of the wavy flagella was very great
but so uneven that an average wavelength has little significance.
The most distinct waves measured around 5 microns in length.
146
Fig. 61. a. Rhodospirillum ruhrum, Hopkins Marine Station ATR 1.1.1.
Tufts of flagella at one or both poles. Flagella rarely have more than one
curve. From E. Leifson, /. Bacteriol. 62, 377-389 (1951).
Fig. 62. a. Nocardia sp., Orskov. Peri-
trichous flagella with irregular waves. Most
flagellated individuals in the culture had
only one or two flagella.
147
56. Borrelia
Two types of Borrelia were studied, neither in artificial cul-
ture. The one organism, Borrelia noviji, was stained directly from
the blood of an infected mouse. The blood was carefully collected
into citrated saline solution, the blood cells removed by slow
centrifugation, and the spirochetes washed in the usual manner.
The other organism studied came from the mouth of a human
male with Vincent's angina. This organism may be labeled Bor-
relia vincentii (Fig. 63).
Flagellar Characteristics
B. noviji was actively motile and flagella stain showed numerous
peritrichous flagella of normal curvature. No variants were ob-
served. The average wavelength was 1.9 microns. B. vincentii
showed numerous flagella with peritrichous arrangement and nor-
mal curvature. No distinct variants were observed. The average
wavelength of the flagella was 2.0 microns.
14S
rv,/^
Fig. 63. a. Borrelia noviji. Peritrichous flagella of normal curvature.
Stained directly from mouse blood.
b. B. vincentii. Peritrichous flagella of normal curvature. Stained directly
from material from the human mouth.
a. From E. Leifson, /. Bacteriol. 60, 678-679 (1950).
149
5 7. Treponema
Several unsuccessful attempts were made to secure material for
direct study of Treponema pallidum from syphilitic chancres. The
number of spirochetes were too few and got lost in the wash. At-
tempts at direct staining of spirochetes from infected rabbit testicle
were also unsuccessful. Several strains of cultivated Treponema
were stained and some of these are illustrated. Included among
these were strains labeled T. pallidum, Reiter, Nichol, Kazan, and
Noguchi. Some oral strains of Treponema were also studied.
Flagellar Characteristics
The Reiter strains of T. pallidum showed the best and most
unequivocal flagellation. The flagellation was subpolar multitri-
chous as illustrated in Figs. 64c and d. This appears to be the basic
flagellation of Treponema. The Kazan strain of T. pallidum also
showed this type of flagellation on some individuals. Many in-
dividuals of the strains named, and also the Nichol and Noguchi
strains, showed subpolar monotrichous flagellation. This type is
probably the same as subpolar multitrichous.
Many individuals in most strains showed a polar monotrichous
flagellum-like structure illustrated in Fig. 64a. If this had been
the only flagellum-like structure seen in Treponema it would cer-
tainly have been labeled a polar flagellum. In the literature it is
referred to as a polar filament. A similar type of filament some-
times may be seen connecting two organisms as in Fig. 64b. These
polar and intersomal filaments have about the same wavelength
as the soma. So, for that matter, also have the subpolar flagella.
The average wavelength of the subpolar flagella is 1.2 microns.
150
Fig. 64. a. Treponema paUiditm, Reiter strain. The polar structures on
tliese spirochetes are commonly referred to as terminal filaments and may
not be flagella.
b. Treponema sp., Oral type, Hampp. The upper organism has a single
subpolar flagellum. Connecting the two organisms is a filament probably of
the same nature as the terminal filament on the lower organism.
c, d. T. pallidum, Reiter strain. Characteristic subpolar multitrichous flag-
ellation. In c is shown this type of flagellation particularly well.
a-d. From E. Leifson, /. Bacterial. 62, 377-389 ( 1951 ).
151
5 8, Bartonella
Two cultures of Bartonella bacilliformis were received from
Aristidis Herrer, Lima, Peru. Transfers to various blood media
grew the organisms rather scantily. The best preparations were
obtained from blood agar slants by suspending the growth in dis-
tilled water and washing in the usual manner. The organisms
showed much clumping and none of the preparations were par-
ticularly good.
Flagellar Characteristics
In spite of the frequent location of the flagella at the poles of
the soma the organism appears definitely to be peritrichous. The
flagellation was poor, the organisms usually in clumps, which left
few isolated, flagellated individuals on the slides. The flagellar
wavelength was very short, averaging 0.95 micron with an average
amplitude of 0.25 micron (Fig. 65).
152
Fig. 65. a. Bartonella bacilliformis, Herier
VS 306. The picture shows what appears to be
two individuals more or less end to end. Although
the flagella are somewhat concentrated at the
poles they are definitely peritrichous in arrange-
ment. Note the tiny wavelength.
M
153
59. Selenomonas
The genus Seleno7nonm apparently has a ubiquitous distribu-
tion in nature. It appears to be a common inhabitant of the cow
rumen, the human throat, dog intestine, river water, etc. It is
anaerobic and somewhat fastidious in its growth requirements.
Morphologically it is unique, with a slight curvature to the soma
and unusual flagellar arrangement (Fig. 66).
One culture labeled Selenomonas ruminantium was received
from Marvin Bryant of the U.S.D.A. It was isolated from cow
rumen. Similar appearing organisms were seen by the author in
material from dog intestine and in river water but these were not
isolated. A culture labeled Spirillum spiitigenum was received
from J. B. Macdonald of Harvard University. Growth of this
organism was rather unsatisfactory but sufficient organisms were
present for staining.
Fig. 66. a, b, c. Selenomonas ruminantium, Bryant. These are typical ex-
amples of single individuals. The soma is slightly curved and the flagella orig-
inate as a tuft from the concave side.
d, 6. S. ruminantium, Bryant. These organisms appear to be in the process
of cell division. In d the left half is starting to develop flagella. In e each
half has a distinct tuft of flagella.
f. S. ruminantium, Bryant. This organism appears to have been turned
so the concave side is up. The flagella appear to orignate from a disklike
structure.
g. S. ruminantium, Bryant. This individual shows a single \ariant flagellum,
which might be labeled curly, in addition to the normal flagella. This was
the only flagellar variant observed in all the slides examined. The wavelength
of this curly flagellum is 1.2 microns.
h. S. ruminantium, Bryant. The several flagella which can be seen emanat-
ing from the soma are twisted into a single strand.
i. S. ruminantium, Bryant. Flagella of two organisms twisted together.
What else could it be?
j. Selenomonas sp. Organism stained directly from water of the DuPage
River in Illinois.
k. Selenomonas sp. Organism stained directly from the intestinal content
of a dog. Note the smaller somatic size of this organism compared to S. rumi-
nantium and the strain from the DuPage river.
1. Spirillum (Selenomonas) sputigenum, Macdonald. The flagellar ar-
rangement and wavelength classifies this organism as Selenomonas. The
soma is smaller than that of S. ruminantium and comparable to that of the
organism from the dog intestine.
154
b-
c
f
•
•
J '.
1
155
Flagellar Characteristics
The flagella originate as a tuft from the concave side of the
organism. In some individuals the flagella appear to originate
from a single point while in others the origin is more diffuse.
Sometimes only a thick, solid appearing, structure is seen which
probably represents several flagella twisted together. The flagellar
wavelength was somewhat variable and very long, averaging about
4.0 microns. In all the slides examined only one organism was
seen with a single flagellum of shorter wavelength. This curly
flagellum had a wavelength of 1.2 microns. The organism labeled
Spirillum sputigenum appears somewhat smaller than the or-
ganism from the cow rumen and the one seen in river water, but
of about the same size as the one from the dog intestine.
60, Caryophanon
Only one motile culture of this genus was obtained for study.
A culture labeled Caryophanon latum was received from Owen D.
Weeks of the University of Idaho. At the suggestion of Dr. Weeks
the organism was cultured in peptone media with 1% sodium
acetate at pH 7.6. Best growth and flagellation was obtained on
agar slants. In the liquid medium the growth was rather light.
Flagella stains were made from agar slants incubated at 20, 30,
and 37° C. Best flagellation was found at 20° C, almost as good
at 30° C, but definitely poorer at 37° C. Good motility was not
observed in any of the cultures in spite of good flagellation. Dur-
ing the period the culture was studied only the smooth phase of
growth was observed. The organisms were mainly short, ovoid
rods, frequently arranged in short chains or irregular groups. Only
occasionally were seen the longer forms with the characteristic
banded appearance.
Flagellar Characteristics
Numerous peritrichous flagella were observed at incubation
temperatures of 30° C. and below. Most frequently the flagella
were normal in curvature but individuals with curly flagella were
common (Fig. 67). Individuals with both normal and curly
flagella were occasionally seen. Change of pH of the bacterial sus-
pension did not change the wavelength of the flagella. The normal
flagellar wavelength averaged 2.14 microns, and the curly 1.09
microns.
156
Fig. 67. a. Canjophanon latum showing typical peritrichous flagella of
normal curvature.
b. C. latum from tlie same culture as in a showing mainly curly flagella
with a few normal flagella.
157
61. DuPage River Organism
In the early course of the study of the flagellation of the bac-
teria in the DuPage river west of Chicago a most unusual organism
was observed. In flagella stains directly from the water the
organism appeared as a large rod, usually curved, often in a semi-
circle, and sometimes straight. From the soma protruded numerous
spines, much like toothpicks stuck into a wiener. A typical or-
ganism is illustrated in Fig, 68f. Typical flagella were never seen
on these organisms and their bacterial nature was questioned.
After considerable effort the organisms were isolated in pure cul-
ture and were found to be flagellated with predominantly polar
monotrichous flagella. The flagella are typically very long with
relatively short wavelength, averaging 1.37 microns. To date the
organism in pure culture has not developed the spines so char-
acteristic of the organism in the river water. The identity of the
flagellated and the spined forms was established by the observa-
tion of spined forms with the typical flagella in the original enrich-
ment cultures made by adding small amounts of various nutrients
to the river water. Only in these mixed cultures of bacteria, pro-
tozoa, and algae have the organisms been seen with both spines
and flagella on the same individual. The organisms vary greatly
in size and are typically capsulated. In certain media and under
certain cultural conditions branching forms may be seen. What
is the nature and function of the spines remains to be determined.
158
d
b •
Jk V
J^
f
Fig. 68. a. A typical organism as seen in broth culture. Polar niono-
trichous flagella.
b. This picture is from a mixed culture in a very dilute medium. The
curved soma is very characteristic under such conditions.
c. The organism illustrated shows a short branch. Other and more
branched individuals were also observed in some media but most of these
were nonflagellated.
d. e. The two organisms illustrated show the spined soma and the charac-
teristic polar flagellum. Both spines and flagella were only observed in the
mixed cultures obtained by adding small amounts of nutrients, such as yeast
extract, to the river water.
f. The tvpical spined but nonflagellated form seen in river water. The
soma was typically curved, often in a semicircle. The nature and function of
the spines has not been determined.
159
62. Appendix
Mlxed Flagellation
Cultures of bacteria in which are constantly found individuals
with both polar and lateral flagella are not common. A typical
example was illustrated in Chromobacterium. Another example is
that of an unidentified organism from water illustrated in Figs.
69a to 69f inclusive. In this mixed type of flagellation the polar
flagella have invariably shown a greater wavelength than the
lateral flagella. If the wavelengths were the same the phenomenon
would probably be overlooked and the organisms regarded as of
the ordinary peritrichous type.
Filaments of Polar Flagellates
Filamentous mutants of polar flagellated bacteria may have the
appearance of peritrichous flagellation. If the cellular units in the
filament are long and the organism is polar multitrichous or lopho-
trichous, the true nature of the flagellation is usually obvious. Tufts
of lateral flagella at regular intervals is not characteristic of peri-
trichous flagellation. An example of this type is shown in Fig.
69g. Other examples are the Fseudomonas sp. illustrated in Fig.
9s and the Lophomoiias illustrated in Fig. 16c. If the cellular units
in the filaments are very short the true nature of the flagellation
may be difficult to recognize.
Filaments of polar monotrichous organisms may be very diffi-
cult to recognize for what they are. In Figs. 69h and 691 are il-
lustrated the nonfilamentous and filamentous form of an unidenti-
fied, nitrogen-fixing, soil organism. This organism was originally
described in the literature as peritrichous or showing mixed flagel-
lation with a polar flagellum which appeared to be thicker than
the lateral flagella. This culture was obtained and, on plating, both
rough and smooth colonies were found. The smooth colonies were
composed of polar monotrichous short rods, illustrated in Fig. 69h.
The rough colonies were composed of long and short filaments
with flagella which had the appearance of being peritrichous. A
short filament is illustrated in Fig. 69i. In pure form this fila-
mentous mutant might be difficult to recognize for what it is.
161
Flagella on Protozoa and Algae
Reference has been made in the preface to the flagella on
protozoa and algae. The author has not studied these organisms
in detail using flagella staining techniques. With many of these
organisms fixation techniques different from those used with bac-
teria have to be employed not to damage the soma. The typical
appearance of flagella of algae is illustrated in Fig. 69j showing
Chlamydomonas sp. This type of flagella is similar to the undulant
flagella found on some bacteria. Protozoa such as Trichomonas
have the same type of flagella. The typical helical flagella found
on bacteria evidently are not characteristic of the flagella on pro-
tozoa and algae. The organism shown in Fig. 69k is unidentified
but definitely nonbacterial. Except for the long wavelength of
about 6 microns the flagellum is very similar to those on some bac-
teria. In Fig. 691 is illustrated a most unusual(?) and interesting
type of flagellation on a protozoan or algal organism. CiHated
flagella of the type shown may not be so unusual if proper staining
methods are used.
Fig. 69. a, b, c , d, e, f . Mixed polar monotrichous and peritrichous flagel-
lation.
Note the longer wavelength of the polar flagellum. In f the polar flagellum
is missing. This organism was isolated from water, showed a cream colored
or slightly yellowish growth on agar, and was nonfermentative. If regarded
as peritrichous it could be classified as either Achromobacter or as Flavo-
bacterium. If regarded as polar flagellated it would be classified as Pseudo-
monas or Xanthomoruis. Mixed flagellation poses a difficult taxonomic prob-
lem but fortunately it is rather rare.
g. This illustration shows two short filaments of a basically polar multi-
trichous organism. It may be mistaken for a peritrichous type but such types
rarely show several flagella originating from one point on the soma as in the
illustration. This organism was stained directly from river water.
h, i. These two illustrations are of a nitrogen-fixing organism received
from E. Gray in England. The short filament shown in i is a mutant of the
polar monotrichous organism illustrated in h. In pure culture the filamentous
form could readily be mistaken for a peritrichous type.
j. Chlamydomonas sp. Note the undulant and somewhat irregularly shaped
flagella.
k. An unidentified, nonbacterial organism from water. Except for the long
flagellar wavelength the flagellum is not unlike some found on bacteria.
1. A nonbacterial organism from water with a most unusual (?) type of
flagellation. The trunk of the flagellum has the typical undulant shape. The
cilia-like structures covering the flagellar trunk are not artifacts.
162
0 ' b . c . d e f
1 If
I p
163
Index
Names of genera are set in bold face, as are page numbers indicating first page of
the chapter dealing with the genus.
abortivoequina (Salmonella) , 113
ahortiisbovis (Salmonella) , 113
Acetobacter, 44, 46
aceti, 46, 47
orleanense, 46
rancens, 47
acetobutylicnm (Clostridium), 131, 136,
139
Acetomonas, 44
melanogena, 4 5
suboxydans, 45
suboxydans var. roseitm, 45
Achromobacter, 40, 4\, 92
cycloclastes, 40, 41
adelaide (Salmonella) ,113
Aerobacter, 100
cloacae, 100, 101
aerofoetidum (Clostridium), 132, 134, 139
Aeromonas, 48, 122, 123
formicans, 5 0
hydrophila, 48, 5 0
harveyi, 122, 123
liquefaciens, 5 0
if/)/<?e, 122, 123
aeruginosa (Pseudomonas) , 2 5, 26
agilis (Azotobacter), 63, 64
agilis (Nitrobacter), 22, 2 3
Agrobacteritim, 74
gypsophilae, 74
pseudotsugae, 75
radiobacter, 74, 75
rhizogenes, 74, 7 5
tumefaciens, 74, 75
albensis (Photobacterium) , 120, 122, 123
al ben sis (Vibrio), 122, 123
alboflavus (Protaminobacter) , 34
Alcaligenes, 26, 90
bookeri, 26, 27
bronchisepticus, 90, 91, 93
denitrificans, 90, 91, 93
faecalis, 90, 91, 93
faecalis var. radicans, 26, 17
\
Algae, 162, 163
alliicola (Pseiidomonas) , 3 3
tf/i/f/ (Bacillus), 126, 129
amaranthicola (Xanthomonas) , 36, 39
amylovora (Erwinia), 102, 105
ananas (Erwinia), 105
anafutn (Salmonella), 113, 114
angulata (Pseudomonas), 28, 3 2
anolium (Serratia) , 106, 107
Arizona (Escherichia) , 99
arizona (Salmonella), 114
aroideae (Erwinia), 102, 104, 105
arsenooxydans (Pseudomonas) , 26, 27
arsenooxydans (Xanthomonas) , 3 6, 3 8
Arthrobacter, 86
citreus, 86, 87
simplex, 86
atroseptica (Erwinia), 104, 105
aurantibutyricum (Clostridium) , 13 6, 139
Azotobacter, 62
agilis, 63, 64
chroococcum, 62, 63, 64
indicum, 63, 64
macrocytogenes, 62, 63, 64
vinelandii, 61, 63, 64
Azotomonas, 66
insolita, 66, 67
bacilliformis (Bartonella), 152, 153
Bacillus, 124
A- J, 129
rf/j'«, 126, 129
Wt'WJ, 126, 129
rerews, 124, 128, 129
circulans, 126, 129
coagulans, 116, 129
firmus, 129
laterosporus, 119
lentus, 126, 129
licheniformis, 129
viacerans, 124, 129
megaterium, 124, 129
265
pantothenicus, 129
pasteurii, 124, 126, 129
polymyxa, 124, 129
pulvifaciens, 129
pumilus, 124, 129
sphaericus, 126, 12 8, 129
stearothermophilus, 124, 129
sub t His, 124, 129
technicus, 129
Bacterium tardicresccns, 3 8
Bartonella, 152
bacilUformis, 15 2, 153
begoniae (Xanthomoiias) , 39
beijerinckii (Clostridium), 136, 139
^f r/a (Salmonella) ,113
Bethesda (Escherichia) , 99
bibula (Cellulamotias), 96, 97
bifermentans (Clostridium), 132, 139
blegdam (Salmonella), 113
booker i (Alcaligenes) , 26, 27
bookeri (Pseudomoiins), 26, 27, 32
Borrelia, 148
novyi, 148, 149
liuccntii, 148, 149
botiiliiinm (Clostridium), 132, 138
brcdciicy (Salmonella), 113
bronchisepticus (Alcaligenes), 90, 91, 93
budapest (Salmonella), 113
bullata (Mycoplana) , 40, 41
bullata (Pseudomonas), 40, 41
butylicnm (Clostridium), 134, 139
butyricnm (Clostridium), 134, 138
California (Salmonella), 113
campcstris (Xanthomonas) , 36, 39
capitoialc (Clostridium), 132, 138
<:(/)•;?« (ClosfriJium), 134, 139
carotovora (Ericinia) , 102, 105
Caryophanon, 156
latum, 156, 157
cattleyac (Pseudomonas) , 3 2
Caulobacter, 140
t'ibrioides, 140, 142
Celhilomonas, 96
bibula, 96, 97
per lurid a, 96, 97
rossica, 96, 97
Cellvibrio, 56
/«/i7/.v, 5 6
vulgaris, 56
centrosporogenes (Clostridium) , 13 2, 138
rfrfz<s (Bacillus), 124, 128, 129
rerro (Salmonella), 113
chauvoei (Clostridium) , 13 2, 138
Chlamydomonas, 162
chlororaphis (Pseudomonas) , 16, 32
cholerae (Vibrio), 52, 54, 5 5
choleraesuis (Salmonella) , 113
Chromatium, 144
Chromobacteritim, 76
laurentium, 76, 77
manilae, 76, 77
violaceutn, 76, 77
chroococcum (Azotobacter) , 61, 63, 64
chrysanthemi (Erwinia), 102, 105
cichorii (Pseudomonas), 3 3
circulans (Bacillus), 116, 129
citreum-mobile (Corynebacterium) , 82, 8 5
cifreus (Arthrobacter), 86, 87
cloacae (Aerobacter) , 100, 101
Clostridium, 131
acetobutylicum, 131, 136, 139
aerofoetidum, 132, 134, 139
aurantibutyricum, 136, 139
beijerinckii, 13 6, 159
bifermentans, 13 2, 139
bofulinum, 13 2, 138
butylicum, 134, 139
butyricum, 134, 138
capifoiale, 132, 138
rar»/s, 134, 139
centrosporogenes, 13 2, 138
chauvoei, 13 2, 138
cochlcarium, 134, 138
difficile, 13 2, 138
felsineum, 131, 13 2, 138
/f-jcn, 1 3 2
histolyticum, 134, 139
lentoputrescens, 132, 139
nigrificans, 131
«0f>7, 131, 138
parabotulinum, 134, 138, 139
pasteurianum, 136, 139
perfringens, 1 3 1
roseum, 131, 13 6, 139
septicum, 13 2, 138
sphenoid es, 134, 138
sporogenes, 13 6, 138
166
tcrtiiiw, 13 6, 139
tetaiii, 131, 138
tetanomorphum, 132, 134, 139
tbermosaccharolyticiiiii, 134, 138
coagnlans (Bacillus), 126, 129
cochlearium (Clostridium), 134, 138
coli (Escherichia) , 99
coli (Vibrio), 52, 54, 5 5
Corynebacterium, 82
citreum-mohilc, 82, 8 5
fimi, 82, 84, 8 5
ftaccutnfaciens, 82, 84, 8 5
michiganense, 82, 84
poinsettiae, 82, 84, 8 5
tritici, 82, 84
cuneatus (Pseudomonas) , 26, 32, 52
cuneatus (Vibrio), 26, 52
cuniculi (Noguchia), 118
cycloclastes (Achromobacter) , 40, 41
cypripcdii (Erwinia), 105
deititrificaiis ( Alcaligcites) , 90, 91, 93
i/erZ'jj (Salmonclhi), 113, 114
Desulfovibrio, 5 6
dextrinosolvens (Succinovihrio) , 5 8
</ij^r/7e (Clostridium), 132, 138
diminuta (Pseudomonas) , 26
dimorpha (Mycoplana) , 40, 41
duesseldorf (Salmonella), 113
DuPage River organism, 158
eiifcritidis (Sctlmoiiclla) , 113, 114
Erwinia, 102
amylovora, 102, 105
ananas, 105
aroideae, 102, 104, 105
atroseptica, 104, 105
carotovora, 102, 105
chrysanthcmi, 102, 105
cypripedii, 1 0 5
/tf/z&r^/, 102, 105
millet iae, 10 5
nimipressuralis, 104, 105
phytophthora, 104, 105
rhapontica, 105
sa/icis, 102, 105
solanisapra, 102, 104, 105
trachciphila, 102, 105
Escherichia, 98, 101
Arizona, 99
Bcthesda, 99
co//, 99
freundii, 99
«scH (Salmonella), 113
europaca (Nitrosomonas) , 20, 21
faecalis (Lophomonas) , 42, 43
faecalis (Alcaligenes) , 90, 91, 93
faecalis var. radicans (Pseudomonas) , 26,
32
faecalis var. radicans (Alcaligenes), 26
felsineum (Clostridium), 131, 132, 138
/fieri (Clostridium), 132
/f/7« (Vibrio), 52, 54, 5 5
Filaments of polar flagellates, 161
/!wi (Corynebacterium), 82, 84, 8 5
firm us (Bacillus), 129
fischeri (Photobacterium) , 120, 122, 123
/;sr/jfri (Vibrio), 120, 123
fiaccumfaciens (Corynebacterium) , 82, 84,
85
Flavobacterium, 94
marinotypicum, 94, 9 5
suaveolens, 94, 9 5
f-uorescens (Pseudomonas) , 26, 3 3
formicans (Aeromonas) , 50, 51
freundii (Escherichia), 99
fuhum (Rhodospjr/lhim), 146
////rz/j (Cellvibrio) , 5 6
gallinarum (Salmonella), 112
gelatinosa (Rhodopseudomonas) , 144, 145
geranii (Xanthomonas) , 3 9
glycinca (Pseudomonas), 28, 3 2
granulosis (Noguchia), 118, 119
gypsophilae (Agrobacterium) , 74
habana (Salmonella), 113
/xiri'O'i (Photobacterium), 120, 122, 123
harieyi (Aeromonas), 122, 123
hederae (Xanthomonas) , 3 9
histolyticum (Clostridium) , 134, 139
hyacinthi (Xanthomonas), 39
Hydrogenomonas 22
panfofropha, 22, 2 3
hydrophila (Aeromonas), 5 0, 51
167
indica (Serrafia), 106, 107
indicum (Azotobacter) , 63, 64
insolita (Azotomonas) , 66
itersonii (Spirillum) , 60
jejjitii (Vibrio), 52
juglattdis (Xanthamonas) , 39
kilicnsis (Serrafia), 106, 107
Lachnospira, 5 8
multiparus, 58, 59
lachrymans (Pseudcymonas) , 3 2
Lactobacillus, 82
plan f am III, 82
laterosporus (Bacillus), 129
lathryi (Erwinia) , 102, 105
latum (Caryophanon), IS 6, 157
laurentium (Chromobacterium) , 76, 77
letitopufrescens (Clostridium), 132, 139
/fM/«j (Bacillus), 126, 129
licheniformis (Bacillus), 129
lindneri (Pseudomonas), 46
lindneri (Zymomonas), 46
linum (SpirilUim) , 60, 62
liquefacietis (Aeromonas), 50, 51
Listeria, 86
monocytogenes, 86, 88
Lophomonas, 42, 161
faecalis, 42, 43
macerans (Bacillus), 124, 129
macrocytogenes (Azotobacter) , 61, 63, 64
malvacearum (Xanthomotias) , 39
vianihotis (Xanthomonas) , 3 6, 39
manilae (Chromobacterium) , 76, 77
marcescens (Serratia) , 106, 107
marginata (Pseudomonas), 2 8, 32
viarinotypicum (Flavohacterium) , 94, 9 5
megaterium (Bacillus), 124, 129
Methanomonas, 34
methanica, 34, 3 5
michiganense (Corynebacterium) , 82, 84
milletiae (Erwiuia), 105
mirabilis (Proteus), 108, 110, 111
Mixed flagellation, 161
monocytogenes (Listeria), 86, 88
montevideo (Salmonella), 113
moscow (Salmonella) , 113
Mycoplana, 40
bullata, 40, 41
dimorpha, 40, 41
newport (Salmonella), 113
nigrifaciens (Pseudomonas) , 26, 3 2
nigrificans (Clostridium), 131
nimipressuralis (Erwinia), 104, 105
Nitrobacter agilis, 22, 23
Nitrosotnonas europaea, 20, 21
Nocardia, 146
Noguchia, 118
cuniculi, 119
granulosis, 118, 119
siiniac, 118
woi'j'i (Borrelia), 148, 149
novyi (Clostridium), 13 1, 138
pallidum (Treponema), 150, 151
palustris (Khodopseudoinonas) , 144, 14 5
pantothenicus (Bacillus), 129
papavericola (Xanthomonas) , 36, 39
Pasteurella, 116
pseudotuberculosis, 116, 117
pasteurianum (Clostridium), 13 6, 139
pasteurii (Bacillus), 124, 126, 129
pelargonii (Xanthomonas), 39
percolans (Vibrio), 42, 43, 52
perfringens (Clostridium), 131
perlurida (Cellulomonas) , 96, 97
phosphorescens (Photobacteriinii ) , 120
phosphoreum (Photobacterium) , 120, 122,
123
phosphoreum (Vibrio), 111, 123
Photobacterium, 120
albensis, 120, 122, 123
fischeri, 120, 122, 12 3
harieyi, 120, 122, 123
phosphorescens, 120
phosphoreum, 120, 122, 123
i?/)/W, 120, 122, 123
splendid urn, 120, 122, 123
phytophthora (Erwinia), 104, 105
plantarum (Lactobacillus), 82, 8 3
plymuthica (Serratia), 106, 107
poinsettiae (Corynebacterium), 82, 84, 8 5
poly color (Pseudomonas), 2 8, 32
polymorphum (Spirillum), 60, 62
polymyxa (Bacillus), 124, 129
265
Protaminobacter, 34
alboflains, 34
ruber, 34, 3 J
Proteus, 108
mirabilis, 108, 110, 111
morganii, 108, 110, 111
rettgeri, 108, 110, 111
vulgaris, 108, 110, 111
proteus (Vibrio), 5 2, 5 5
Protozoa, 162
Providence group, 108, 110, 111
prtini (Xanthovionas) , 39
pseudomallei (Pseudonionas) , 26, 32
Pseudomonas, 25
aeruginosa, 26, 32, 33
alliicola, 33
angulata, 28, 32
arsenooxydans, 26
booker i, 26, i 2
bullata, 40, 41
cat Hey ae, 28, 32
chlororaphis, 26, 32
cichorii, 3 3
cuneatus, 26, 32
diminuta, 26, 28, 32
faecalis var. radicans, 26, 3 2
fluorescens, 26, 3 3
glycinea, 28, 3 2
Halophilic types, 30, 31, 3 3
lachrymans, 32
lindneri, 46
marginal a, 28, 32
methanica, 34, 3 5
nigrifaciens, 26, 28
Phytopathogenic types, 28, 32 3 3
poly color, 2 8, 30, 32
pseudomallei, 26, 32
r/^«, 2 8, 3 3
saccharophila, 26, 32
savastanoi, 28
savastanoi var. fraxiiii, 2 8, 32
synxantha, 26, 3 2
washingtoniae, 2 8, 3 0, 32
pseudomonas, filaments, 161
pseudotsugae (Agrobacterium) , 75
pseudotuberculosis (Pasfeurella) , 116,
pullorum (Salmonella), 112
puh'ifacicns (Bacillus), 129
pumilus (Bacillus), 124, 129
radiobacter (Agrobacterium) , 74, 75
rettgeri (Proteus), 108, 110, 111
rhapontica (Eruinia), 105
Rhizobium, 68
rhizogencs (Agrobacterinw ) , 74, 7 5
Rhodopseiidomonas, 144
gelatinosa, 14 5
palustris, 14 5
spheroid cs, 145
Rhodospirillum, 146
full urn, 146
ruhrum, 146, 147
r/te (Pseudomonas) , 28, 3 3
ricinicola (Xanthomonas) , 3 6, 3 9
roseum (Clostridium), 131, 136, 139
rossica (Cellulomonas) , 96, 97
rostock (Salmonella) , 113
rzi^e^ (Protaminobacter) , 34, 3 5
rubicundus (Pseudomonas), 5 2
rubicundus (Vibrio), 52, 54, 5 5
rubrilineans (Xanthomonas) , 36, 38, 39
rubrum (Rhodospirillum), 146, 147
ruminantium (Selenomonas) , 154, 155
saccharophila (Pseudomonas), 26
salicis (Erwinia), 102, 105
Salmonella, 112
abortiiocquina, 113
abortusbovis, 113
adelaide, 1 1 3
anatum, 113, 114
arizona, 113, 114
tfr/<?, 113
blcgdam, 113
bredeney, 1 1 3
budapesf, 1 1 3
California, 1 1 3
cerro, 113
choleraesuis, 1 1 3
i/fr^3', 113, 114
duesseldorf, 1 1 3
enteritidis, 113, 114
esjew, 1 1 3
gallinarum, 112
habana, 1 1 3
17 montevideo, 113
moscow, 1 1 3
newport, 1 1 3
pullorum, 112
169
rostock, 113
schleissheim, 113
schottmuelleri, 1 1 3
senftenberg, 1 1 3
simsbury, 1 1 3
thompson, 1 1 3
typhimurinm, 112, 113, 114, 116
typhosa, 113, 114
Virginia, 113, 114
Wichita, 112, 113, 114
Sarcina, 78
iircae, 78, 79
savastanoi (Pseinloiiionas) , 2 8, 32
schleissheim (Salmonella) , 113
schottmuelleri (Salmonella), 113
Selenomonas, 154
ruminantiiim, 154
senftenberg (Salmonella) , 113
iejiw (Photobacteriutn), 120, 122, 123
sepiae (Acromonas) , 122, 123
scpticiim (Clostridium), 13 2, 138
serpens (Spirillum) , 60
Serratia, 106
anolium, 106, 107
indica, 106, 107
kiliensis, 106, 107
marcescens, 106, 107
plymuthica, 106, 107
nrinae, 106
simiae (Noguchia), 118
simplex (Arthrobacter) , 86
simsbnry (Salmonella) , 113
solanisapra (Erivinia) , 102, 104, 105
sphaericus (Bacillus), 126, 128, 129
sphenoiJes (Clostridium), 134, 138
spheroides ( Rhodopseudomonas) , 144, 145
Spirillum, 60
itersonii, 60
linum, 60, 62
polymorphum, 60, 62
serpens, 60
sputigenum, 1 54
virginianum, 60
splendidum (Photobacferium) , 120, 122,
123
splendidum (Vibrio), 122, 123
sporogenes (Clostridium) , 13 6, 138
Sporosarcina, 78
sputigenum (Spirillum) , 154
170
stearothermophilus (Bacillus), 124, 129
Streptococcus, 80
suaieolcns (Flaiobacterium) , 94, 9 5
w/'/z/ii (Bacillus), 124, 129
Succinovibrio, 58
dextrinosolvens, 5 8, 5 9
synxantha (Pseudotnonas) , 26, 32
taraxaci (Xanthomonas) , 3 9
tardicrescens (Xanthomonas) , 3 6, 3 8
technicus (Bacillus) , 129
tertium (Clostridium), 13 6, 139
/f/awi (Clostridium), 131, 13 2, 138
tetanomorphum (Clostridium) , 132, 136,
139
thermosaccharolyticum (Clostridium) , 134,
138
Thiobacillus, 22
thiooxidans, 22, 23
thioparus, 22, 2 3
thompson (Salmonella) ,113
tracheiphila (Erwinia), 102, 105
translucens (Xanthomonas) , 3 9
Treponema, 150
oral types, 15 0, 151
pallidum, 15 0, 15 1
Trichomonas, 162
tritici (Corynebacterium) , 82, 84
tumefaciens (Agrobacterium) , 74, 7 5
typhimurium (Salmonella), 112, 113, 114,
116
typhosa (Salmonella), 113, 114
tyrogenes (Vibrio), 52, 54, 5 5
«rcrtf (Sarcina), 7i, 79
urinae (Serratia), 106, 107
vesicatoria (Xanthomonas) , 39
Vi^'rio, 26, 27, 52, 122, 123
albensis, 122, 123
cholerae, 5 2, 54, 5 5
ro//, 52, 54, 5 5
cuneatus, 26, 52
/f/«i, 52, 54, 5 5
^ir/jm, 122, 123
halophilic types, 54, 5 5
;(•;««/, 5 2
percolans, 42, 5 2
phosphorcum, 122, 123
prof ens, 5 2, 54, 5 5
rubicund Its, $2, 5 4, 5 5
splendid urn, 122, 123
tyrogeiics, 52, 54, 5 5
librioides (Caulobacter) , 140, 142
i/giiicola (Xanthomoitas) , 3 6, 3 9
lincoitii (Borrelia), 148, 149
tiiiclaudii ( Azotobactcr) , 62, 63, 64
liolaccuw (Chrowobacterinm) , 76, 77
Virginia (Salmonella), 113, 114
virginianum (Spirillum) , 60
vulgaris (Cclhibrio) , 5 6
vulgaris (Proteus), 108, 110, 111
washingtoniac (PsenJomonas) , 2i
Wichita (Salmonella), 112, 113,
Xanthomoitas, 3 6
amaranthicola, 3 6, 3 9
arsenooxydans, 3 6, 3 8
begoniae, 3 9
be ti cola, 3 9
campestris, 3 6, 3 9
, 32
114
geranii, 39
hederae, 39
hyacinthi, 39
juglandis, 39
waliacearum, 39
manihofis, 36, 39
papavcricola, 36, 39
pelargonii, 3 9
/)r«M/, 39
riciiiicola, 36, 39
rubrilincans, 36, 38, 39
taraxaci, 3 9
tardicrescens, 3 6, 3 8
translucens, 39
vignicola, 36, 39
vesicatoria, 3 9
zinniae, 3 6, 39
zinniae (Xanthomonas) , 36, 37, 39
Zytnotnonas, 46
anaerobia var. poinaceac, 46, 47
Undneri, 46
172
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