ON SECONDARY COLONY DEVELOPMENT IN BACTERIA
AND AN ANALOGY WITH TUMOUR PRODUCTION
IN HIGHER FORMS

BY

ALEXANDER HADDOW M.D.
working with the aid of a full-time grant from the British Empire Cancer Campaign.

(From the Department of Bacteriology, University of Edinburgh,
and the Research Institute of the Royal Cancer Hospital (Free), London).

I. INTRODUCTION.
II. SECONDARY COLONY FORMATION.

IIT. PossiBLE INTERPRETATIONS.

1. Contamination hypothesis.

2. Primary selection hypothesis.

2a. Biochemical changes involved, in
relation to primary selection.

3. ‘Transformation and secondary
selection.

3a. Specificity of transformation.

IV. THEORIES CONCERNING THE MECHANISM
INVOLVED IN TRANSFORMATION.
1. Hybridisation.
2. Segregation of characters in non-
equational fission.
3. Dedifferentiation, dissociation,
adaptive reduction.
4. Bacterial mutation.
5. The side-chain theory.

V. CELLULAR INHIBITION AND THE ORIGIN OF
DISCONTINUOUS VARIATION.
1. The general concept.
2. The nature of the inhibitory pro-
cess and adaptive response.

VI. 'THE ANALOGY BETWEEN SECONDARY COLONY
FORMATION IN BACTERIA AND TUMOUR PRO-
DUCTION IN HIGHER FORMS.

VII. Summary.

VIII. REFERENCES.

I. INTRODUCTION

In a recent review (Haddow 1937) and in
other papers (see Haddow 1936, Haddow and
Robinson 1937) the author propounded the
theory that the cancer cell represents a discontin-

uous and irreversible variant of the corres-
ponding somatic cell, differing from the latter in
the characters of fission-rate and metabolic
behaviour. It was suggested that the problem of
the origin of cancer might therefore be regarded
as a special case in the origin of cellular varia-
tion, and suggestions were made as to the
fundamental nature of the environmental
changes leading to tumour production. In par-
ticular, it was postulated that these changes
are frequently inhibitory in nature, and that
the increase in growth-rate which marks the
emergence of malignancy is due in many cases
not to any primary stzmulation of growth but
represents the emancipation of potentially
variable cells from chronic inhibition. Thus,
evidence was presented to show that a number
of the carcinogenic hydrocarbons exhibit growth-
inhibitory properties not possessed by a con-
siderable number of related non-carcinogenic
substances. It was further found (Haddow,
Scott and Scott 1937) that the inhibitory effect
thus produced differed from that manifested
by diverse toxic substances in its relative pro-
longation even after a single administration and
in its primary independence of toxic action in
the non-specific sense. The stages in the pro-
duction of malignancy by the carcinogenic
hydrocarbons (and possibly by other tumour-
producing agents such as x- and gamma-radiat-
ion) were therefore conceived to be (1) the
enforcement of a sustained retardation in the
rate of growth of the treated normal cells;
and (2), an irreversible dedifferentiation of
the affected but viable cells accompanied by
permanent metabolic alterations, increase in

 
 

ON SECONDARY COLONY DEVELOPMENT IN BACTERIA

growth-rate, and functional release from en-
vironmental inhibition.

It may be asked whether there is any other
instance of the operation of such a principle of
variation, and it is the object of the present
paper to draw attention to such a case. In the
author’s opinion the environmental origin of
organic change can nowhere be so clearly
deciphered, or studied with such a degree of
experimental control, as in the bacteria, and
although the investigation of variation in bac-
teria possesses inherent disadvantages as com-
pared with certain aspects of the same subject
in other living forms, these are partly absent in
the present example. The phenomenon of
secondary colony formation has long been re-
cognised by bacteriological workers, e. g., by
Giinther 1895, Hartmann 1903 in Torula cel-
liculosa n. sp., Preisz 1904, and Eisenberg 1906.
But it is almost certainly not familiar to the
generality of investigators in other fields of
biology, and it is clear that relatively little
attention has been concentrated on its wider
significance. It is for these reasons that the
present account is submitted.

II. SECONDARY COLONY FORMATION

The growth of a bacterial colony is in every
case limited by the quantity of available food-
stuff in the surrounding medium, and in certain
cases it is also retarded by the Jocal accumu-
lation of inhibitory products of bacterial meta-
bolism. Ordinarily therefore the increase of
such a colony is brought to a close by the oper-
ation of these factors in differing degrees, the
major influence being mostly a decrease in
available nutriment to such a level as to render
protoplasmic synthesis — and cell-division —
no longer possible. In normal circumstances
no further colonial change of any significance
takes place. It is however a matter of old-
standing observation that for certain bacterial
strains the colony evolution is more complex
than the above, since in these cases development
is accompanied or followed by the appearance
of so-called papillae or secondary colonies
(daughter colonies, Knépfe), which apparently
arise from the substance of the primary or mother
colony. It is, further, of the greatest interest
that the cells of these secondary colonies can
frequently be shown to metabolise some con-
stituent of the medium which the cells of the
primary colony are unable to attack. An im-

 

377

portant factor is obviously the inherent capacity
for variation of any given strain, and it seems
clear that “‘ an organism which has no capacity
for spontaneous variation to a carbon compound
is not caused to vary to it by cultivation in
contact with the compound no matter how long
the contact may be prolonged ” (Lewis 1934).
It is nevertheless probable that daughter colo-
nies may be observed at times in cultures of
all species of bacteria, a view which was sup-
ported by Enderlein.

Klotz (1906) showed that a non-lactose-
fermenting organism B. perturbans, if grown on
solid media containing lactose, produced colo-
nies which developed lactose-fermenting papil-
lae. A further and important example of this
phenomenon was provided in a strain of B. coli
studied by Neisser (1906) and Massini (1907)
and referred to by them as Bact. colt mutabile.
When this organism was grown on solid media
containing lactose and a suitable indicator such
as neutral red, it formed primary colonies which
were unable to split the available lactose and
which therefore remained pale. During devel-
opment, however, secondary colonies appeared
which frequently possessed the property of
fermenting lactose. The work of Massini
was repeated by Burk (1908), and the succeeding
years saw a number of papers confirming and
extending these observations. ‘Thus Miller
(1909, 1911; see also Benecke 1g09) described
the production of papillae by certain strains of
coliform bacilli grown in the presence of ara-
binose. and by B. typhosus when grown on
rhamnose or iso-dulcite. Similary, Burri and
Duggeli (rg09) and Burri (1910) described a
coliform bacillus (Bact. imperfectum) not
fermenting saccharose but yielding a saccharose-
fermenting race (B. perfectum) after growth in
medium containing this sugar. Other studies
at that time included those by Kowalenko (1910)
with single-cell technique, Jacobsen (1910),
Thaysen (1911), Penfold (1911, 1912), Bernhardt
and Markoff (1912), Mandelbaum (1912),
Dobell (1912-13) and Baerthlein (1913), while
among more recent contributions may be
mentioned those of Stewart (1926, 1927, 1928),
Haag (1927), Hadley (1927), Ramchandani
(1929), Kennedy, Cummings and Morrow
(1932), Lewis (1933, 1934), Kritschewski and
Ponomarewa (1934), Hall (1935), Dulaney and
Michelson (1935) and Deere, Dulaney and
Michelson (1936). ,

Lewis (1933) classified his variable strains
378

— of B. mycoides — in three groups as follows :
(1) those obtaining carbon principally from
nitrogen-containing compounds but capable of
variation in relation to sucrose; (2) those giving
secondary colonies on agar containing protein
alone, variation apparently being related to
some unused fraction of the split protein; (3)
strains giving secondary growth both in the
presence and absence of sucrose but varying
to sucrose in preference to protein. ‘The addit-
ion of non-utilisable carbon compounds in
respect of which the organism lacked the
capacity to vary (organic acids as well as
carbohydrates and alcohols) was without effect
in all groups, nitrogen metabolism proceeding
unhindered. The same author (1934) used
eleven strains of colon bacteria variable to
lactose or sucrose, two strains of B. paratyphi B
variable to raffinose, and three strains of
B. typhi variable to rhamnose, and his work
is of special importance as showing that secon-
dary colony formation may occur in synthetic
media containing lactose as the sole source
of carbon. According to Hall (1935) — in
Bact. coli mutabile — “the ‘ mutant ’ type
rarely, if ever when pure, reverts to the parent
type, and, as a rule, is indistinguishable from
it morphologically, culturally, or serologically,
except by its more rapid fermentation of lactose,
and by its inability to form characteristic mul-
tiple papillae or secondary colonies.”

Figs. 1 and 2 show the appearance of secon-
dary colony formation in strains of B. para-
typhosus B in which the secondary variant
possessed the property of fermenting lactose.
Miller (1909) discovered that B. paratyphosus B
(although not B. aertrycke) produces secondary
colonies when grown in the presence of raffinose,
and Figs. 3-14 trace the development of a single
colony of another strain of the same organism
on raffinose-neutral-red-bile-salt-agar.

III. PossiBLeE «INTERPRETATIONS

1. Contamination hypothesis.

It is clear therefore that there is no doubt of
the reality of the phenomenon as one affecting
many strains of a considerable number of bac-
terial species. It must however be recognised
that several interpretations are theoretically poss-
ible, and these may now be examined. It has
been suggested inthe first place that the appear-
ances might depend on a simple contamination of

A. HADDOW M. D.

the culture by the organism appearing in the
papillae. ‘There is however a considerable body
of evidence to show that no ordinary form of
the contamination hypothesis is sufficient to
account for the facts observed. In particular
it must be stressed that not infrequently the
cells of the secondary colonies can only be shown
to differ from those of the primary colony in
one biological character, that of utilising a
constituent of the medium which the primary
is unable to attack. This at least tends to
indicate genetic relationship between the two
forms. Again, secondary colony formation in
a suitable strain is very commonly found to
occur in every colony in each of repeated plat-
ings, and possesses a definite relation in time —
as will be shown — to the development of the
primary colony. Further, admixture of dis-
crete colonies of the two types is not ordinarily
met with ; hence purification from the alleged
contaminant is not possible. It is therefore
apparent that contamination in the usual sense
is hardly an acceptable explanation. |

yas Lbaay woe ee”

2. Primary selection hypothesis.

Even when the close relationship of the two
forms be admitted, there is however the alter-
native possibility that they co-exist in a natural
mixture or a symbiotic relationship, and that
preponderance of one over the other is brought
about by certain selective factors in the environ-
ment. But any simple selection hypothesis is
greatly weakened by the fact that the behaviour
of strains such as Bact. coli mutabile as des-
cribed above, remains unchanged even after
long continued purification (as by repeated
plating of broth cultures derived from single
colonies). It is further disproved by the results
obtained from single cell isolations (e. g., Kowa-
lenko 1910; Penfold 1912, p. 215) which Lewis
(1933) regarded as excluding selection in many
cases.

The problem is not unlike that in the work
of Stephenson and Stickland (1933), in which
experiments were conducted to test whether
the hydrogenlyases produced during the 20-hour
period of a culture were the result of selection.
It was shown that when formate is introduced
into a growing culture of Bact. coli the enzyme
appears in full strength before the lapse of a
single generation, i. e., before any demonstrable
increase in the total or viable count. In this
as in the case under discussion, it seems that
ON SECONDARY COLONY DEVELOPMENT IN BACTERIA

the phenomenon is a transformative rather then
a selective process, and that the cause is repre-
sented by environmental changes in the med-
ium. In any discussion of the possible
selective origin of secondary colony forms it
must be remembered that these variants can
almost invariably be shown to possess a meta-
bolic advantage in that they are capable of
utilising a constituent of the medium not
utilisable by the primary form. If their origin
were purely selective therefore one might hope
to detect signs of a continuous utilisation of the
substance in question (e. g., lactose) from the
beginning of the culture. As will be seen sub-
sequently in another connection, this probably
does not occur. While evidence is thus strongly
against simple or primary selection as an expla-
nation of secondary colony formation, there is

no possible doubt that once variation hasaccurr- .

ed the altered environmental circumstances are),
such as must exert powerful selective forces,
leading to a relative preponderance of the variant}
and in many cases to overgrowth of the primar
colony.

2a. Biochemical changes involved, in relation
to primary selection.

At this stage some attention must be devoted
to the evidence which is available regarding
the biochemical changes involved in secondary
colony formation. In the first place it is ob-
vious that one is not justified in assuming that
carbohydrates are not utilised simply because the
medium fails to become acid. This was pointed
out by Merrill (1930) and, in addition, Jones,
Orcutt and Little (1932), working with strains of
atypical colon bacilli, concluded from quantita-
tive determinations that lactose in their experi-
ments was utilised from the beginning of culture,
although the lactose broth remained alkaline for
periods varying from 3 or 4 days to 2 or 3 weeks.
It is a possibility therefore, as has been pointed
out by several workers, that both the parent
and variant strains of Bact. coli mutabile are
able to utilise lactose and that the difference in
their behaviour is due to a difference in type of
sugar metabolism. In 1934 Lewis remarked
‘“ much has been written on the problem of
whether the specific sugar or alcohol is attacked
from the beginning of a culture or only after a
period of contact with it, but no very definite
experimental evidence is available and opinions
differ.’ In 1927 however Stewart had investi-

379

gated the same problem by a method employing
B. colt communis as an indicator of the presence
of lactose. He found that before the emergence
of the secondary form Bact. coli mutabile con-
sumed either no lactose or only minute quanti-
ties, e.g., less than 0.0325 per cent. in four days.
On the other hand the primary form varied to
the secondary form in a discontinuous manner,
leading to an abrupt and steep increase in
sugar consumption. In 1936 moreover Deere,
Dulaney and Michelson carried out experiments,
using the official gravimetric method for
lactose determination of the Association of Agri-
cultural Chemists, to determine whether the
primary form of Bact. coli mutabile utilises
lactose from the beginning of growth or only
after the appearance of the secondary form.
The results showed that the parent form of the
organism uses only very small quantities of
lactose, if any, before the secondary variant can
be detected. On the other hand, the primary
form showed similar ammonia production and
pH changes whether grown in plain broth or
lactose broth, indicating that it uses nitrogenous
compounds as its source of energy. Further,
the primary and secondary forms when grown
in plain broth produced like changes in ammonia
concentration and pH. These authors pointed
out however that their results did not eliminate
the possibility that minute amounts of lactose
might be utilised by Bact. coli mutabile, since
the lactose determination was not sufficiently
accurate to estimate the disappearance of traces
from a concentration of one per cent. It is
of interest that for FE. coli Hershey and Bronfen-
brenner (1936) found no indication of a lactose-
fermenting mechanism other than preliminary
hydrolysis to the constituent monosaccharides.
Real changes in lactose fermentation may be
attributed to variation in the cellular activity
of lactase, an enzyme which Hershey and
Bronfenbrenner found to be intracellular and
not liberated to any appreciable extent after
death and autolysis of the bacteria.

3. Transformation and secondary selection.

Quite apart from the simple selection hypo-
thesis already considered, the view was put
forward by Smith (1913) and others that variat-
ion occurs in the absence of any specific sugar
and that the sugar when present merely acts as
a selective agent favouring the “ spontaneous ”
variant. Stewart (1927) however thought there

 
380

was no basis for any suggestion that variation
of this type is constantly occurring without the
appropriate stimulus. As has already been
mentioned, there seems little doubt that selec-
tion must operate immediately transformation
has taken place, so that the next phase of the
problem concerns the relation of the sugar to
the induction of variation. Writing in 1933
Lewis thought there could be little or no differ-
ence of opinion that enhancement of fermen-
tative power takes place only when the organism
is cultivated in contact with the specific com-
pound with which the variation is associated...
“ ability to attack unused nutrients is acquired
through variation due to the specific stimulus
exerted by the substance concerned.’’ But
this author later modified his opinion (1934), and
described experiments which he claimed showed
that variation in certain coliform strains occurred
in the absence of lactose, the variant cells being
however so few as to escape detection by conven-
tional methods of plating. He then regarded the
evidence as conclusive “‘ that variation of muta-
inle strains to a given specific carbon compound
occurs spontaneously in media from which the
compound is absent’, and looked on sugars
and alcohols as acting as specific selective agents
rather than as stimulators of variation. While
Lewis still correlated the occurrence of variation
with limitation of growth due to depletion of the
medium, he felt himself obliged to abandon
the theory of a specific inciting stimulus to
variation in mutabile strains of bacteria. In the
writer’s opinion this problem of the relation
between the carbon compound and the induc-
tion of variation is still not solved, and there
appears to be a need for further critical
experiments with purified synthetic media con-
taining no trace of the substance in question.

3a. Specificity of transformation.

It is of interest that the study of training and
adaptation in bacteria gives considerable support
to the theory that primarily non-utilisable
compounds in the substrate may influence
the induction of variation in a more or less
specific fashion. A good account of this subject
is given by Knight (1936), according to whom
‘‘ the enzymic constitution of bacteria and yeasts
can change under the stimulus of changed
composition of the media in which the cells are
grown, and can respond to different substrates
by production of the appropriate enzyme. ”’

A. HADDOW M. D.

Among examples of this type of change may be
quoted the work of Dienert (1900) who showed
that a yeast usually unable to ferment galactose
became able to do so after being grown in the
presence of this sugar. Similarly Twort (1907;
see also Penfold 1911, 1912) carried out impor-
tant experiments on the induction of the pro-
perty of lactose-fermentation (normally absent)
in B. typhosus. ‘Twort found that the long-
continued propagation of this organism in a
lactose-containing substrate might be followed
by the appearance of variants capable of fer-
menting this sugar, and in addition he described
a number of similar variations in carbohydrate
fermentation when other organisms were grown
for various periods of time in the presence of
different sugars. Prolonged growth under such
circumstances was thus shown to tend to the
production of variants which fermented the
particular substances with which the strain was
cultured. Although this process was referred
to as ‘training’, Penfold suggested that the
modus operand: of the sugar might again be
largely selective. Penfold also studied the
fermentation of dulcitol by B. typhosus. Late
fermentation of dulcitol is however recognised
as a frequent “normal” attribute of this
organism, and this author found that of several
different strains of B. typhosus grown in litmus-
dulcitol-peptone-water some formed acid in
one or two days, although the majority did not
do so till the fourth or fifth day or even later.
When such cultures were plated out daily on
agar containing dulcitol and neutral red, the
proportion of red colonies to white showed a
gradual increase from the second to about the
fifth day, reaching a maximum when the
peptone water culture had become acid.
Arkwright (1930) regarded the history of this
variation as ‘‘ a typical case of the transformation
of a culture which is unable to ferment a
certain sugar or alcohol into one capable of
rapidly fermenting it.” In such a case the
opportunities for the operation of natural selec-
tion are obvious in so far as a few individual
bacteria capable of fermenting the alcohol are
afforded an excess supply of available foodstuff
as compared with the non-fermenters, a pre-
ferential situation which is likely to result in
the overgrowth of the latter by the former.
But the critical point in the underlying biology
is once more the question of the precise mode
of emergence of the fermenting strain, and in
this connection it is of importance that in this
 

 

ON SECONDARY COLONY DEVELOPMENT IN BACTERIA 381

case also the white colonies of the original
culture on dulcitol-neutral-red-agar in the course
of development produced red papillae composed
of active dulcite-fermenters.

Karstrom (1937) investigated the relation
between the presence of specific carbohydrates
in the substrate and the development of the
corresponding carbohydrases in bacteria. As a
result he classified the cell enzymes as (1) con-
stitutive enzymes produced constantly and inde-
pendently of the composition of the media in
which the cells were grown; and (2) adaptive
enzymes produced only in the presence of,
and in response to, specific substrates in the
culture medium. According to Virtanen (1934),
‘* in general it seems that the metabolic enzymes
proper are always constitutive, whereas the
enzymes (especially hydrolases) which convert
the nutrients into suitable form for the actual
metabolic reactions may be adaptive’’. The
apparently adaptive production of hydrogen-
lyases by organisms of the colon group
was examined by Stephenson and Stickland
(1932, 1933) and Yudkin (1932). The last
author discussed the alternative hypotheses of
selection and adaptation as possible explanations
of the phenomenon, and decided in favour of
the latter. Similar evidence was provided by
Stephenson and Stickland, who showed that
while the addition of formate to Bact. coli
growing in tryptic casein-digest medium pro-
duced no simple selective action in either
direction, adaptive production of formic hydro-
genlyase occurred more quickly than would
be expected on a purely selective basis. In a
later study of the formation of galactozymase
by Saccharomyces, Stephenson and Yudkin
(1936) further weakened the selection hypothes-
is by demonstrating that specific and adaptive
enzyme production occurred in the complete
absence of cell growth or multiplication.
According to Knight (1936), “ the training of
an exacting strain to become non-exacting
involves a change in the enzymic constitution
of the organisms. An exacting strain which
requires, for example, tryptophan as an essential
nutrient, when it is trained and becomes non-
exacting and able to use ammonia as sole source
of nitrogen, must have acquired enzymes to
enable it to synthesise tryptophan using ammonia
as nitrogen source. In general, in the training
of exacting strains to become non-exacting,
enzymes required for the synthesis of proto-
plasm from the simpler nutrients have to be

produced. The suggestion is, therefore, that
the new enzymes are produced as a direct
reaction to the chemical stimulus of the new
nutrients, in the absence of the normal nu-
trients.”’ Ina study by Gladstone (1937) of the
nutrition and nitrogen requirements of Staphylo-
coccus aureus, the great majority of strains
grew well on media of known chemical com-
position which included 16 amino-acids. Initial
differences in amino-acid requirements were
found to exist among these strains, but as the
organisms were adapted to utilise fewer amino-
acids such differences tended to disappear.
Finally, by a process of training, strains were
produced which could grow on a medium from
which all amino-acids were excluded, and in
which the main source of nitrogen was ammonia.

It must be mentioned that in most cases of
the adaptive production of enzymes by bacteria
the alterations are of the nature of fluctuational
modifications, withdrawal of the specific sub-
strate leading to atrophy or disappearance of
the appropriate enzyme system. But the in-
formation they yield is sufficient at least to
suggest that similar specific relationships may
exist between organism and substrate in the
induction of irreversible and heritable changes.
In the view of Hershey and Bronfenbrenner
(1936) phenomena suggesting the specific ela-
boration of enzymes corresponding to a defined
substance present in the medium indicate no
more than the realisation of a potential and
perhaps latent physiological capacity of the
bacterial species in question. This may be
compared with the earlier suggestion by Grey
(1924) that the bacterial enzymes concerned
in the decomposition of carbohydrates are few
compared with the number of substances
which may be attacked. Grey postulated
five systems which in various combinations
might cause the degradation of such substances :
an enzyme removing hydrogen, an enzyme
removing oxygen, and enzymes splitting off
groups of 1, 2 and 3 carbon atoms respectively,
the last three being interchangeable or at any
rate of common origin.

IV. THEORIES CONCERNING THE MECHANISM
INVOLVED IN TRANSFORMATION

From the evidence detailed above it is reason-
able to conclude that the phenomenon of
secondary colony development involves bio-
logical transformation, and that the main role of
 

382

selection is confined to the period following
emergence of the variant. It now becomes
necessary to consider the various possibilities
concerning the mechanism of change. ‘In
order that two differentiated halves may be
produced, some event must take place by which
a chemical distinction between the two halves is
effected.” (Bateson 1913). Morphological
appearances alone are of little or no value in
deciding this question. The component organ-
isms of secondary colonies may present no
apparent structural divergence from the normal,
although the writer has frequently found evi-
dence of pronounced disintegrative changes
in such cells. In a study of impression films of
the secondary colonies produced by B. para-
typhosus B (Schottmiiller) on raffinose-agar,
Kritschewski and Ponomarewa (1934) described
and depicted an extraordinary series of
large-celled variants of spindle- and trypano-
some-like morphology. But such changes are
irregular in occurrence and difficult of inter-
pretation, and the mechanism of variation can
best be studied from physiological rather than
morphological data.

1. Hybridisation.

Attention must first be directed to the hypo-
thesis which posits a sexual process in bacteria
and hybridisation as the source of such variants.
While the study of variation in bacteria is
undoubtedly subject to certain disadvantages,
it has been widely assumed that since such cells
divide as a rule by asexual binary fission with
a complete absence of the fundamentally non-
essential influence of sex and conjugation,
variation by hybridisation can well be excluded.
But although one must always entertain the
possibility of an autogamous or even syngamous
fusion in bacteria as the lowest order of sexuality,
it is curious to find that the mere occurrence
of discontinuous and heritable variation has
been regarded as evidence of sexual reproduct-
ion in these forms, a view dismissed by Henrici
(1928) as a quite unwarranted assumption.
From time to time however certain authors
have attempted to describe or interpret the
phenomena of bacterial variation in the terms
employed for higher forms. Thus in a study
of diphasic variation in the Salmonella White
(1925) thought there could be no doubt that
specific and non-specific qualities were in
general alternatives comparable almost to a

A. HADDOW M. D.

pair of Mendelian allelomorphs. Stewart (1926,
1927, 1928) is however perhaps the only author
to attempt a description of secondary colony
formation in Mendelian terms. He chose the
paracolon bacilli and the colon bacilli as exam-
ples of stable and true-breeding non-lactose-
fermenters and lactose-fermenters respectively,
with Bact. colt mutabile as an unstable non-
lactose fermenting organism regularly giving off
a fermenting daughter-race; and he brought
forward evidence which he interpreted as
showing that these three groups are Mendelian
variants of one species, of which paracolon is
the homozygous dominant, mutabile the hetero-
zygote, and colon the homozygous recessive.
Since the positive character for fermentation is
recessive he regarded the dominant factor —
for Bact. coli mutabile probably correctly as
will be seen later — as an inhibitor, and claimed
that while all three groups have the power
of fermenting lactose, the dominant and hetero-
zygote also carry a factor which inhibits this
property. Representing the inhibitory factor
as I, its absence as i, and the factor for lactose-
fermentation as F, he described the constitu-
tion of the three groups as follows : paracolon
TIFF, Bact. coli mutabile FF, colon iFF.
The regularly recurring variation of mutabile
Stewart represented as Mendelian variation
brought about by the segregating of allelomorphs
in a heterozygote, iFF dividing to TIFF, iFF
and iFF. The most powerful objection to
such a hypothesis is the fact that the IIFF form
is not found among the descendants of the
papillae of mutabile in culture, a difhculty which
Stewart suggested might be explained by an
overgrowth of this form by the FF form, or
by the association of a lethal factor with the
IIFF combination. In discussing the objection
that hybridisation must depend on gametic
union, Stewart pointed out the lack of positive
evidence that bacteria do not in fact conjugate
and the possibility that, even if they do not,
a heterozygote may arise by mutation affecting
one member only of an allelomorphic couple.
As a result of his studies Stewart drew up the
following as the laws governing variation in
native heterozygous bacteria : “1. A bacterium
heterozygous in a given character (e. g., power
of fermenting lactose), if exposed to the appro-
priate stimulus, will give descendants of two
kinds : (a) heterozygous like the parent, (b)
homozygous recessive (fermenting lactose). Since
the positive character is recessive to the negative,
 

 

ON SECONDARY COLONY DEVELOPMENT IN BACTERIA 383

the formulae of the two must be written HFF/L
for the heterozygote, and iiFF/L for the reces-
sive. The homozygous dominant, IIFF/L, a
paracolon, has not been found among the
offspring of native heterozygotes. 2. The
homozygous recessive or red race breeds strictly
true, even after many subcultures on media
indifferent for the character concerned. 3. If
a race is heterozygous for two or more characters
(e. g., fermentation of lactose and dulcite),
and if it is exposed to the stimulus appropriate
for one character it will segregate in this char-
acter only. 4. Segregation will only take place
in answer to a definite stimulus and in adaptat-
ion thereto. There is no basis for the sugges-
tion that variation is constantly taking place
without the appropriate stimulus, but that the
varying forms only survive when selected by
appropriate surroundings, and that in conse-
quence the variation falsely appears adaptive. ”’

Stewart’s hypothesis has not received any
degree of support, although it represents a very
necessary and complete examination of one of
the more important possible mechanisms
involved in this type of variation. Henrici (1934)
however thought there was no justification
for regarding these variations as evidence of
sexual reproduction, and opinion is general
that the facts can be interpreted on a simpler
basis. According to Henrici ‘‘ we have ample
evidence that variations similar to those exhib-
ited by bacteria occur in other organisms
known not to exhibit sexual reproduction.
We may observe variants arising in secondary
colonies or in sectors, in cultures of moulds
and yeasts and unicellular green algae. In
moulds, little localised tufts of aerial mycelium
differing from the parent type in colour or spore
formation may be considered homologous with
secondary colonies. Now when moulds and
yeasts reproduce sexually they form typical
sexual spores, ascospores or zygospores, which
may be readily observed and recognised. Cul-
tures of moulds and yeasts may be found to
exhibit all of the types of variation encountered
in colonies of bacteria, without exhibiting any
sign of sexual spores. The author has observed
a dissociation of the yeast Torula pulcherrima
into red and white strains and into smooth and
rough forms without any trace of ascospore for-
mation. Moreover, it has been possible to
demonstrate typical variations of this type in
cultures of a fungus (one of the smut parasites)
which was started from a single spore known

to be in the haploid state. Were there could
be but one sex in the culture, yet permanent
variations occurred ” It is of further interest
that Erikson (1935), in a study of the aerobic
streptothrices, and Sherman and Wing (1937),
for the coli-aerogenes group, were unable to
obtain any evidence of a sexual mode of repro-
duction.

2. Segregation of characters in non-equational
Jission.

Topley and Ayrton (1923-24) pointed out
that if the variations of bacteria are to be inter-
preted on the basis of genetics, the facts may
be fitted to the theory more readily if we assume
that a segregation of characters has occurred
during a non-equational cell-division, rather
than assuming that they have resulted from a
re-comlnnation of characters in a sexual fusion of
cells (see Henrici 1934). ‘“ The biological
factors discussed must have a material basis,
and the suggestion that the variations observed
are due to an unequal distribution of the sub-
stances concerned, at the moment when division
of the bacillus occurs, is so obvious that it may
well be provisionally accepted as a working
hypothesis. Such acceptance does not imply
any specific view as regards the structure or
mode of reproduction of the bacilli. We know
that multiplication by binary fission occurs, and
this form of growth gives ample opportunity
for such a segregation as we have suggested.”

Variation by non-equational division is pro-
bably best exemplified by the phenomenon of
colonial sector-formation. It has long been
observed that whole sectors of a colony may
show obvious variation from the remainder
in one or more of a number of characters (e. g.,
microscopic or gross structure, opacity or con-
sistency; biochemical activity such as pigment
production or specific fermentations). A gener-
al study of the subject was given by Nirula
(1928) and a mention of sector-formation in
Ascomycetes by Dodge (1936), while Punkari
and Henrici (1935) described interesting variat-
ions in a chromogenic asporogenous yeast
(T. pulcherrima) involving both secondary colony
formation and sectoring. It is of some
interest that the angulation of the sector, and
the distance of its apex from the centre of the
colony, give a measure of the time of emergence
of the variant in relation to the growth of the
colony as a whole. It is also striking that such
384
sector-formation in colonial organisms is the
simplest possible example of modification within
the “ Woodger’s cell-cone ” of developmental
biology (see Woodger 1931; and Needham 1936,
Fig. 13 and p. 63). Fig. 15 illustrates the ap-
pearance of lactose fermenting sectors in a non-
lactose-fermenting organism, and other examples
have been observed by the writer from time to
time. Thus when certain plate-cultures of
B. paratyphosus B (Tidy) were examined under
the Leitz Ultropak at a magnification of about
50 diameters and in conditions of dull illumin-
ation, it was found that the colonies, which
under other conditions of magnification and
illumination appeared perfectly uniform, were
sharply divided into two types according to
colour. While some were dull white, others
possessed a delicate olive-green colour, and in
addition a number of colonies showed remar-
kably beautiful sector-variation in these two
colours. Such appearances were probably de-
pendent on differences in the surface structure
of the two colonial types.

It is found as a rule that the cells of the sector
breed true, while the bulk of the colony gives
rise to further colonies which may show sector-
formation in turn. That is, the relation be-
tween primary cell and variant is the same as in
the case of secondary colony formation. This
physiological similarity is of interest in the
study of secondary colonies themselves, since
the possible influence of selection in the induc-
tion of these variants can be discounted much
more readily in sector-formation, for obvious
reasons, than in the case of secondary colony
formation. It is also of importance that Nirula’s
studies on sectoring were carried out with
isolated cells obtained by a modification of
the Burri single-cell technique.

3. Dedifferentiation,
reduction.

dissociation, adaptive

It is probable from the relation between
normal and variant strains both in secondary
colony formation and in sectoring that the variant
is derived from the parent by a process involving
dedifferentiation or loss. Since the change
results in the manifestation of a character
(e. g., lactose-fermentation) previously appar-
ently absent, it follows that the material lost
must be regarded as an inhibitor of the reaction
or enzyme-system concerned. Such changes
are usually discussed under the heading of

A, HADDOW M. D.

“ bacterial dissociation ”’, a term however which
Gardner (1935) regarded as inept, and to which
he preferred ‘ adaptive reduction”. If the
change under discussion is indeed a metabolic
dedifferentiation which permits the utilisation
of an otherwise inaccessible substrate, it may
be compared with the metabolic reversions
described by Fildes (1934). ‘‘ It appears poss-
ible to assume that when an organism adjusts
itself to a simple substrate with which normally it
cannot deal, it is because at one time in the
history of the species it had been in the habit
of using that substrate, that the process is a
process of atavistic reversion ”’, (as in the syn-
thetic adjustment of the typhoid bacillus in the
presence of ammonia, with which it cannot
normally deal, but absence of tryptophan, on
which it normally is dependent).

It is important that more than one degree of
dedifferentiation may be shown. Thus Stewart
(1927) described a typhoid race “ Bucknall ”’
which achieved full dulcite-fermenting capa-
city by two discontinuous steps, each of which
took place in papilla formation on dulcite.
The race thus formed pale red papillae from
which came white and pale red colonies. The
pale red colonies in their turn formed dark red
papillae, which gave pale red and dark red
colonies on subculture. ‘he white, pale red
and dark red forms were quite distinct and
remained so after growth on dulcite-free media.
Stewart interpreted the phenomenon as due
to the successive segregation of two inhibitory
factors. Lewis (1934) also noted tertiary colony
formation on continued ageing.

4. Bacterial mutation.

The question now arises whether the emer-
gence of secondary growth can justifiably be
referred to as bacterial mutation. Although
the term has been freely employed in bacterio-
logical literature, note must be taken of certain
objections and qualifications to its use. Jollos
was among the first to point out that the term
“‘mutation”’, coined for sudden changes occurr-
ing in the forms of life with sexual reproduc-
tion (metaphyta and metazoa), is not applicable
to asexual bacteria in which testing of the muta-
tion process by crossing experiments is impos-
sible. Again, in the opinion of Haldane (1933)
a given difference between two somatic cell
types cannot be proved or disproved, by
genetical methods, to be due to chromosomal
aj

 

ON SECONDARY COLONY DEVELOPMENT IN BACTERIA 385

aberration or gene mutation, since such cells
do not reproduce sexually and it is only by sex-
reproduction that the geneticist can distinguish
nuclear from plasmatic changes. ‘ The proof
that a change is due to a gene mutation can only
be given if the altered gene segregates from its
normal allelomorph at meiosis according to
Mendel’s laws... ”’ (Haldane (1934). But ac-
cording to the same author (1932) “ there is
no reason to think that bacterial mutation is a
phenomenon essentially different from muta-
tion in higher organisms, and it is not even clear
that it is commoner. ”

Much of the difficulty is due to the use of
the term without adequate definition, and con-
fusion has arisen through insufficient appre-
ciation of, or respect for, its technical use,
with all its added implications derived from
plant and animal genetics. Further, many
geneticists appear disinclined to allow the word
in any but its artificial or technical sense. The
situation is exactly similar to that in the
description of the malignant cell as a somatic
mutation (see Haddow 1937). It may be noted
on the other hand that a definition of mutation
sufficiently wide to include micro-organisms
(and somatic cells) was given by Gates (1915).
According to Bernhardt (1915) the term
‘* mutation ’’ must be used in bacteriology in
a sense other than that of de Vries, and as
implying that the descendants show new

heritabie characters, regardless of the means by |
which their hereditary constitution has been:

influenced, or of the extent of the change.
Assuming that the conception of mutation is
valid in the present example, the simpler
mechanisms may briefly be reviewed. The
possibility of mutation by factorial loss (and
especially by the loss of an enzyme inhibitor
in non-equational fission) has already been con-
sidered as a probable interpretation in some of
the cases at least. It is naturally of especial
importance in circumstances where dediffer-
entiation or degradation of various kinds (e. g.,
serological) have been experimentally demon-
strated, and it permits less scope for reversi-
bility than other explanations. But apart from
the loss (or gain) of individual factors, variation
may be due to the temporary inhibition of
one by another, a conception referred to by
Toenniessen (1921) as ‘‘ Valenzwechsel der
Erbfaktoren ”. The changes occurring in the
cell were also likened (by Gates) to the shift
of a polygon of forces moving to a new position

of stability. Lastly, according to Henrici (1934)
the assumption of a tautomeric structure of
the living protoplasm of bacteria may explain
the changes occurring in the genetic elements
by means of mutation, and the resultant per-
manent modification forms or mutants. Thus
Gotschlich (1929) considered it possible that the
genes behave in a manner similar to tautomeric
forms which exist in equilibrium while
their substitution forms are unequivocal and
definite.

5. The side-chain theory.

Miller (1911) regarded the type of variation
under discussion, and particularly that shown
by B. typhosus in the presence of iso-dulcite,
as an adaptation to a substance which otherwise
inhibits growth. Further, he sought to inter-
pret such changes on the basis of Ehrlich’s
side-chain theory. According to this view
B. typhosus possesses receptors which anchor
iso-dulcite : growth is therefore inhibited since
the bacillus is not able to use these receptors for
assimilation. ‘There is considerable likelihood
that this conception is fundamentally correct,
and experimental evidence is available to
support it. Thus Knight (1936) discussed
the inhibition of growth in nitrifying bacteria
(Nitrosomonas, Nitrobacter) produced by organic
compounds such as glucose, urea and asparagine,
with special reference to the work of Wino-
gradsky on inhibition of growth and of Meyerhof
on inhibition of respiration. In such experi-
ments respiration and growth are inhibited in
parallel, growth-inhibition resulting from a
respiratory inhibition which is due in turn to
displacement of the normal respiratory substrate
(in this case ammonia or nitrite) or to a block
of the respiratory catalyst (as by glucose when
applied in the absence of normal substrate).
Similarly, Sobotka, Holzman and Reiner (1936)
found that non-utilisable pentoses, and parti-
cularly xylose, have a retarding effect on the
rate of fermentation of hexoses by brewer’s
yeast, an effect due mostly to competitive diffus-
ion. Miiller’s hypothesis, that certain non-
utilisable compounds in the substrate may
inhibit growth through interference with the
assimilation of utilisable compounds, is there-
fore seen to be an entirely reasonable one.
Assuming its correctness he postulated that the
mutation consists in the emergence of a race
capable of overcoming this receptor-block,
386

V. CELLULAR INHIBITION AND THE ORIGIN OF
DISCONTINUOUS VARIATION

1. The general concept.

There can be no doubt that the cellular chan-
ges under discussion are intimately bound up
with antecedent or coincident changes in the
bacterial environment. In this regard Miiller’s
application of Ehrlich’s hypothesis is of much
greater interest than any of the theories already
discussed, since it suggests both cause and
mechanism, while the others are concerned with
alternative mechanisms alone. The mere proof
of the correctness of any one of these alterna-
tives (e. g., simple segregation, sexual segre-
gation, or the various possibilities included
under bacterial mutation) might in no way
elucidate the real problem, viz., the nature of
the force which induces such change. ‘To the
writer, the side-chain hypothesis seems of par-
ticular interest as representing a special case
of what may be suggested as a principle of
wider application, namely, that cellular inhibit-
ion of various kinds and degrees is the initial
process in the induction of dedifferentiating,
irreversible and therefore heritable changes in
asexual cells. The source of inhibition appears
to be mainly if not exclusively environmental
in origin, so that knowledge of the nature of
the significant environmental alterations gives
an opportunity to correlate these with the
resultant biological change. In this connection
Table I shows the development of raffinose-
fermenting secondary colonies in B. para-
typhosus B (Tidy) — on 1 per cent. raffinose-
neutral-red-bile-salt-agar —- in relation to the
growth of the primary (five colonies). The
ageregate of these data is presented in Fig. 16,
and data based on Figs. 3-14 are shown
graphically in Figs 17 and 18. Lemon (1933)
suggested that the maximum expansion rate in
a bacterial colony be indicated by plotting the
actual increase in unit intervals of time. If for
each period of time (T) the bacterial count or
area is Ny, N,, No... at times ty, ty, ty,...,
the following formula is used :

ANy-No) (NeNy)
(ert) (Ty (rt) PT) +Ty

. etc.,

where the suffix of the denominator indicates
the mid-point of the period during which the
increase has occurred. Fig. 18 shows the advan-

A. HADDOW M. D.

tage of this method in demonstrating the rela-
tion of the emergence of secondary colonies
to inhibition of the growth of the primary
colony.

For the successful induction of discontinuous
and permanent variation in any cellular character
two main factors thus appear to be necessary : (1)
an environmental source of interference with,
or inhibition of, the given character, of such
a nature as to be long-continued but never-
theless admit of the survival of the majority of
the affected cells; (2) an organism possessing the
inherent capacity for variation in respect of the
character in question. The general concept
has been well expressed by Noel Paton (1926)...
‘If changes in environment lead to changes
in the chemical processes in the living units
they may so alter the conditions that continued
existence is impossible, or may lead to adaptation
to the chemical changes, provided that the
modification is not detrimental to existence under
the altered conditions.”” In the theoretical
case of an organism growing in a completely
unimpeded manner in a medium to which it
is ideally adapted, no variation of any kind will
be expected to occur. Such a state is most nearly
approached in the logarithmic phase of bacterial
growth, and Winslow (1935) has shown that if
one transfers a culture in this phase of growth
to a fresh medium of the same kind, the cell
characters remain strikingly constant. It has
already been suggested that depletion of food-
stuff is the most important factor in the inhibit-
ion of growth of a bacterial colony growing in a
limited medium, and it is certainly significant
that the influence of increased density of inoc-
ulation is to accelerate secondary colony form-
ation and the effect of frequent subculture to
postpone both papillation and variation. “ If
subcultures are made daily on to fresh media,
the change which initiates papilla formation can
be deferred indefinitely in the bacteria carried
on, for variation does not appear in them even
if the appropriate sugar is continuously pre-
sent.’ (Stewart 1927). Papillation may also
be prevented by the addition of utilisable
substrate : thus Lewis (1933) described a pro-
tein-sparing effect in an interesting strain of
B. mycoides in which, although secondary colo-
nies appeared on agar containing protein alone,
the variation apparently being related to some
unused fraction of the split protein, no
secondary growth occured on the addition of
sucrose.

 
 

387

ON SECONDARY COLONY DEVELOPMENT IN BACTERIA

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3-14. The development of secondary colonies in B. paratyphosus B on neutral-red-bile-salt-raffinose-agar.

Fies.
Appearance of a single colony at 12, 18, 24, 30, 42, 54, 66, 78, 90, 114, 162 and 222 hours after inoculation.
388

2. The nature of the inhibitory process and
adaptive response.

Speaking of the induction of heritable varia-
tion in general terms, it seems probable that
identical end-results may be produced through
inhibitory effects of different kinds. It may be
regarded as certain that the gradual inhibition
in growth-rate of a developing bacterial colony
is not dependent on intrinsic causes, as was
assumed by Stewart (1927; cf. Winslow 1935,
above). The effect appears to be conditioned
entirely by environmental changes, and the
nature of these may now be discussed.

The physiological results of these changes
constitute senescence. In relation to the induc-
tion of variation it is therefore important that
secondary colony formation is closely depend-
ent on the ageing of the primary colony, and
that papillae appear first on the older or more
central portions, where inhibitory changes are
likely to be more intense. Inhibition of growth
is almost certainly dependent on a metabolic
inhibition, and it is of interest that Wooldridge,
Knox and Glass (1936), in a study of the effect
of age of culture on bacterial enzymes, found
that the activity of most bacterial dehydrogen-
ases appears to increase at first, to reach a
maximum -— usually within 24 hours — and
subsequently to diminish. The same close
relation between enzyme activity and phase of
growth was further studied by Wooldridge and
Glass (1937; see also the data given by Bach
1936 for decline in activity of various dehy-
drogenases in B. proteus in the first 96 hours of
culture).

Lewis (1933) recognised the arrest of growth
as due not to intrinsic but to extrinsic factors,
the chief being depletion of nutrients. But
he also described experiments to show that
metabolic changes in the medium, quite apart
from simple depletion, influence secondary
colony formation. Lewis regarded it as improb-
able that any general law could be applied
to all cases but thought that different extrinsic
factors might cause arrest of growth in different
organisms. In some cases for example retard-
ation is probably assisted by alkaline changes
in the medium — a circumstance which must
imply an additional advantage to the acid-pro-
ducing organisms of the secondary colonies —
while in others inhibition is known to be due
in part to accumulation of peroxides. Gause
(1934) found that the growth of populations

A. HADDOW M. D.

of Saccharomyces cerevisiae ceased before ex-
haustion of the energy sources of the medium,
the effect being due to the establishment of
a definite ratio between the concentrations of
alcohol and sugar. Brown (1937) has recently
discussed the production of growth-inhibiting
substances by fungi. For a general account of
the causes of inhibition of bacterial growth,
whether by depletion or intoxication of the
medium or by physical crowding, see Gay
(1935).

In an interesting study of a luminous organism
in relation to bacterial nutrition on agar Cruick-
shank (1934) investigated the question whether
growth is limited by the accumulation of toxic
substances or is the result of exhaustion of the
nutrient substances of the medium. The acti-
vity of light production appeared to bear a
direct relation to the state of growth (see also
Harvey 1928, Shoup 1929 and Johnson 1936 on
oxidation intensity and luminescence in bac-
teria. Harvey found exact agreement in the
oxygen consumption of luminous bacteria as
determined by the Thunberg microrespirometer
and by the time which elapses before the lumin-
escence of an emulsion of the bacteria in sea
water begins to dim, when over 99 per cent.
of the dissolved oxygen has been consumed).
By using the light production of the colonies,
recorded photographically, as a measure of
metabolic activity, Cruickshank found that
diffusible food substances are rapidly consumed
from the agar in the neighbourhood and sub-
sequently from a distance. It is of interest
that the rate of diffusion of essential food sub-
stances was found to correspond approximately
to that of haemoglobin in agar. Since the
same principle appeared to apply in the case
of other organisms, it was concluded that the
important factor in determining the final amount
of growth is the volume of agar from which the
organisms may ultimately draw their food
supply. Experiments were also described to
show that whereas luminosity declined with
exhaustion of the medium, it became restored
following replenishment by agar patching.
Cruickshank found it difficult therefore to
explain his findings on the basis that arrest of
growth is due to inhibition by metabolic pro-
ducts, but concluded that from the time that
growth commences there is set up a gradient
of concentration of nutritive material, and
that with continued growth the concentration
throughout the agar surrounding the colony

 
ON SECONDARY COLONY DEVELOPMENT IN BACTERIA

progressively falls. He recognised the impor-
tant fact that although nutrition might fall
below the level adequate for cell-division, it
might still be sufficient for some measure of
metabolic activity.

In a study of factors influencing bacterial
populations Cleary, Beard and Clifton (1935)
found that growth is first restrained by the
changing ratio of total cells to available
building material. This view was supported
by Clifton, Cahen and Morrow (1936), who
concluded that ‘‘ growth-rate decreases as the
concentration per cell of materials essential
for growth decreases in cultures of bacteria
in which a relatively high population has been
established.” In a similar study Dambovi-
ceanu and Roth (1936) also attributed arrest
of development to disappearance of foodstuff,
and were unable to detect the presence of inhib-
itory substances. From experiments described
by Hershey and Bronfenbrenner (1937) it
appears that under the usual conditions of
cultivation, rates of growth and respiration of
Bact. coli are limited by the rate at which
oxygen can reach the cells, and that this limi-
tation prevents rapid exhaustion of the nutrient
materials. If however oxygen is available in
excess, growth soon ceases as a result of oxidative
removal of foodstuffs. They also stated that
their work did not lend support to the view that
accumulation of growth-inhibitory metabolic
products is responsible for cessation of growth
in bacterial cultures.

But although inhibition is very largely due
to the negative influence of depletion or depri-
vation, there is no doubt that the same result
may also be produced to some extent, as has
already been indicated, by the positive effects
of various harmful substances in the medium.
These may be classified as (1) toxic substances
foreign in nature, i. e., possessing no natural
relation to the metabolism of the cells in ques-
tion; (2) toxic products of metabolism (for the
mathematical aspects of intoxication of a
medium by catabolic substances see Kostitzin
1935, 1937); (3) potentially nutrient substances
which are however not utilisable but cause
inhibition by competitive interference with
assimilation of the normal substrate. Similar
to substances in class (3) are compounds which,
while possessing high affinity for specific en-
zymes, cannot be attacked and therefore inhibit
(e. g., the competitive inhibition of succinic
dehydrogenase by malonic acid). And it is

389
probably of related interest that in at least a
number of cases (e. g., xanthine oxidase, Dixon
and Thurlow 1924) the velocity of enzyme acti-
vity may be appreciably retarded by even
moderate increase in the concentration of
specific substrate.

Whereas acute inhibition or injury is followed
either by death or by recovery, the type
of mild yet chronic inhibition described in
the present paper is such as does not cause
death in any high proportion of the affected
cells, at least for a considerable time. The
conditions are probably similar to those in an
ageing population of Paramecium caudatum (see
Gause 1934, Fig. 5), where decline in repro-
duction-rate is accompanied by increasing en-
vironmental resistance, increase in the intensity
of competition, and decrease in the capacity
to realise potential growth. Secondly, gradual
recovery is impossible on account of the uniform
persistence of the inhibition. In the case of
a bacterial culture allowed to age without
renewal of medium or removal of inhibitory
substances, it is obvious that inhibition must
continue without remission, provided the organ-
ism itself does not vary. But the cell may
undergo adaptive variation if such a process
is inherently possible for any given strain, and
there can be little doubt of the relative physio-
logical advantages of such a change as is exem-
plified by the formation of secondary colonies.
In the face of a condition of gradually increasing
stasis we find the multicentric development of
areas showing vigorous proliferation, and these
proceed to increase both in size and in numbers
at a time when the growth of the primary
colony continues to be retarded or has alto-
gether ceased.

Since an inhibition of the kind suggested may
be produced either by the prolonged non-lethal
action of certain “‘toxic”’ substances or by
the constant presence of non-utilisable sub-
stances competing with the normal substrate,
it is probable that the corresponding adaptive
reactions are (1) a change whereby absorption
of the toxic substance is lessened, the cell thereby
becoming relatively refractory to its action;
and (2) a change enabling the cell to metabolise
the previously non-utilisable inhibitor and so
emancipate itself from the mhibition due to
this cause. Secondary colony formation is an
ideal example of the latter mechanism, and
it is of the greatest possible interest that proven
examples of the former are also available. It
390

has long been recognised for instance that the
exposure of pathogenic trypanosomes to non-
curative concentrations of various trypanocidal
drugs frequently leads to the appearance of
new strains exhibiting a more or less specific
drug-resistance (see Schlossberger and Schiiffner
1934; Yorke and Murgatroyd 1935a, b; von
Jancso and von Jancso 1935) and Ehrlich ori-
ginally advanced the hypothesis that acquired
resistance of protozoan strains is due to a loss
of binding capacity. In an important paper
Hawking (1937; see however Pedlow and Reiner
1935) investigated the absorption of arsenical
compounds by normal and_atoxyl-resistant
trypanosomes by exposing the organisms in
vitro at 37° C. to suitable concentrations of
these compounds. The parasites were then
centrifuged out and the distribution of the
arsenical determined, either by measuring the
trypanocidal activity of the supernatant fluid
for fresh trypanosomes or by chemical esti-
mations of the arsenic content of the super-
natant fluid and of the deposited trypanosomes.
Hawking found that while normal trypanosomes
absorbed all the available drug from suitable
concentrations of typical trivalent compounds
(reduced tryparsamide, halarsol and novarseno-
billon), living resistant trypanosomes absorbed
little or none from similar concentrations of
the same compounds, although absorption
occurred if stronger concentrations were used
or if the trypanosomes were dead. A similar
difference in the behaviour of normal and
resistant trypanosomes was observed when the
parasites were exposed to reduced tryparsamide
in vivo. Compounds to which the atoxyl-fast
trypanosomes showed no resistance, e. g.,
phenyl-arsenoxide, sodium arsenite or tartar
emetic, were absorbed to the same extent by
both types of organism. It therefore appears
that the comparative failure of atoxyl-fast try-
panosomes to absorb typical trivalent arsenical
compounds constitutes the explanation of the
drug-resistance of these parasites. In a dis-
cussion of these results Hawking suggested their
further interpretation on the hypothesis of
receptor modification, the change being directed
primarily against the side-chains on the benzene
ring. Although drug-resistance in the trypano-
somes is one of the best examples of adaptive
transformation in the face of a dysgenic in-
fluence, there are others of considerable interest.
Thus Neuschlosz (1919, 1920, quoted by
Gay 1935) found quinine-fast paramecia capable

A. HADDOW M. D.

of destroying 80 per cent. of the drug in solution
while non-adapted organisms in the same solu-
tion produced a disappearance of less than
5 per cent. Again, according to Pett (1936,
1937) the effect of continuous subculture of
yeast in cyanide indicates the possibility of
developing a strain which will remain constant
with or without cyanide.

The principles above defined are strikingly
exemplified by the methods used to induce
dedifferentiation — frequently irreversible or
associated with renewed power of growth — in
bacteria and protozoa generally. These include
prolonged ageing, starvation, or the growth of
an organism in its own antiserum or in suitable
concentrations of bacteriostatic dyes, and are
patently such as result in a profound interference
with metabolism and growth. The mechanism
of the bacteriostatic action of dyes was studied by
Huddleson (1937) in the case of the Brucellae.
‘*When a dye is added to culture media, a chem-
ical or physical union occurs between the
dye and certain food substances in the media
necessary for bacterial growth. When united
with certain dyes in the proper concentration,
the food substances cannot be utilised for
growth energy by Brucella. Thus, there fol-
lows no visible growth on the medium... The
mechanism of dye bacteriostasis in the case of
Brucella would appear to lie in inherent
differences in the capacities of certain of the
species to assimilate for growth energy the
necessary food substances that are combined
with certain dyes in the proper concen-
tration. ” It is of interest that H -> O changes
similar to those produced by weak con-
centrations of antiseptics have also been
described in cultures grown under conditions of
deprivation, as in dilute broth or on agar with
a diminished concentration of nutritive sub-
stances. ‘The essential principle governing bac-
terial variation has been well expressed by
Rettger and Gillespie (1935) on the basis of
their observations of the process in B. mega-
thertum. ‘ The factors which stimulate cellular
variation are apparently unfavourable to con-
tinued normal growth. When they accumulate
very rapidly in a culture, development ceases
before variation can take place. In other words,
variation is possible only when favourable
and unfavourable influences are so balanced as
to permit slow growth in the face of untoward
circumstances. ’’ It is also important that the
natural occurrence of serological variations or

 
ON SECONDARY COLONY DEVELOPMENT IN BACTERIA

relapse strains in trypanosomiasis and in relap-
sing fever (see Cunningham, Theodore and
Fraser 1934; Russell 1936) is probably to be
interpreted as a response to the inhibitory
properties of the serum which develop in the
course of the infection. In a study of infections
with Trypanosoma lewisi Taliaferro (1929; see
also Coventry 1925) demonstrated the presence
of a reaction product which inhibited the repro-
duction of the parasites without affecting their
viability (see also Dingle 1936 on the growth-
inhibitory powers of specific antisera).

VI. THE ANALOGY BETWEEN SECONDARY COLONY
FORMATION IN BACTERIA AND TUMOUR PRODUC-
TION IN HIGHER FORMS

The appearances described above, and in
particular the generation of a vigorously growing
strain of cells in a senescent colony, seemed to
the writer to present a striking resemblance to
the emergence of a malignant variant in the
cells of ageing animal tissues. Such 4 compa-
rison raises the question as to how far one can
be justified in any attempt to transfer principles
governing the behaviour of bacterial cells to a
study of the somatic cells of higher forms.
In discussing another aspect of the same problem
White (1926) wrote : “It is a very far cry from
the genetic phenomena of the higher animals and
plants to the serology of the Salmonella types.
But when the same type of explanation, which
has been found to hold good in the case of the
former, is found readily applicable to the latter,
criticism based simply on grounds of complexity
is devoid of intrinsic force.’’ There is little
doubt that certain cellular attributes are so
fundamental that the principles which govern
them must apply throughout the whole range
of living matter. This seems most true of
the laws pertaining to growth and its variations,
and if so the change to which we allude as
cancer in man and animals must be more basic
than sexual differentiation itself. Again, it is
a notable circumstance that various important
functions in different forms, as for instance the
fermentation of carbohydrate by yeast and
sugar-utilisation by mammalian tissues, may
show chemical similarity or even identity (see
Kluyver 1932). It is probable then that cer-
tain laws, and especially those relating to
growth, operate in a continuous fashion through-
out the whole field of biology. As a further
case may be chosen the similarity or identity

391
(logistic law of Verhulst) of such varied examples
as the sigmoid curve of growth of a bacterial
colony in a restricted medium (Figs. 16, 17),
the curve of human population growth (Pearl
1925), the curve of human body growth as
estimated by increase in height (see Kostitzin
1937 p. 181), that of the growth of the blue
whale as measured by increase in length (see

Report on the progress of the Discovery
Committee’s investigations p. 18 : Colonial
Office 1937), and the general statement

that “ populations of organisms of the most
diverse kinds, ranging from bacteria and yeast
to man, are found statistically to follow in their
growth the logistic curve” (Pearl 1927).

If it be allowed that secondary colonies repre-
sent tumours of the primary colony, then we
assume in the latter some element of unity or
primitive organisation. It is interesting that
Willmer (1935) postulated such colony-whole-
ness in tissue cultures of metazoan cells, draw-
ing attention to ‘...the suggestion that a
tissue culture should not be regarded simply
as a group of cells growing independently in a
medium away from their natural position,
but rather that it should be compared to a
primitive organism, with a certain regulative
capacity, that is to say, with a tendency to
behave as a whole.” “It might be natural
to suppose that the cells of a culture grow out-
wards into the medium because, by so doing,
they move to areas of higher oxygen tension,
lower CO2 tension, and a weaker concentration
of metabolites. This in a solid medium would
tend to give the culture a spherical form,
which in practice it does assume when embedded
in the medium far away from any surface, and
it would seem possible to imagine other fairly
simple forces, such as those caused by the prox-
imity of the glass surface of the coverslip or
flask, which might tend to flatten the sphere
into the disk shape characteristic of well-grown
cultures; but the necessity for an explanation
of the curious distribution of the layers of cells
away from the glass and the evidence obtained
from a study of the repair of injured cultures
indicate that other more complex forces enter
into the situation, and that the culture must
be regarded not as a collection of individual
cells but rather as a colony which shows evi-
dence of polarity, and of the existence of definite
axes.”’ A similar principle is probably repres-
ented, in its very simplest form, by the isolated
bacterial colony. Conversely, the fundamental
392

nature of the cancer process is shown by the
fact that, although the countless cells of the
mammalian organism are constrained or aligned
to play their part in the division of labour
within the cell-state, they still retain sufficient
individuality to enable them to shed their differ-
entiation in the face of adverse local changes.

As the most constant and striking feature of
cancer is rapid and continuous or uncontrolled
growth of the malignant cells, so the outstanding
characteristic of bacterial papilla formation is
the more vigorous growth of the secondary
variant. In both cases we have the apparent
paradox that, in circumstances which lead to
ageing or inhibition of the organism as a whole,
there may appear a new cell type with a per-
manently increased rate of growth and, pro-
bably, with other physiological properties which
enable it to resist, or to escape from, the in-
creasing stranglehold brought about by environ-
mental changes. It is important to emphasise
that the alteration does not consist in the acquis-
ition of a new function so much as in variation
in the property of growth which is normally
possessed by all cells excepting those of extreme
differentiation, There is no doubt that different
strains of malignant cells have genetically
characteristic rates of growth, and these are
probably the reflection of a similar condition
in the corresponding normal somatic cells.
Again the same principle probably applies for
bacteria : thus Mason (1935), as the result of a
comparison of the maximal growth rates and
generation times of various bacteria under
optimal conditions, looked on growth rate as
often characteristic of the genus.

Aside from the general aspect, the argument
may now be briefly recapitulated and the ana-
logy continued in points of detail. It is a remark-
able fact that the theories which from time to
time have been evolved to deal with these phen-
omena are strikingly similar, or even identical,
in their application. So we find that selection
hypotheses (for selection in the origin of cancer
see Fischer 1936, 1937), various aspects of the
mutation theory, and even cellular hybridisat-
ion, have all been invoked in attempts to elu-
cidate the origin of variations of this kind.
Even from the beginning therefore it must
be acknowledged that papilla-formation and
tumour-formation have much in common, since
they both represent a similar type of variation in
an asexual cell. Again, it is clear that the
cellular change in both cases is of the nature of

A. HADDOW M. D.

a dedifferentiation which is mostly irreversible.
It has been mentioned that variable bacteria may
undergo more than one dedifferentiation, giving
rise to secondary and tertiary colonies in suc-
cession, and: similar changes have been shown
to occur in the cancer cell. Thus Bittner (1931)
showed that hereditary genetic changes might
occur in the tumour cell during the process of
transplantation, and that the cell might deviate
from the genetic constitution of the individual
which gave rise to it. Cloudman (1932) also
demonstrated that a tumour in the course of
propagation might change its character so as
to become transplantable in a larger propor-
tion of a mixed population, and found that in
these circumstances one or more genes less were
required for susceptibility in the host. Such
changes are obviously in the direction of further
dedifferentiation or change by loss.

As regards causation, it is the writer’s belief
that the origin of a malignant race of cells is not
to be attributed to any process of direct stimul-
ation of growth, as is so often assumed. From
present knowledge it appears improbable that
any primary stimulatory process will produce
an increased amount of growth for any period
of time much longer than the duration of its
action. On the contrary, the increase in rate
of growth of malignant tissue is maintained
quite indefinitely, and long after it has escaped
from the pathological environment, localised
in time as well as space,in which it was engend-
ered. When the application of a carcinogenic
hydrocarbon is followed some months later
by the appearance of a rapidly growing neo-
plasm, the result may suggest some process
of direct stimulation. But such a view is not
necessarily correct, and it seems less likely
that the continued proliferation of malignant
cells is due to any formative stimulus than that
it represents the unmasking or release, in an
adaptive dedifferentiation, of the capacity for
growth which they always possessed, albeit
in an inhibited sense, in their differentiated or
integrated state. In the early history of cellular
pathology a somewhat similar problem centred
round the nature of inflammatory response,
and according to Welch (1897; see Welch 1937),
“the doctrine of Virchow was long accepted
without question, that inflammatory cell-growth
is the result of the action of external stimuli,
the so-called inflammatory irritants, upon the
cells, which are thereby directly incited to
grow and multiply. The attack upon this

 
Se terrae a eens = a

 

ON SECONDARY COLONY DEVELOPMENT IN BACTERIA 393

doctrine has been vigorously led by Weigert,
who denies absolutely the power of any external
agencies to stimulate directly cells to prolif-
eration. He considers that to concede such a
bioplastic power to external agents is equivalent
to the acceptance of a kind of spontaneous
generation of living matter. Weigert’s views
upon this subject have undoubtedly had a most
powerful influence upon pathology. It has
been such an influence as a good working
hypothesis, whether finally demonstrated to
be true or not, has often had in the develop-
ment of science. In putting to the test of actual
observation Weigert’s hypothesis, we have been
led to recognise the frequency and the impor-
tance of primary injuries to cells inflicted by
external agencies. Not only various degener-
ations and necroses of entire cells, but more
subtle and partial damage, of cytoplasm and
nucleus have been made the subject of special
study. It has been recognised that our older
methods of hardening tissues reveal often only
very imperfectly the finer structure of cells,
and new and better methods have been intro-
duced which enable us to detect more delicate
lesions of cell-substance which formerly escaped
attention... Weigert’s postulate of some primary
injury to the tissues as the immediate effect of
mechanical, chemical and other external agencies,
which were formerly regarded as the direct
stimuli of cell-growth and multiplication, has
been fulfilled in many instances where such
damage had previously been overlooked or
unsuspected. It is his belief that in cases
where we cannot now detect such primary
injury more thorough search and better methods
will enable us to do so. ”’

With regard to malignant variation the pre-
sent suggestion is that the adaptation is an
irreversible one in response to chronic inter-
fering changes in the environment of the type
defined above, and it is a striking fact that
agents such as x-rays and the gamma rays of
radium, which the majority of workers now
regard as inhibitory in their biological effects
under all conditions, should rank next to the
carcinogenic hydrocarbons in tumour-producing
activity.

Pursuing still further the analogy between the
formation of tumours and colonial papillae,
attention must be directed to certain physio-
logical similarities. First is the important cir-
cumstance that in both cases the variant cells
show permanently altered metabolic properties

which must confer distinct survival value and
which probably furnish the additional energy
demanded by the increased rate of growth.
Secondly, both types of variation give indicat-
ions of the importance of relative degrees of
cell-stability (probably genetically determined)
in the face of dysgenic changes. The signi-
ficance of this conception has already been
demonstrated for experimental carcinogenesis
(ce. g., Kreyberg 1936), and it is probably equally
valid in the induction of mutation in bacteria.
Thus the environmental changes which induce
secondary colony formation in the case of cer-
tain cultures are quite without this effect in
the great majority of bacterial strains, indicating
the essential importance of cellular instability
or an intrinsic capacity for variation of this par-
ticular type. Whereas adverse circumstances
mostly produce a continuous cellular modifi-
cation which is reversible in its earlier stages
but later ends in death, the same changes pro-
duce in mutable strains a discontinuous alterat-
ion in cell characters with the emergence of
a strain of increased relative viability.

Much discussion has centred round the ques-
tion whether malignant change occurs initially
in a single cell or in a number of cells almost
simultaneously, and of considerable general
interest is the fact that while secondary colony
formation may affect but a single focus within the
primary colony, it is more commonly multi-
centric in origin, and within a short time not
infrequently affects several hundreds of foci
in a colonial area of say 5-15 sq. mm. (Figs. 7-14,
Table I). The outcome must obviously be
the resultant of the regional distribution of
inhibition and the degree of homogeneity of
the affected bacterial population. Although
inhibitory conditions at any given time must
vary from point to point in the colony, and in
general be more intense in the centre than at
the periphery, the individual cells of the pri-
mary colony must exhibit a relatively high
degree of biological uniformity. Assuming an
essential similarity between bacterial papillae
and tumours, and disregarding whether the
exact analogy is between a single tumour and
an individual papilla or the mass which may result
from the fusion of several, it seems probable
that while animal tumours may arise from a
focus so small as to involve only a single cell,
multicentric development is at least a possi-
bility in cases where identical environmental
conditions affect a number of biologically
394

comparable
tissue.

It is of interest to find to what extent bacterio-
logists have been impressed by this general
analogy. Massini himself acknowledged the
resemblance (1907, p. 264 and Fig. 1) : in the
words of Marie’s abstract-review (1907)...
“‘@ la fagon d’une tumeur maligne, ces granules
traversent les différentes couches de chaque
colonie ou bien se développent dans le voisinage
du point ou celle-ci a offert trop de résistance. ”’
While the present paper was in course of pre-
paration the writer was interested to learn
through Sir John Ledingham that Dr W. J.
Penfold had many years ago been impressed by
the resemblance between the formation of
secondary colonies and tumours. In a sub-
sequent correspondence Dr Penfold was kind
enough to provide full details. Penfold took
the analogy seriously as early as 1912 — when
he first drew attention to the subject — and
still later in 1922, when he published a paper
(Penfold 1922) embodying his main views.
After describing papilla-formation in B. col
when grown on monochloracetate-agar and in
B. typhosus on iso-dulcite agar, he pointed out
that the causative substances can generally be
classified in two groups, potential food sub-
stances on the one hand, and growth-inhibitory
poisons on the other. In view of the discussion
of possible mechanisms of variation, it must be
noted that Penfold was fully aware of the im-
portance of the corresponding types of adaptive
response, viz., acquisition of the capacity to
metabolise a previously non-utilisable foodstuff,
and, secondly, development of resistance to
a poison to which the cells were formerly sus-
ceptible. Penfold apparently recognised the
organisms of the secondary colonies as variants
derived from the original bacteria, although
in his interpretation he probably attributed
undue primary importance to the role of selec-
tion. Further, he traced the fundamental
resemblances between a_ bacterial colony and
a composite tissue, and between the nodules
appearing as secondary colonies in the one and
as neoplasms in the other. Lastly, while
interested mainly in extrinsic factors in the
causation of tumours and bacterial papillae, he
also indicated the significance of intrinsic
(cellular) factors as influencing the variation
process in each case. Penfold concluded with
a short account of preliminary and projected
experiments on the influence of aniline, toluid-

cells in a small volume of

A. HADDOW M. D.

ine, benzidine and naphthylamine on secondary
colony formation in B. coh, B. dysenteriae
Flexner and other species.

This concludes the discussion of evidence
on which the general thesis, of biological
analogy or even homology between secondary
colonies and metazoan tumours, is based. It
need hardly be pointed out that the basic pheno-
mena of growth, senescence and variation can
be studied with greater accuracy in the bacterial
colony than in almost any other living material,
and the developing colony is particularly suited
to the study of the changes brought about
by depletion and other inhibitory influences.
Technical conditions are such as could scarcely
be realised under any circumstances in the
culture of differentiated tissues, and the main
advantages are due to the high rates of growth
and metabolism exhibited by many bacterial
species growing in standard media, to the rela-
tive ease with which cultures can be manipulated
in a pure condition, and to the facility with
which the composition of the medium can be
experimentally controlled.

TABLE I
Development of raffinose-fermenting secondary colonies in

B. paratyphosus B in relation to the growth of the
primary colony.

 

 

Hours :
after Mean diameter of Number of
inocul- primary col. in secondary colonies
. micrometer units

ation

col.r 2 3 4 5 coll 2 3 4 5
17 g 10 8 10 5 o 0 e@ 9 90
24 20 20 21.5 19 16 o 0 6 6 Oo
28 26 25 24 25 23 0 0 0 08 oO
40.5 37 35 34 41 40 o 0 9 08 0
48 43 40 40 8647 46 o 0 0 0 0°
52 42 40 40 50 50 ? ? ? 0 ?
65 - 43 42 40 58 55 ir 18 20 Ir 2
72 43 42 40 «60 63 14 27 20 12 4
78.5 43 43 40 65 65 22 30 25 14 6
gt 48 50 46 75 74 32 48 39 35 33
100 5045.45 7870 45 52 39 41 47
118 50 48 45 = 78 93 65 79 68 91 76
137 50 50 50 80 85 1oo 102 115 148 108
160 53 52 50 100 95 95 134 115 160 130

VII. SuMMARY

The development of our knowledge of second-
ary colony formation is summarised. Various
possible interpretations are surveyed, and it is
concluded that the phenomenon is usually an
irreversible dedifferentiation and represents an
ON SECONDARY COLONY DEVELOPMENT IN BACTERIA 395

adaptation to unfavourable or inhibitory con-
ditions. Inhibition may be due (1) to gradual
depletion of the medium, (2) to the action of
toxic substances in the environment, and (3)
— possibly — to the competitive effect of
non-utilisable substances in the substrate.
It is suggested that the respective substances
may impress some degree of specificity on the

 

Pic. 1,» 30.

transformation in cases (2) and (3), and that
selection must play an important part in case (3)
once the variation has appeared. After a des-
cription of the nature of the adaptive response,
attention is drawn to the analogy between
secondary colony formation and tumour pro-
duction in higher forms, and this is discussed
in points of detail as well as on general grounds.

 

Fic. 2. X 10.

Fics. 1 and 2. Secondary colony formation in strains of B. paratyphosus B on neutral-red-bile-salt-lactose-agar.

 

Fic. 15. Sector formation and appearances suggesting
variation by non-equational fission in an organism
growing on neutral-red-bile-salt-lactose-agar. Note

lactose-fermenting sector at X. xX 30.

350 700

 

BACT. PARATYPHOSUM. B (Toy)

SECONDARY COLONY FORMATION ON
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Fic. 16. Development of raffinose-fermenting secondary
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growth of the primary colony. Data from five colon-
les.
396

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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growth of the primary colony. Data based on Figs.

3-14.

VIII. REFERENCES

ARKWRIGHT, J. A., 1930 : Variation. Jn A system
of bacteriology. (Medical Research Council).
London 1930.

Bacu, D., 1936 : L’évolution des déshydrogenases
du Proteus vulgaris. C. rend. soc. de biol., 122,
1068-1070.

BAERTHLEIN, K., 1913: Untersuchungen tber Bact.
coli mutabile. Centr. f. Bakt., 1, 66, 21-35.
Bateson, W., 1913: Problems of genetics. New

Haven 1913.

BENECKE, W., 1909 : see Zetts. f. indukt. Abstammungs.
u. Vererbungslehre, 2, 215-218.

BERNHARDT, G., 1915 : Uber Variabilitat pathogener
Bakterien. Zeits. f. Hyg., 79, 179-248.

BERNHARDT, G., and Marxorr, W. N., 1g12 : Ueber
Modifikationen bei Bakterien. Beitrag zur Frage
der sogenannten “ Mutation ’’ bei Bakterien.
Centr. f. Bakt., 1, 65, 1-4.

Bittner, J. J., 1931 : A genetic study of the trans-
plantation of tumors arising in hybrid mice.
Amer. Four. Cancer, 15, 2202-2247.

Brown, W., 1937: The production of growth-in-
hibiting substances by fungi. Report of proceed-
ings, IInd. Internat. Congress for Mucrobiol.,
London, 37-38.

Burk, A., 1908 : Mutation bei einem der Koligruppe
verwandten Baktertum. Arch. f. Hyg., 65,
235-242.

Burri, R., 1910: Uber scheinbar plotzliche Neuer-

A. HADDOW M. D.

 

BACT. PARATYPHOSUM B (TIDY)

RATE_OF INCREASE IN AREA OF PRIMARY

COLONY AND SECONDARY COLONIES ON
(% RAFFINOSE - AGAR AT 35°C
(LEMON'S FORMULA)

 

 

om

 

 

A: PRIMARY

©: seconpary

 

 

2

 

  
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Z og ~ |
6
Z
Do
bl
04
9g
wh :
Fo
< i \
a
" oe
® |
I A a I
| 4&4 “~~ os " a
I i i j
0 20 40 CT) BO 100 120 ray 160 180 200
TIME IN MOURS iia
Fic. 18. Development of raffinose-fermenting secondary

colonies in 8B. paratyphosus B in relation to the
growth of the primary colony. Interpreted according
to the formula of Lemon (1933).

werbung eines bestimmten Garungsvermogens
durch Bakterien der Coligruppe. Centr. f. Bakt.,
HI, 28, 321-345.

Burrei, R., and Ducceii, M., 1909 : Beitrige zur
Systematik der Coli-aerogenes-Gruppe nebst
Beschreibung einer neuen Methode zur Unter-
suchung der Garungsgase. Centr. f. Bakt., I,
49, 145-174.

Cueary, J. P., Bearp, J. P., and Cuirron, C. E.,
1935 : Studies of certain factors influencing
size of bacterial populations. Four. Bact., 29,
205-213.

CLirTon, C. E., CaHEN, S. F., and Morrow, G.,
1936 : Metabolic activities of Escherichia coli
in a synthetic medium. Proc. soc. exp. biol.
med., 35, 40-44.

CLoupman, A. M., 1932 : A comparative study of
transplantability of eight mammary gland tum-
ors arising in inbred mice. Amer. Four.
Cancer, 16, 568-630.

Coventry, F. A., 1925 : The reaction product
which inhibits reproduction of the trypano-
somes in infections with JT. lewisi, with refer-
ence to its change in titre throughout the course
of infection. Amer. Four. Hyg., 5, 127-144.

CRUICKSHANK, J., 1934: A study of a luminous
organism in relation to nutrition on agar.
Four. Path. Bact., 39, 141-148.

CUNNINGHAM, J., THEODORE, J. H. and Fraser,
A. G. L., 1934 : Further observations on Indian
relapsing fever. Pt. I. Types of spirochaetes
 

Soe

ON SECONDARY COLONY DEVELOPMENT IN BACTERIA 397

found in experimental infections. Indian Four.
Med. Res., 22, 105-156.

DaMBovIcEANu, A. and Rotu, H., 1936 : Contri-
bution 4 l’étude du metabolisme des bactéries.
(milieux vaccinés, milieux épuisés). C. rend.
soc. de biol., 723, 61-63.

Deerg, C. J., DuLaney, A. D. and Micuetson, I. D.,
1936: The utilisation of lactose by Escherichia
coli-mutabile. Four. Bact., 31, 625-633.

Drenert, F., 1900 : Sur la fermentation du galactose
et sur l’accoutumance des levures 4 ce sucre.
Ann. Inst. Past., 14, 139-189.

Dincie, J. H., 1936 : Growth-inhibiting power of
specific antisera. Amer. Four. Hyg., 23, 1-9.

Drxon, M. and TxurLow, 5., 1924 : Studies on
xanthine oxidase. II]. The dynamics of the
oxidase system. Biochem. Four., 18, 976-988.

Dose t, C., 1912-13: Some recent work on muta-
tion in organisms. our. Genetics, 2, 324-350.

Doncg, B. O., 1936 : Reproduction and inheritance
in Ascomycetes. Science, 83, 169-175.

Duraney, A. D. and Micuetson, I. D., 1935 : A
study of B. coli-mutabile from an outbreak of
diarrhoea in the new-born. Amer. Four. Publ.
Health, 25, 1241-1251.

EIsenserc, P., 1906 : Uber sekundare Bakterien-
kolonien. Centr. f. Bakt., I, go, 188-194.
Erixson, D., 1935 : The pathogenic aerobic orga-
nisms of the actinomyces group. Medical
Research Council Spec. Rep. N° 203. London

1935.

Fives P, 1934 : Some medical and other aspects
of bacterial chemistry. Proc. Roy. Soc. Med.,
28, I-12.

Fiscuer, A., 1936 : Kraeftcellen som en ubikvitaer
Varietet af normale Vaevsceller. Hospitalstidende,
79, 1064-1070.

Fiscuer, A., 1937 : The theory of the developmental
physiology of malignant tumors. Amer. Four.
Cancer, 31, 1-20.

Garpner, A. D., 1935 : see Brit. Med. Four., 1, 1318.

Gates, R. R., 1915 : The mutation factor in evolu-
tion. London 1915.

GausE, G. F., 1934 : The struggle for existence.
Baltimore 1934.

Gay, F. P., 1935 : Agents of disease and host resis-
tance. London 1935.

GuapsTong, G. P., 1937: The nutrition of Staphy-
lococcus aureus; nitrogen requirements. Brit.
Jour. Exp. Path., 18, 322-333-

GorscHLicH, E., 1929 : Allgemeine Morphologie
und Biologie der pathogenen Mikroorganismen.
In Handbuch der pathogenen Mikroorganismen.
Jena 1929.

Grey, E. C., 1924: The latent fermenting powers of
bacteria, I-III. Proc. Roy. Soc., B, 96, 156-170.

GUntuer, C., 1895 : Einfihrung in das Studium der
Bakteriologie. Leipzig 1895.

Haac, F. E., 1927 : Der Milzbrandbacillus, seine

Kreislaufformen und Varietaten. Arch. f. Hyg.,
98, 271-321.

Happow, A., 1936 : Ann Rept. Brit. Empire Cancer
Campaign, 292-302.

Happow, A., 1937: Cellular inhibition and the origin
of cancer. In press.

Happow, A., and Ropinson, A. M., 1937 : The
influence of various polycyclic hydrocarbons on
the growth rate of transplantable tumours.
Proc. Roy. Soc., B, 122, 442-476.

Happow, A., Scott, C. M. and Scott, J. D., 1937:
The influence of certain carcinogenic and other
hydrocarbons on body growth in the at.
Proc. Roy. Soc., B, 122, 477-507.

Hapuey, P., 1927 : Microbic dissociation. our.
Infect. Dis., 40, 1-312.

Hatpang, J. B. §., 1932 : The causes of evolution.
London 1932.

Hatpang, J. B. S., 1933 : Genetics of cancer.
Nature, 132, 265-266.

Hapang, J. B. S., 1932 : see Four. Path. Bact., 38,
507-508.

Hax., I. C., 1935 : Metabolic “ mutation’ and
colonial dissociation in the genus Bacterium.
Jour. Bact., 29, 13.

Hartmann, M., 1903 : Eine rassenspaltige Torula-
Art, welche nur zeitweise Maltose zu vergaren
vermag (Torula colliculosa n. sp.) Woch. f.
Brauerei, 20, 113-114.

Harvey, E. N., 1928 : The oxygen consumption of
luminous bacteria. Your. Gen. Physiol., 11,
469-475.

Hawkine, F., 1937 : Studies on chemotherapeutic
action. I. The absorption of arsenical com-
pounds and tartar emetic by normal and resis-
tant trypanosomes and its relation to drug-
resistance. four. Pharm. Exp. Ther., 59, 123-156.

Henrici, A. T., 1928 : Morphologic variation and
the rate of growth of bacteria. Baltimore 1928.

Henrici, A. T., 1934 : The biology of bacteria.
New York 1934.

HersHey, A. D. and BRONFENBRENNER, J. B., 1936:
Dissociation and lactase activity in slow lac-
tose-fermenting bacteria of intestinal origin.
Four. Bact., 31, 453-464.

Hersuey, A. D. and BRONFENBRENNER, J. B., 1937 :
On factors limiting bacterial growth. 1. Proc.
Soc. Exp. Biol. Med., 36, 556-561.

Huppueson, I. F., 1937 : The mechanism of the
bacteriostatic action of dyes on the growth of
the Brucellae. Report of proceedings, IInd. Inter-
national Congress for Microbtology, London, 35-37.

Jacospsen, K. A., 1910 : Mitteilungen tiber einen
variablen Typhusstamm (Bact. typhi mutabile).
Centr. f. Bakt., I, 56, 208-216.

Jancso, N. von and Jancso, H. von, 1935 : The role
of the natural defence forces in the evolution
of the drug-resistance of trypanosomes. II.
The rapid production of Germanin-fast T.
398 . A. HADDOW M. D.

brucei strains in animal with paralysed defence.
Ann. Trop. Med. Parasitol., 29, 95-109.

Jounson, F. H., 1936 : Oxidation intensity and
luminescence in bacteria. Four. Cell. Comp.
Physiol., 8, 439-463.

Jones, F. S., Orcutt, M., and Lirtrz, R. B., 1932 :
Atypical (slow) lactose-fermenting B. coli.
Jour. Bact., 23, 267-279.

Karstrom, H., 1937 : The problem of adaption
in bacteriology. Report of proceedings, IInd. Inter-
national Congress for Microbiology, Londsn,
473-474-

Kennepy, J. A., Cummines, P. L., and Morrow,
N. M., 1932 Atypical lactose-fermenters
belonging to the genus Bacterium (Bergey) :
Cultural and biochemical reactions. Jour. Infect.
Dis., 50, 333-343-

K1orz, —., 1906 : see Four. Infect. Dis., 2, (supp.)

35-

Kiuyver, A. J., 1932 : Microbial metabolism and
its bearing on the cancer problem. Science, 76,
527-532.

Knicut, B. C. J. C., 1936 : Bacterial nutrition :
material for a comparative physiology of bac-
teria. Medical Research Council Spec. Rep.
Ne 210. London 1936.

Kostitzin, V. A., 1935 : Sur l’intoxication d’un
milieu par les produits cataboliques d’une
population. C. rend. Acad. sci. 201,516-518.

Kostirzin, V. A., 1937 : Biologie mathématique.
Paris 1937.

KOWALENKO, A., 1910 : Studien ber sogenannte
Mutationserscheinungen bei Bakterien unter be-
sonderer Berucksichtigung der Einzellenkul-
tur. Zeits. f. Hyg., 66, 277-290.

KREYBERG, L., 1936 : Genetic factors in the develop-
ment of spontaneous and induced tumours in
animals. Repts. of the IInd. Internat. Congress
against Cancer, Brussels 1936, 1, 115-121.

KRrITSCHEWSKI, I. L., and Ponomarewa, I. W., 1936:
On the pleomorphism of bacteria. I. On the
pleomorphism of B. paratyphi B. Four. Bact.,
28, 111-126.

Lemon G. C., 1933 : An interpretation of bacterial
growth-rate curves. Four. Hyg., 33, 495-496.

Lewis, I. M., 1933 : Secondary colonies of bacteria
with special reference to Bac. mycoides. Jour.
Bact., 25, 359-386.

Lewis, I. M., 1934 : Bacterial variation with special
reference to behaviour of some mutabile strains
of colon bacteria in synthetic media. Your.
Bact., 28, 618-638.

MANDELBAUM, M., 1912 : Ueber das Bact. metatyphi.

Centr. f. Bakt., 1, 63, 46-53.

Marie, A., 1907 : see Bull. Inst. Pasteur, 5, 791-793-

Mason, M. M., 1935 : A comparison of the maximal
growth rates of various bacteria under optimal
conditions. Four. Bact., 29, 103-110.

Massint, R., 1907 : Uber einen in biologischer

Beziehung interessanten Kolistamm (Bacterium
colt mutabile)}. Ein Beitrag zur Variation bei
Bakterien. Arch. f. Hyg., 61, 250-292.

Merritt, M. M., 1930 : Carbohydrate metabolism
of organisms of the genus Mycobacterium. Your.
Bact., 20, 235-286.

Miter, R., 1909 : Ueber mutationsartige Vorgange
bei Typhus-Paratyphus- und verwandten Bak-
terien. Centr. f. Bakt., I, Ref. 42, Bhft. 57-58.

Motter, R., 1911 : Mutation bei Typhus- und
Ruhrbakterien. Mutation als spezifisches Kul-
turmerkmal. Centr. f. Bakt., 1, 58, 97-106.

NEEDHAM, J., 1936 : Order and Life. London
1936,

Neisser, M., 1906 : Ein Fall von Mutation nach
de Vries bei Bakterien und andere Demon-
strationen. Centr. f. Bakt., I, Ref. 38, B.
98-100.

NiRuLA, R. L., 1928 : Saltations in bacteria. Ann.
Bot., 42, 431-476.

Paton, D. NoEL, 1926 : The physiology of the
continuity of life. London 1926.

Peart, R., 1925 : The biology of population growth.
New York 1925.

PeaRL, R., 1927 : The growth of populations. Quart.
Rev. Biol., 2, 532-548.

Peptow, J. T., and Rerner, L., 1935 : On the
mechanism of chemotherapeutic action. XII.
Comparison of the binding of chemotherapeutic
agents by normal and resistant trypanosomes.
jour. Pharm. Exp. Ther., 55, 179-199.

Penrotp, W. J., 1911 : Studies in bacterial variation
with special reference to thk.chemical functions
of the members of the typhoid-coli group. Four.
Hyg., 11, 30-67.

PENFOLD, W. J., 1912: On the specificity of bacterial
mutation. Four. Hyg., 12, 195-217.

PENFOLD, W. J., 1922: A contribution to a discuss-
ion on the causation of cancer. Med. Four.
Australia, 1, 460-462.

Pett, L. B., 1936 : Studies on yeast grown in cyanide.
Il. Biochem. Four., 30, 1438-1445.

Pert, L. B., 1937 : Observations on yeast grown in
cyanide. Report of proceedings, IInd. International
Congress for Microbiology, London, 475-476.

Preisz, H., 1904 : Studien uber Morphologie und
Biologie des Milzbrandbacillus. Centr. f. Bakt.,
I, 35, 280-293.

Punkari, L., and Henrici, A. T., 1935 : Further
studies on spontaneous variations of TYorula
pulcherrima. Four. Bact., 29, 259-267.

Ramcuanpant, J. C., 1929 : Saltations in bac-
teria. II. Bacillus prodigiosus. Ann. Bot., 43,
579-586.

Retrcer, L. F., and Gitvespre, H. B., 1935 : Bac-
terial variation : an inquiry into the underlying
principles governing the cell morphology of
Bac. megatherium. four. Bact., 30, 213-232.

RussELL, H., 1936 : Observations on immunity in

mee
ON SECONDARY COLONY DEVELOPMENT IN BACTERIA

relapsing fever and trypanosomiasis. Trans. Roy.
Soc. Trop. Med. Hyg., 30, 179-190.

SCHLOSSBERGER, H., and SCHUFFNER, R., 1934: Drug-
resistance experiments with arsenopyridine de-
rivatives on trypanosomes. Four. Chemother.,
II, 81-92.

SHERMAN, J. M., and Wine, H. U., 1937 : Attempts
to reveal sex in bacteria : with some light on
fermentative variability in coli-aerogenes group.
Four. Bact., 33, 315-321.

SHoup, C. §., 1929 : The respiration of luminous
bacteria and the effect of oxygen tension upon
oxygen consumption. Your. Gen. Physiol., 73,
27-45.

SmitTH, J. H., 1913 : On the organisms of the typhoid-
colon group and their differentiation. Centr.
ft. Bakt., I, 68, 151-165.

Sopotka, H., Hoizman, M., and Rerner, M., 1936:
Selective fermentation. III. Fermentation of
hexose-pentose mixtures. Biochem. Four., 3o,
933-940.

STEPHENSON, M., and STIcKLAND, L. H., 1932 :
Hydrogenlyases : Bacterial enzymes liberating
molecular hydrogen. Biochem. Four., 26, 712-
724,

STEPHENSON, M., and Sticktanp L. H., 1933 :
Hydrogenlyases. III]. Further experiments on
the formation of formic hydrogenlyase by
Bact. col. Btochem. Four., 27, 1528-1532.

STEPHENSON, M., and YuDKIN, J., 1936 : Galacto-
zymase considered as an adaptive enzyme.
Biochem. JFour., 30, 506-514.

Stewart, F. H., 1926 : Mendelian variation in the
paracolon-mutabile-colon group, and the applic-
ation of Mendel’s principle to the theory of
acquired virulence. Four. Hyg., 25, 237-255.

Stewart, F. H., 1927: Segregation and autogamy
in bacteria : a contribution to cellular biology.
London 1927.

Stewart, F. H., 1928 : The life cycle of bacteria.
Alternate asexual and autogamic phases. Four.
Hyg., 27, 379-395: .

TALIAFERRO, W. H., 1929 : The immunology of
parasitic infections. New York 1929.

Tuaysen, A. C., 1911 : Studien tiber funktionelle
Anpassungen bei Bakterien. Centr. f. Bakt., I,
60, I.

TOENNIESSEN, E., 1921 : Ueber die Variationsformen
der Bakterien und ihre Uebereinstimmung mit
den Variationsformen der Metazoen. Centr. f.
Bakt., I, 86, 353-380.

Top.ey, W. W. C., and Ayrton, J., 1923-24 : The
segregation of biological factors in B. enteritidis
(Aertrycke). Jour. Hyg., 22, 305-313.

Twort, F. W., 1907 : The fermentation of glucosides
by bacteria of the typhoid-coli group and the
acquisition of new fermenting powers by Bacillus
dysenteriae and other micro-organisms. Proc.
Roy. Soc., B. 79, 329-336.

 

399

VirTANEN, A. I., 1934 : On the enzymes of bacteria
and bacterial metabolism. Your. Bact., 28,
447-460.

Wetcu, W. H., 1937 : Adaptation in pathological
processes. Baltimore 1937.

Wuite, P. B., 1925 : Jn An investigation of the
Salmonella group, with special reference to
food poisoning. Medical Research Council Spec.
Rep. N° gr.

Wuitt, P. B., 1926 : Further studies of the Salmonella
group. Medical Research Council Spec. Rep.
N° 103.

Witimer, E. N., 1935 : Tissue culture : the growth
and differentiation of normal tissues in artificial
media. London 1935.

WinsLow, C.-E. A., 1935 : What do we mean by a
bacterial life cycle? Science, 81, 314-315.
Woonerr, J. H., 1913 : The concept of organism
and the relation between embryology and gen-

etics. Quart. Rev. Biol., 6, 178-207.

Wootprince, W. R., and Giass, V., 1937 : Variability
in the activity of bacterial enzymes. II. Factors
associated with viability and growth. Biochem.
Jour., 31, 526-531.

Woo pribcr, W. R., Knox, R., and Grass, V.,
1936 : Variability in the activity of bacterial
enzymes. I. The effect of the age of the culture.
Biochem. Four., 30, 926-931.

Yorke, W., and Murcatroyp, F., 19354 : Biological
problems in chemotherapy. Lancet, 1, 191-193.

Yorke. W., and Murcarroyn, F., 1935b : Biological
problems in chemotherapy. Trans. Roy. Soc.
Trop. Med. Hyg., 28, 435-457.

YupKIN, J., 1932 : Hydrogenlyases. II. Some factors
concerned in the production of the enzymes.
Biochem. Four., 26, 1859-1871.

SUMMARY

The development of our knowledge of secondary
colony formation is summarised. Various possible
interpretations are surveyed, and it is concluded
that the phenomenon is usually an irreversible
dedifferentiation ard represents an adaptation to
unfavourable or inhibitory conditions. Inhibition
may be due (1) to gradual depletion of the medium,
(2) to the action of toxic substances in the envi-
ronment, and (3)... possibly... to the competitive
effect of non-utilisable substances in the substrate.
It is suggested that the respective substances may
impress some degree of specificity on the trans-
formation in cases (2) and (3), and that selection
must play an important part in case (3) once the
variation has appeared. After a description of the
nature of the adaptive response, attention is drawn
to the analogy between secondary colony formation
and tumor production in higher forms and this is
discussed in points of detail as well as on general
grounds.

 
400
ZUSAMMENFASSUNG

Der Autor setzt nacheinander die Entwicklung
unserer Kenntnisse tiber die Entstehung der ,,se-
kundaren Kolonieen” auseinander. Er zieht die
verschiedenen méglichen Auffassungen in Betracht
und schliesst, dass das Phaenomen allgemein be-
trachtet eine irreversible Entdifferenzierung ist und
eine Anpassung an ungiinstige und hemmende Beding-
ungen darstellt. Die Hemmung kann bedingt sein :

1. Durch eine stufenweise ,,Entleerung” des
Milieu’s.

2. Durch die Einwirkung von toxischen Subs-
tanzen auf die Umgebung.

3. — Vielleicht — durch die Mitwirkung von
nicht brauchbaren Substanzen in dem Substrat.

Der Autor nimmt an, dass die entsprechenden
Substanzen in gewissen Grade eine Spezifitat der
Umwandlung hervorrufen kénnen (im Fall 2 u. 3)
und dass die Selektion eine wichtige Rolle spielen
miisse im Fall 3, wenn einmal die Variation ein-
getreten ist.

Nachdem er die Natur der Anpassungsreaktion
beschrieben hat, richtet der Autor das Augenmerk
auf die Analogie, welche zwischen der Bildung
einer sekundaren Kolonie und der Tumorenstehung
bei den héheren Tieren besteht; er diskutiert ebenso
die besonderen wie die allgemeinen Seiten des
Problems.

RESUMEN

Ej Autor expone sucintamente el desarrollo de
nuestros conocimientos tocante a la formacién de
colonias secundarias. Examina diversas interpreta-
ciones posibles e infiere que el fenédmeno es general-
mente una desdiferenciacion irreversible y representa
una adaptacién a condiciones desfavorables o
inhibitorias.

La inhibicién puede ser debida :

A la deplecién gradual del medio.

2. A la accién de substancias tdxicas en los
alrededores.

. .. quiza... al efecto competitorio de substancias
no utilizables en el substrato.

El Autor sostiene que las substancias respectivas
pueden provocar algunos grados de especificidad
en la transformacion en los casos (2) y (3), en cuanto
la variacién ha aparecido.

Después de haber descrito la naturaleza de la
respuesta de adaptacién, el Autor llama la atencién
sobre la analogia que hay entre !a formacién de una
colonia secundaria y la produccién de tumor en los
organismos superiores; discute tanto el aspecto
general del problema como los detalles.

RESUME

L’Auteur expose succinctement le développement
de nos connaissances concernant la formation

A. HADDOW M. D.

de colonies secondaires. [1 envisage diverses inter-
prétations possibles et conclut, que le phénomene
est généralement une dédifférenciation irréversible
et représente une adaptation 4 des conditions défa-
vorables inhibitrices.

L’inhibition peut étre due :

1. A la déplétion graduelle du milieu.

2. ATlaction de substances toxiques aux alentours.

3. ... peut-étre... a Veffet competitif de substances
non utilisables dans le substrat.

L’Auteur suggére que les substances respectives
peuvent provoquer quelques degrés de spécificité
sur la transformation dans les cas (2) et (3), et que
la sélection doit jouer un réle important dans le cas
(3), une fois que la variation est apparue.

Aprés avoir décrit la nature de la réponse d’adap-
tation, YAuteur, attire l’attention sur l’analogie
qu’il y a entre la formation d’une colonie secondaire
et la production de tumeur chez les organismes
supérieurs; il discute aussi bien les détails que l’aspect
général du probléme.

RIASSUNTO

FE’ riassunto lo sviluppo delle nostre conoscenze
sulla formazione di colonie secondarie. Sono prese
in considerazione varie possibili interpetrazioni
e si conclude che il fenomeno e, generalmente,
uno sdifferenziamento irreversibile e rappresenta
un adattamento a condizioni sfavorevoli o inibitrici.

L’inibizione pud essere dovuta :

1. Allo esaurimento graduale del mezzo.

2. Ad azione di sostanze tossiche nell’ambiente
circostante.

3. ... pud darsi... ad effetto competitivo di sostanze
non utilizzabili nel substrato.

Viene avanzata Videa che le rispettive sostanze
possano imprimere un certo grado di specificita
nella trasformazione nel caso 2 e 3 e che la selezione
deve avere una parte importante nel caso 3, pot che é
avvenuta la variazione.

Dopo aver descritto la natura della risposta di
adattamento, viene rivolta l’attenzione alla analogia
tra formazione di colonie secondarie e produzione
di tumori, e cid viene discusso sia nei dettagli che
da un punto di vista generale.

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