University of Southern California Dissertations and Theses

Studies on the cytology of bacteria and yeasts by means of ultraviolet photolysis

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Studies on the cytology of bacteria and yeasts by means of ultraviolet photolysis

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Content STUDIES ON THE CYTOLOGY OP BACTERIA AND YEASTS
BY MEANS OP ULTRAVIOLET PHOTOLYSIS
A Dissertation
presented to
the Faculty of the Graduate School
University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
by
Tod Mittwer
June 1952
Ph, O . Bcu 'S2
This dissertation, written by
............
under the guidance of h.%9..J^acuity Committee
on Studies, and approved by a ll its members, has
been presented to and accepted by the Council
on Graduate Study and Research, in partial fu l
fillm ent of requirements fo r the degree of
D O C T O R O F P H IL O S O P H Y
..
Dean
Committee on Studies
Chairman
A CKNOWLEDGMENTS
The author wishes to express appreciation of the
invaluable suggestions and advice given him by the members
of his guidance committee. Especially appreciated was the
counsel and encouragement of the chairman. Dr. James W.
Bartholomew.
It is an additional pleasure to acknowledge the
cooperation of Dr. Richard P. Baker, Department of Experi
mental Medicine, in granting facilities for the use of the
electron microscope.
TABLE OP CONTENTS
CHAPTER PAGE
I. INTRODUCTION ...................................... 1
II. REVIEW OP THE LITERATURE............. 4
Biological applications of photochemistry . . 4
The gram stain................................. 9
The cytology of yeasts........................ 10
The work of Wager and Peniston............ 12
The work of Guilliermond................. 13
The work of Lindegren.................... 17
The work of DeLamater.................... 18
Recent contributions of others ............ • 19
III. MATERIALS AND METHODS........................... 31
Source of organisms........................... 31
Fixing procedures ...................... 31
Staining procedures ..... 32
Ultraviolet irradiation ...................... 32
Optical microscopy ............................. 34
Electron microscopy ................ ..... 34
IV. RESULTS........................................... 35
Effect of ultraviolet irradiation on the
gram and acid fast stains ............... 35
Ultraviolet irradiation used to study
bacterial cytology............. 42
V
CHAPTER PAGE
Ultraviolet irradiation used to study
yeast cytology............................ 46
Electron microscopy of yeast after dry
irradiation............................. 51
The yeast nucleus during budding ......... 54
Structure of the yeast nucleus ........... 67
Cytology of yeast ascospores.............. 81
Cytology of bud scars...................... 91
V. DISCUSSION ................................... 97
Effect of ultraviolet light on staining • . 97
Bacterial cytology .......................... 99
Yeast cytology ............................. 105
VI. SUMMARY AND CONCLUSIONS...................... 115
BIBLIOGRAPHY .......................................... 121
LIST OP TABLES
TABLE PAGE
I. Interpretations of yeast organelles by various
investigators . . . . . . . . . . . 26
II. Survey of species losing gram positivity after
exposure of heat fixed smears to ultraviolet
light for 30 hours or l e s s ................. 36
III. Effect of ultraviolet light on the gram stain
ing characteristics of the endospores of
Bacillus subtilis ............................. 41
LIST OP FIGURES
FIGURES PAGE
1. Diagram of the resting yeast cell.................. 24
2. Influence of time, species, and method of fixation
on loss of gram positivity after exposure of fix
ed smears to ultraviolet light................... 38
3. Correlation of loss of gram positivity with loss
of acid fastness after exposure of Mycobacterium
sp. to ultraviolet light.......................... 40
4. Effect of ultraviolet irradiation on morphology of
Bacillus subtilis. Electron micrographs. . . . 43
5. Saccharomyces cerevisiae, electron micrographs.
Various conditions of irradiation# ............. 47
6 . Saccharomyces cerevisiae, electron micrographs.
Dry irradiation.................................... 52
7. Sa c char omyce s cerevisiae, electron micrographs.
Resting cells...................................... 55
8 . Saccharomyces cerevisiae, electron micrographs.
Early stages of budding........................... 57
9. Saccharomyces cerevisiae, electron micrographs.
Early stages of budding........................... 59
10. Sac charomyce s cerevisiae, electron micrographs.
Intermediate stages of budding................... 61
11. Saccharomyces cerevisiae, electron micrographs.
Intermediate stages of budding................... 33
FIGURE
12. Sac char omyce s cerevisiae. electron micrographs
Intermediate stages of budding.............
13. Sac char omyce s cerevisiae, electron micrographs
Late stages of budding......................
14. Saccharomyces cerevisiae, electron micrographs
Late stages of budding......................
15. Sa c char omyces cerevisiae « electron micrographs
Final stages of budding.....................
16. Sa c char omyce s cerevisiae, electron micrographs
Structure of nucleus........................
17. Sac char omyce s cerevisiae, electron micrographs
Structure of nucleus........................
18. Sa c char omyce s cerevisiae, electron micrographs
Structure of nucleus. ......................
19. Sa c char omyce s cerevisiae, electron micrographs
Preliminary stages of ascospore formation.
20. Saccharomyces cerevisiae, electron micrographs
Single-spored asci..........................
21. Sa c char omyce s cerevisiae, electron micrographs
Two-spored asci........................
22. Sa c char omyce s cerevisiae. electron micrographs
Three-8pored asci...........................
viii
PAGE
65
68
70
72
74
76
78
82
84
86
88
ix
FIGURE PAGE
23. Saccharomyces cerevisiae, electron micrographs.
Bud scars............. 92
24# Saccharomyces cerevisiae, electron micrographs.
Bud scars........................................ 94
CHAPTER I
INTRODUCTION
It has long been known that ultraviolet light initi
ates chemical reactions of many kinds. Among organic and
biological compounds these reactions are chiefly of a dé
gradât ive type known as photolysis. Physico-chemical
studies have shown that all types of organic compounds are
degraded to smaller molecules under the influence of ultra
violet irradiation, and that different types of organic
compounds are photolysed at greatly different rates.
Since the classic studies of Nadson and his collab
orators (1926, 1928, 1931, 1933) on yeast and other cells,
it has also been known that ultraviolet irradiation acts
on the various organelles within a cell at different rates
of speed--one structure may be destroyed before another is
visibly affected.
These two fundamental facts indicate the possibility
of using ultraviolet photolysis as a cytological tool. The
present experimental study has explored this possibility
and has shown that ultraviolet is a powerful aid in inves
tigations of nuclear and cell wall structures, since these
remain after irradiation has cleared the cell of other
substances. This technique has for the first time made the
great magnifications of the electron microscope available
2
for study of the interior of yeast cells. This is because
untreated cells have heretofore been observed to be com
pletely opaque, or nearly so, to electrons at the usual
voltages used in the electron microscope. The most strik
ing result of these investigations was the demonstration of
the behavior of the nucleus of the yeast cell during the
budding process.
The many inclusions in yeast cells have made the study
of these cells most difficult, and interpretations of in
ternal structures have accordingly varied considerably.
Today two diametrically opposed theories of the yeast
nucleus are current. One, sustained chiefly by Lindegren
(1949), holds that the large cell vacuole is the nucleus
and contains the chromosomes. This concept is contrary to
a more classic viewpoint, that of Guilliermond (1940b),
which maintains that the small object appearing at the end
of the vacuole is the nucleus. That both concepts are
widely held today, is indicated by a survey of recent
(since 1946) textbooks of microbiology. Of those available
at the University of Southern California, two explained the
nucleus according to Guilliermond, three according to
Lindegren, three discussed both major theories, and eight
avoided any discussion of the yeast nucleus. Other in
vestigators have propounded concepts which differ from these
3
two in various details.
In view of the prevailing uncertainty, it was decided
that an independent investigation of this question, using
photolytic techniques, might help to clarify yeast cytology
and would be decidedly worth while.
CHAPTER II
REVIEW OP THE LITERATURE
The technique used in these investigations is, as far
as the author is aware, entirely new. Consequently there
can be no literature review of the method as such. The
principles upon which this technique is based, however,
have been amply studied, as have the specific questions to
which this technique has been applied. These matters will
therefore be reviewed as concisely as possible in the
following paragraphs.
Biological applications of photochemistry. Photo
chemistry is a large branch of science and a vast amount of
literature has accumulated in this field. Only such por
tions as apply to the problem at hand will be reviewed here.
Kistiakowsky (1928) and Rollefson and Burton (1939) have
reviewed the general principles of the subject in monograph
form, and Ellis and Wells (1941) have compiled an exhaustive
treatise on the chemical action of ultraviolet light.
Recent advances can be found in the symposium conducted by
Lind (1948). Briefly, the principles of photochemistry can
be summarized in two laws :
1. Only light which is absorbed can act chemically.
Prom this it follows that the amount of material transform
ed depends upon the nature of the material (i.e. its absorp
tion characteristics), and the wave-length of the radia-
5
tion; and is proportional to the intensity of irradiation
multiplied by the time of irradiation,
2, The absorption of light is a quantum process in
volving one quantum per absorbing molecule or atom. The
photochemical yield is determined by the thermal reactions
of the system produced by the light absorption. This sec
ond law, which we owe to Einstein, provides for exact math
ematical description of photochemical reactions. The de
tails need not be set forth here, since the experiments to
be described deal with whole cells, i.e. a mixture of
highly organized compounds, a system too complex for an
analysis of the individual component chemical reactions.
It should be noted, however, that a photochemical reaction
proceeds in two steps: (a) an activation, or primary step,
which is due to the absorption of a quantum of energy by
the molecule, an alteration in the configuration of the
valence electrons, and which is independent of temperature,
and (b) a secondary (chemical) reaction which is accelerate
ed by an increase in temperature, like any ordinary chem
ical reaction.
No organic compound so far studied has failed to be
come transformed under the influence of ultraviolet light.
The following list contains a very small fraction of the re
actions discussed by Ellis and Wells (1941), and serves to
6
illustrate the types of reactions which can be expected in
biological material. It will be observed that practically
all the reactions are oxidative and result in cleavage to
smaller, usually volatile, molecules.
Substrate Products
alcohols, aldehydes 00, Eg, hydrocarbons
organic acids COg, CO, Hg, hydrocarbons
dibasic acids decarboxylation
polysaccharides monosaccharides, lactic acid
glucose in solution COg# 00, Hg
proteins amino acids, decomposition
amines NH3
NH3 nitrite
fats hydrolysis, oxidative decomposition
cyclic compounds more stable, but decompose
(substituents readily attacked)
Fortunately for the use of photolysis in cytology,
different substances are attacked at greatly different
rates of speed. This difference is indicated by the
following examples, selected from Kistiakowsky (1928).
Molecules reacting
Reaction per quantum absorbed
Formation of Ng and Hg from NH3 0.25
Formation of HCL from Hg and Clg 1,000,000
Bleaching of dyes 0.02 to 0.05
Hydrolysis of acetone 200
Ultraviolet and other radiation has been applied to
microorganisms and the effects observed for many years.
Although these studies were almost exclusively performed
7
on living organisms, and usually with a view to studying
the genetic changes or bactericidal effects induced, a few
investigators have observed and described cytological
changes caused by ultraviolet light. Reviews concerned
with genetic and cytological effects of radiations have
been prepared by Catcheside (1948), Duggar (1936), and
Rossignol (1946).
Nadson and his collaborators, over a period of many
years* observations, were the first to observe and report
a very important phenomenon: the extraordinary resistance
of the nucleus to destruction by ultraviolet rays. Their
studies were made on yeast, bacteria, higher fungi, and
plants (Nadson and Philippov, 1928; Nadson and Rochiin,
1933; Nadson and Rochlin-Gleichgewicht, 1926, 1928; Nadson
and Stern, 1931). In the case of cells of higher plants
and of Saccharomyces cerevisiae. these investigators found
that internal structures were sensitive to radiation in the
following order: first the chondriome, then chloroplasts,
then the cytoplasm, and lastly the nucleus. During the
process the permeability of the cell increased. These
findings become of importance in the demonstrations of
yeast nuclei in the electron micrographs to follow.
Nadson*3 work has been confirmed and considerably ex
tended by more recent investigations. Lacassagne (1930),
8
Lacassagne and Holweck (1930), and Latarjet (1944) obtained
essentially identical results to those of Nadson, and
Lacassagne regarded radiation as "a sort of microdissec-
tion.”
The investigations of Davidson (1940) shed more light
on the effects of radiation of yeast cells from a chemical
point of view. This worker found that ultraviolet irradia
tion frees a large swnount of nitrogenous material. A
small amount of this was in the form of protein, while a
high proportion was present as amino nitrogen. Consider
able amounts of nucleotides, nucleosides, and purines were
also found. Irradiated yeast also yielded growth promoting
substances, which were identified as "nucleic acid like
substances," by Loofbourow, Eng1ert, and Dwyer (1941) and
Loofbourow and Morgan (1940).
Another important advance has recently been made by
Errera (1951). He found that the Peulgen positive reaction
of cell nuclei is not modified by irradiation with ultra
violet light at 2537 A, although the nuclear affinity for
methyl green is appreciably diminished. In subsequent
efforts to elucidate the mechanism of this phenomenon.
Errera (1952) conducted experiments using purified sub
stances. He found that photolysis of calf thymus desoxy-
nucleic acid occurred with a very low quantum yield, on the
9
order of 10"®. Photolysis of nucleoproteins of the same
origin took place at a comparable speed. On the other hand
the quantum yield for peptide bonds is of the order of
10"^. This would mean that protein is hydrolyzed about
1000 times as fast as DNA and this affords a reasonable ex
planation for the resistance of cell nuclei to destruction
by ultraviolet rays. This does not mean, of course, that
nuclei are not altered by irradiation, for it is well known
that ultraviolet readily induces mutations.
The gram stain. The mechanism of the gram stain has
been reviewed in detail by Bartholomew and Mittwer (1952).
This differential stain is believed by some workers to re
sult from a dye-iodine-ce11 substance complex which is
formed in gram positive cells containing a special compound.
Others believe the differentiation is due primarily to dif
ferences of permeability of the cell membranes to one or
more of the reagents used. Although the mechanism involved
has not been definitively explained, it appears probable
that permeability plays a major role, since a cell must be
intact to stain gram positively. That chemical factors
also are involved is demonstrated by the fact that gram
positive cells can be rendered gram negative by the re
moval of certain compounds. It is possible that these
chemical factors act by causing a change in the molecular
10
architecture of the cell membrane*
The experimental data to be reported in the following
chapters cannot be used to support either major theory of
the gram stain mechanism. However this work does give
additional precise information on this important phenome
non.
The cytology of yeasts. The importance of yeasts in
human culture and economy has inspired a prodigious amount
of investigation into their activities. These researches
have produced a more than abundant literature which has
been frequently reviewed. Because of the controversial
nature of the yeast nucleus, review treatment of cytologi
cal aspects occasionally become polemic, and the interested
reader should read several if he wants to obtain an im
partial viewpoint. Valuable recapitulations can be found
in Cutter (1951), Guilliermond (1920, 1940b), Henrici
(1941, 1947), Lindegren (1945d, 1949), and Martens (1946).
The last named has been called "monumental . . . thorough
and unbiased” by Cutter, and the present author concurs.
However Cutter’s review, covering the period since 1946,
contains sharp criticism of Lindegren’s work. The reader
obviously would be wise to consult original publications on
any specific point which interests him. Unless otherwise
noted, the following review material is concerned only with
11
Saccharomyees cerevisiae.
The first thorough and systematic investigations of
the yeast nuclear apparatus were those of Wager (1898),
later reinvestigated and extended by Wager and Peniston
(1910). These studies confirmed in the authors* opinion
the earlier views of Janssens (1893) who was the first to
claim that the yeast vacuole was actually the nucleus* The
subsequent work of Janssens and LeBlanc (1898) and of
Janssens (1902) crystallized this "nuclear vacuole" concept
in their own minds* Similar observations of Beijerinck
(1894, 1898) undoubtedly gave additional prestige to this
viewpo int•
However shortly after Wager’s first report appeared,
Guilliermond (1903) published his doctorate thesis claiming
that the small deeply staining body at the edge of the
vacuole was in fact the yeast nucleus* This was the begin
ning of some forty years’ labor on yeast cytology by
Guilliermond (1904, 1910ab, 1913ab, 1915, 1917, 1920,
1938ab, 1940ab). This work was so thorough and convincing
that Guilliermond’s ideas became universally accepted until
Lindegren’3 interpretations of yeast cytology led him to
revive Wager’s theory with some modifications* The
question thus remains open, and the main points will be re
viewed here in greater detail, as groundwork for the ex-
12
périmental results and discussion to follow*
The work of Wager and Peniston* These authors (1898,
1910) observed living cells, stained preparations, and
paraffin sections stained with carmine and nigrosin* They
described the "nuclear vacuole” as containing a chromatin
network of fine threads attached to the deeply staining
excentric body adjacent to the vacuole. This deeply stain
ing body they considered to be the nucleolus and it was
pictured as bearing a peripheral layer of chromatin and a
thicker chromatin patch on one side. Within the vacuole
was what they termed the "central volutin granule," a
structure now frequently referred to as the "dancing body."
Other structures not concerned with reproduction are also
described*
It is a remarkable fact that yeast cytologists, no
matter how divergent their interpretations might be, all
diagram the cell in an almost identical manner and show the
same structures* Only the labeling of parts is different*
Thus the division of the deeply staining excentric body
(the amitotic nuclear division of the Guilliermond school)
has been seen and recorded by the earliest investigators*
Wager and Peniston considered that the budding process did
not show stages of karyokinesis, but had to be regarded as
a direct division of their "nucleolus" into two equal or
13
nearly equal parts, accompanied by division of the chroma
tin in the vacuolar network# This "nucleolus" divided
either in the neck joining the bud to the mother cell, or
occasionally in the mother cell itself. Such division was
considered amitotic.
The general concepts of Wager and Peniston were sup
ported, with some differences, by the work of Janssens
(1893, 1902) and of Janssens and LeBlanc (1898). The
latter took the vacuole to be in reality the nucleus. How
ever these authors considered the appropriate place for a
nucleolus to be within the nucleus, and so in their de
scriptions the "central volutin granule" of Wager is called
the nucleolus. Janssens claimed in addition to have seen
true mitosis with the formation of spindles and asters.
This latter claim has been doubted by later workers on the
grounds that it was not reproducible in budding cells.
The work of Guilliermond. This indefatigable investi
gator has summarized his findings in monograph form (1920)
and more recently in a review (1940b). He used a wide
variety of the common cytological fixatives, usually stain
ing the nucleus with hematoxylin. He was, however, con
stantly aware of the value of observations of living cells,
and many of his conclusions are based on data obtained by
vital staining with a dilute solution of neutral red. He
14
early came to the conclusion that Wager’s views were inex-
act--the "nuclear vacuole" was simply a vacuole containing
metachromatic granules (reserve material) (Guilliermond,
1903, 1910ab, 1917). The staining of the vacuolar granules
with neutral red was considered strong evidence for their
non-nuclear nature (Guilliermond, 1938ab, 1940a).
The nucleus described by Guilliermond (1920) was
"relatively large in comparison to the cell," about 1
micron in diameter, and occupied a variable position de
pending upon the form and stage of development of the cell.
In any case it was often closely associated with the vacu
ole. The nucleus presented a well differentiated struc
ture : it was surrounded by a membrane and contained a
nucleolus and a chromatic framework which in Saccharomyces
cerevisiae was abundant and distinct.
Guilliermond observed that the bud was composed of a
very dense cytoplasm containing a few basophilic granules
which had emigrated from the mother cell. When a certain
size had been reached, a little vacuole containing meta-
chromatic corpuscles appeared in the bud cytoplasm. This
bud vacuole was often seen to result from the entrance of
a little of the vacuole from the mother cell. The nucleus
underwent no modification until the bud acquired its de
finitive dimension. At this time the nucleus elongated and
15
took the shape of a dumb-bell, one of the heads of which
entered the bud. The two heads then separated, one remain
ing in the mother cell, the other in the bud. Guilliermond
saw no evidence of karyokinesis, and considered the nuclear
activity to consist simply of a direct division (amitosis).
However he conceded (1917) that in one case, the nuclear
division in the aseus of Schizosaccharomyces octosporus, a
mitotic division probably occurred, although the chromo
somes were too small to be clearly defined or to be count
ed.
The general concepts of Guilliermond have been con
firmed by a number of workers and were quite generally ac
cepted until Lindegren reopened the question in 1945. Many
workers, however, disagreed with Guilliermond in various
details, particularly with regard to mitosis. Among these
were Dangeard (1893) who described the division of the
nucleolus during a direct division of the nucleus.
Swellengrebel (1905) presented drawings showing a true
mitotic process with 4 chromosomes evident. Puhrmann
(1906) also was able to obtain mitotic figures in his pre
parations and considered the chromosome number to be 4,
Katar (1927) confirmed that the bud attained consider
able size before any change in the nucleus was visible.
Eventually a spindle (without astral radiations) was formed.
16
Kater was unable to count the chromosomes on account of
their small size, but Judged their number to be at least 8 .
Gaumann (1928) and also Henneberg (1916) obtained re
sults very similar to those of Guilliermond, with no chro
mosomes evident. Badian (1937), while claiming that his
nucleus was identical with that of Guilliermond, stained
what he considered to be 2 rod-shaped chromosomes in vari
ous stages of division. These 2 chromosomes when in the
resting state formed a chromâtinic mass at one side of the
vesicular nucleus. His nucleus contained no nucleolus in
the resting stage. During the division of Saccharomyces
cerevisiae the components of each pair of chromosomes came
together by their proximal ends to form two heart-shaped
bodies connected by a thread. Badian considered this
appearance had led previous workers to believe the division
was amitotic.
Renaud (1938ab), a student of Guilliermond, stained
Saccharomyces ellipsoideus with hematoxylin and discovered
a small centrosome associated with the nucleus. His draw
ings show typical mitotic figures and he interpreted the
division as a true mitosis. The mitotic figures were small
and the chromosomes were often united in confused aggre
gates. Renaud believed it was impossible to think of
counting the chromosomes, but he estimated that there were
17
at least 8 .
The work of Lindegren. In a series of papers Lindegren
and his co-workers (Lindegren, 1945cd, 1949, 1951ab;
Lindegren and Lindegren, 1951; Lindegren and Rafalko, 1950)
have evolved a concept of the yeast nucleus which is simi
lar in many respects to that of Wager and Peniston.
Lindegren also considered the vacuole as the "nuclear
vacuole" and believed it to contain 6 pairs of chromosomes
in the vegetative diplophase of Saccharomyces cerevisiae.
The deeply staining excentric body which Wager and Peniston
took to be the nucleolus (the nucleus of Guilliermond) he
considered to be the centrosome. At the same time
Lindegren considered the granule in the vacuole to be the
nucleolus, as did Janssens*
Since the introduction of the Peulgen nuclear stain,
the nucleus of Guilliermond (Lindegren*s centrosome) has
been known to be strongly Peulgen positive (Margolena,
1932; Rochlin, 1933; Im%ene cki, 1936; Wing'd and Lausten,
1939). This has greatly strengthened the Guilliermond
school of thought. Lindegren based his concept largely up
on his findings that certain delicate granules in the
vacuole (his chromosomes) also were Peulgen positive when a
special sensitive modification (Rafalko 1946) of the
Peulgen stain was used. Lindegren has developed a large
18
number of special fixing and staining procedures which he
claims stain differentially and specifically the various
organelles. He has published many excellent photographs
(see especially Lindegren, 1949) which are more credible
than his predecessors* diagrams.
Lindegren reproduced the work of Renaud and obtained
essentially identical results except that he did not see
the tiny centriole described by Renaud. Lindegren refer
red to this division as a division of the centrosome.
Since this division appeared to be transverse, he felt it
could not be a division of chromosomes. This author has
recently revised his conception of the budding process in
some details (Lindegren 1951b). His amended version of the
mechanics of budding is as follows. It should be noted
that Guilliermond*s nucleus (the centrosome of Lindegren)
plays a leading part ;
The yeast cell contains a nucleus whose rigid centro
some carries a band of Peulgen-positive chromatin
(centrochromatin) on its surface. The first step in
budding is the formation of the bud by an extension of
the centrosome over which the cell wall persists.
Next the nuclear vacuole extends a process into the bud
bud which contains the chromosomes. Pinally the cen
trochromatin divides directly and the cells separate;
a plug either of centrosome or cytoplasm sealing the
bud pore. The cytoplasm, the centrosome, the centro
chromatin and the nuclear wall are autonomous non
genic organelles which never originate de^ novo.
The work of DeLamater. Using an improved Peulgen
technique and other specially developed staining procedures.
19
DeLamater (1949ab, 1950) succeeded in producing a set of
photographs of what appeared to be true mitosis in
Saccharomyces cerevisiae. Although the preparations were
not sufficiently clear to distinguish individual chromo
somes, DeLamater believed their number to be probably four.
This investigator identified his mitotic figures with the
nucleus of Guilliermond and the photographs showed the
figures to be outside the vacuole. Thus these results are
incompatible with the concept propounded by Lindegren,
especially since no chromosome-like figures were observed
in the vacuole.
The photographs presented by DeLamater showed a cen
triole which divided into two, the two halves of which mi
grated to opposite sides of the chromosomal mass to form
a characteristic metaphase spindle. As the chromosomal
mass divided, the chromosomes became stretched. Eventually
the daughter nucleus was drawn into the bud. Although the
pictorial evidence is not perfectly clear, the stages of
mitosis shown by DeLamater strongly resemble mitosis in
higher organisms. The evidence seems convincing that the
mechanism of budding in yeast is essentially as demon
strated by Guilliermond and further developed by DeLamater.
Recent contributions of others. Improved Peulgen and
other techniques have led a number of workers to reinvesti-
20
gate yeast cytology within the last decade or so. Most,
but by no means all, have tended to confirm the Guilliermond
idea. SinotB and Yuasa (1941) demonstrated mitotic figures
containing 4 chromosomes. Levan (1946, 1947) produced
mitotic disturbances in yeast by means of benzene and
camphor, and stained with Peulgen. His photographs showed
nuclei and chromosomes. He counted at least 10, possibly
more, chromosomes. Brandt (1941) and Oaspersson and Brandt
(1941) studied the absorption of ultraviolet light by dif
ferent regions of the yeast cell. They found cytoplasmic
granules containing ribose nucleoprotein and suggested
that these might be the equivalent of the nucleus. Such an
interpretation seems a little far-fetched. Cutter (1951)
also believed their report lacked conviction, because of
vague terminology.
Subramaniam (1947) and bubramaniam and Ranganathan
(1945ab, 1946abc) have advanced the Guilliermond concept
with new Peulgen and hematoxylin preparations of budding
yeast. They often observed amitosis-like figures, but also
succeeded in demonstrating two chromosomes in Saccharomyces
cerevisiae. Occasionally three or four chromosomes appear
ed and the authors considered such cells to be in tripleid
or tetraploid states.
Lindegren*s proposals have found some support in the
21
work of Naidu and Bakshi (1946ab) and of Snirath (1946ab).
The former found 12 chromosomes in Saccharomyces cere vis iae
in addition to two small bodies which they presumed to be
centrioles. Their pictures give the impression that the
chromosomes are in or on the vacuole, often occupying
nearly the entire area of the cell. Snirath (1946a)
found one to five Peulgen positive bodies in the vacuole
of Saccharomyces cerevisiae. He refrained from interpret
ing these bodies as chromosomes, and later (1946b) identi
fied them as centrioles. At the same time he counted six
pairs of chromosomes, using Lindegren’s toluidine blue
chromosome stain. He considered the nucleus to be a com
pound structure containing a hemispherical centriole at
tached to the nuclear vacuole. A patch of chromatin was
found to lie in intimate contact with the centriole.
Richards (1928, 1932, 1934, 1938), in a series of in
vestigations using a number of widely different techniques
(Peulgen and other stains, observation with the Ultropak
lens system, ultraviolet photography, colchicine stimula
tion) failed to find any grouping of chromatin that might
be interpreted as chromosomes . He considered that the
budding of yeast probably occurred by amitosis, but admit
ted that future investigations with new techniques might
alter this concept. This work was given further weight
22
by the studies of Beams, Zell, and Bulkin (1940). These
workers stained budding cells with hematoxylin and Peulgen
and came to the conclusion that "all cytological evidence
shows that the division of the yeast cells during the bud
ding process is amitotic."
In a paper which deserves serious consideration,
Nagel (1946) has reinvestigated and analyzed the whole
question of yeast cytology. To attain objectivity and to
avoid carrying along the connotations of 75 years of con
tradictory opinions and terminology, she has coined new
names for the principal yeast structures. The vacuole was
termed the "magnicorp," the deeply staining excentric
body became the "parvicorp," and the chromatin patch on
the latter was called the "companion body." Using a wide
variety of techniques, Nagel was unable to observe any
mitotic figures. She noted that division of the parvicorp
may be completed in the mother cell and then pass into the
bud, but more often it appeared to divide directly into
the bud. The magnicorp was seen to burst rapidly out into
the bud. This occurred before the parvicorp entered the
bud, as had been previously observed by Lindegren and
Guilliermond, and when visible particles were present in
the magnicorp they also entered the bud, at times assuming
paired forms and positions difficult to interpret as
23
"reserve stuff*" Nagel concluded that inasmuch as both the
parvicorp and magnicorp seemed to be associated with vege
tative cell division, a complete interpretation would have
to take cognizance of both. An analysis of the stainable
particles in the magnicorp, however showed them to vary in
number from zero to over ten, making it difficult to cor
relate them with chromosomes. At the same time, the
Peulgen positive nature of the parvicorp was indicative of
its nuclear nature.
Figure 1 is a diagram of yeast organelles. The
letters are keyed to Table I (expanded from Nagel) which
summarizes the various interpretations that have been given
to yeast cytology.
24
FIGURE 1
DIAGRAM OF THE RESTING YEAST CELL
See Table I for interpretation
TABLE I
25
INTERPRETATIONS OF YEAST ORGANELLES
BY VARIOUS INVESTIGATORS.
Letters In first column refer to Figure 1
Structure
Janssens
and
LeBlanc Guilliermond
Wager
and
Peniston Henneberg
A peripheral
layer of
chromât in
B nucleolus
C part of
nucleus
nucleus nucleolus nucleus
D chromatin
patch
E chromatin
network
F nucleus va cuo le nuclear
vacuole
va cuo le
G nucleolus central
volutin
granule
H volutin
I cytoplasm
J fatty
granules
K volutin
granules
L glycogen
No. of amitotic amitotic amitotic amitotic
Chromo
somes
26
TABLE I (continued)
Structure Kater
A
B
C
D
E
Lindegren
Subramaniam
and
Ranganathan Dangeard
hetero
chromatin
nucleolus
nucleus centrosome
centrioles
nucleus
nucleolus
nucleus
vacuole nuclear
vacuole
vacuole
G nucleolus
or balled
up chromo
somes
H
I
J
K
No. of
Chromo
somes
chromosomes
meta-chro- mitochondria
matic
granules
8
glycogen
6 pairs
protoplasm
27
TABLE I (continued)
Structure Swellengrebel Puhrmann Renaud DeLamater
A
B nucleolus nucleolus
nucleus nucleus nucleus nucleus
(with cent-(with
rosome) centiole)
D
E
P
G
vacuole
H
I
J
K
L
not seen
No. of
Chromo*
some 8 at least
8
probably 4
28
TABLE I (continued)
Sinot6 Naidu
and and
Structure Yuasa Levan Bakshi Snirath Richards
A
B
C
D
nucleus nucleus centriole uncertain
chromatin
patch
E
P vacuole nuclear
vacuole
nuclear
vacuole
G
H
I
J
K
L
No • of
Chromo
somes at least 12
10
6 pairs
amitotic
29
TABLE I (continued)
Structure Henrici
Winge*,
et al Badian Nagel
A
B
C nucleus nucleus nucleus parvicorp
D chromo
somes
companion
body
E
P vacuole vacuole va cuo le magnicorp
G dancing
body
central
granule
H volutin particles
in magni
corp
I
J
K
fat
vacuoles
VO lut in non-fatty
cytoplasmic
granules
No. of
Chromo
somes
unknown
possibly
amitotic
30
TABLE I (continued)
Structure Beams, et al Oaspersson and Brandt
A
B
C
D
E
nucleus euchromatin of nucleus
P
G
H
I
J
vacuole vacuole
me t a chroma tic granules
K volutin
No. of
Chromo
somes
amitotic
CHAPTER III
MATERIALS AND METHODS
Source of organisms. All cultures of bacteria and
yeast used wore obtained from the stock culture collection
of the Department of Bacteriology, University of Southern
California. Unless otherwise specified all bacterial cul
tures were grown on nutrient agar slants for eighteen hours
at 37° C. Yeasts were grown for four or eighteen hours on
wort agar slants at room temperature. Yeast ascospores
were obtained after five to seven days* growth at room
temperature on a modified Henrici vegetable juice medium
(Wickerham, Plickinger, and Burton, 1946). This medium was
found to give a higher percentage of ascospores in a short
er length of time than did gypsum blocks or Gorodkawa* s
medium. Protozoa were obtained from the Department of
Zoology, University of Southern California, and from grass
infusions.
Pixing procedures. Pixatives used were heat, 1% for
maldehyde, 1% HgClg, 5% phenol, 1% picric acid, osmic acid
vapor, Lugol*s iodine, 0.5^ NaNg, Carnoy*s solution,
Bouin* s solution, and Schaudinn* s fixative (heated, as used
by De Lamater, 1950). Por fixation of the yeast nucleus it
was found that osmic acid vapor and Bouin*s solution gave
the best results, and these were used almost exclusively in
32
the later experiments. Formulas for the above solutions
are given in detail by Guilliermond (1920) and by Lindegren
(1949). Fixation times for fluids varied from two minutes
to several hours. It was found, in general, that ten to
twenty minutes fixation was ample. Osmic acid vapor fixa
tion was for two minutes.
Staining procedures. The Hucker and Kopeloff-Beerman
modifications of the gram stain and the Ziehl-Neelsen acid
fast stain were conducted according to the Manual of Methods
for Pure Culture Study of Bacteria (1943). Peulgen stains
were prepared according to Coleman (1938) and Rafalko
(1946). Cell wall stains used were those of Dyar (1947)
and Welshimer and Robinow (1949). The toluidine blue stain
which Lindegren (1949) claims is specific for volutin was
also used. Ascospore formation in yeast cultures was fol
lowed with the modification of the Schaeffer-Pulton spore
stain developed by Bartholomew and Mittwer (1950). The
special staining and fixing procedure which enabled
DeLamater and Mudd (1951) to demonstrate chromosomes in
bacteria was not found to be adaptable to electron micros
copy.
Ultraviolet irradiation. A Westinghouse sterilamp
WL-15 was used as a source of ultraviolet light. This
lamp has an output at 2537 A of 32 microwatts per cm^, at
33
a distance of one meter. Exposures for the following work
were made at a distance of 12 cm. Heat produced by the
lamp was negligible.
Por determination of the effect of radiation on stain
ing characteristics, smears were prepared on glass slides,
fixed, and exposed to the ultraviolet lamp for various
periods of time.
To study the effects on morphology, cells were exposed
under various conditions: (a) dry, as smears on glass
slides; (b) anaerobically, in suspension in a shallow cell
with a quartz cover, and (c) aerobically, in suspension in
shallow dishes without covers. Depending upon whether the
preparation was dry or in suspension, the final results
differed considerably. This was probably due to a differ
ence in the type of photolytic reactions predominating. It
was found that a combination of wet and dry irradiation
cleared the cells best for electron microscopy. Times of
irradiation varier^ from about half an hour to several days,
as indicated in the chapter devoted to results.
Two types of electron microscope preparations were
used: (a) cells were exposed to ultraviolet and, using a
suspension of these cells, collodion films on wire screens
were prepared in the usual manner; (b) dry cells were ex
posed by preparing smears on glass slides which had been
34
dipped in 0.2% collodion and the preparation floated off on
water. This film was then picked up on screens.
Optical microscopy. After irradiation treatments, the
cells were observed with the ordinary oil immersion micros
cope, in unstained wet mounts, and on fixed smears stained
according to the procedures previously mentioned. These
preparations were also observed with the Spencer phase
contrast microscope. Both the Bright M and Dark M objec
tives were found to be useful.
Electron microscopy. The electron microscope used
(RCA model EMU) was that of the Department of Experimental
Medicine, University of Southern California. Preparations
were observed both with and without the use of a chromium
shadowing technique.
CHAPTER IV
RESULTS
The effects of irradiation were studied from the point
of view of staining characteristics and of cytological
aspects of cells. For convenience the results will be
divided into sections and presented separately.
Effect of ultraviolet irradiation on the gram and acid
fast stains. A total of 28 species of gram positive micro
organisms were subjected to irradiation (see Table II) on
heat fixed slides. All of these became gram negative after
the treatment, and none was found which resisted conversion
to the gram negative state. The number of species used in
the test justifies the conclusion that all gram positive
organisms would probably lose this characteristic upon
sufficient exposure to ultraviolet light.
A wide variation existed in the rates of conversion to
the gram negative state. Bacillus megaterium lost its gram
positivity in less than one hour of irradiation. Bacillus
subtilis required six hours. Micrococcus pyogenes var.
aureus required 24 hours, and some of the yeasts required
nearly 30 hours. The exact time necessary for conversion
was not determined for each species listed in Table II.
However, all became gram negative in 30 hours or less.
The method of fixation greatly influenced the results.
TABLE II 36
SURVEY OF SPECIES LOSING GRAM POSITIVITY AFTER EXPOSURE
OF HEAT FIXED SMEARS TO ULTRAVIOLET LIGHT
FOR 30 HOURS OR LESS.*
Bacillus subtilis var. aterrimus
Bacillus macerans
Bacillus megaterium
Bacillus mesenterieus
Bacillus' me tiens
Bacillus" cere us var. mycoides
Bacillus subtilis var. nlger
Bacillus polymyxa
Bacillus subtilis
Bacterium globifome
ferynebacterium fimi"
üorî^ebacteriûm pseudodiphtheria
Lactoba ci1lus casei
Lactobacillus plantarum
MicroCOecus pyogenes var. aureus
Mycobacterium jacticola
Sarcina lutea
Streptococcus liquefaciens
Mycobacterium smegmatis
Saccharomyces carlsbergensis
Sa c char omyce s cerevisiae
Sa c char omyce s ludwigii
Sciii z os a c char omy ce s octosporus
Éansenula saturnus
Hanseniospora melligeri
^ygos ac charomyce s halmenbranis
Endomyces ohmeri
Mycoderma cerevisiae
* No negative results were obtained.
37
Osmic acid vapor fixation greatly hastened the loss of gram
positivity for B. subtilis. while formalin fixation retard
ed it, as compared to heat fixation. However, osmic add
vapor fixation did not significantly speed the loss of gram
positivity for Saccharomyces cerevisiae.
An intriguing but unexplainable effect of osmic acid
vapor was observed. #ien heat fixed smears of B. subtilis
had been exposed to irradiation for one and a half hours
they were still entirely gram positive. If these smears
were exposed to osmic acid vapor after this irradiation,
the cells then stained entirely gram negatively.
Figure 2 presents a detailed study of four organisms.
It shows the difference in rates of conversion from gram
positive to gram negative dependent upon species and man
ner of fixation. It will be noted that in each instance
the percentage of cells converted to the gram negative state
increased with time of irradiation in such a fashion as to
yield a typical sigmoid curve. These curves were accurate
ly reproducible if sufficient cells (800 to 100) were
counted for each coordinate. Figure 2 shows the results
for irradiation of dry fixed smears. Essentially similar
results were obtained by irradiation of organisms suspended
in distilled water in a quartz cell.
Observations of gram stained smears showed that an in-
38
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FIGURE 20
SA CCHAROMYCES OEREVISIAE, ELECTRON MICROGRAPHS,
SINGLE-SPORED ASCI.
85
EXPLANATION OP FIGURE 20.
Sac charomyce a cerevislae. electron micrographs
All cells fixed in Bouin*s solution.
Chromium shadows d « 14, OOOX.
(A) Irradiated 48 hours in aerobic
suspension, and 48 hours dry.
(B) Same as (A).
( 0) Same as (A)•
(D) Same as (A).
86
I
«■ 3
B
FIGURE 21
SACCHAROMYCES OEREVISIAE. ELECTRON MICROGRAPHS.
TWO-SPORED ASCI.
87
EXPLANATION OP FIGURE 21.
Saccharomycea cerevislae. electron micrographs.
All cells fixed in Bonin*s solution.
Chromium shadowed. 14,000X.
(A) Irradiated 48 hours in aerobic
suspension, and 48 hours dry.
(B) Same as (A).
( C) Same as (A).
(D) Same as (A).
88
B
FIGURE 22
SACCHAROMYCES CEREVISIAB. ELECTRON MICROGRAPHS.
TîîREE-SPORED AS CI.
89
EXPLANATION OP PIQURE 22.
Sac charomyce s cere visiae. electron micrographs.
All cells fixed in Bouin* s solution.
Qiromium shadowed. 14,000X.
(A) Irradiated 48 hours in aerobic
suspension, and 48 hours dry.
(B) Same as (A).
( C) Same as (A).
(D) Same as (A).
90
Figure 20 shows asci with single ascospores. The
spores in these instances do not appear to be surrounded by
membranes, but seem to consist of a nucleus surrounded by a
dense area of protoplasm. In (D) there appears to be a
second nucleus in the process of division or disintegration
outside the dense protoplasmic area.
Two-spored asci are illustrated in Figure 21. Some of
the spores here appear to have definite limiting membranes,
especially evident in the overlapping of the spores in
in Figure 21 (B). In the others, the spore wall appears
much denser in the area where the two spores touch each
other.
Figure 22 shows asci containing three spores. The
two on the right in (A) appear to have just been formed by
a division, separation being not quite complete. The upper
spore on the left in (D) appears to contain a dividing
nucleus, and this ascus may have been about to become four-
spored.
Most investigators of the sporulation problem recom
mend that a sporulation medium be inoculated with a
young actively dividing culture for maximum spore produc
tion (see for example Lindegren, 1949; Wickerham, et al,
1946). It will be noticed however, that frequently
(Figure 20 (B, D), Figure 21 (A), Figure 22 (C)) ascospores
91
were formed in cells bearing a number of bud scars. This
indicates that a cell often attained a considerable age
(with respect to the number of offspring it has borne) be
fore it formed as cospores.
Cytology of bud scars. To the knowledge of the pres
ent investigator, only one published paper has dealt
specifically with the question of bud scars on yeast cells
(Barton, 1950). This has undoubtedly been due to the dif
ficulty of seeing the scars in stained or wet mounts pre
parations, rather than to the lack of interest. A count of
bud scars is an accurate indication of the age of the cell
in terms of generations.
Figure 23 illustrates several cells, one of which
contains twelve countable scars. It will be noticed that
the scars on the cell in Figure 23 (A) occur only on the
two ends, as though budding has alternated, first on one
end, then the other. The other two cells bear scars over
the entire surface with no discernable pattern. A
comparison of these cells with those in Figure 24, shows
that, in general, those cells with many scars are con
siderably larger than younger cells.
Barton (1950) has termed the "birth scar" that scar
which results from the separation of a cell from its own
mother cell. He considered that the birth scar was
92
I
A B
FIGURE 23
sacciiaromyces cerevisiae. ele ctron mi CROGRAPHS
BUD SCARS.
93
EXPLANATION OP FIGURE 23.
Sa c chapomy ce a cerevisiae. electron micrographs
All cells fixed In Bonin's solution#
Chromium shadowed. 14,OOOX#
(A) Irradiated 48 hours In aerobic
suspension, and 48 hours dry.
(6) Same as (A).
( C) Same as (A).
94
A
FIGURE 24
SACCHAROMYCES CEREVISIAE, ELECTRON MICROGRAPHS
BUD SCARS.
95
EXPLANATION OP FIGURE 24.
Saecharomyees cerevisiae, electron micrographs.
All cells fixed In Bonin's solution.
Chromium shadowed. 14,000%.
(A) Irradiated 48 hours In aerobic
suspension, and 48 hours dry.
(B) Same as (A).
(C) Same as (A).
96
always the largest one on the cell, due to stretching as
the cell grew. Observation of the photographs in Figures
23 and 24 shows that it would often be difficult to deter
mine with any degree of assurance which scar was the birth
scar, even in the younger cells.
Irregularities in the locations from which new buds
arise are evident from a comparison of the cells in Figure
24. In Figure 24 (A) a daughter cell is being formed at
a site very close to the birth scar. In Figure 24 (B) a
daughter cell had been formed at the end opposite the birth
scar, and a second budding is taking place at a point near
ly midway between the two ends. Figure 24 (C) illustrates
an obviously irregular arrangement of bud scars with a
single scar on one end of the cell, and five soars and a
young bud bunched together on the other half of the cell.
CHAPTER V
DISCUSSION
Effect of ultraviolet light on staining. Very few in
vestigations of the effect of radiation on staining of
microorganisms have been made. Cernovodeanu and Henri
(1910) noted that ultraviolet light influenced the stain
ing characteristics of bacteria and protozoa but gave no
details of methods used. Their work was not confirmed by
later investigations (Mayer and Dworski 1932, Smithburn
and Lavin 1939). Dharmendra and Mukerjee (1949) recently
found that a twelve hour exposure to ultraviolet light
caused the human leprosy bacillus to lose its acid fastness
but had no effect on the acid fastness of the rat leprosy
bacillus. In view of the similarities between the gram and
acid fast stains (Bartholomew and Mittwer 1952), the in
vestigations reported in the first section of Chapter IV
were undertaken.
In efforts to determine the mechanism of the gram re
action, many methods have been discovered which convert
gram positive cells to a gram negative state (Bartholomew
and Mittwer 1952). In contrast to many of these methods,
which are often difficult or unreliable, the technique of
irradiation is simple and affords definite and certain re
sults. A practical implication immediately suggests it
self. Smears should not be left lying about exposed to
98
sunlight or other sources of ultraviolet light, as this
would influence the reaction shown by subsequent gram or
acid fast stainihg.
The loss of gram positivity on the exposure of fixed
smears of bacteria to ultraviolet light could be explained
in two ways. First, it is known that ultraviolet light
influences cell membrane permeability characteristics
(Davidson 1940, Rahn and Barnes,1933, Loofbourow, et al
1941, Nadson and Stern 1931). Secondly, it is well known
that ultraviolet light is photolytic to the substances con
tained within the cell (Ellis and Wells 1941). Therefore
the results presented in this thesis do not help directly
to prove or disprove either the cell membrane permeability
or the chemical theories as to the cause of gram positivity.
It is probable, however, that this method of inducing loss
of differential staining characteristics will be helpful in
future experimental study of the gram and acid fast stain
mechanisms. For example, a Mycobacterium species might be
irradiated just to the point where acid fastness was lost,
and the cells then analyzed to see if they still contained
mycolic acid.
The effect of ultraviolet light on the stainability of
spores is especially interesting. The ability of the spore
to stain in the gram procedure would indicate that impor-
99
tant changes had occurred in the permeability characteris
tics of the spore or the spore coat. It would be difficult
to explain this appearance of staining by a change only in
the chemical nature of the spore. The change from gram
positive to gram negative spores could be explained by a
further increase in the permeability of the spore coat, or
by additional chemical degradation of the spore substance.
It is also obvious that any chemical change in the
constitution of a cell or spore membrane will have an
important influence on its permeability characteristics.
The experiments reported above do not allow the separation
of the effects produced by chemical degradation of sub
stances in the cell membrane, and in the cell as a whole.
Bacterial cytology. The differences were considerable
between the results obtained by irradiation of cells on
slides and those in suspension. The empty cell areas ap
pearing on irradiation of cells in suspension can be ex
plained on the basis of a leaching out of cell material,
due to a change in cell membrane permeability and/or to
the production by photolysis of substances of increased
solubility. The discussion above and the earlier review
of the literature have shown that both phenomena occur.
The sequence of events occurring on irradiation of
cells in suspension could be reconstructed as follows.
100
First, material begins to leave the cell or its membrane
which would not leave in the absence of ultraviolet light.
Secondly, the cell becomes gram negative but maintains its
normal size and shape. Third, the cell begins to flatten,
due to material loss, and a large cell wall area becomes
spread out and easily seen. Lastly, a residual small body
is left which is within the old cell wall and which is
only about one-half the size of the normal cell.
The small rods seen in Figure 4 (I, J) resemble the
pictures presented by Miller and Birch-Andersen (1951) to
show the effects of plasmolysis by salt, phenol, and merc
uric chloride. It is impossible at the present time to
state whether these objects are actually morphological
structures or simply altered physical states of protoplasm.
It seems illogical to believe that plasmolysis would result
in such neat rod-like structures, or even that plasmolysis
would occur under the given conditions. The small rod
shaped bodies could represent the gram negative medulla
area of Churchman (1927). This theory of Churchman has
been attacked, but it has not been proved that the medulla
area does not exist. These small rods are also analogous
to the small gram negative rod often seen in a chain of
large gram positive rods (Bartholomew and Mittwer 1951),
and are similar to the structures left in enzymatically
101
digested cells (Weidel 1951).
As seen above, irradiation of cells in a dry state re
sults in a gradual change from an opaque to a transparent
condition with respect to electrons, and must be due to the
breakdown of cell substances to volatile products which
then leave the cell. The series of pictures seen in Figure
4 shows that the effect of ultraviolet light is very simi
lar to that reported for electron beams by Knaysi (1950)
and by Mudd, et al (1951) and which they attribute to
volatilization. Ultraviolet light as used in these expri
mants is, however, a more sensitive and easily controlled
method of revealing internal structure than is an intense
electron beam.
Irradiation of dry slides gives no evidence of the
small opaque rod observed in the irradiated cell suspen
sions. A different type of action takes place, revealing
transparent granules in the bacteria. These transparent
areas strongly resemble the Type C bodies described by
Knaysi (1950) and are probably not nuclei, since nuclei
would be more likely to appear dark as do Knaysi's Type A
(nuclear) bodies. Knaysi found that excessive electron
bombardment disintegrated first the cytoplasm, then the
Type C (transparent) bodies, and the cytoplasmic membrane.
Meanwhile the typ^^^di^^^re^jery.^i^elstant to such
102
treatment# This sequence of events is practically identi
cal to that described earlier by Nadson and Rochlin (1933)
for ultraviolet and X-rays. Knaysi found that Type A
bodies stained with basic dyes but lost this ability after
treatment with desoxyribonuclease. This fact, plus
evidence of multiplication and other chemical tests, led
him to the conclusion that Type A bodies were of nuclear
nature•
On the other hand, Mudd, et al (1951) have published
electron micrographs showing dark bodies which appear to
be identical to the Type A bodies of Knaysi, but these
authors present evidence purporting to show that they are
not nuclei but mitochondria. These authors state that
light areas of low density to electrons were the nuclear
sites (see also Mudd, Smith, Hillier, and Beutner, 1950)*
Mudd and Smith (1950) determined that the pattern of
light nuclear sites with a darker cytoplasm is completely
reversed by treatment with HCL. They state that HCL ap
parently coagulates the nuclear contents into a very dense
material which then appears dark against a relatively
light cytoplasm. Consequently it appears that there are
two schools of thought among bacterial cytologists as to
the nature of the light areas appearing in electron micro
graphs of bacteria. Nadson and Stern 1931b) observed cells
103
of Bacillus mycodles during ultraviolet irradiation and saw
vacuoles form in a previously homogeneous cytoplasm. It is
thus possible that the clear areas in the cells in Figure 4
are vacuoles produced by photolysis of cell contents, and
electron micrographs in the literature may contain arti
facts produced by the electron beam itself.
Yeast cytology. The yeast structure revealed by ir
radiation of dry smears (Figure 5 (C), 6) agrees fairly
well with the composite picture developed by previous in
vestigators (Figure 1). The electron micrographs reveal,
however, an additional structure not previously reported:
a thin line between the vacuole and the cell wall. This
structure could not be identified with certainty. It
could be a cell vacuole or a vacuolar membrane, or it could
be the result of shrinkage of the vacuole during drying.
This structure could also be seen with the phase microscope
during observation of irradiated cells which had been re
suspended in water. The phase microscope also revealed
that the central vacuole sometimes had continuity with a
similar structure in a young bud, confirming the observa
tions of Guilliermond (1920), Nagel (1946), and Lindegren
(1949).
Of greater interest to yeast cytologists is the be
havior of the nucleus during the budding process, illus
104
trated by Figures 7 through 15. An important question
immediately poses itself. Is the dark structure in these
photographs really the nucleus? A preponderant body of
evidence indicates that it is.
In the first place, Nadson and his collaborators
(1926, 1928, 193lab, 1933) determined that the nucleus was
the last internal structure to be destroyed in cells of
plants, fungi, and yeasts (Guilliermond*s nucleus) upon ex
posure to ultraviolet and X-rays. These results were con
firmed by Lacassagne (1930), Lacassagne and Holweck (1930),
and Latarjet (1944). A similar resistance of bacterial
nuclei to electrons was found by Knaysi, Hillier, and
Fabricant (1950). The work of Errera (1951, 1952), cited
in Chapter II, showing the very low quantum yield for
photolysis of desoxyribonucleic acid by ultraviolet light,
makes this resistance of the nucleus understandable from
a physico-chemical point of view. The present investiga
tions confirmed the resistance to ultraviolet radiation of
the nucleus of several species of protozoa. In these
organisms the nucleus was unmistakable, and the demonstra
tion of its resistance to photolysis was completely con
vincing.
Additional evidence for the nuclear nature of the
dark structures in the yeast cells pictured here is af-
105
forded by the fact that these structures took a positive
Peulgen stain, even after irradiation. This is in accord
with the findings of Errera (1951) with regard to desoxy
ribonucleic acid. The specificity of the Peulgen nuclear
stain has occasionally been questioned (Gulland, Barker,
and Jordan 1945) where lipoids are present. However, at
the present time cytologists are practically unanimous in
agreeing that this stain does specifically reveal the pre
sence of desoxyribonucleic acid in situ (Brachet 1946;
DeLamater, Mescon, and Barger 1950; Li and Stacey 1949;
Overend and Stacey 1949). On the basis of all the evi
dence, it can reasonably be concluded that the structures
presented here are in fact nuclei, and that their behavior
during the budding process has been demonstrated.
How then can these apparently "amitotic" divisions of
the nucleus be reconciled with the photographs of DeLamater
(1950)? DeLamater*s photographs (of modified Peulgen pre
parations) sometimes markedly resemble the electron micro
graphs of Figures 7 to 15. At other times they show,
though not too clearly, figures that resemble the mitotic
spindle of metaphase. Assuming DeLamater's inferences to
be correct, and that the yeast cell divides by a mitotic
process comparable in all respects to the cells of higher
organisms, two sources of error are possible in the pres
106
ent investigations. First, it is conceivable that the
centrioles were small enough to have been completely photo-
lysed. This does not seem probable, inasmuch as the
"nuclear bud," corresponding to DeLamater's centiole, is
very much in evidence. Secondly, it has been reported
( CatCheside 1948) that irradiation causes an alteration in
the surface properties of chromosomes, probably by depo
lymerization of nucleic acids, causing them to stick to
gether. A phenomenon of this nature might tend to ob
scure the individuality of chromosomes and make the divi
sion of the nucleus appear falsely amitotic. Such an in
terpretation could explain the appearance of the nuclei in
Figure 16, which appear to consist of a bunch of balled up
chromosomes, and also the diffuse appearance of the inter
nal structure of the nuclei in Figure 18. However certain
clear-cut internal structures are found in Figures 11 (A)
and 17 (A) would be less readily explainable on the basis
of sticky chromosomes.
On the other hand, stained preparations made after
acid hydrolysis (i.e. the Feulgen stain and DeLamater's
or Robinow's modifications of it) subject the cells to
the probability of serious artifacts caused by the hydro
lytic procedure itself. This liability to artifact pro
duction has not been noted by DeLamater or others using
107
the Peulgen stain, although it would appear to be a serious
source of error in staining internal structures of micro
organisms. Both the heating and the acid are detrimental
to precise cytological study. Hedën and Wyckoff (1949)
found that heating at 60®C, as in the Peulgen and ribon-
nuclease techniques, caused a coarse and irreversible gran
ulation in protoplasm. Their electron micrographs demon
strate dramatically the havoc that can be wrought by heat
ing and they concluded that "this fact must be given care
ful consideration when conclusions are drawn from the ap
pearance of heated protoplasm concerning the distribution
of nucleic acids."
Also, Mudd and Smith (1950) and Mudd, Smith, Hillier,
and Beutner (1950) demonstrated with electron micrographs
that nuclear contents are densely coagulated by treatment
with HCL. These authors futher found that SchaUdinn's fix
ative (which was used by DeLamater) shrank the cells and
was unsuited for electron microscopic observation of in
ternal structure, a finding confirmed by the present in
vestigations. There are then, three definite sources of
artifacts to be reckoned with in interpreting DeLamater*s
photographs, in addition to the fact that they are not
clear. Since DeLamater himself was unable to count chromo
somes (nor do others agree ; see Table I) it is obvious
108
that his interpretation is not the last word on the subject.
As far as can be determined, the present photolytic tech
nique is less apt to produce artifacts than Peulgen stain
ing, although it is of course not immune to criticism. It
can logically be stated that the photographs presented in
this thesis are at least as valid, and probably more so,
than those of DeLamater. In addition the former have the
great advantage of clarity at high magnifications.
It would appear from the photographs that vegetative
reproduction of 8_. cerevisiae takes place amitotically, as
Gullliermond maintained for 40 years. This is not nec
essarily the case. Cell reproduction might be termed
amitotic merely because of the inability of the investi
gator to see what was taking place inside the nucleus. As
a matter of fact, the word amitosis has never been ade
quately defined. Kater (1940), in his review of amitosis,
stated that
Originally . . . amitosis was a synonym for direct
cell division which was interpreted as division of
the cell by constriction following constriction of
a resting nucleus . . . For some years, however,
it has been evident that amitosis must be consider
ed under three heads, namely, a constrictional div
ision of the nucleus not associated with cytodieresis,
or fission of the cell, the problematical one in
which also the cytoplasm supposedly divides, and the
division of the ciliate macronucleus.
Kater paid special attention to the problem of
amitosis in yeasts, since he (1927) had previously made
109
cytological studies of S. cerevisiae. He doubted that
amitosis occurred in yeasts and believed that amitosis had
not been proved for any type of cell reproduction. He con
ceded that in the final analysis, however, the burden of
proof rested with either side of the controversy.
The present writer must concede that the question re
mains open. Although most of the picutres presented here
suggest amitosis, certain ones among them appear to leave
no doubt that the nucleus is not homogeneous and probably
contains chromosomes or their equivalent (Figures 5 (D),
11 (A, D), 12 (A, B), 14 (B), 15 (B), 16, 17, 18). Since
investigators have been practically unanimous in pronounc
ing a mitosis-like process (meiosis) to be the mode of
ascospore formation (Kater 1940), it must be accepted that
chromosomes exist in the yeast cell and therefore probably
take part in budding. It is possible that division of
chromosomes occurs within the nucleus before the nuclear
bud appears.
The observations of Lindegren (1949) cannot be dis
missed entirely. Even Gullliermond (1920) observed the
close association between the vacuole and the nucleus dur
ing budding. This constant correlation has also been ob
served by Snirath (1946ab), Nagel (1946), as well as by
Lindegren (1951b). The microcinematographic studies of
110
Bayne-Jones and Adolph (1932) suggest a similar occurrence.
In the last named paper, it was observed that granules of
the mother cell moved into the bud, but none moved in the
opposite direction. Nagel made the reasonable observation
that since this association exists, both the nucleus and
the vacuole (parvicorp and magnicorp) must be taken into
consideration in any interpretation of the budding process.
She further considered that the presence of the magnicorp
in the fully developed spore indicated its importance as a
cell entity.
Hence, if the parvicorp and magnicorp are accepted as
an entity involved in cell division, the two together would
have to be termed the nucleus, and Lindegren's interpreta
tion would be a correct one. The disagreements then would
be reduced to a question of terminology and the location of
the chromosomes. It is the opinion of the present writer
that, while this association between nucleus and vacuole
exists, the role of the vacuole is a subsidiary, probably
non-genetIc one. On the other hand, the structure which
has been termed the nucleus in this thesis has been amply
demonstrated to be of nuclear nature and to contain desox
yribonucleic acid.
There is a little disagreement as to the meiotic
nature of as cospore formation, and the photographs present
Ill
ed in Figures 19 and 22 resemble rather well the descrip
tion of the process given by Nagel (1946). Preceding spore
formation the nucleus became approximately twice its normal
diameter and somewhat resembled a conventional prophase#
Also as found by Nagel, only a few cells were in meiotic
division at any one time, and some of the stages of divi
sion were so rapid as to be missed entirely.
Some new and interesting observations can be made,
however, regarding the ascospores of Figures 19 and 22.
It is evident at a glance that cells of widely different
ages can produce ascospores, as judged by the varying
number of bud scars on the as ci. The aseus in Figure 20
(D) has not over five bud soars, and the one in Figure 20
{ C) probably has even fewer. On the other hand, the coll
in Figure 20 (B) bears at least eleven bud scars. The
cell in Figure 22 ( C) probably contains still more, but not
all are visible, because of the density of the ascospores.
A second point of interest is in the physical nature
of the ascospore. The single ascospores in Figure 20
appear to consist chiefly of a nucleus surrounded by an
area of dense cytoplasm. No limiting membrane for the
latter is evident, but the dense cytoplasm of the spore
seems to blend gradually into the less dense cytoplasm of
the as eus. The spores in Figure 21 appear to have mem^
112
branes, or at least sharp boundaries, It is possible that
this membrane or boundary is formed only after all the
spores have been formed that are to be formed by that
particular cell. This would seem to be indicated by the
fact that while the double ascospores in Figure 21 have
sharp boundaries, those in the three-spored as eus (in the
process of division) in Figure 22 (A) do not yet appear to
have developed membranes.
The bud scars make an interesting study in themselves.
The location and order of appearance of bud soars has been
described in Oiapter IV (Figures 23 and 24), and is found
to differ considerably from that described by Barton (1950).
Barton found a definite and constant sequence for the site
of origin of the first eight buds. The sequence of budding
sites was found in many cases to be highly irregular in
the present investigations, and thus Barton's results are
not confirmed. In agreement with Barton a "birth scar"
could often be identified as a scar which was obviously
larger than the "bud scars." Barton attributed this
larger size to stretching as the cell grew after its
separation from the mother cell. It appears fairly certain
from the photographs presented in this thesis that a cell
keeps on growing, since cells with many scars always ap
pear larger than those with few scars. This observation
lis
accounts for the common knowledge that different individ
uals in a yeast culture vary greatly in size.
Barton observed a cell to bud twenty-three times, and
no bud was observed to appear at a previous site of forma
tion. The present investigator observed no cells with as
many as twenty-three bud scars, although with sufficient
searching some could undoubtedly be found. In accord with
Barton, no evidence of budding at an old site could be
found and it is reasonable to assume this does not occur.
This assumption raises an interesting philosophical ques
tion: since possible budding sites are limited, is a
Sa c char omyce s cell mortal even under the most favorable
growth conditions, provided it does not have conditions
sufficiently unfavorable as to form ascospores? Calcu
lations based upon the typical cell illustrated in Figure
23 (A) show that the maximum geometrically possible number
of bud sites is of the order of 100. The actual order of
magnitude is of less importance than the fact that it is a
finite number, since a maximum possible number of buds
marks the cell's mortality. This sharply differentiates
the life cycle of Saccharoymces from such forms as
S chi z o s a c char omy ce s or bacteria, which are theoretically
immortal, barring accidents, since each daughter cell in
these latter organisms is a new individual and neither of
114
a pair can be considered the original mot)ier cell. This
mortality as described above for Sa c char omyce s probably
accounts for the fact that it is difficult to obtain a
yeast culture without an appreciable proportion of dead
cells.
CHAPTER VI
SUMMARY AND CONCLUSIONS
The investigations reported in the preceding chapters
have shown that ultraviolet irradiation destroys the
ability of gram positive microorganisms to retain the gram
stain. They have also confirmed that these radiations des
troy acid-fastness in Mycobacteria. Since it is known that
ultraviolet photolysis effects a chemical degradation of
all cell substances and also increases membrane permeabil
ity it is impossible to bolster either the cell membrane
permeability or the chemical theories of the gram or acid
fast stain mechanisms. It was found that a wide variation
existed among species in the rates of conversion to the
gram negative state, and that the method of fixation also
significantly altered those rates. Radiation also influ
enced the staining of bacterial spores. Normally non-
st ainabie in the gram procedure, under the influence of
ultraviolet the spores became first gram positive, then
gram negative. Such changes in staining characteristics
obviously result from the chemical changes induced by the
radiation, changes which probably affect the permeability
of the cell and spore walls. It was pointed out that it
would be unwise to allow smears to be exposed to sunlight
or other sources of ultraviolet light, as this would alter
116
their reaction to subsequent gram or acid fast staining.
The advantages of using ultraviolet photolysis for further
investigations into the mechanism of the gram or acid fast
stains were also indicated.
No morphological changes in cells just converted to the
the gram negative state were apparent. However, continued
irradiation resulted in obvious cytological changes. Ir
radiated bacteria in suspension were found to lose material
gradually, leaving a small residual rod within the cell
wall. The nature and meaning of this residual rod are
problematical. Irradiation of bacteria in the dry state
revealed transparent areas or vacuoles in the cell. It was
concluded that the vacuoles were probably caused by the
photolysis of the cell contents and that previous investi
gators might have been misled by similar artifacts.
Dry irradiation of yeast cells demonstrated the pos
sible existence of a new vacuolar membrane not heretofore
described. Ultraviolet photolysis of yeast cells by a
combination of wet and dry irradiation resulted in cleared
preparations suitable for demonstrating nuclear behavior in
the electron microscope. Overwhelming evidence for the
identity of the yeast nucleus in the electron micrographs
was presented. Including observation of preparations stain
ed with the Peulgen technique, other stains, phase micro-
117
copy, and controls of protozoa.
A complete series of photographs was presented show
ing the development of yeast cells during the budding pro
cess. The process can be described as follows: the nucleus
of a resting cell develops a small protruberance which the
author has termed the "nuclear bud." As a bud appears on
the cell, the nuclear bud enlarges and moves toward the cell
bud, which it then enters. The nuclear bud enlarges, be
comes the "daughter nucleus" and eventually becomes dis
joined from the original nucleus. The bud and its nucleus
grow to approximately the same size as the mother cell and
its nucleus, and both cells acquire the appearance of rest
ing cells. Later the two cells separate.
The yeast nucleus was di own to be non-homogeneous, but
chromosomes could not be identified. Certain serious
sources of error in interpretation of Peulgen preparations
of earlier investigators were pointed out.
In comparing the results obtained in these investiga
tions with the results of other yeast cytologlsts, it was
concluded that the nucleus is identical to the structure
which Gullliermond considered to be the nucleus. In con
tradistinction to Gullliermond, but In agreement with
DeLamater, It was also reasoned that vegetative multiplica
tion probably occurs by means of a mitotic process. It was
118
agreed that Lindegren and Nagel were correct in their ob
servations that the vacuole was closely associated with
the nucleus in cell division; but it was considered doubt
ful that the vacuole itself had a nuclear function. Al
though there has been little disagreement regarding asco
spore formation, preparations were made and studied* In
general the structures found confirmed the observations of
Nagel and others. At the same time, certain new and
Interesting observations were made* Although most authors
recommend the use of young cultures for producing asco
spores, it was found by counting bud soars that many cells
reach a relatively advanced age before producing spores.
Ascospores were found to consist, in the early stages, of
a nucleus surrounded by an area of dense cytoplasm. The
latter appeared to shade off gradually into the less dense
cytoplasm of the ascus. After the meiotic divisions had
taken place, the individual ascospores appeared to develop
a membrane around themselves.
Bud scars have heretofore been little studied* They
offer some interest, however, as indicators of the age of
the yeast cell* The sequence of budding sites was found
to be often irregular, contradicting the observations of
Barton. It was found that the age of the cell, as deter
mined by the number of bud scars, correlated with an
119
increase in size. This phenomenon accounts for the common
observation that individual yeast cells in a pure culture
vary greatly in size.
Confirming Barton, it was found that a yeast cell
probably never buds twice in the same place. Since the
available area for budding is limited, it can be concluded
that the yeast cell is mortal (in contradistinction to
bacteria) and its life span in terms of generations of
offspring is limited. The phenomenon probably accounts for
the consistent finding of appreciable numbers of dead cells
in yeast cultures.
In conclusion, it can be stated that a new technique
for experimental cytology has been developed, a technique
which makes the high magnifications of the electron micro
scope available for investigations hitherto impossible.
Much of the present investigation has of necessity been
exploratory, and the new technique cannot be considered to
have been perfected, although a solid groundwork has been
laid. It is to be hoped that future investigations will
result in improvements and in the clarification of many
cytological questions, both in microbiology and other
biological and medical sciences.
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Asset Metadata

Creator Mittwer, Tod Edwin (author)

Core Title Studies on the cytology of bacteria and yeasts by means of ultraviolet photolysis

School Graduate School

Degree Doctor of Philosophy

Degree Program Bacteriology

Degree Conferral Date 1952-06

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biological sciences,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c30-261967

Unique identifier UC11226760

Identifier DP23893.pdf (filename),usctheses-c30-261967 (legacy record id)

Legacy Identifier DP23893.pdf

Dmrecord 261967

Document Type Dissertation

Rights Mittwer, Tod

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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