ISOLATION AND PARTIAL CHARACTERIZATION

OF A BACTERIUM

WHICH GIVES OFF FILTRABLE CELLS

APPROVED:

Major Professor

f- „ •/_ jTu Minor Professor

A /Q \ C L

a X A J U Director of the Department! of Biology

Dean of the Graduate School

QJ.

Siu, Henry Hon-wan, Isolation and Partial Characterization

of a Bacterium Which Gives Off Filtrable Cells. Master of

Science (Biology), May 1973, 50 pp., 6 tables, 2 illustrations^

literature cited, 50 titles.

The present study was on the isolation of a bacterium

capable of producing filtrable forms which passed through the

0.45H filter membrane and regeneratedthe parental form; the

testing of the true filtrability of the isolate; the relation-

ship between growth of the isolate and appearance of filtra-

bility; partial characterization of the isolate and visuali-

zation of the elements that passed through the membrane filter

with electron microscopy and shadow casting technique.

This is a report of a bacterium which yielded filtrable

forms. This bacterium was isolated from a culture of Azotobacter

vinelandii passed through 0.45y filter membranes. The isolated

bacterium was tentatively identified as a species closely

resembling Pseudomonas reptlvora. The data showed that the

production of filtrable forms was independent of mechanical

forces but was related to culture age. The effective size

of the filtrable elements was, between 0.30^ and 0.45H.

Electron microscope studies revealed that the elements which

passed through the filters had various forms, possibly due to

/') .v /• / /// is !

high cell plasticity. The exact mechanisms which enabled the

filtrable forms to pass through filters were not determined.

ISOLATION AND PARTIAL CHARACTERIZATION

OF A BACTERIUM

WHICH GIVES OFP FILTBA3LE CELLS

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OP SCIENCE

By

Henry H. ¥. Siu, B. S,

Denton, Texas

May, 1973

TABLE OP CONTENTS

Page

LIST OP TABLES It

LIST OP ILLUSTRATIONS v

Chapter

I. INTRODUCTION 1

II. MATERIAL AND METHODS 10

Organisms Media Filtration of Cultures Culturing of Piltrates Piltrability Determination at Different Stages of Growth

Isolation of the Piltrable Elements

III. RESULTS 17

II. DISCUSSIONS 34

V. SUMMARY 46

iii

LIST OP TABLES

Table Page

I. Composition of Burk's Nitrogen-free Salts Solution 11

II. Appearance of Filtrable Oells in Cultures of the Isolated Bacterium ........... 23

III. Filtrability of Azotobacter vinelandil, Serratia marcescens, and the Filtrable Organism 24

IV. Filtrable Forms in Sonified and Unsonified Cultures 28

V. Presence of Filtrable Forms in Filtrations Using the Wall Vacuum and Gravitation 29

VI. Characterization of the Filtrable Organism 31

iv

LIST '0? ILLUSTRATIONS

Figures Page

I. Growth curve of culture containing filtrable bacteria measured, by viable cell counts and by optical density 21

II. Percentage of samples containing filtrable forms shown as a function of culture phase or age 22

CHAPTER I

INTRODUCTION

The existence of a ftitrable form of bacteria was first

reported early in the 1900*s (15). These bacteria were shown

to be viable and able to pass through filters which held back

normal bacterial cells. Early workers claimed that during

the growth cycle of certain bacteria, there were stages in

which they dissociated into minute viable fragments which were

different not only in size but also in morphology from the

usual form (16, 17). The subsequent growth of these fragments

resulted in the reappearance of the usual cell form. This

idea was shared by many workers who offered much evidence to

support it, but it was also forcefully rejected by others.

Among those early workers who supported the idea of filtrable

forms of bacteria were P. Lohnis and N. R. Smith, who claimed

that all bacteria live in an organized and also in an amorphous

stage; the latter they called the 1symplastic stage'1 (26). In

their theory, at a certain stage in the growth cycle of a

bacterium, the living matter in separate cells would mix

together by the disintegration of the cell wall to form a

symplasm. The symplasm would later develop into regenerative

units which increased in size and formed regenerative bodies

which they called gonidia. These transient forms, they claimed,

were smaller than the normal bacterial cell. These bodies would

then grow and stretch out to the full cell size of the normal

bacterial form (25).

It

Lohnis and Smith based their conclusions on many obser-

vations which they made on cultures of various species of

bacteria, including such diverse forms as Bacillus subtilis,

Stretococcus lactis, Lactobacillus bulgaricus, and,- especially

members of the genus Azotobacter.which became their favorite

organism for the demonstration of filtrable forms in the life

cycles of bacteria.

Jones observed the same changes an,d agreed with the concept

of a symplastic stage in the life cycle of Azotobacter (19)

but he failed to confirm filtrability. Along the same line of

investigation -was. the work of Kahn (20) on acid-fast bacteria.

He observed the development of small numbers of cells and of

single cells of tubercle bacilli in microdroplets of growth

medium and confirmed the occurrence of the filtrable symplasmic

stage in these organisms (20, 21).

During the same period, other experiments designed to

test the phenomenon of filtrable bacteria were based on experi-

ments performed with Salmonella typhosa, spirochetes, and acid-

fast bacteria (22). Usually, cultures were filtered through

Berkefeld or Chamberland candles and the filtrates tested for

the presence of living organisms either in animals or on lab-

oratory media. These experiments indicated that some bacteria

were filtrable^ but the results were not conclusive, since some

of the cultures from the filtrates did not always give rise to

the original type of bacterium used. Furthermore, there were

also many well performed experiments which gave unequivocally

negative results (28, 3, 27).

In 1931, Hadley,:Delves, and Elimek (18) were the first

to induce dissociation in the Shiga bacillus by treatments

such as aging, metabolic products accumulation, temperature

changes, and pH changes. Ihsy were able to isolate filtrable

forms which formed minute colonies designated G type colonies.

Four years later, Klieneberger demonstrated for the first

time the existence of L forms, bacteria with faulty cell walls,

in Streptobacillus moniliformis (2). She implied that the &

type colonies of Hadley and her coworkers were intermediate

forms between the bacillary structure and the L forms (22).

Klieneberger did not regard the 1 forms as mutants genetically

different from the baeillary forms from which they were

obtained. Later workers such as Brown and Hunemaker (2),

Dawson and Hobby (4), and Dienes (6) supported the validity of

the growth phase phenomenon and the existence of L forms.

Ten years after the discovery of L forms, observations by

Dienes (6, 8) and Dienes and Smith (14), with the use of the

constant temperature microscope stage, showed the development

of the L elements and their transformation into bacilli. Hence

they established evidence that the L forms are a phase or

morphological stage in the growth of the organisms studied by

Kleineberger (22). Following this, Dienes (7) further demons-

trated the L phase of bacteria in a large variety of organisms

such as E. coli, Flavobacterlum, Haemophilus Influenzae (7-11),

Proteus vulgaris (9> 10), Bacillus (12), and Clostridium (13).

L phase mophology was also reported in Salmonella and Shigella

by" Klieneberger (22).

In her original description of the L phase, Klieneberger

described the formation of granules within the bacterium,, which

later fused to form the L bodies. These bodies could transform

directly into bacilli or could reproduce their own kind,as seen'

in the stabilized L forms. She claimed that the amorphous It

stage of symplasm observed by Lohnis and Smith (25, 26) corres-

ponded to the L forms but she failed to demonstrate the filtrable

n

gonidia included in lohnis .life cycle cf Azotobacter. After

the recognition and acceptance of L form as a phase in the

growth cycle of bacteria, the idea of filtrable gonidia capable

of reverting to bacterial form was abandoned until the recent

works of Bisset and Hale (1). They described the production

of gonidia arising from the rupture of mature vegetative

cells in cultures of Azotobacter• The gonidia described by

these authors were capable of reverting to typical Azotobacter

cells. Similar findings were reported by Lawrence (23) in

cell-free filtrates of aged cultures of Rhlzobium and Azotobacter.

Further evidence of these gonidia was put forth by Yan Schreven

(29), who studied the morphology of penicillin-treated cells of

Azotobacter by carbon shadow casting techniques and electron

microscopy. All these workers suggested that gonidia were not

identical but closely resembled the L forms previously described.

They appeared to possess adequate material resources to revert

to the normal form if conditions were suitable? this resembles

the reproductive method of the asexual spores found in algae

and fungi.

In 1950, Ward claimed that Azotobacter agile could develop

into long filaments which would swell at one or more points into

balloon-like forms, spheres or, bud-like swellings, and might

Ai+.hAT dosrenerate or reproduce normally-shaped cells by a

pinching off of these swollen ends (30). The descriptions

given by Ward are not unlike those generally accepted for the

genus Saccharomyces in many respects.

The present investigation deals with the isolation of a

bacterium from cultures of Azotobacter vlnelandli and its

identification. This bacterium was isolated in the filtrate

(0.45H) from cultures of Asotobacter vlnelandli. The filtrable

bacterium grew up in pure culture after filtration. Since then,

repeated experiments show the true filtrability of this organism

and attempts have been made to identify and visualize it in its

filtrable form.

CHAPTER BIBLICGRAPHY

1. Bisset, K. A. and C. M. Hals, "The Cytology and. Life Cycle of Azotobacter chroococcam," J. of G-en. Microb., 38 (Aug. 1964), 329-338.

2. Brown, T. and J. C. Hunemaker, Hat-bite Fever, A Review of the American Cases wlth Revalution of Ethiology« Bull John Hopkins Hosp., 70 (1942), 201-327.

3. Cooper, F. B. and S. A. Petroff, "Filtrable Forms of the Tubercle Bacillus," Infections Diseases, 43 (1928), 200-214,.

4. Dawson, M. H. and G. L. Hobby, Pleuropneumonla-llke Organisms as a Variant Phase of Streptobaclllus Moniliformis, 3rd. Internal, Oongr. Microbiol.Abstr. 1939, 177-178.

5. Dienis, 1. "A Peculiar Reproductive Process in Colon Bacillus Colonies," Proc. Soc. Exp. Biol. Med., 42 (1937), 773-778.

6. Dienes L., "Further Observation on the L organism of Klieneberger," Proc. Soc. Exp. Biol. Med., 42 (1937), 778-779.

7. Dienes L., "The Significance of the Large Bodies and the Development of L type of Colonies in Bacterial Cultures," LL Bact,, 44 (19Vr), 37-73.

8. Dienes L., "Further Observation on the L Organism of Klieneberger," Proc. Soc. Exp. Biol. Med., 39 (1938), 365-367.

9. Dienes, L. "Reproductive Processes in Proteus Cultures," Proc. Soc. Exp. Biol. Med., 63 (1946), 265-270.

7

8

10. Dienes, L., "Complex Reproductive Processes in Bacteria," Cold Spring Harbor Symposia on Quant. Biol.. XI (1946), 51-59.

11. Dienes, L., "Isolation of Pleuropneumonia-like Organisms from influenzae with the Aid of Penicillin," Proc. Soc. Exp. Biol. Med., 68 (1949), 589-590.

12. Dienes, L., "Isolation of L Type Colonies from a Gram Positive Spore-tearing Bacillus," Proc. See. Exp. Biol. Med., 68 (1949), 589-590.

13. Dienes L., "Isolation of L Type Cultures from Clostridia," Proc. Soc. Exp. Biol. Med., 75 (1950), 412-415.

14. Dienes, L. and ¥. E. Smith, "Reproduction of Bacteria from the Large Bodies of Bacteroides funduliformis," Proc. Soc. Exp. Biol. Med., 51 (1942), 297-298.

15. Gloyne, S. R., Glover, R. E. and A. S. Griffith, "Experi-ment to Determine Whether there is a Filtrable form of the Tubercle Bacillus," 3^ Path Bact., 32 (1929), 775-785.

16. Hadley, P., Microbio. Dissociation, "The Instability of Bacteria species with Special Referance to Active Dissociation and Transmissible Autolysis," Infect. Diseases, 40 (1927), 1-312.

17. Hadley, P., "Further Advances in the Study of Microbio. Dissociation," Infections Diseases, 60 (1937), 129-192.

18. Hadley, P., E.'Delyes, and G. iClimek, "The Filtrable Forms of Bacteria," J. Infect. Diseases, 48 (1931), 1-159.

19. Jones, D. H., "Further Studies on the Growth Cycle of Azotobacter," J. Bact., 44 (1920), 325-333.

20. Kahn, M. C., "A Developmental Cycle of the Tubercle Bacillus as Revealed by Single-cell Studies," Am. Rev. Tuberc., 20 (1929), 150-200.

21. Kahn, M. G., "A Growth Cycle of the Human Tubercle Bacillus as Determined by Single-cell Studies," Tubercle, 11 (1930), 202-217.

22. Klieneberger U. E., "Filtrable Forms of Bacteria," Bact. Review, 15 (1951), 81-88.

23. Lawrence, J. C., "Filtrability of the Swarmers of Rhizobium and Azotobacter," Nature, Vol. 176, Ho. 4491, (1955), 1033-1034.

24. Lewis, I. M., "Dissociation and Life Cycle of B^ Mycoides," Bact., 24 (1932), 381-420.

If

25. Lohnis, P., "Studies Upon the Life Cycles of the Bact.," Mem. Hat. Acad.,Sci., 16, 2nd Memoir, Washington (1921), 1-335. t*

26. Lohnis, F. and I. R. Smith, "Life Cycles of Bacteria," J. ACT. Res., 6 (1916), 675-720.

27. Pinner, R. and M. Voldrich, "The Disease caused by Filtrates of the Tubercle Bacillus Cultures," Am. Rev. Tubere., 24 (1931), 73-94.

28. Soltys, M. A. and A, W, Taylor, "The Filtration of Mycobacterium tuberculosis and Mycobacterium stercusls through C-radocol Membranes," Path Bact., 56 (1944), 173-180.

29. Van Schreven, D. A., "Effects of Penicillin on the Morphology and Reproduction of Azotobacter chroococcum," Antonie Van Leeuwenhoclc J. Microb. Serol», 32 (Ja'& 1966), 67-93.

30. ¥ard, C. B. Jr., "The Effect of nutrition and Toxic Agents on the Cytology on an Azotobacter Species," Masters' Thesis, Oklahoma A & M College, 1950.

31. Weinberger, H. J., S. Madoff and L. Dienes, "The Pro-perties of L forms Isolated from Salmonella and the Isolation of L forms from Shigella," J. Bact., 59 (1950), 765-775.

CHAPTER II

MATERIALS AID METHODS

Organisms

The organism used in this study was isolated from an

Azotobacter culture and was tentatively identified as a strain

of Pseudomonas reptlvora according to Bergey's Manual of

Determinative Bacteriology. The organism used as an indicator

of the integrity of all filter membranes used was a strain of

pigment—producing Serratia marcescens obtained from the stock

culture collection of the Biology Department, North Texas State

University. For purposes of comparision, Azotobacter vlnelandll

ATCO 12837> the culture from which the bacterium studied was

isolated, was also used. All cultures were incubated on a

rotary shaker at moderate speed at 30° C unless otherwise stated.

Media

Serratia marcescens was grown in Nutrient Broth (Difco

Laboratories, Detroit, Michigan).

The organism under investigation was grown in either

Trypticase Soy Broth (Difco) or Nutrient Broth. When solid

10

11

media were required, agar was added to the liquid medium to a.

final concentration of two percent (¥/¥),„

Azotobacter vinelandil ATCC 12837 was grown in Burlc's

nitrogen-free medium (5) supplemented with glucose. The com-

position of Burk's nitrogen-free salts solution is given in

Table I.

TABLE I

COMPOSITION OF BtJRK'S NITROGEN-FREE SALTS SOLUTION

Component Concentration

(Grams/Liter)

KHgPO^ : .. . 0.2

KgHPO^ 0.8

MgS04 . 7 H20 0.2

CaClg . 2 H20 . 0.085

FeSO^ . 7 HgO . 0.005

Na2MQ04 . 2 E20 0.0003

An aqueous glucose solution was sterilized separately

and added to the sterile salts solution to a final concentration

of 1% glucose (W/V) to obtain the growth medium

cultivation of Azotobacter.

used for the

The above complete medium was supplemented with two

percent agar when solid media were required. All media used

were prepared by sterilization at 121°C for fifteen minutes (2).

12

Filtration of Cultures

All filtrations were performed with MF-Millipore mem-

brane filters (Mlllipore Corporation, Bedford, Mass.) with a

pore size of 0.45 unless otherwise stated. Two types of

filter holder units were used: a sterile plastic disposable .

type (Nalgene Sybron Corporation, Rochester, New.York) and a

glass type (Millipore XX 5 047 00). When glass filter holders

were used, membrane filters were sterilized separately in

covered petri dishes filled with distilled water. The sterile

membrane filters were then placed on the holder with sterile A

forceps. In all cases, disc prefilters (Millipore type Ap 25)

were used to minimize clogging. For all filtrations 10 ml

of the culture to be filtered were always mixed with 1 ml of 7

a culture of Serratia marcescens containing 10 cells per ml

and the filtrations were carried out using the water aspirator

or the wall vacuum.

Culturing of Filtrates

Filtrates were collected and cultured by one of two methods:

in one method (glass filter holders), the 10 ml of filtrate

were collected aseptically into a 250-ml stoppered flask with

30 ml of new sterile media. This was then incubated and aerated

by shaking. The other method (disposable unit) involved

13

pouring 30 ml of sterile media through the same filter used

for filtering the culture; and then the whole apparatus was

incubated and aerated by continuous shaking on the rotary

shaker.

Filtrability Determination at Different Stages of Growth

Viable Gell Counts

Viable cell counts were performed by using spread plates

and sterile distilled water dilution blank,s. Triplicate

plates were spread for each dilution, incubated for forty-

eight hours,and counted (4). Any plate on which Serratia

marcescens colonies appeared was assumed to have resulted from

a sample passed through a damaged filter membrane, and there-

fore the result fudged to be invalid. The criterion..for

flltrability rested entirely on the fact that filtrable bacteria

should pass through filter membranes capable of retaining

Serratia marcescens.

Optical Density Measurements

The optical density of cultures was measured at a wave-

length of 520 nm using a Bausch and Lomb Spectronic Twenty

spectrophotometer; optical density — cell- number correlations

were established previously by viable cell counts using serial

dilutions and spread plates.

14

Stages of PIltrability

Filtrability was determined by filtering aliquants of

cultures at varying times during the growth curve, and

culturing the filtrates. Growth curve stages were obtained by

viable cell counts.

Isolation of the Filtrable Elements

Collection by Ultraoentrlfugatlon •

Five hundred milliliters of culture filtrates were obtained

from various cultures under study by the method previously des-

cribed for obtaining 10 ml of filtrates. These filtrates•were

loaded immediately and aseptically into sterile 75~®1 polyal-

lomer ultracentrifugation tubes, capped, and prepared for

centrifugation in a Beckman Model L3-40 ultracentrifuge with

a Southwest $42 angle head rotor. The filtrates were centri-

fuged for two hours at a force of 100,000 X g at a temperature

of 5°C. The supernatants were poured off carefully, each

residue was resuspended in a drop of supernatant, and all super--

natants then pooled. All manipulations were performed at 5°C •

or less in order to retard growth.

15

Electron Microscopy of the Filtrable Elements

Tiie residue obtained by centrifugation was washed twice

with phosphate buffered saline (PBS) and pelleted again by

centrifugation. The washed residue was resuspended anew in PBS,

and, transferred to two hundred mesh copper grids (Ernest

Fullam Incorporation, Schenectady 1, Sew York) previously coated

with collodion and prepared for chromium shadow casting (3).

The grids so obtained were .examined with an EGA EMU 3G- elec-

tron microscope at an initial magnification of 11,500 X*

Characterization of the Filtrable Bacteria

Identification of the bacterial growth resulting from

the filtrable forms was performed according to the criteria

given in Bergey1s Manual of Determinative Bacteriology (1).

These included morphology, culture characteristics, fermen-

tation tests, biochemical tests, and staining reactions.

CHAPTER BIBLIOGRAPHY

1. Breed, R. G.» T. D. Murray and I. 5. Smith, Berge.y 's Manual of Determinative Bacteriology, 7th ed., Baltimore, The Williams & Wilkins Co., 1957.

2. Collins, C. H., Microbiological Methods, Butterworth & Co., London, 1964.

3. Dawes, C. J., Biological Techniques in Electron Microscopy, Barnes & Noble Inc., N. I., 1971.

Quanti-4. Pelczar, M. Jr., P. A. Hansen and W. A. Konetzka, •tative Bacterial Physiology Laboratory Experiments, Burgers Publishing Co., Minneapolis, 1955»

5. Wilson, P. W. and S. G. Knight, Experiments in Bacterial Physiology, Burgers Publishing Co., Minneapolis 1952.

16

CHAPTER III

RESULTS

The isolated filtrable organisms under investigation

grew very well both in Tryptica.se Soy Broth and in Nutrient

Broth. The cells were usually singular or were joined at the

ends to give paired cells. In several instances, short chains

of three or four organisms were also observed. This bacterium

grew very well aerobically. In young cultures, the cells,

appeared as slender rods with a mean cell size of 1 x 2 pm as

determined by visual measurements with a microscope fitted

with an occular micrometer. Cell size and morphology appeared

identical to those of pseudomonas raptivora and no "gonidia"

or other small bodies could be detected even after extensive

examination by transmitted light or phase contrast microscopy.

On solid media, the filtrable organism formed small, '

round, and smooth colonies after 24 hours of growth at 30°0.

When grown in liquid cultures, the cells tended to stick togeth-

er and form aggregates. The cells produced a yellowish-brown

pigment and also produced a putrid smell as they aged. The

17

%

I

Plate I

" ' f"<' \-,-ft.T-* ;% s *^V-%'a?V£-'". - ' i -*v

•m

• V

/

<0*

" : - ' ^ *&*;> %6?\ - j i g - - m - f '

Sight-hour-old Trypticase Soy Eroth culture of bacteria which yield filtrable forms; 2,000 X

18

19

pigment produced was soluble in water but not in chloroform.

Plate I is a photomicrograph of an eight-hour culture grown

in Trypticase Soy Broth.

When cultures aged, autolysis of the cells was commonly

observed microscopically, and the media bacame viscous.

Microscopic"examination of these aged cultures revealed cell

fragments and debris.

As the cells aged in solid or in liquid culture, they

also produced extracellular debris and slime. Very often,

cells were seen to attach to large pieces of this material. If

large volumes of old cultures were filtered, the slimy extracel-

lular material tended to clog the filters and made filtration

difficult and sometimes impossible. Plate ..-II is a photograph

of an old culture and shows the extracellular material (indi-

cated by arrows) and entrapped cells. The cells were gram

negative and the typical mor^ology was as shown in Plates I and

II.

In liquid cultures, these bacteria could grow to a

9 concentration of 10 cells per milliliter in a period of three

4

days from an inoculum of 5 x 10 cells per milliliter, figure I •

shows the growth curve of the bacteria in Trypticase Soy Broth

cultures, measured by viable cell counts and by optical density.

20

'%':l\^ «T 3-:; ''^X1^'

• * * /

f A -- j*.

t".

VJt J> 'i Vs.

^'JS mt «» ^

% - : . * " *«

*-—*' , """'"

W ; ir-_;

#' ' g8i ;

t \ « • " t'V>v

- : :

Plate II

Extracellular sline ( —a ) In old cultures of which yield filtrable forms; 2,000 I. The slime is Indicated

by arrow. " • '

21

O •©

rS p

w

TIME IN HOURS

Figure I

Growth curve of culture containing filtrable bacteria measured by viable cell counts g——Xand by optical density©-—~©as a function of time.

22

Microscopic observation indicated cell lysis at the onset

of the stationary phase of growth in over two hundred filtration

experiments examined. Approximately' eighty percent of these

cultures yielded filtrable forms when tested. (Table II)

In every case, the results were assumed to be valid because

of retention of Serratia marcescens by the filter membranes.

On the other hand, the filtrable form of organism under study

passed through the 0 .45 | i filters and yielded subcultures

indicative of normal bacterial cells. However, in almost all

of these experiments, the filtrate had to be incubated for

more than forty-eight hours before appreciable growth appeared.

When the filtrate was first collected, it was clear, and

phase contrast microscopy revealed no whole cells or cell

fragments. These two observations indicate that the total

number of cells which passed through the filters in each

culture tested was actually very small.

The next series of experiments was designed to determine

the effective cell size of the filtrable bacterial form.

Azotobacter and Serratia were used, as control organisms as

previously described. Azotobacter was used as a control because

of the previous history of filtrability (1), and also because

of the great variability of its size. The results obtained

23

TABLE II

APPEARANCE OP FILTRABLE CELLS IN CULTURES OP THE ISOLATED BACTERIUM#

Experiment Number a/b

1 15/20

2 19/20

3 17/20

4 16/20

5 10/20

6 16/20

7 20/20

8 18/20

9 19/20

10 17/20

*A11 cultures were tested at the end of 40 hours growth at 30°C.

a. Number of cultures yielding filtrable forms. b. Number of cultures tested.

24

TABLE III

FILTRABI1ITT OP AZOTOEAOTER VINEMNDII. SBRRAT'IA MARCESOENS, AHD THE FILTRABLE ORGANISM

Organism Pore Size of Membrane in (J

Organism 0.65 0.45 0.30 0.22

Azotobacter vinelandii - - - -

Serratia marcescens + - - -

Filtrable isolate + + + -

According to Table III, the filtrable organism has a size

of less than 0.3Qp in effective diameter. However, it is not

possible to say that the cells had a size between 0.22 and

0.3^ssince millipore filter membranes can retain particles of

size less than the pore size owing to electrostatic charges

acquired during filtration (Milipore filter Corp., Bedford, Mass.).

It can be stated that the effective particle size is probably

0.22 to 0.3 [i .

To determine whether the filtrable forms were produced

only during certain stages in the growth cycle of the mother

cultures, these were sampled by filtration at different phases

of growth. The results obtained are represented in Figure II.

S s.o

60 60 W>

time lift m m

Figure II

Percentage of samples containing filtrable forms shown as a function of culture phase or age. Each bar represents the percentage of positive cultures obtained from 20 samples of culture tested at the time indicated; the numbers show this ratio•

$

m

%jn

5 •o m ut

Q

35.

3 0* r* r*

in

26

The data in Figure II show clearly that filtrable forms

are found in all stages of the growth cycle in the majority

of cultures tested, but that a higher proportion of cultures

contain filtrable cells near the end of the log phase of growth.

This time corresponds very well with the onset of autolysis of

the majority of cells in the culture. It is not implied that

these data offer quantitative criteria, only that a higher

portion of culture aliquants give filtrable forms in older

cultures.

It was observed also that aged cultures were more difficult

to filter* and it was assumed that this was due to the presence

of large amounts of extracellular material produced by the

growing cells. In order to minimize interference from this

material, cultures were often diluted by shaking with sterile

media or phosphate buffered saline. This treatment increased

the percentage of recovery of filtrable forms from samples

noticeably. Numerous attempts to filter cultures of more than

120 hours of age did not give positive results. It is inferred

from this that filtrable cells are bound by the cell lysate

or extracellular material and retained on the filter membranes

by this means. It is suggested that neglect of this consider-

ation will give spurious results.

27

In view of the fact that filtrability seemed to coincide

with autolysis, young cultures -sere sonified and filtered to

determine whether ftitrable elements were present inside the

cells at all times during the growth curve but released only

when the cells lysed, or, to the contrary, whether they were

produced only at certain stages during growth. This question

was tested by breaking the cells mechanically by ultrasonic

waves and filtering the lysed cells so obtained. Cultures

tested were taken at various intervals during the first forty

hours of growth as measured by optical density, and sonified

with a Branson Sonifier (Branson Instrument Corporation,

Stamford, Conn.) for fifteen minutes with an audio frequency

current of 9.2 to 9.3 amperes at 18,000 cycles per second.

Control experiments were performed using cultures not exposed

to ultrasonic waves. The cells were then filtered and cultured

separately, as previously described. The results are shown in

Table IV.

According to the results shown in Table IV, ultrasonic

disruption of the cells did not show any significant difference

from controls with regard to the presence of ftitrable forms.

Thus it appears proper to say that this organism was filtrable

or could produce filtrable elements only in cultures older than

28

TABLE I?

FILTRABLE FORMS IB SONIFIED AND UNSONIFIED CULTURES

Age of Culture in Hours

Presence of Filtrable Forms Age of Culture in Hours Sonified Unsonified

5 - -

10 - -

15 - -

20 - -

25 - -

30 + -

35 + •f

40 + +

thirty hours of age. This corresponds to the stationary growth

phase, as shown in the growth curve in Figure I. The data in

Table IV also show that the filtrable forms are not inactivated

by soni cation.'.

Since all the filtrations were performed by the application

of pressure on the liquid surface, it was assumed that perhaps

cells were being "pushed" through the filters, although it was

obvious that Serratia marcescens could not be "pushed" through

the same filters. In order to rule out this possibility,

29

experiments were performed in which no external force other

than gravitation was applied. Results from these experiments

are shown in Table V. Results obtained by using gravitation

alone did not vary from those obtained when suction was applied.

TABLE V

PRESENCE OP FILTRABLE FORMS IN FILTRAT10NS

USING THE WALL VACUUM AND GRAVITATION

Age of Cultures in

Presence of Filtrable Forms When Using

Hours Wall Vacuum Gravitation

10 -

20 -

30 + +

40 4- +

In ultracentrifugation experiments, 75 ml of filtrate were

used in each tube, yet it was very difficult to obtain an appre-

ciable amount of residue even after ultracentrifugation at

100,000 X g for two hours. Therefore, to obtain enough residue

for electron-microscopy, residues in several tubes had to be

pooled together in a total volume of about half a milliliter.

Plates 17 to VII are electron photomicrographs of bacteria-like

bodies found in the residue. The initial magnification is

11,500 X diameters. Plate III shows the background of the

30 * %

photographic surface OIL which the specimens were placed, and is

included here as a control. The arrows on Plate III indicate

chromium particles on the grid resulting from the shadowing

procedure. Plates 17 tc VII show the different forms of bodies

obtained.

The forms obtained varied in shape from round, as - shown on

Plate 17, to rod-like, as in Plate VII. The bacteria-like forms

shown in these electron photomicrographs did not reveal a complete,

well-defined boundary. This might indicate the absence of a

rigid cell wall and might account for the variety of shapes

observed. However, it is acknowledged that the presence or

absence of cell wall could not be unequivocally determined by

means of the shadow casting techniques employed here, smd, con-

sequently, no claims regarding cell anatomy are made.

The fact that these residues were capable of yielding

copious growth of the organism from which the filtrable forms

were originally obtained was considered an adequate criterion to

state that the forms observed by electron microscope were

transition forms of the bacterium under study. As with the

growth experiments, in no case was Serratia marcescens observed

in these preparations.

31

TABLE VI

CHARACTERISATION OF THE FILIRABLE ORGANISM

Pigment production .....

Microscopic morphology

Temperature

yellowish-brown pigment soluble in water but not in chloroform.

motile rod with polar flagellation (Plate 71II) and gram negative reaction.

this organism grew readily at 30°C and 37°0 but failed to grow at 42°C,

Reactions t

Isolate Pseudomonas Isolate reptivora

Biochemical Tests Reduction of nitrates + Methyl red test + Acetylmethylcarbinol production - -

Hydrogen sulfhide prod. -

Citrate utilization + -

Urease production - •

Reaction on litmus mills; alkaline and alkaline and peptonization peptonization

Casein hydrolysis Starch hydrolysis - -

Gelatin Hydrolysis + Sheep blood cells hemolysis hemolysis Oxidase - —

Fermentation Tests* Glucose no acid no acid Fructose it Maltose ft tt Mannitol If n

Dextrin I! II Galactose *t Arabinose it Xylose fS it Lactose If tt Cellulose not attacked not attached

-;*As determined by color reaction of brom cresol purple indicator incorporated in the media.

32 .

Characterization of the isolated bacterium according to

the criteria set forth by Bergey1 £ Manual of Determinative

Bacteriology indicated that it was of the pseudomonas family,

and it was tentatively identified as a species closely related

'to Pseudomonas reptivora. The results of these tests are

shown in Table VI.

By comparing results of the tests shown in Table VI, it

is obvious that the isolate closely resembled reptivora

except that the new isolate reduced nitrates and grew on

citrate as a sole source of carbon, whereas Pj_ reptivora did

not. Prom the information given in Bergey1s Manual of

Determinative Bacteriology, the only other species in the

same family that closely resembles I\_ reptivora is Pseudomonas•

aeruginosa. However, the fact that the isolate could not

grow at 42°C set it apart from P^ aeruginosa. ' Furthermore,

many P^ aeruginosa produce pigments extractable by chloroform,

whereas the pigment from the isolate was not chloroform"extract-

able.

35

Plate VIII

Leifeson flagella stain of the bacterial cells from which, flltrable forms were obtained; 2,000 X.

OEAPTEE IT

DISCUSSION

The filtrability of bacteria has been reported ever since

the beginning of the century (8). In modern perspective, most

of the reports indicate that the filtrable forms were actually

L forms (5). The present worlc reports the isolation and char-

acterization of a bacterium capable of passing through 0.45 H

filter membranes.

As can be seen from Table VI, there was a. tendency for

filtrability to occur during autolysis, which suggests to the

author that filtrability was due to viable elements or bodies

inside the vegetative cells of this particular bacterium.

Since filtrability of young cultures was not evident even in

cultures disrupted by sonication, it could be further inferred

that the filtrable elements were not produced in all cells but

were produced in a small percent of the population only as a

consequence of aging of individual cells. Whether these

elements were produced only under adverse environmental con-

ditions or whether these were part of the normal life of the

organism was not determined.

34

35

Similar granules or bodies have been shown in L form

culture of many bacteria by light microscopy (1, 17), elec-

tron microscopy (2, 6, 9, 13)> and differential centrifuga-•

tion (16). These bodies range in size from less than 0.45 to

1.0n in diameter and numerous reports have been written on

their abilities to pass through filters and to regenerate

L forms (4, 3, 15» 7). The filtrable elements of the organism

reported here have properties similiar to those of L forms of

other bacteria. However, the results presented here show that

these elements pass through 0.3 H filter membranes and regenerate

intact bacterial cells, whereas those of L forms described in

the literature give rise only to L forms but, normally, not

the parental bacteria from which they were derived (5» 7» 4).

Another difference was the size of the elements. Weibul and

Ludin reported in 1961 that in Proteus» L form elements with

size less than 0.6 could not regenerate (17). Boven et al.also

reported the same findings in group A streptococcal L forms (1).

Thus it is generally accepted that elements less than 0.6 H

cannot produce viable cells. The filtrable elements reported

here were able to pass through 0.3 H filters. This fact could

be due to one of three possibilities:

a) The filtrable forms had a size of 0.6|ior more but

could "squeeze" through the pore of the filter membrane.

. • 36

b) They grew,through the filter during filtration, as.

reported in several cases (18, 12).

c) They did not have a rigid body and were able to deform

easily even when only slight pressure was applied.

The last possibility appeared to be easily excluded, since

filtrability occurred even when no pressure was applied. Size

determinations from electron microscopy showed that all elements

were larger than 0.3 . At the same time, the fact that they

do not have rigid boundaries indicated that these elements

could possibly pass through pores of filters because of their, extreme

plasticity. The growth of the elements through filter^ was

not feasible here since the filtrations were completed within

relatively short times. Furthermore, all filtrations were

performed in media incapable of supporting even minimal growth.

(18).

Recently, protoplasts have been reported to be able to

pass through 0.22(i filters and form L forms (18). However,

protoplasts were usually formed artificially and not obtained

naturally; throughout the entire investigation reported here,

no protoplasts were seen in any of the many cultures examined,

although these were searched for diligently by light transmis-

sion and phase contrast microscopy. Thus, the filtrable

characteristics of this organism indicate that it is not an

-37

L form according to the descriptions of L forms generally

accepted in the literature.

The only forms of this organism seen throughout the entire

investigation were the vegetative form, the lysed vegetative

cells, and the filtrable forms shown by the electron photo-

micrographs. Therefore, the filtrable elements in this parti-

cular organism could not be L forms in the commonly understood

sense.

As indicated in Table III, sonication of young cultures

did not produce filtrability. Hence filtrable elements must

have been produced when the culture reached a certain "age of

growth, while further aging resulted in lysing of the cells and

release of the filtrable elements. The above facts have some

fl

resemblance to the filtrability theory of Lohnis and Smith (1)^

but it is reported here in a new lightt i.e., milliponje membranes

of well-determined size rather than filter candles of undeter-

mined pore size, and use of Serratia marcescens as an indicator

of filter integrity. Actually, up to the present time, repro-

duction by filtrable granules was considered to be confined to

L forms only. Since the L form was regarded as one of the

forms produced during the growth cycle of bacteria, it is logical

to assume that some bacterial cells may deviate from the normal

way and exhibit the L type of reproduction without having an

38

1 stage In their life cycle. Therefore, the filtrable stage t!

described by Lohnis and Smith and others could possibly be

attributed to cases in which organisms were able to undertake

regeneration with or without the appearance of the L form

(11, 10, 14). The data present here strongly suggested this

possibility; however, further investigation are necessary in

order to fully understand the phenomena. In any case, it is

clear that the filtrable forms observed here are not identical

with L forms described in the literature in that reversion to

the parental form is uniform. It appears reasonable to assume

that an aberrant L form has been isolated or that a truly

"filtrable form" of bacteria has been observed for the first

time.

39

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40

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Electropiiotoralorograph of spherical particle in the ultracentrifugation residue of filtered, bacterial cultures giving growth of typical ceils. Specimens were shadow oast with chromlua; the bar represents 1}! . ' . '

41

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43

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Plate 711

Electrophotoaicrograph. of irregularly shaped body

- J f e ^ i f ° e a t r l f U 6 e d c u l t u r« titrate, shadow Uj,i3l> u~e »-'8.r represents l|j.

*

CHAPTER BIBLIOGRAPHY

1. Boven, 0. van, H. 1. Ensering, and W. Hijmans, "Size Determination by Phase Microscopy of the Reproductive Elements of Group A Streptococcal L Forms," J. Gen. Microbiol.. 52 (1968), 413-420.

2. Cole, R. M., The Structure of the Group A Streptococcal Cell and its L Form, Current Research on Group A Streptococcus. Excerpta Medica Foundation, (lew York, 1968), 5-42.

3. Conssons, R. T. and R. M. Cole, The Size and Repllcatlve .Capacities of Small Bodies of Group A Streptococcal L Forms, Ed. by R. Caravans, Amsterdam, Excerpta Medica Foundation, (New York, 1968).

4. Dienes, L., "Morphology and Reproduction Processes of the L Forms of Bacteria I, Streptococci and Staphylococci," J. Bact.> 1967•

5. Dienes, L. and S. Bullibant, "Morphology and Reproductive Processes of the L Form of Bacteria," Bact. , (Feb.-1968),.672-687.

6. Ghosh, B. K., and R. G„ Murray, "Fine Structure of Listeria monocytogenes in Relation to Protoplast Formation," J. Bact.« 93 (1967), 411-426.

7. Fodor, M., and L. Miltenyi, "Studies on L Forms of Staphy-lococcus aureus Strains of Different Antibiotic and Phage Sensitivity," Acta Microbiol., 11 (1964), pl55»

8. Hadley, P., "The Instability of Bacterial Species with Special Reference to Active Dissociation and Transmissible Autolysis," J_j_ Infectious Diseases, 40 (1927)» 1-312.

44

45

9. Hoof, A. van, and ¥. Hi3 maris, "An Electron Microscopy-Study of I» forms of Group A Streptocacci," Antonle Van Leeuwenhock, J. Microbiol., 25 (1959), 88-96.

10. Lawrence, J. C., "Filtrability of the Swarmers of Rhlzoblum Azotobacter," Nature, Vol. 176, No, 4491, (1955)?

1033-1034.

11. Lolmis, F. and Hv R. Smith, "Life Cycles of Bacteria/' J. Agr. Research, 6(1916), 675-720.

! ' 12. Molander, C. ¥., H. J. Weinberger, and B. M. Kagan, "Growth

of Staphyloccoccal L forms on and through Membrane Discs of Various Pore Sizes," Jj. Bac

15.

It.. 89-3 (1965), 7-9»

13. Page, M. I., 0. A. Ashley and L. Roberts, "Ultrastructure of Meningococcal L forms," Antimicrob. An;r. Chemother., . (1968), 127-131. 1

14. Schreven, D. van, "Effects of Penicillin on the Morphology r Chroococcum," J. Microb. and Reproduction of Azotobacte

32 (Jam. 1966.), 67-93.

Weibull 0., "Size of Minimal Reproductive Units of Bacterial L forms," Proc. Soc. Exp. Biol. Med., (1961), 113-132. .

emical and Metabolic Pound in Cultures of a • Microbiol., 24 (1961),

16. Weibull, C., and H. Beclonan, "Ch Properties of Various Elements Stable Proteus L form," ch, Gen 379-391.

17. ¥eibull, C., and B. Lundin, "Grojwth of Elements of Yarious Sizes Formed in Cultures of a jStable Proteus L form," JL. Gen. Microbiol.. 27 (1962), 241-248.

18. "rfyrick, P. B. and H. Gooder, "Gdowth of Streptococcal Protoplasts and L colonies on jMembrane Filters," Bact.. 105 (1971)j 646-656, 1

CHAPTER 7

SUMMARY

The present study reports the isolation of a bacterium

from a culture of Azotobacter vinelandil. This bacterium

was capable of producing forms that passed through 0.45 w

millipore filter membrane and regenerated the parental form.

The study showed the true filtrability of the bacterium

and the appearance of the filtrable forms. Various identifi-

cation procedures showed that the isolate belonged to the

genus Pseudomonas and closely resembled Pseudomonas reptivora

except that the isolate reduced nitrates and grew on citrate

as a sole source of carbon. Electron microscopy and shadow

casting techniques revealed the actual forms that passed through

the filters. Size determination of these filtrable elements

showed that they were not the same as those regenerative elements

found in L forms of other bacteria as described in the literature,

However, the exact mechanism responsible for their filtrability

needs to be further investigated.

46

BIBLIOGRAPHY

Books

Breed, R. G., T. D. Murray and N. R. Smith, Bergey's Manual of Determinative Bacteriology. 7th ed., Baltimore, The Williams & Wilkins Co., 1957.

Collins, C. H., Microbiological Methods, Butterworth & Co., London, 1964.

Conssons, R, T. and R. M. Cole, The Size and Replicative Capacities of Small Bodies of Group A Streptococcal L"Forms,• Ed. by R. Caravans, Amsterdam Excerpta Medica Foundation, IJew, York, 1968,.

Dawes, D. J., Biological Techniques in Electron Microscopy. Barnes & Noble Inc., i. X., 1971-

Pelcsar, M. Jr., P. A. Hansen and ¥. A. Konetzka, Quantitative Bacterial Physiology Laboratory Experiments. Burgers Publishing Co., Minneapolis, 1955. -

Wilson, P. W. and S. G. Knight, Experiments in Bacterial Physiology, Burgers Publishing Co., Minneapolis, 1952.

Articles

Bisset, K. A. and C. M. Hale, "The Cytology and Life Cycle of Azotobacter chroococcum," of Gen. Microb.. 38 {Aug. 1964), 329-338.

Boven, C. van, H. L. Ensering and «. Hi jmans, "Size Determi-nation by Phase Microscopy of the Reproductive Elements of Group A Streptococcus L Forms," Gen. Microbiol.. 52 (1968), 413-420.

48

Cooper, P. B. and 3. A. Petroff, "Piltrable Forms of the Tubercle Bacillus," J. Infections Diseases, 42 (1928), 200-214. ' *

Dienis, L., "A Peculiar Reproductive Process in Colon Bacillus Colonies," Proc. -Soc. Exp. Biol. Med., 42 (1937), 773-778.

Dienes L., "further Observation on the L Organism of-Klieneberger," Proc. Soc. Exp. Biol. Med., 42 (1937), 778-779.

Dienes L., "Purther Observation on the L Organism of Klieneberger," Proc. Soc. Exp. Biol. Med., 39 (1938), 365-367.

Dienes, L., "Reproductive Processes in Proteus Cultures," Proc. Soc. Exp. Biol. Med., 63 (1946), 265-270.

Dienes, L., "Complex Reproductive Processes in Bacteria," Cold Spring Earbor Symposis on Quant. Biol., 11 (1946), 51-59.

Dienes L., "The Significance of the Large Bodies and the Develop-ment of L Type of Colonies in Bacterial Cultures," _J. Bact., 44 (1947), 37-73.

Dienes, L. , "Isolation of Pleuropheursionia-like Organisms from H. influenzae with the Aid of Penicillin,"Proc. Soc. Exp. Biol. Med., 68 (1949), 589-590. L •

Dienes,.!., "Isolation of L Type Colonies from a Gram Positive Spore-bearing Bacoillus," Proc. Soc. Exp. Biol. Med., 68 (1949), 589-590.

Dienes L., "Isolation of L Type Cultures from Clostridia," Proc. Soc. Exp. Biol. Med., 75 (1950), 412-415.

Dienes, L., "Morphology and Reproduction Processes' of the L Porms of Bacteria streptococci and Staphylococci," J. Bact., -(1967).

Dienes, I. and S. Bullibant, "Morphology and Reproductive Process of the L Form of Bacteria," Bact., (Peb. 1968), 672-687.

Dienes, L. and ¥. E. Smith, "Reproduction of Bacteria from the Large Bodies of Bacteroides funduliformis," Proc. Soc. Exp. Biol. Med.. 51 (1942), 297-298. " *

49

FodorM., and L. Kiltenyi. "Studies on L Forms of Staphylococcus aureus Strains of Different Antibiotic and Phage Sensitivity," Acta. Microbiol., 11 (1964), pl55.

Ghosh, B. K., and R. G. Murray, "Fine Structure of Listeria monocytogenes in Relation to Protoplast Formation," J. Bact., 93 (1967), 411-426.

Gloyne, S. R., R. E. Glover and A. S. Griffith, "Experiment to Determine Whether there is a Filtrable form of the Tubercle Bacillus," J. Path Bact., 32 (1929), 775-785.

Hadley, P., "The Instability of Bacterial Species with Special Reference to Active Dissociation and Transmissiole Autolysis," J. Infect. Diseases, 40 (1927)1-132.

Hadley, P., "Further Advances in the Study of Microbic Dissociation," Infect. Diseases,60 (1937), 129-192.

Hadley, P., E. Delves and G. Xlimek, "The Filtrable Forms of Bacteria," infect. Diseases, 48 (1931), 1-159.

Hoof, A. Van and W. Hi 3mans, "An Electron Microscopy Study of L Forms of Group A Streptocacci," Antonie Van Leeuwenhock, Is. Microbiol., 25 (1959), 36-96.

Jones, D. H., "Further. Studies on the Growth Cycle of Azotobacter," Is. 3act. t 44 (1920), 325-333.

Kami, M. C., "A Developmental Cycle of the Tubercle Bacillus as Revealed by Single-cell Studies," Am. Rev. Tuberc., 20 (1929), 150-200.

Kahn, M. 0., "A Growth Cycle of the Human Tubercle Bacillus as Determined by Single-cell Studies," Tuoercie, 11 (1930), 202-217.

Klieneberger, U. E., "Filtrable Forms of Bacteria," Bact. Rev., 15 (1951), 81-55. " ~

Lawrence, J. C., "Filtrability of the Swarmers of RnizoDium and Azotobacter," Mature. Vol. 176, Ho. 4491 (1955), 1033-1034.

50

Lewis, I. M., "Dissociation and Life Cycle of B. Myeoides." J. Bact., 24 (1932), 381-420.

tl

Lohnis, P., "Studies Upon the Life Cycles of the Bact.," Mem. Hat. Acad. Soi., 16, 2nd Memoir, Washington (1921), 1-335.'

fi

Lohnis, P. and N. R. Smith, "Life Cycles of Bacteria," Agr. Res., 6 (1916), 675-720.

Molander, C. W., H. J. Weinberger and B. M. Kagan, "Growth of Staphylococcal L Forms on and through Membrane Discs of "Various Pore Sizes," J. Bact., 89-03 (1965), 7-9.

Page, M. I., 0. A.. Ashley and L. Roberts, "infrastructure of Meningococcal L Forms," Antlmicrob. Agr. Chemother, (1968), 127-131.

Pinner, R. and M. Voldrich, "The Disease Caused by Filtrates of the Tubercle Bacillus Cultures," Am. Rev. Tuberc. 24 (1931), 73-93.

Schreven, D. A. van, "Effects of Penicillin on the Morphology and Reproduction of Aaotobacter chroococcuia," Antonie Van Leeuwenhock J. Microb. Serol., 32 (Jan. 1966), 67-93.

Soltys, M. A. and A. ¥. Taylor, "The Filtration of Mycobacterium tuberculosis and Mycobacterium stercusis through G-radocol Membranes," Path Bact., 56 (1944), 173-190.

Weibull C., "Size of Minimal Reproductive Units of Bacterial L Forms," Proc. Soc. Bro. Biol. Med., (1961), 113-132.

Weibull, C. and H. Beckman, "Chemical and Metabolic Properties of Various Elements Found in Cultures of a Stable Proteus L Form," Gen. Microbiol.. 24 (1961), 379-391.

Weibull, C., and B. Lundin, "Growth of Elements of Various -Sizes Formed in Cultures of a Stable Proteus L Form," Gent. Microbiol.. 27 (1962), 241-248.

Weinberger, H. J., S« Madoff and L. Dienes, "The Properties of L Forms Isolated from Salmonella and the Isolation of L Forms ' " from Shigella." J*. Bact.. 59 (1950), 765-775.

51

Wyrick, P. B. and H. Gooder, "Growth of Streptococcal Protoplasts and L colonies on Membrane Filters," Eact., 105 (1971), 646-656.

Reports

Brown, T. and J. 0. lunemaker, Bat-bite fever, A Review of the .American Oases witii Revalution of Ethiology t . Bull John Hopkins Hosp.» 70 (1942), 201-327.

Cole, R. M., The Structure of the Group A Streptococcal Cell and its L Form, Current Research on Group A Streptococcus, Excerpta Hedica Foundation, (N. I., 1968), 5-42.

Dawson, M. H. and G. L. Hobby, . Pleuropneumonia-jj-M Organisms as a Variant Phase of Streptobacillus Moniliformis, 3rd. Internat. Congr. Microbiol. Abstr., 1939 > 177-178*

Onblished Materials

Ward, C. B. Jr., "The Effect of Nutrition and Toxic Agents on the Cytology on an Azotobacter Species," Masters' Thesis, Oklahoma A & M College, 1950.