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THE MORPHOLOGY OF AZOTOBACTER VINELANDII
GROWN IN DIALYZED SOIL MEDIUM
THESIS
Presented to the Graduate Council of the
University of North Texas in Partial
Fulfillment of the Requirements
For the degree of
MASTER OF SCIENCE
By
Hoda A. Jradi, B.A.
Denton, Texas
August, 1992 Hoda, Jradi A. , The Morphology of Azotobacter vinelandii Grown in Dialyzed Soil Medium. Master of Science
(Biology), August, 1992, 57 pp, 13 Illustrations, List of
references, 6 Titles.
This research describes the changes in cell morphology of Azotobacter vinelandii cells cultured in dialyzed soil medium. This particular culture medium was assumed to provide the bacteria with an environment similar to their natural habitat, the soil. Cells were grown in the medium for 4, 8 and 16 days and fixed with glutaraldehyde and osmium tetroxide. Sections were cut to a thickness of 60 to 90 nm. Observation of the cells was performed using electron microscopy. Electron micrographs of cells in young cultures showed morphological differences from cells grown in chemically-defined, nitrogen-free media. Electron micrographs of cells in older cultures revealed the presence of a cell form not previously described in the literature.
These are cells approximately 0.5 pm in diameter surrounded by a thick, rigid membrane. TABLE OF CONTENTS
Page
LIST OF IllUSTRATIONS...... iv
INTRODUCTION...... 1i
Morphology Pleomorphism and Monomorphism Cyst Formation Conclusion
MATERIALS AND METHODS...... 13
Cultures and Media Microscopic Observations Light Microscopy Electron Microscopy Negative Staining RESULTS...... 18 DISCUSSION...... 48
LIST OF REFERENCES...... 54
iii LIST OF ILLUSTRATIONS
Figure Page
1. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing peritrichous flagellation...... 22
2. Light micrograph of A. vinelandii grown in dialyzed soil medium for two days ...... 24 3. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing double cell form and peritrichous flagellation-...... 26
4. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing long rod shaped cells...... 38
5. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing electron dense small cells, ghost cells and large spherical cells-...... 30
6. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing rigid limiting membrane...... 32
7. Electron micrograph of A.vinelandii grown in dialyzed soil medium for four days showing small cells with thick cell walls...... 34
8. Electron micrograph of A. vinelandii grown in dialyzed soil medium for four days showing multi-layered cell wall...... 36
9. Electron micrograph of A. vinelandii grown in dialyzed soil medium for eight days showing thick limiting edge of the cell, ghost cells and large rod shaped cells...... 38 10. Electron micrograph of A. vinelandii grown in dialyzed soil medium for eight days Showing internal membrane of the cell ...... 40 iv LIST OF ILLUSTRATIONS--CONTINUED
Figure Page
11. Electron micrograph of A. vinelandii grown in dialyzed soil medium for eight days showing internal structure of the small cell...... 42
12. Electron micrograph of A. vinelandii grown in dialyzed soil medium for 16 days showing small spherical cells, large oval cells and bizarre cells...... 44
13. Electron micrograph of A. vinelandii grown in dialyzed soil medium for 16 days showing separation of the cell wall...... 46
V INTRODUCTION
In 1890, Winogradsky's (48) concern with the nitrogen
cycle, specifically the fixation of atmospheric nitrogen,
together with his interest in the existence of
oligonitrophiles, brought him success in isolating
anaerobic, spore-forming bacteria capable of fixing
atmospheric nitrogen which were placed in the genus
Clostridium. The method that he employed depended on the
removal of oxygen from the culture by aerobic organisms,
making it possible for the development of anaerobic ones.
His observations led him to believe that he had also
encountered aerobic oligonitrophiles, but he was unable to obtain them in pure culture.
In 1901, by applying the techniques used by Winogradsky
in discovering the anaerobic nitrogen-fixing bacteria,
Beijerinck (5) isolated pure cultures of aerobic nitrogen
fixing bacteria that he called azotobacter. These came from
the soils and canal waters of the city of Delft, Holland. He
established the genus Azotobacter with two species, chroococcum and agile. Following this, the taxonomy and physiology of these bacteria became a popular subject of
1 2 intensive study that led to an extensive literature and
several lasting controversies. Beijerinck (5) noted the
similarity of characteristics between the two species of the
genus Azotobacter and the blue-green alga which he had
previously studied. Because of this resemblance, Beijerinck
(5) named A. chroococcum after the cyanophytan of the
family Chroococcacea. Jensen (19) and Kyle and Eisentark
(23) disagreed with much of the work reported up to 1950 and
considered Azotobacter a non-pigmented, blue-green alga.
Imshenetski (18) also noted such similarities as
nitrogen-fixation, cell dimensions and structures, division
pattern, and capsule formation between Azotobacter and the
blue-green alga, now cyanobacteria. By 1930, considerable
disagreement surrounded the taxonomic position of these
bacteria, their morphology, role in nature, ecology, and
relationship to the plants.
The description of the genus Azotobacter was surrounded
by confusion as mentioned above, and the list of pleomorphic
types was so lengthy that it was difficult to distinguish
among different forms and also to understand existing
terminology, especially when authors failed to publish
photographic evidence for some of the descriptions (5, 14,
18, 19, 23).
More than sixteen species of Azotobacter have been proposed by various authors, including Beijerinck's initial
designations. This includes the following: Azotobacter 3 chroococcum, A. agile, A. vinelandii, A. woodstownii, A. svrmii, A. nigricans, A. araxi, A. lacticogenes, A. insigne,
A. macrocytogenes, and A. paspali. Most of these species have been disregarded and Bergey's Manual of Systematic bacteriolocw (37) lists only: A. chroococcum, A. vinelandii, A. beierinckii, A. nigricans, A. armeniacus, and
A. paspali.
Green and Wilson (14) showed by biochemical analyses that major similarities between the two species chroococcum and beiierinckii existed. Moreover, he discovered that A. chroococcum and A. beilrinckii were practically identical, but differed significantly from A. vinelandii and A. agile.
These results were confirmed by the work of De Ley and Parks
(12), who studied deoxyribonucleic acid homologies and base ratio composition in the Azotobacter, and found antigenic similarities between the two species chroococcum and beiierinckii. Many differences between Azotobacter species have been made , sometimes on the basis of cultural characteristics (5, 17, 18, 20, 23).
The description and morphological characteristics of
Azotobacter are given in the of Bergey's Manual of
Systematic Bacteriology (37) as follows: "Large ovoid cells
1.5-2.0 pm or more in diameter. Pleomorphic, ranging from rods to coccoid cells. Occur singly, in pairs or irregular clumps, and sometimes in chains of varying lengths. Do not produce endospores, but form cysts. Gram negative. Motile 4 by peritrichous flagella, or non-motile. Aerobic, but can also grow under decreased oxygen tensions. Water-soluble and water-insoluble pigments are produced by some strains of all species. Chemoorganotrophic, using sugars, alcohols and salts of organic acids for growth. Nitrogen-fixers; generally fix nonsymbiotically at least 10 mg of atmospheric nitrogen/g of carbohydrate (usually glucose) consumed.
Molybdenum is required for nitrogen fixation but may be partially replaced by vanadium. Non-proteolytic. Can utilize nitrate and ammonium salts (all but one species) and certain amino acids as sources of nitrogen. Catalase positive. The pH range for growth in the presence of combined nitrogen is 4.8-8.5; the optimum pH for growth and nitrogen-fixation is 7.0-7.5. Occur in soil and water; one species occurs in association with plant roots. The mol% G
+ C of the DNA is 63.2-67.5 (Tm)-"
Morphology
A variety of morphological forms of the cells of bacteria in the genus Azotobacter have been reported by many investigators. This is a clear indication that there are many morphological variations in Azotobacter cells, and it is obvious that these were deemed of prime importance by several investigators including Lohnis and Smith (27) and
Bisset and Hale (6). In 1913, Jones (20, 21) reported the presence of intracellular granules in the Azotobacter life- 5 cycle. He came to the conclusion that the filtrable
inclusions represented reproductive bodies that were
liberated from the mother cell and that these eventually
gave rise to normal Azotobacter cells. Many authors (6, 16,
21, 26, 27, 28, 44, 49) considered the varied morphology of
Azotobacter to be an expression of a complex life-cycle,
with the variation in form representing stabilized stages of
the cycle. The life-cycle they described involved some
thirteen different morphological stages, which included
gonidia (27), microcysts (27, 49), and fungoid forms(44).
Using cultures of A. chroococcum, Lohnis and Smith (27),
also reported the presence of filterable inclusions capable
of regenerating the normal cells of Azotobacter.
In 1922, Almequist (2) reported the finding of granules
in cultures of Azotobacter. He wrote, " We must expect to
find similar forms in our environment, probably most of them
are difficult to culture."
In 1937, Lewis (26) claimed that the cultures studied
in reports of the life-cycle of Azotobacter consisted of
empty cells and fragments of membranes, as well as fat and volutin granules. Jones (20, 21, 22) also, presented data which showed that involutions were present in aging cultures. Bisset and Hale (6) reported on the similarity between the involution forms observed by Jones and the endospores of Bacillus. Mellon (31) also described a variety of similar forms including bodies of six to seven 6 micrometers found in Bacillus coli.
In 1927 Hadley (15) examined the available data with
regard to occurrence and possible significance of the
various forms reported previously (2, 19, 27, 49) and tried
to make a conclusive statement when he wrote, "Whether as
suggested by Mellon, the zygospores are the mother cells of
the filtrable forms of bacteria, for the existence of which
in many species sufficient evidence may now be said to
exist, or whether the filtrable bodies are the micro-gonidia
or the gonites as suggested by Enderlein and Almequist . . .
cannot at present be stated. . . But, whatever the actual
significance of these minute bacterial forms may eventually
prove to be, we may be certain of three things; they occur
regularly, consistently, and in great numbers of many
bacterial cultures under certain growth conditions and at a
certain stage of development; they do not always long endure
as such, but after a brief development often appear
apparently passing into other developmental stages; and
although they may closely resemble certain artificial
structures on the slide, they are not artifacts."
Hadley's (15) conclusive statement did not end the
debate that began in the early days of bacteriology. The
question of the existence of minute forms in the bacterial
life-cycle has been going on since that time. Gonzalez and
Vela (28), reported in 1980 that the descriptions given by previous investigators are inadequate in that they refer 7 only to one phase of the organism's morphology, and that as such, do not adequately describe the true nature of these bacteria.
Monomorphism and Pleomorphism
As early as 1877, Nageli (in Smith and Conant, 36) proposed that all bacteria belonged to one species and that there was only one cell type, a highly variable form capable of passing from one state to another both morphologically and biochemically. That concept was designated as the conservative view of monomorphism. Those cells, especially in old cultures, which did not conform to the normal were said to be contaminants. The extent and variety of cell forms (pleomorphism) were not acknowledged as a common phenomena in the life-cycle of bacteria (36).
Despite the lack of correlation and consistency among the findings of various workers, many different descriptions, as mentioned above, have been published of the genus Azotobacter (3, 8, 13, 20, 21, 22, 23, 26, 28).
Jensen (19) noted that many morphological variations existed in pure cultures of Azotobacter. He presented evidence of the existence of rod-shaped or oval cells measuring about 2 to 4 Lm; spherical cells about 2 to 3 tm in diameter; very small rod-shaped cells or spherical cells of less than 1 pn in diameter; cysts; and large cells.
Jensen (19) also assumed that these forms were affected by 8 the culture medium or by its composition. In his work, he
recognized the already known and reported shapes: the oval
rods which were subject to great variation in size;
spherical cells; and the cysts, previously called
"arthrospores" by Jones (20, 21, 22). All reported morphological variants were generally associated with an udetermined life-cycle of the bacterium.
Many different cell types occur as discrete stabilized stages in the growth cycle of the organism (2, 6, 13, 20,
22, 26). Frequently encountered forms included large, non spore-forming rods; coccoid budding cells; Gram negative rods; and arthrospores (cysts, microcysts, gonidia, and spores).
Some investigators have suggested that morphological variability should be attributed to environmental influences
(20, 21, 30, 39). However, Eisentark et al. (13) postulated that pleomorphic cells could, in fact, be stages in the bacterium's life-cycle, and that they could be deteriorating forms resulting from depletion of nutrients in the growth medium. Also, that they could be contaminants, or cells unable to divide or synthesize a cell wall. It is well known from previous investigators (17, 44, 45, 48) that A. vinelandii grown in the laboratory produces morphological variants. When grown in Difco peptone by Vela and
Rosenthal (44), A. vinelandii produced fungoid cells, which were described as having irregular shapes and no structural 9 rigidity. Vela and Rosenthal (46) assumed that glycine present in a concentration of 38 to 75 mg/l in Difco peptone induced the formation of those fungoid cells. They were described as large, lacking in complete cell division, and sometimes dividing by budding. Eisentark et al. (13) observed similar giant cells of A. agile grown on media containing beef extract or soil extracts. Also, van Shreven
(39) has noted transparent forms of A. chroococcum when grown on mannitol agar with peptone. These autolyze spontaneously according to van Shreven.
The Cvst Form
In 1938 Winogradsky (48) succeeded in converting the vegetative cells of Azotobacter chroococcum into the cyst form. He used organic acids and alcohols as oxidizable substrates and obtained an entire population of Azotobacter in the cyst form. He claimed that the formation of those cysts might be permanently suppressed when the bacteria were grown on media containing glucose or sucrose as a carbon source. Wyss et al. (49, 50) observed that encystment occurred in vegetative cells of A. vinelandii grown on
Burk's medium without glucose. Those cysts had previously been described by Omelyanski et al. (31) and Batchinskaya
(3) described the formation of two-layered capsules surrounding the cells of Azotobacter and found those to be similar to cysts. 10
Not only Azotobacter, but many other bacteria are
characterized by the ability to form cysts. It is reported
that such cysts are generally smaller than the vegetative
cells. Other bacteria capable of producing cysts in special
media belong to the genera Myxobacter, Cystobacter, and
Methanococcus. The cyst is a protective coat which covers
the entire cell, making it resistant to heat and many other
harsh conditions (9, 38, 46, 47). Cysts are structurally
different and easily distinguished from vegetative cells and
from the endospore of other bacteria (38). Each cyst is the
product of a vegetative cell and possess a thick, double
layered cyst coat (38, 50).
In 1964 Vela and Wyss (42), found laboratory cysts to
be more sensitive to gamma rays than were the soil
Azotobacter in situ in the soil. In 1974 Vela et al.(46)
exposed soils to microwaves, both in the laboratory and in
the field, and found that the soil microflora were more
resistant to microwave energy than were laboratory-grown
cells. Also, they found that Azotobacter became susceptible
to microwaves when they were removed from their natural
habitat.
Notwithstanding the numerous reports of the last eighty
years, there is no evidence that natural cyst formation
occurs in the soil, although cyst formation in response to
organic compounds such as n-butanol is common (45, 47).
Many investigators (1, 4, 38, 41, 49, 50) have studied the I1
physiology and morphology of cysts in order to understanOd
their functions . In 1974, Vela (47) reported that
Azotobacter could survive for 15 years in dry soil in glass
containers. Later, Moreno et al. (30) reported that
Azotobacter could survive in dry soil in glass containers
for periods of time in excess of 24 years. Despite all the
evidence of the resistance of the laboratory grown cysts,
the survival form of Azotobacter in nature has not been yet identified.
Conclusion
Relying on previous work, it is valid to say that the morphology and survival form of bacteria of the genus
Azotobacter is quite dependent on the age of the culture and growth conditions.
Many different descriptions of the bacteria of the genus Azotobacter have been published, as well as of the species vinelandii. The morphology of this bacterium is the object of this investigation. The research presented here is designed to elucidate the morphology of these very interesting organisms when they are grown in a medium resembling their natural habitat.
The question regarding the form of A. vinelandii is associated with the growth condition. Small rod shapes and oval cells observed in nitrogen-free media can be, as mentioned before, a laboratory artifact. Growth in a 12
chemically defined, nitrogen-free medium is probably quite
different from that which occurs in the soil (28, 45). For
this reason, the question concerning naturally occurring
cells cannot be resolved unless Azotobacter can be directly
observed in their natural habitat.
It has been assumed that microorganisms which survive
in the soil have a morphology quite different from that of
microorganisms grown in the laboratory. There is no
evidence to support the assumption that laboratory grown
organisms resemble in any way those found in nature.
The thrust of this work is to describe the cells of A.
vinelandii as they appear in nature. For this purpose, a
medium composed of soil and water is used on the assumption
that this is more like the natural habitat of A. vinelandii
than is a nitrogen-free medium containing 0.5 to 2.0% (w/v)
glucose.
Soil dialysate supports the growth of A. vinelandii,
and it is evident that the nutrients required for the growth
of this organism are found in soil. The medium used for the
growth A. vinelandii is very likely to resemble the natural
habitat. It is composed of soil chemicals and water (28).
It is assumed that the results of this investigation will yield a clearer view of the morphology of A. vinelandii
as it exists in nature than what can be obtained by looking
at the literature or at cultures grown in laboratory media. 13
MATERIALS AND METHODS
Cultures and Media
The culture of Azotobacter vinelandii used in this
study was obtained from the stock culture collection at the
University of North Texas. It was maintained on slants of modified Burk's Agar medium, and periodically checked for
purity and strain confirmation by streaking on Burk's medium
and on nutrient agar.
All cultures used in this study had the same origin,
and all experiments were started with inoculum from cultures grown on Burk's medium plates incubated at 260C. The
composition of Burk's nitrogen-free medium is given below.
Components Concentration Grams/Liter Sucrose...... 20.0
KH 2 PO4 ...... 0.20
K2 HPO4 ...... 0.80
MgSO4 .7H20...... 0.20
CaCI2.7H20...... 0.08
FeSO4 .7H20...... 0.005
Na2 MoO4 .2H2 0...... 0.0003
Water...... ILit pH adjusted to 7.0 wiLh either K2 HPO4 or KH2 PO4
Soil Medium Culture
Soil samples were collected from various locations on 14
the campus of the University of North Texas when the ground
was dry. This was done by removing litter from the soil
surface and taking the exposed soil to a depth of one or two
centimeters. Several samples were collected and combined in
polyethylene bags. In the laboratory, the soils were broken
into fine dust with a mortar and pestle and passed through a
100 mesh sieve. Dialyzed soil medium was prepared by
pouring 15 g of the finely powdered garden soil into pre
boiled and washed dialysis tubing. Dialysis tubes were
closed by tieing the ends into secure knots leaving
sufficient empty space for air expansion when autoclaved.
Tubes with soil were then placed in 25 ml of distilled water
in Erlenmeyer flasks and left to sit for two hours. After
this time, the flasks were sterilized in the autoclave at
1210C for 20 minutes and left to sit for 24 hours.
Dialysis tubes containing the soil where then
discarded, bacterial inoculum from the cultures grown on
Burk's Agar plates was added to the clear dialysate.
Azotobacter vinelandii grown in this clear soil dialysate
was used to study the morphology as the cultures aged.
All cultures were grown on the shaker at 260C. Samples for microscopic observation were obtained from these cultures as
needed. Before each preparation was made for electron microscopy, samples of the growing cultures were streaked on plates of Burk's agar and nutrient agar. 15
Microscopic Observation
Light Microscopy Microscopic observation of A. vinelandii was performed with a light microscope at 100OX (oil immersion) in order to confirm the presence of cells in the culture. These were prepared for oil immersion light microscopy by obtaining a very thin film of culture between the slide and the coverslip.
Transmission Electron Microscopy Chemical fixation is the most extensively used method for preparation of biological tissues and cells for both transmission and scanning electron microscopy. Its main purpose is to preserve the structure of the cell with minimum alteration from the living state and to prepare the cells for subsequent treatment, including staining and exposure to the electron beam.
Employing the recommended method in Introduction to
Biological Electron Microscopy: Theory and Techniques(11), the cells were collected from the soil dialysate medium, centrifuged at 3000 rpm for 3 minutes, and pre-fixed with 2 percent glutaraldehyde in 0.2 M cacodylate buffer for 15 minutes. The strength of the fixative was set to minimize extraction and shrinkage of cells. Also, the use of 0.2 M cacodylate buffer was mainly to avoid fluctuations in the pH of the cells and the formation of acidic waves as the 16 fixative entered the cell. Following pre-fixation, the cells were centrifuged at 3000 rpm for one minute, the supernatant decanted and the pellet washed three times with the same buffer. The cells were then embedded in 1.5 percent agar. The resulting agar block was cut into small pieces with a sharp blade and transferred into a test tube for post-fixation. A 2 percent concentration of osmium tetroxide was used as the post-fixative. This preparation was washed in buffer then dehydrated by passing through 30,
50, 75, 85, 95 and 100 percent aqueous acetone before embedding in Epon 812.
Epon 812 resin was used in this experiment because it is more electron transparent and offers a higher contrast for embedded specimens than other embedding agents. Two solutions, A and B, were prepared and kept in the refrigerator until needed:
A B
Epon resin 812 62 ml 100 ml
Hardener DDSA 100 ml (dodecenyl succinic anhydride)
Hardener NMA - 98 ml (nadic methyl anhydride)
Accelerator DMP-30 1.5 to 3% [2,4, 6, tris (dimethyl aminomethyl phenol)
The recommended mixture (solution A and B) is as follows:
Solution A 7 ml 17
Solution B 3 ml
DMP-30 0.15 ml
Sections were cut with an MTC-6000 Ultramicrotome
(Sorvall Instruments, Norwak, Conn.) equipped with a glass knife. All sections were stained with uranyl acetate (2 g of uranyl acetate in 20 ml of 50% ethanol) for 15 minutes and with lead citrate (0.01 to 0.04 g of lead citrate in 10 ml of distilled water and 0.1 ml of ION NaOH) for another 15 minutes and examined with the electron microscope (JOEL
100CX, Tokyo, Japan). Pictures of the bacteria were taken, using Kodak SO-163 film.
Negative Staining
Negative staining was also used in this study because it produces a dark background that delimits particulate specimens. Cells were harvested, fixed, and washed as described above. The bacteria, which do not interact with electrons were mixed with phosphotungstic acid which scatters electrons very effectively. A 2 percent aqueous solution of phosphotungstic acid (PTA) was adjusted to the desired pH (4.5 to 7.2) with 5N KOH. The solution was then filtered (Whatman#1 paper) and stored in the refrigerator.
Prior to its use, the PTA stock was mixed with 0.1 percent aqueous bovine serum albumin (BSA), to avoid accumulation of stain and in order to obtain maxamal dispersion of 18 particulate matter including bacterial cells. A thin layer of the cell suspension with the electron-scattering substance was mixed and dried on a collodion carbon-coated grid. Such grids were examined with the electron microscope
(JOEL 100CX, Tokyo, Japan) and pictures of the bacteria were taken. Kodak SO-163 film was used in making prints.
RESULTS
The data obtained from these studies suggest the existence of a cell form of Azotobacter vinelandii not previously recognized. Bergey's Manual of Systematic
Bacteriology describes these bacteria as large, rod shaped organisms which become extremely pleomorphic in aged cultures (37). Studies carried out in this laboratory
(Cagle, Chang, Hartnett, Vela) agree in every particular with the descriptions given in Bergey's Manual. These, and other published and unpublished studies on the morphology of
A. vinelandii cells were based on cultures grown in chemically defined, nitrogen-free media.
The objective of this research was to show that cells of A. vinlandii grown in chemically defined, nitrogen-free media are probably not representative of this organism as it exists in nature. Since soil dialyzate is more like the natural habitat of these organisms (28), it is assumed that the cells grown in it more closely resemble the bacteria in 19 in nature. Cell morphology could not be determined in cultures less than four days old since cell densities were not sufficiently great to make usable pellet. Figure 1 shows flagellated cells typical of those observed in soil dialyzate cultures during the first 2 days of growth. The cells in Figure
1 are large, flagellated, and rod shaped. Peritrichous flagella is evident in the majority of cells and wet mount observation shows them to be motile. Figure 2 is a light photomicrograph of cells grown for two days in soil dialysate. Figure 3 also shows a flagellated cell. This cell is in the double cell form typical of A.vinelandii. No internal structures are evident in the cells in figure 1 and 3 because these are negatively stained preparations. The morphology of four day old cells of A. vinelandii in soil dialyzate is similar from that described in
Bergev's Manual of Systematic Bacteriology (37).
By the fourth day of culture in soil dialysate, cells become rounded and non-motile. Those in Figure 4 are seen only rarely but are shown here to illustrate the transition from long, rod shaped cells to the spherical form.
Figure 5 shows the typical morphology of cells grown in dialyzed soil for four days. Arrow number 1 points to the predominant cell form seen in four day old cultures of A. vinelandii in soil medium. Arrow number 2 shows a "hole" in the section. These holes are quite common in preparations of four day old cultures. The cell indicated by arrow number 3 is similar to the rounded cell form of A. vinelandii found in soil 20 dialysate and also in chemically defined nitrogen-free media.
The same cells are shown in Figure 6 for the purpose of illustrating the cell walls, or limiting membranes of these small cells. At different post-staining times, interior details of the small cells (Fig.7) becomes evident. A thick, rigid cell wall or outer barrier was observed. The internal structure of the small cells was fully revealed making it obvious that these small cells of 0.25 to 0.75 pm in diameter are fully formed and organized as are the other cells in these cultures.
The cell in Figure 8 shows a multi-layered cell wall. This cell wall is characteristic of Gram negative cells and is the same as seen in cells grown in Burk's chemically defined, nitrogen-free medium although the morphology of the cell is quite different.
Figure 9 shows the morphology of A.vinelandii cells grown on dialyzed soil for eight days. Arrow number 1 points to the predominant cell form seen in eight day old cultures, it must be noted that the arrow points directly at the limiting edge of the cell. This gives the appearance of a thick rigid cell wall.
Other cells in this picture show the same structure. Arrow number 2 points to "ghost" cells or possibly holes left in the sections when the cells that were there first fell off or were pulled off by the microtome knife. The third arrow points to the cell form not frequently encountered in the eight day old cultures. Figure 10 illustrates the interior details of cells in eight day old culture. The arrows point to the internal membrane 21 of the small cells. Figure 11 shows more detailed internal structures and also the difference between the external membrane and the inner one. The latter is plainly separated and pulled away from the outer one (arrow number 3).
By the sixteenth day of culture in soil dialysate, cells of
A. vinelandii tended to be different from those observed during the first eight days. Cells of widely varied morphology including spherical and oval shapes (Fig. 12) appeared. Arrow number 1 in Figure 12 points to an oval cell representative of a large part of cells in older cultures. Arrow number 2 points to a small cell, similar to the ones observed in young cultures. It was observed that as the culture ages, the number of small spherical cells decreases and the number of oval cells increases proportionately. The third arrow points to a bizarre cell. This bizarre cell is typical of A. vinelandii grown in dialyzed soil medium for 16 days.
Figure 13 shows cells of A.vinelandii in 16 day old culture.
These cells are considerably smaller than those seen in Burk's medium or in dialyzed soil medium during incubation periods of less than four days. The morphology of these cells appears to be the same as that of cells grown in Burk's medium. The arrow in this picture points to a separation between the cell wall and the cell membrane. 22
FIG. 1. Electron micrograph of negatively stained four day old cells of Azotobacter vinelandii grown in dialyzed soil medium. The micrograph shows peritrichous flagellation. Bar= 0.5 pm. 23
.4
- op
.*.... . 24
FIG. 2. Light microscopy of A. vinelandii cells grown for two days in dialyzed soil medium. 25 26
FIG. 3. Electron micrograph of negatively stained four day old cells of A. vinelandii grown in dialyzed soil medium. Micrograph shows double cell form and flagellation. Bar=0.5 Em. 27
4 28
FIG. 4. Electron micrograph of a thin section (60 to 90 nm) of four day old cells of A. vinelandii grown in dialyzed soil medium. The micrograph shows long rod shaped cells. Bar=0.5 gm. 29
0
*
b w 30
FIG. 5. Electron micrograph of a thin section (60 to 90 nm) of four day old cells of A. vinelandii grown in dialyzed soil medium. Arrow number 1 points to an electron dense small cell of 0.25 pm diameter. Arrow number 2 points to a "hole" in the supporting medium. Arrow number 3 points to a cell of 1.2 pm diameter. Bar=1.0 gm. 31
*.1' p 3 r 32
FIG. 6. Electron micrograph of four day old cells of A. vinelandii grown in dialyzed soil medium. Arrow number 1 points to the thick cell wall of the small cell. Arrows number 2 and number 3 point to the remains of the limiting membrane left behind when the cell was removed by the microtome knife. Bar=1.0 p m. 33
1
{t 34
FIG. 7. Electron micrograph of a thin section (60 to 90 nm) of four day old cells of A. vinelandii grown in dialyzed soil medium. Arrows point to the thick rigid cell wall. Bar=1.0 pim. 35
s
2 4 4$
) I 4.. * sri r ej. t 36
FIG. 8. Electron micrograph of a thin section of a four day old cell of A. vinelandii grown in dialyzed soil medium. Arrow points to the multi-layered cell wall. Bar=1.0 m. 37 38
FIG. 9. Electron micrograph of a thin section (60 to 90 nm) of eight day old A. vinelandii cells grown in dialyzed soil medium. Arrows number 1 point to the thick limiting edge of the predominant cell type. Arrows number 2 point to the "ghost" cell. Arrow number 3 points to a cell form not frequently encountered. Bar=1.0 pm 39
p .70, U 4 40
FIG. 10. Electron micrograph of a thin section (60 to 90 nm) of eight day old cells of A. vinelandii grown in dialyzed soil medium. Micrograph shows internal structures of the cells. Arrows point the internal membrane of the cell. Bar=l.0 Lm. 41
0wo 42
FIG. 11. Electron micrograph of a thin section (60 to 90 nm) of eight day old cells of A. vinelandii grown in dialyzed soil medium. Micrograph shows internal structures of the cells. Arrow number I points to the external membrane of the cell while arrow number 2 points to the internal membrane of the cell. Arrow number 3 points to a separation space between the two membranes. Bar=1.Q gpm. 43
6* 0 44
FIG. 12. Electron micrograph of a thin section (60 to 90 nm) of 16 day old cells of A. vinelandii grown in dialyzed soil medium. Arrow number 1 points to an oval cell, representative of a large population of cells in the culture medium. Arrow number 2 points to a small spherical cell. Arrow number 3 points to a "bizarre" cell. Bar=1.0 pm. 45
.4p4 LMr to401 i) *AI6
Mun0i. 46
FIG. 13. Electron micrograph of a thin section (60 to 90 nm) of 16 day cells of A. vinelandii grown in dialyzed soil medium. Arrow points to the separation of the cell wall.Bar=0.25 gm. 47
t
id 48
DISCUSSION
This study was designed to observe the cell morphology of Azotobacter vinelandii grown in dialyzed soil medium.
Observations of the cells were performed using electron microscopy. Cells of A. vinelandii were grown in the soil medium for a period of 4, 8 and 16 days. Glutaraldehyde and osmium tetroxide were used as fixatives following the procedure described in the Materials and Methods. Sections were cut to a thickness of 60 to 90 nm and observed under a
transmission electron microscope.
Extensive investigation has been focused on the morphology.of A. vinelandii cells. Despite this fact, only very few studies were concerned with the cell morphology of
this organism in its natural habitat, the soil. Since soil
dialysate is more like the natural habitat of the organism
(28), it is assumed that the cells grown in it are similar
to those of the bacteria in nature.
The micrographs presented here show morphological
differences between the cells grown in chemically defined
nitrogen-free media and those grown in the soil medium. It must be noted however, that no other known type of microscopy has ever led to reports of cells like the ones
encountered in this study.
Negative staining of cells from a four day old culture
of A. vinelandii, showed peritrichous flagellation and 49 morphology similar to that of cells grown in chemically defined media (Figs. 1, 3). These cells also fit the description of A. vinelandii given in Bergey's Manual of
Systematic Bacteriology (37). Thin sectioning of the cells revealed morphological characteristics also consistent with those of cells grown in chemically defined nitrogen-free medium and described in Bergev's Manual.
Figure 5 shows that the majority of cells were small and spherical measuring some 0.25 to 0.75 .m in diameter.
These are light refractile (16) and also refractile to electrons in the electron transmission microscope. They are the predominant cell form in four day old cultures and the size is fairly constant in the range from 0.25 to 0.35 pn.
Many electron micrographs show a separation between the outer wall (cell wall) and inner limiting membrane (cell membrane). It appears that the outer wall is hard (Figs. 6,
7, 9, 10, 11), and probably complex in both chemical and structural composition (Fig. 13). It is not difficult to imagine that these are the survival forms of the Azotobacter in nature since they meet the criteria described by Vela in
1964 (40), Vela and Wyss in 1965 (42), and Lopez-Gonzalez and Vela in 1981 (28). The small round cells seen at four days in soil dialysate cultures continue to grow and change as previously described (Figs. 4, 5, 6, 7, 9, 10, 11, 13).
By the eighth day of culture, the average cell size ranges from 0.5 to 0.75 pm and the cells appear elongated and 50 pleomorphic (Figs. 9, 10, 11).
Holes (Figs. 5, 6, 9) in the sections were frequently encountered in the four day old cultures as well as in the eight day old cultures. These are cell-shaped holes which often contain fragments of cell material attached to their periphery (Fig. 9). The constancy of this observation strongly suggests that such holes are the result of cells
"lifted off" by the microtome knife and removed from the embedding material leaving a hole in the section. All the holes observed were the exact shape of the cells present in the micrographs examined; an observation which supports the idea that the cells were "bumped" out of the supporting medium because they were too hard for the glass knife.
Since some cells were sectioned, it must be assumed that the glass knife could cut them into sections but only under certain conditions. The following sketch helps to explain this: 51
Resulting section
Knife
The process of ultrathin sectionning of plastics is described as a splitting of a section from the surface of the block, the knife acting as a wedge. 52
Knife stroke 1 Resulting thin section
Knife stroke 2
Knife stroke 1 Cell is I ed off* Resulting hole
Knife stroke 53
Micrographs of the eight day old cultures showed the small round cells observed in the four day old cultures and also larger cells similar to those grown in chemically defined, nitrogen-free media. The outer wall of the small round cells appeared thinner and less. This implies that he outer wall is not as hard as at four days.
Micrograph of 16 day old cells showed spherical and oval cells. These cells did not vary much in size and ranged between 0.75 and 1.5 ptm in diameter. It was noted that as the culture aged, the number of small spherical cells seen in the younger cultures decreased. When sizes were compared, it was noticed that the small cells with dense outer walls were approximately 3/4 the size of large cells. The large cells are similar to those grown in chemically defined media. It is assumed that the small cells observed earlier continue to grow in the presence of water and eventually become full grown, mature cells. One can assume that in nature, the small cells would remain as such in the absence of water but would continue growing in its presence.
The objective of this work, to examine the morphology of A. vinelandii in a soil medium, was accomplished. A new cell form was described for the first time although its
existence had been postulated by Vela (45, 47), Vela and
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