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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

KH2PO4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .0.20

K2 HPO4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.80

MgSO4 .7H20.....................................0.20

CaCI2.7H20......................................0.08

FeSO4 .7H20......................................0.005

Na2MoO4 .2H20...................................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

2

)4..

4 4$

I

* sri

tr

ej.

s

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

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.

Knife

Knife stroke 1

Knife stroke 2

Knife stroke 1 Cell is I ed off*

Knife stroke

Resulting thin section

Resulting hole

52

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

Wyss (41, 42) and Lopez-Gonzalez and Vela (28).

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57

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