• TABLE OF CONTENTS
HIDE
 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Introduction
 Literature review
 Materials and methods
 Experimental results
 Discussion
 Summary
 Appendix
 Bibliography
 Biographical sketch
 Copyright














Title: osmotic fragility of cells and spheroplasts of a marine Vibrio
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Title: osmotic fragility of cells and spheroplasts of a marine Vibrio
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
    List of Tables
        Page iv
        Page v
    List of Figures
        Page vi
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
    Literature review
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
    Materials and methods
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
    Experimental results
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
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    Discussion
        Page 75
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    Summary
        Page 87
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    Appendix
        Page 90
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        Page 103
        Page 104
    Bibliography
        Page 105
        Page 106
        Page 107
        Page 108
    Biographical sketch
        Page 109
        Page 110
    Copyright
        Copyright
Full Text










THE OSMOTIC FRAGILITY OF CELLS AND


SPHEROPLASTS OF A MARINE


By
JOHN RUTLEDGE BORING, III


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY









UNIVERSITY OF FLORIDA


August, 1961


VIBRIO




























ACKNOWLEDGMENT


The author wishes to express his appreciation to

Dr. Darrell B. Pratt and Dr. Max E. Tyler for the helpful

guidance which made this study possible.
















TABLE OF CONTENTS


Page

LIST OF TABLES . . . . . . . . . . . iv

LIST OF FIGURES . . . . . . . . . ... vi

INTRODUCTION . . . . . . . . . . . . .

LITERATURE REVIEW . . . . . . . . .. . . . 5

Osmolysis of marine bacteria . . . .... . 6

Penicillin-induced spheroplasts of Gram-
negative bacteria . . ........... . 14

MATERIALS AND METHODS . ... . . . . . . . . 17

EXPERIMENTAL RESULTS . . . . . . .. . . . 29

The morphological and physiological characteristics
of the marine bacterium M.B. 22 . .. . . 29

Spheroplasts of Vibrio M.B. 22 ........ .. 39

The osmotic lysis of cells and spheroplasts of
Vibrio M.B. 22 . . .. . . . . . . 54

DISCUSSION ......... .. ... . . . . 75

SUMMARY . . . . . . . .. . . . 87

APPENDIX A . . . . . . . . . . . . 90

APPENDIX B . . . . . . . ....... .. . .100

BIBLIOGRAPHY .. . . . . . . .105















LIST OF TABLES


TABLE Page

1. EFFECT OF INCUBATION TEMPERATURE ON THE GROWTH
OF VIBRIO M.B. 22 . . . . . . . . 31

2. PHYSIOLOGICAL CHARACTERISTICS OF VIBRIO M.B. 22 . . 36

3. FERMENTATION OF CARBOHYDRATES BY VIBRIO M.B. 22 . . 37

4. SENSITIVITY OF VIBRIO M.B. 22 TO ANTIBIOTICS. . . . 40

5. GROWTH OF VIBRIO M.B. 22 IN GRADED PENICILLIN
CONCENTRATIONS . . . . .. . . . 42

6. MORPHOLOGICAL FORMS OBSERVED FOLLOWING THE ADDITION
OF PENICILLIN TO GROWING CULTURES OF VIBRIO M.B. 22 . 43

7. THE RELATION OF SPHEROPLASTS FORMED PER CELL AND THE
GROWTH OF SPHEROPLASTS 4 4 ...... .i . 47

8. GROWTH OF SPHEROPLASTS IN RESPONSE TO NUTRIENT
CONCENTRATION . . .. . 4 50

9. EFFECT OF WASHING SPHEROPLASTS . . . . . 52

10i STABILITY OF SPHEROPLASTS IN ARTIFICIAL SEA WATER 4 53

11. EFFECT ON VIBRIO M.B. 22 AND PSEUDOMONAS AERUGINOSA
OF SUSPENSION IN DISTILLED WATER . . 56

12. LYSIS OF CELLS AND SPHEROPLASTS OF VIBRIO M.Bi 22
ESTIMATED BY OPTICAL DENSITY AND VIABILITY . . 58

13. LEAKAGE OF PROTEIN FROM VIBRIO M.B. 22 . . 4 59

14. RELATION OF COLONY COUNT TO DIRECT COUNT OF VIBRIO M.B. 22 61

15. RELATION OF COLONY COUNT TO DIRECT COUNT OF
SPHEROPLASTS OF VIBRIO MB. 22 ... . . 63

16. LYSIS OF VIBRIO M.D. 22 in GRADED CONCENTRATIONS OF NaC1
AS ESTIMATED BY INDOL FORMATION . . . . ... 64











LIST OF TABLES--Continued


TABLE Page

17. LYSIS OF SPHEROPLASTS IN GRADED CONCENTRATIONS OF
NaCI AS ESTIMATED BY INDOL FORMATION . . . . 65

18. LYSIS OF VIBRIO M.B. 22 IN GRADED CONCENTRATIONS OF
NaC1 AS ESTIMATED BY TETRAZOLIUM REDUCTION . . . 68

19. LYSIS OF SPHEROPLASTS IN GRADED CONCENTRATIONS OF
NaCI AS ESTIMATED BY TETRAZOLIUM REDUCTION .. . . .. 69

20. LYSIS OF VIBRIO M.B. 22 AS ESTIMATED BY DIRECT COUNT . 72

21. LYSIS OF SPHEROPLASTS AS ESTIMATED BY DIRECT COUNT . 74

22. COMPARISON OF THE LYTIC PATTERNS OF CELLS AND
SPHEROPLASTS OF VIBRIO H.B. 22 . . . . . . 84

23, STANDARD CURVE: OPTICAL DENSITY OF GRADED
CONCENTRATIONS OF FORMAZAN . . . . . . 91

24. ANAEROBIC VERSUS AEROBIC REDUCTION OF TETRAZOLIUM
BY VIBRIO M.B. 22 . . . . * . 93

25. TETRAZOLIUM REDUCTION AS A FUNCTION OF TETRAZOLIUM
CONCENTRATION . . . . . . . . . . 94

26. TETRAZOLIUM REDUCTION AS A FUNCTION OF GLUCOSE
CONCENTRATION . . . . . . . . 96

27. TETRAZOLIUM REDUCTION BY GRADED CELL CONCENTRATIONS
OF VIBRIO M.B. 22 . ... . . . 97

28. RATE OF TETRAZOLIUM REDUCTION BY A DILUTE CELL
SUSPENSION . . ..... . .. . .. . . 98

29. STANDARD CURVE: OPTICAL DENSITY OF GRADED CONCENTRATIONS
OF INDOL . . . . . . . . * 101

30. INDOL PRODUCTION AS A FUNCTION OF TRYPTOPHAN
CONCENTRATION .... . . . . . . 102

31. INDOL PRODUCTION BY GRADED CELL CONCENTRATIONS OF
VIBRIO M.B. 22 ........ . . . . . 103















LIST OF FIGURES


Figure Page

1. Phase contrast micrograph of cells of Vibrio M.B. 22 32

2. Electron micrograph of Vibrio M.B. 22 . . . 33

3. Phase contrast micrograph of spheroplasts of
Vibrio M.B. 22 . . . . . . . . 45















INTRODUCTION


Marine bacteria are microorganisms found in nature as inhabi-

tants of the sea. They are found in coastal waters, in waters distant

from land, and on marine fishes. Marine bacteria may readily be iso-

lated from the sea by cultivation on media containing organic nutrients

and sea water. However, it has often been found that a high proportion

of such isolates fail to develop when transferred to media containing

organic nutrients dissolved in distilled water (Johnson, 1936;

Bielling, 1958). These isolates are characterized by a marked suscep-

tibility to osmotic lysis and when suspended in distilled water or

dilute salt or sugar solutions lose many of the physiological capa-

bilities they are able to achieve as integral cells. The adaptation

to a marine habitat has markedly limited the environment which is

suitable for the development of these organisms, and this charac-

teristic distinguishes many marine bacteria from most non-marine

species, for the latter organisms survive in dilute salt solutions and

grow in media prepared with distilled water,

The osmotic damage to cells occurring when marine bacteria are

exposed to distilled water has been examined in detail: the luminous

species fail to produce light, fail to respire, and lose their
motility (Johnson and Harvey, 1938); other species are irreversibly
inhibited in their ability to respire (Boring, 1955). Physical damage

1










to cells also occurs: the turbidity of cell suspensions is less in

distilled water than in sea water (Riley, 1955), and the suspensions

are foamy. Foaminess is believed to be due to the leakage of cell

constituents (Hill, 1929). Electron micrographs of cell suspensions

in distilled water reveal cell wall "ghosts" with slit-like areas on

their surfaces believed to be cracks in the cell wall (Johnson,

Zworkin and Warren, 1943). The evidence supports the postulate made

by Hill (1929) that at a critical difference in osmotic pressure

between cell and medium the cell wall ruptures exuding the cell con-

tents. The cells then lose those capabilities which are dependent on

cellular integrity.

The basis of the osmotic fragility of marine bacteria is

not known. Salton (1956) has suggested that the fragility of lumi-

nous and halophilic species may reside in the nature of the cell

walls of the organisms, He considered it conceivable that the cell

walls of these species are mechanically weak due to thinness, chemical

composition, or physical structure and, whereas the cell walls of

non-marine bacteria protect the cells against osmotic lysis, the walls

of marine species may not have this ability. The studies in the

experimental research for this thesis were concerned with the osmotic

fragility of one marine bacterium. In particular the studies were

undertaken to add more information to the knowledge of the role of

the cell wall in protecting cells against osmotic shock.

The minimum salt concentration necessary to protect cells of











one marine bacterium was determined by estimating the degree of lysis

of cell suspensions in a series of graded concentrations of sodium

chloride. Several indirect methods and one direct method were developed

for estimating lysis. It was believed that a comparison of the lysis

of normal cells and spheroplasts was an effective method for estimating

the role of the cell wall in protecting the marine bacterium against

osmotic shock. Accordingly, spheroplasts of the marine bacterium were

prepared, some of their properties examined, and their lysis pattern

in graded salt concentrations compared with the lysis of normal cells.

Any difference in the lytic patterns of cells and spheroplasts was

considered to be attributable to the rigid penicillin-sensitive

component of the cell wall.

In the course of the investigation some of the characteristics

of the marine bacterium were studied and the organism was found to be

a species of Vibrio. The formation and properties of spheroplasts of

the bacterium were also examined and spheroplasts were found to be

formed one for one from cells. Lysis of cells and spheroplasts was

estimated by turbidity, the reduction of 2,3,5-triphenyltetrazolium

chloride, indol production, and a direct microscopic method. Cells

and spheroplasts grown in sea water media were found to lyse rapidly

when placed in solutions less than 0.1 M in sodium chloride. The

lytic patterns of cells and spheroplasts were determined to be similar

and this was interpreted to mean that the rigid penicillin-sensitive







4


component of the cell wall gave little protection to the cells against

osmotic lysis. The evidence supported Salton's suggestion that the cell

walls of some bacteria are sensitive to osmotic damage.














LITERATURE REVIEW


The osmotic lysis of a few species of marine bacteria has

received considerable attention in efforts to analyze the effects

on both function and structure of the cells accompanying the exposure

of the bacteria to solutions dilute in salt. Past experiments have

not, in general, been concerned with the nature of osmotic fragility

but rather with the effects which occurred when cells were lysed.

Few species have been studied but experiments have demonstrated that

some aspects of osmotic lysis may be common to many marine bacteria.

These aspects are considered in this review.

In the present study it was believed that a comparison of the

lysis of normal cells with the lysis of spheroplasts was an effec-

tive method for estimating the protection against osmotic shock

afforded the marine bacterium by the rigid component of its cell wall.

Accordingly, spheroplasts were induced by penicillin from cells and

some of their properties were examined. However, it was not within

the limits of the experiments to examine in detail either the specific

function of penicillin in inducing spheroplasts or the chemical compo-

sition of the spheroplasts. Some assumptions concerning the sphero-

plasts of the marine bacterium were based on experimental results

reported for other Gram negative bacteria. Experimental evidence

considered pertinent to this point is given in this section.











Osmolysis of Marine Bacteria

Investigators have, in general, examined the salt requirement

of obligate halophilic bacteria by attempts to determine the kinds

and concentrations of salts necessary to support growth (Flannery,

1956). This approach has probably stemmed from the suggestion by

LeFevre and Round (1919) that halophilic bacteria require a suitable

osmotic pressure rather than specific salts. These investigators

believed that the successful replacement of sodium chloride by other

salts would demonstrate a requirement for osmotic pressure for the

growth of halophiles. Subsequent experiments have not definitely

established this point: various salts were substituted for sodium

chloride and supported the growth of some halophiles (Flannery, et al.,

1952), but not of others (Robinson and Gibbons, 1952). For several

marine bacteria Macleod, Onofrey, and Norris (1954) demonstrated a

specific nutritional requirement for the sodium ion. A sodium ion

requirement for the growth of 100 marine isolates was also shown by

Tyler, Bielling, and Pratt (1960). In this regard, however,. Sistrom

(1960) suggested that some fresh water bacteria may also require sodium

ion for growth.

Pratt and Austin (1961) demonstrated experimentally that for

one marine species sodium chloride had two functions; it was required

for growth, and it was needed as an osmotic stabilizer. It was the

latter function which could be replaced by other salts. This evidence

supported the suggestion of Richter (1928) that the sodium chloride











requirement of marine bacteria serves two purposes: one of nutrition,

and one of osmotic regulation. It is the fragility of marine bacteria

and, in consequence, their requirement for an osmotic stabilizer with

which the present investigation is concerned.

The studies of the osmolysis of marine bacteria have involved,

for the most part, two species of luminous bacteria,. Photobacterium

fisher and Photobacterium harveyi. Interest in the osmotic fragility

has evolved from the studies on luminescence of the bacteria. Harvey

(1915) used osmotic fragility of a luminous bacterium (unidentified)

in demonstrating the dependence of luminescence on cellular integrity.

He found that a dense cell suspension gave a bright light in oxy-

genated sea water, but no light in oxygenated tap water. It was

believed that the bacterium lysed in the tap water and, as a conse-

quence, the system responsible for luminescence was disrupted. Harvey

suggested that all marine cells can be cytolysed by distilled water.

The osmolysis of Bacillus fisher (Photobacterium fisher)

in hypotonic solutions was investigated by Hill (1929) who used lumi-

nescence and its inhibition to estimate lysis. The procedure used

was to place one drop of dense cell suspension in 10.0 ml of test

solution, to shake the tube vigorously, and to watch for the disap-

pearance of luminescence. Cell suspensions in diluted sea water were

first examined, In 12.5 per cent sea water luminescence was greatly

decreased but persisted for 6 hr, while in 6 per cent sea water lumi-

nescence lasted only 5 min. In 0.125 M sodium chloride luminescence











was unaffected; in 0.0625 M it was greatly dimmed, and in 0.0312 M the

light lasted only 5 min. Hill also demonstrated that luminescence was

protected by appropriate concentrations of sucrose. He felt that the

failure of the cells to luminesce in the diluted sea water was an

osmotic effect and was not due to the absence of the customary salts

as such. He demonstrated as well that a dense suspension of cells in

6.0 per cent sea water became clear and foamy. Microscopic examina-

tion with dark field illumination revealed little change in the struc-

ture of the cells. The results support, but do not necessarily estab-

lish, Hill's conclusion that the bacteria are surrounded by a rigid

membrane and that the membrane does not swell freely but ruptures,

exuding the cell contents at a critical difference in osmotic pressure

between cell and medium, He was careful to point out, however, that

inhibition of luminescence did not necessarily indicate cytolysis.

Korr (1935) found that luminescence was never obtained

from cells which had been cytolysed in hypotonic solutions, but he

pointed out that lack of luminescence by cells suspended in distilled

water may not be a true indication of complete lysis. Some of the

bacteria may remain viable for several hours as he demonstrated by

inoculating sea water medium with portions of such suspensions. Growth

was obtained from suspensions of cells which had been exposed for

3.5 hr to distilled water. He also emphasized that test solutions

must be added rapidly and all at once to the bacteria if luminescence

was to be destroyed in hypotonic solutions,











Johnson and Harvey (1937) made a careful and detailed study of

the lysis of the luminous bacterium Achromobacter fisher (Photobac-

terium fisheri. When dense suspensions of the bacterium were placed

in distilled water the suspension became clear and foamy, the luminescence

and motility ceased, and the bacteria were difficult to centrifuge.

Densitometer experiments (measurement of light transmittance) revealed

progressively more light transmitted in diluted sea water suspensions

reaching a maximum in distilled water. Small amounts of calcium or

magnesium salts caused a decrease in light transmittance but did not

prevent foaminess or loss of luminescence or motility. As sea water

was progressively diluted the volume of cells which could be packed

by centrifugation also,became less. These results were thought to

support the conclusion that luminous bacteria placed in distilled

water undergo cytolysis without even a momentary swelling.

Johnson and Harvey (1938) extended their studies on the

cytolysis of Achromobacter fisher to include quantitative measurements

of viability, luminescence, and respiration in both diluted and con-

centrated sea water. The results indicated there was no great loss

of viability until sea water was diluted to 10 per cent and that

respiration and luminescence disappeared at about the same point. In

dilutions of sea water down to 10 per cent the luminescence of cell

suspensions decreased almost in proportion to dilution. Respiration,

on the other hand, was unaffected in solutions above 50 per cent, sea

water. Luminescence was protected by sea water, 052 M sodium chloride,












0.5 M sodium phosphate; but it was inhibited in 0.73 M sucrose, 0.53 M

potassium chloride, and 0.37 M magnesium chloride. Respiration was

protected by sea water, 0,52 M sodium chloride, and to some degree by

0.73 M sucrose and 0.53 M potassium chloride. Luminescence of cells

in 18 per cent sea water was inhibited but the effect could be reversed

by the addition of salts. The reversal was not complete but was 80 per

cent that obtained in sea water. The viability measurements were not

completely reproducible or consistent and they only indicated that few

bacteria in the more dilute solutions of sea water were capable of

engendering colonies.

With the advent of the electron microscope the opportunity to

visualize lysed marine bacteria was available. Johnson et al(1943)

examined 8 species of luminous bacteria; 6 from marine sources and

2 from fresh water. Cells of the marine specie,& prepared in 3 per cent

sodium chloride were dense and revealed no distinct cell wall structure.

Marine cells placed in distilled water revealed evidence of lysis

involving the exudation of cell contents and partial disintegration of

the cell. Cell wall "ghosts" were readily observed and these revealed

a mosaic density with relatively transparent circular or slit-like

areas. These areas were discussed in detail, and Johnson believed they

might possibly be cracks in the cell wall through which protoplasmic

constituents were exuded.

Using respiration as a measure of cell activity Boring (1955)











studied the kinds and concentrations of salts necessary to support this

function in a marine bacterium (unidentified). He postulated that salt

was required as an osmotic stabilizer. Sodium chloride or magnesium

chloride in the proper concentrations supported respiration but potas-

sium chloride did not. The ability of the organism to respire was

destroyed on placing cells in distilled water, and this ability was

not restored by the subsequent addition of salts to bring the tonicity

of the solution to that of sea water.

Riley (1955) examined the lysis of the same marine bacterium,

Lysis was estimated by measurements of turbidity of cell suspensions

in various concentrations of salts. In appropriate concentrations of

sodium and magnesium chlorides the turbidity remained high and constant.

In progressively lower concentrations of these salts the cell suspensions

became progressively less dense to transmitted light. It was also found

that suspensions with cells placed in potassium chloride solutions,

even relatively concentrated solutions, were almost clear.

Pratt and Happold (1960) studied the requirements for indol

formation by a marine bacterium (M.B. 22). They demonstrated that maxi-

mum indol formation from tryptophan by whole cells occurred in artificial

sea water or in a solution containing 0.42 M sodium chloride and 0,01 M

potassium chloride. The osmotic lysis of cells in distilled water was

used as a method of obtaining a cell-free extract capable of forming

indol. Maximum activity of the extract required tryptophan, potassium

phosphate and pyridr--l phosphate. In such a solution the extract











formed indol in an amount similar to that of cells in artificial sea

water. The cell-free extract required pyridoxal phosphate for activity

whereas the cells did not, However, the cells, but not the extract,

required a suitable amount of sodium chloride for activity.

That marine bacteria are fragile to osmotic shock has been

brought out in this review. The nature of the fragility of these

bacteria has only recently received any attention. Salton (1956) sug-

gested that the fragility of halophilic and luminous bacteria may

reside in the nature of their cell walls, which possibly are weak

due to their.chemical composition or to their physical structure,

In this connection, Brown (1960) has examined the cell walls of a

marine bacterium (not named) which was sensitive to osmotic shock.

The organism was fractionated and the walls were analyzed chemically

for the types of amino acids and for the quantity of amino sugar

(reported as glucosamine). The amount of amino sugar in the cell walls

of the marine bacterium was then compared with the amounts reported to

occur inthe cell walls of several non-marine, Gram negative bacteria.

The per cent of amino sugar in the walls of the marine bacterium was

lower than the amount in the walls of the other bacteria. In regard to

this finding Brown says, "The mechanical weakness of its walls is

possibly reflected in their relatively low content of amino sugar."

This statement gives credence to some interesting speculation which

can be made about the cell walls of marine bacteria. The nature of the

cell wall of one non-marine bacterium, Escherichia coli B, has been

thoroughly examined (Weidel, Frank, and Martin, 1960). The walls of











E coli B were reported to be composed of three layers; the outer layers

were composed of lipoprotein and polysaccharide, the inner layer of a

mucopeptide. The inner layer contained amino acids and amino sugars,

It was the inner layer which was believed to give the walls their

rigidity and mechanical strength. In the light of this result, Brown's

demonstration of a low amount of amino sugar in the walls of the marine

bacterium may support his contention concerning the fragility of marine

bacteria. The cell walls of one non-marine bacterium, Vibrio metachni-

kovii, have been found to be low in amino sugars (Salton and Shafa,

1958). However, it should be noted that Vibrio metschnikovii was

subject to osmotic lysis when cells were suspended in distilled water.

To summarize the results of previous experiments, it may be

said that certain functions, such as luminescence, respiration, and

motility, of many marine bacteria are damaged when cells are placed in

distilled water. The damage is believed to occur as a consequence of

cytolysis of the bacteria, and this view is supported, in the case of

luminous bacteria, by electron micrographs. The cells of marine bac-

teria apparently do not swell and are not completely fragmented upon

lysis, but protoplasmic constituents are believed to be exuded into

the extracellular fluid. The fragility of marine bacteria may reflect

the nature of the cell walls of the organisms. These walls possibly

are mechanically weak and, as a consequence,are subject to osmotic

rupture.











Penicillin-Induced Spheroplasts of Gram Negative Bacteria

Lederberg (1956) described the transformation of Escherichia

coli from rod-shaped to spherical cells in media containing penicillin,

sucrose, magnesium sulfate and organic nutrients. The transition was

complete in 2 to 3 hr. Each rod was found to give rise to a single

sphere and even cells which were about to divide were inhibited from

further growth and division. Spheres incubated in penicillin medium

continued to enlarge but no evidence of multiplication was obtained.

About 50 per cent of the spheres reverted to rods and engendered colonies

in the absence of penicillin, Unlike the normal rods, the spheres were

sensitive to osmotic shock and viability was completely lost by cytol-

ysie in distilled water, Lederberg designated these spheres as

protoplasts.

Objection to the term protoplasts, and its connotations, for

penicillin-induced spheres was raised by Salton and Shafa (1958). They

demonstrated that the penicillin-induced spheres of Gram negative bac-

teria probably contained residual cell wall material, and thus, the

term protoplast was not appropriate. They pointed out that the walls

of Gram negative bacteria were composed of both lipo-protein and muco-

complex components. The mucocomplex contained amino acids, muramic

acid, and glucosamine. It was the mucocomplex which was believed to be

responsible for the structural rigidity of bacterial cells. Cell walls

from normal rods and "walls" from penicillin-induced spheres of Vibrio

metchnikovii and Salmonella gallinarum were analyzed for components of











the mucocomplex. Sphere "walls" of both organisms had smaller amounts

of diaminopimelic acid and amino sugar than did walls from normal rods.

On the other hand, the per cent of polysaccharide and of lipid was

approximately the same for sphere "walls" and rod walls. These results

indicated that it was the mucocomplex structure of the cell wall which

was affected by penicillin. The formation of penicillin-induced spheres

from Gram negative bacteria was the result of interference by the anti-

biotic in the formation of the mucocomplex component of the wall. The

lipoprotein components were probably unaffected by penicillin. The

term spheroplasta has been suggested to describe the penicillin-induced

spherical forms in which the cell wall structure is believed to have

been modified rather than totally removed (McQuillen, 1960). Sphero-

plasts have been described as spherical cells induced by the action of

penicillin (or other methods) which are sensitive to osmotic shock and

incapable of engendering colonies on the usual media.

Lark (1958) described the multiplication of penicillin-

induced "globular" forms of Alcaligenes fecalis which occurred under

special circumstances. In a tryptone medium containing sodium chloride

and 50 units of penicillin per ml the rod-shaped cells were converted

to "globular" forms which could multiply. Multiplication o2 the

"globular" form was dependent on the presence of tryptone in the medium.

Substitution of glutamic acid for tryptone as a nitrogen source resulted

in the formation of "crescents" which were not capable of multiplication.

The active principal in tryptone was not identified.











The preparation and properties of spheroplasts of Aerobacter

aerogenes were examined by Gebicki and James (1960). Cells were

converted one for one into spheroplasts by the action of 1,000 units

of penicillin per ml added to cultures growing in broth containing

0,7 M sucrose, The spheroplasts apparently remained constant in number

for at least 5 hrs. Sucrose solutions of concentrations giving 5 or

more atmospheres of osmotic pressure preserved the spheroplasts; in

less concentrated solutions the spheroplasts lysed as indicated by

direct microscopic counts.

In the present study, penicillin-induced spheres were pre-

pared from the rod-shaped cells of a marine bacterium. The use of

the term spheroplasts to describe the spheres was Justified on the

basis of their properties. The lysis of the spheroplasts was examined

and the results were compared with the lysis of normal cells. Such a

comparison was one method to determine the osmotic strength of the

rigid component of the cell wall of the marine bacterium,














MATERIALS AND METHODS


For the sake of clarity the methods used in this study are

presented in 4 sections. The methods presented in each section

correspond with the different phases of the investigation. The sec-

tions concern, respectively, general methods, methods for the identi-

fication of the marine isolate, methods for the formation and examina-

tion of spheroplasts of the marine bacterium, and methods for esti-

mating the osmotic lysis of cells and spheroplasts.


General Methods

An artificial sea water (ASW) was used in preparing media

and for the washing of cells. It was formulated as follows:

NaCI, 23.5 g; Na2S04*7H20, 11.2 g; MgG12*6H20, 10.2 g; IKC, 0.75 g;

and distilled water, 1,000 ml. The medium used for growth of the marine

bacterium was prepared as follows, unless otherwise stipulated in

individual experiments: trypticase (Baltimore Biological Laboratory,

Inc.), 10.0 g and ASW, 1,000 ml. The pH of the medium was 6.8-7.0.

Trypticase, a tryptic digest of casein, was added as a source of

organic nutrients. A solid medium was prepared by adding 1.8-2.0 per

cent agar (B.B.L.) to the liquid medium. All media were sterilized

by autoclaving at 15 pounds pressure and 121 C for 15 min. The pH was











measured with a meter (Beckman, Zeromatic), or occasionally, with an

aqueous solution of bromthymol blue (0.01 per cent).

Glassware was initially cleaned in a solution of chromous

acid. Thereafter, subsequent to use, it was boiled for about 5 min

in Haemo-sol (Meinecke and Company, Inc.) and rinsed 5 times in tap

water and 5 times in distilled water.

All chemicals used were either of reagent or chemically pure

grade; all salts used were reagent grade. Salt solutions were pre-

pared with double distilled water; the final distillation was from

glass. Occasionally, distilled water that had been deionized was

used.

The bacterium studied was isolated by Bielling (1958) from

coastal waters at Matanzas, Florida. It was one of 100 isolates and

was designated M.B. 22. Based on the results of a series of tests,

reported under EXPER~EIMEQ RESULTS in this study, it was designated

Vibrio H.B, 22. The organism was maintained on slants of trypticase

ASW agar medium; transfer to fresh medium vas made about every four

days. Purity was examined occasionally by streaking organisms onto

the surface of solid media in Petri dishes. Two type of media were

used for this one of trypticase ASW agar and the other of trypticase

distilled water agar medium,

Starter cultures in broth were prepared by transferring

growth from slants to 20 ml of trypticase ASW broth in 125 ml












Erlenmayor flasks. For obtaining large crops of cells to be used in

experiments, 0,06 ml of a starter culture, incubated 3 to 5 hr, was

inoculated into each of a series of Erlenmayer flasks (500 ml) con-

taining 150 ml of 1.0 per cent trypticase ASt broth. All cultures

were incubated at 30 C, except when otherwise designated. All liquid

cultures were aerated by shaking on a machine (New Brunsvick) vith a

rotary motion. Time of incubation was usually 12 hr.

Turbidity measurements were made witha 'Spectronic 20"

spectrophotometer (Bausch and Lomb). Measurements were taken at a

wave loenth of 500 nm.

Centrifugation was done in a Servall apparatus (Type SS-1).

VashinC of cells or spheroplasts was done by sedimentation and

resuspension of cells in the desired solution. One washing consisted

of the following: sedimented cells were resuspended in the appropriate

solution and were re-sedimented; the supernatant was discarded and the

cells were resuspended. Cells were centrifuged at 9,000 ECF (relative

centrifugal force) and spheroplasts at 3,190 RCF., Before centrifuging

sphoroplasts the centrifuge head was precooled at 4 C for about

15 min.


Method for the Identification of Vibrio H.B. 22

The methods used to identify Vibrio H.D. 22 are given in this

section. Unless otherwise stated, the basal growth medium used in all

tests was trypticase (1.0 per cent) ASU. Agar was added to a final

concentration of 2.0 per cent when necessary for the preparation of

a solid medium.












Wet mounts from a young broth culture were examined microscopi-

cally under phase optics for motility, Wet mounts from slant cultures

one week old were examined microscopically for the presence of coccoid

bodies. Cell preparations in sea water were examined by electron

microscopy to determine the type of flagellation. Colonial charac-

teristics were determined from colonies which developed from cells

inoculated on the surface of solid media in Petri plates. The charac-

teristics of growth in liquid cultures, which were stationary, were

determined by inoculating 10.0 ml of liquid medium in a test tube.

To determine the temperature requirements of the bacterium, plates

and slants of the solid medium and broths of the liquid medium were

inoculated and incubated at various temperatures. Uninoculated controls

were also incubated at each temperature. The media were examined for

g;ro:.th periodically for one month.

Certain physiological activities of the bacterium were examined.

The reduction of tetramethylp--phenylene-diamine oxidasee test) was

determined by Kovacs' method (1954). The tests for gelatin liquefac-

tion, nitrate reduction, starch hydrolysis, and hydrogen sulfide pro-

duction were made as described by Bielling (1958). To demonstrate

lipid hydrolysis, the basal medium was modified to contain neutral red

indicator, agar, and 1.0 per cent olive oil. The medium in Petri

dishes was streaked with growth from a slant across a diameter and

incubated. Lipid hydrolysis was indicated by an increased acidity

as shown by a change in the color of neutral red in the zone adjacent












to growth. To demonstrate cellulose decomposition, sterile strips of

filter paper (Jhatman No. 1), suspended in inoculated tubes of the

liquid medium, were examined periodically for decomposition. For the

detection of anaerobic growth, the basal medium was modified to contain

glucose (0.5 per cent) and agar. The solid medium in Petri dishes

was inoculated by streaking growth from a slant over the surface of

the medium and the dishes were incubated in a sealed disiccator jar

placed in a 30 C incubator. Provision for oxygen removal from the jar

was made by the method of Mueller and Miller (1941). The amounts of

the reagents used to obtain anaerobiosis were Na2C03, 0.5 g; chromium

powder, 1.5 g; and H2SO4 (15 per cent by volume), 15 ml. The mixture

of these chemicals brought about the prompt reduction in the sealed

jar of 3.0 ml of methylene blue. The methylene blue (15 ug) was in

an alkaline solution (NaOH) containing 2.0 per cent glucose. The

anaerobic jar was fitted with a one-way valve to allow the escape

of gases. Control plates were incubated aerobically.

The method of Hugh and Leifson (1953) for the demonstration

of oxidative versus fermentative metabolism of carbohydrates was fol-

lowed although the medium was slightly modified: Trypticase, 0.2 per

cent, was substituted for peptone, and ASH for sodium chloride. Other-

wise, the procedure was the same. Duplicate tubes of the solid medium

were inoculated by stabbing. One tube was sealed with a layer of

petrolatum and was designated the "closed tube." All tubes were incu-

bated at 30 C and examined periodically for one month. Acid or alkali

production was indicated by a color change in the acid-base indicator.











The sensitivity of the bacterium to certain drugs was determined

by the method of Shewan, Hodgkiss, and Liston (1954) for the differen-

tiation of asporogenous, non-pigmented rods. The antibiotics used

were penicillin, terramycin, diamino-di-isopropylpteridine (vibrio-

static agent 0-129), and dimethyl-phenanthroline. Solid medium in

Petri dishes was inoculated by spreading about 1.0 ml of a young

broth culture over the surface of the medium. The inoculum was allowed

a short time to dry and tablets containing, respectively, penicillin

and terramycin were placed on the surface of the agar and the other

drugs were added as saturated solutions to wells in the agar. The

dishes were incubated at 30 C for 24 hr. Sensitivity to a particular

drug was indicated by a zone of inhibition of growth around the drug

tablet or well.


Methods used for the preparation of spheroplasts and the examination
of their properties

Spheroplasts were induced from rod-shaped cells of Vibrio

M.B. 22 by the action of penicillin G (Squibb, potassium salt) added

to young cultures growing in ASW containing trypticase, The procedure

used for inducing spheroplasts was as follows: 20.0 ml amounts of

broth (0.1 per cent trypticase in ASW) were inoculated with 0.2 ml of

a liquid culture of Vibrio M.B# 22 (the inoculum was washed once

with ASW subsequent to 12 hr incubation). The cultures were incubated

at 30 C with aeration. When growth was evident as indicated by a

turbidity increase (usually after 2 to 3 hr), 0.1 ml of the appro-

priate concentration of penicillin was added. Samples from incubated











cultures were examined microscopically for spheroplasts at intervals

subsequent to the addition of the penicillin. The turbidity of

penicillin-treated cultures was determined with the "Spectronic 20."

In a number of experiments the rate of turbidity change of

cultures with and without penicillin was examined. In these-experi-

ments special Erlenmeyer flasks contained the cultures: each flask

(250 ml) was fitted with a coloricater tuba to allow the rapid measure-

ment of turbidity without the necessity of transferring the culture.

In some experiments population changes in cultures were estimated by

direct counts. Samples of 1.0 ml each were taken periodically from

the cultures and the number of cells in each sample was determined

microscopically.

To obtain large crops of spheroplasts, cultures of Vibrio

MB. 22 which had been incubated for 8 to 10 hr were diluted five-fold

with fresh broth (1.0 per cent trypticase in ASI) containing peni-

cillin (the final concentration was 100 units per ml). The diluted

cultures were incubated at 30 C with aeration for 5 hr. The sphero-

plasts were harvested by centrifugation and washed twice with ASU.

Concentrated suspensions of spheroplasts in ASH were prepared in this

manner for use in experiments concerning lysis.

Cells and spheroplasts were counted in a Thoma chamber

(HaIvkslcy, London) designed especially for microscopic counts of

bacterial cells. Both cells and spheroplasts were counted with the

aid of a microscope using phase-contrast (dark) illumination. Cells

were observed at a magnification of 100X and spheroplasts at a magnifi-

cation of 400X. Four separate preparations of each sample were











examined and when possible about 100 to 150 organisms were counted from

each preparation. The results reported were an average of these

separate counts. Where pertinent, an estimation of the variation

was reported. The statistics applied follow the recommendations of

Stearman (1955).


Method for the estimation of lysis of cells and spheroplasts of
Vibrio H.B. 22

The cells used in these experiments were harvested exactly

12 hr subsequent to inoculation. Spheroplasts were harvested exactly

5 hr after the addition of penicillin to cultures 8 to 10 hr in age.

Both cells and spheroplast were washed twice in ASW. Concentrated

cell and spheroplast suspensions in ASW were prepared and adjusted

so that 0.1 ml amounts of each diluted with 10.0 ml of ASW gave similar

optical density readings at a wave length of 500 mu. The general

procedure used in all experiments for establishing lysis curves was

as follows. In a 15 mm test tube was placed 0.1 ml of concentrated

cell (or spheroplast) suspension. Ten ml of the appropriate salt

solution was then pipetted directly onto the cells in a manner to insure

the rapid mixing of cells with the salt solution. In this way a

series of cell suspensions in graded concentrations of sodium chloride

was prepared. The cell (or spheroplast) suspensions were incubated

for 5 min at 37 C in a water bath; immediately subsequent to incuba-

tion samples were taken from each suspension and analyzed for residual

activity by the methods used for the estimation of lysis. The











dilution of cells (or spheroplasts) in the suspending fluid and the

incubation at 37 C was arranged so that cells were in solution and at

37 C for exactly 5 min. Salt solutions used for suspending cells all

contained 10-5 H sodium bicarbonate.

Direct evidence of lysis was revealed by an examination of

the supernatants of cell suspensions for protoplasmic constituents.

The supernatants from cell and spheroplast suspensions were examined

for light adsorption at a wave length of 260 mu. Supernatants from

cell suspensions wore also examined for protein. The method used to

estimate the quantity of protein in the supernatant was that of Lowry

et al. (1951). The protein standard used was bovine plasma albumin.

No estimate was made of the total amount of either ultraviolet-

adsorbing material or protein in cell suspensions. The lysis of

cells and spheroplasts in graded concentrations of sodium chloride was

estimated by several methods; turbidity, colony counts, reduction of

2,3,5-triphenyltetrazolium chloride, production of indol, and a

microscopic method. In some experiments several of the methods were

used in examining the same preparation. In these cases the methods

were carried out as rapidly as possible to insure the shortest possible

exposure to the salt solution subsequent to 5 min incubation at 37 C,

Lysis was estimated by viability counts of cells (or sphero-

plasts) suspended in the various salt solutions. Aliquots were taken

from cell suspensions, dilutions were made in trypticase (1.0 per cent)

ASU and 0.1 ml of suitable dilutions was spread with a glass rod over

the surface of a solid medium in Petri diclhes. Triplicate Petri











dishes of each dilution were prepared and the colonies developing

subsequent to 48 hr incubation were counted. The solid medium was a

similar formulation but contained 2.0 per cent agar. Prior to inocu-

lation the solid medium in Petri dishes was dried for several days at

30 C.

Lysis was also estimated by the ability of suspensions of

Vibrio H.B. 22 in various salt solutions to produce indol. Subsequent

to the incubation of cell suspensions at 37 C for 5 min, 2,0 ml of

each suspension was transferred to each of duplicate tubes for the

assay of indol production. Assay tubes contained 250 ug L-tryptophan

(Nutritional Biochemicals Corporation) in 0.2 ml phosphate buffer

(0.01 H, pl 7.2). Subsequent to 20 min incubation at 37 C in a bath,

0.5 ml of 10 per cent trichloroacetic acid was added. Indol was

determined by a modification of the method of Wood et al. (1947).

Two ml of toluene were thoroughly mixed with the entire contents of

the assay tube. One ml of the toluene layer was added to 9.0 ml of

Ehrlich's reagent and after 30 min the optical density was measured

with the "Spectronic 20" at a wave length of 540 mu. The composition

of Ehrlich's reagent was as follows: p-dimethyl-aminobenzaldehyde, 10 g;

ethanol, 800 ml; hydrochloric acid (37 per cent), 200 ml. The ingre-

dients were added in the order listed. Charcoal (Norite A), 5.0 g, was

shaken with the reagent which was subsequently cleared by filtration.

Lysis was estimated by the ability of cell suspensions in











various salt concentrations to reduce the colorless 2,3,5-triphenyl-

tetrazolium chloride to the colored fornazan. Tetrazolium reduction

was assayed by the method of Kun and Abood (1949) vith the exception

that the reduction was carried out under anaerobic conditions (Hanks,

1951) by a modification of the method of Hiunate (1950). Dry 15 mn

tubes were flushed with nitrogen gas and sealed with rubber stoppers.

The tubes were opened and again flushed as cells (or spheroplasts)

were added. After standing for 5 min at 37 C in a water bath, 2.0 ml

were placed in duplicate assay tubes. The assay tubes contained

200 ug tetrazolium and 250 mu moles glucose in 0.2 ml phosphate

buffer (0.01 1, pi! 7.0). The assay tubes were flushed with nitrogen

gas prior to the addition of the cells. After 15 min incubation at

37 C the enzymatic reaction was stopped with 4.0 ml of n-propanol.

After ctan(]ing for 15 to 30 min the tubes were centrifuged and the

optical density of the supernatant was determined with the "Spec-

tronic 20" at a wave length of 485 mu. The nitrogen gas used was

commercial tank nitrogc.a which was freed of oxygen by passage through

a column containing heated threads of metallic copper.

Estimation of lysis by microscopic count of surviving organisms

was also attempted. Cells of Vibrio M.B. 22 lysed in solutions hypo-

tonic to sea water. However, the lysed cells (or spheroplasts) could

not easily be differentiated by microscopic examination from those

which had not lysed. Therefore, a direct count of cells (or sphero-

plasts) would not give a good estimate of the degree of lysis.












To overcome this difficulty the following procedure was adopted.

Cell (or spheroplast) suspensions were prepared in the appropriate

salt concentrations as usual. The tubes were incubated for 20 min

at 37 C in a bath. Then 5.0 ml from each tube was pipetted into a

separate flask (250 al) containing 20 ml of penicillin broth (100 units

penicillin per nl final concentration, 1.0 per cent trypticase ASW

broth). The flasks were incubated for 5 hr at 30 C with aeration.

Samples were taken and the number of spheroplasts were counted

microscopically using a Thorma chamber.

The dry weight of cell suspensions in ASW was determined by

drying samples of known optical density and weighing them. The weight

of cellar was corrected for the weight of salts in the suspensions

(Pratt, unpublished).














EXPERIMENTAL RESULTS


This study was concerned with gaining more knowledge of the

osmotic fragility of marine bacteria, One marine bacterium was se-

lected and some aspects of its fragility were examined. To this

end the organism was described in terms of physiological and morpho-

logical characteristics considered to be of taxonomic significance;

spheroplasts were induced from normal rods and certain of the proper*

ties of spheroplasts were examined; and the lytic thresholds of

cells and spheroplasts were determined by methods developed for the

quantitative estimation of lysis. The lytic thresholds of cells and

spheroplasts were compared in an attempt to determine the role of

the penicillin-sensitive component of the cell wall in protecting the

cell against osmotic lysis.


The Morphological and Physiological Characteristics
of the Marine Bacterium MB. 22

The bacterium examined in this study was isolated by Bielling

(1958) from a sample of sea water obtained at Matanzas, Florida.

The bacterium was designated M.B. 22 at the time of its isolation.

The organism grew rapidly in an artificial sea water (ASW) medium

containing trypticase as a source of organic nutrients but failed

to develop in a medium formulated of trypticase and distilled water.

Maximum growth was obtained in about 12 hr in aerated cultures












growing at 30 C in ASH containing 1.0 per cent trypticase. Growth

developed slower in stationary cultures. Grown for 12 hr in ASW

broth and examined microscopically with phase-contrast optics, the

bacteria were observed to be actively motile and cells were mostly

single with pairs being observed infrequently. The organism was

pleomorphic in morphology and cells were straight to curved rods with

pointed ends.

Colonies on sea water agar containing trypticase were moderate

in size, circular, smooth, entire, slightly raised, homogeneous,

and colorless. Old colonies were yellowish in color and opaque to

transmitted light. Growth on sea water agar slants containing

trypticase was abundant in 24 hr, smooth, homogeneous, and colorless.

Gror:th in trypticase sea water broth in stationary cultures was heavy

and the medium was turbid throughout with a thin pellicle and a

flocculent sediment in old cultures. Growth in aerated cultures was

so heavy that it gave the medium a chalky appearance. Broth cultures

gave a strong odor of indol.

Growth was most pronounced on solid media and in stationary

liquid cultures at an incubation temperature of 37 C (Table 1). This

was not considered unusual although most marine bacteria have a lower

optimum temperature (Bergey's Manual, 1957). However, at least one

species, Photobacterium harveyi, has an optimum between 35 and 39 C.

The optirnua temperature for growth of marine bacteria may reflect the











Table 1

EFFECT OF INCUBATION TEMPERATURE ON THE GROWTH OF
VIBRIO M.B. 22


Temperature of Growth
Incubation

On Plates On Slants In Broth
(OD)*



4 0 0 0.0


30 3+ 3+ 0.48


37 4+ 4+ 0.76


40 2+ 3+ 0.32


50 0 0 0.0


*0D = optical density.
4+ = best growth; growth at
with this standard.


other temperatures was compared


Medium: 1.0% trypticase and 2.0% agar (where necessary) in AS1~.
Plate, slant and broth cultures incubated at appropriate tempera-
tures and readings made after 72 hr. OD determined with
"Spectronic 20" at 500 mu.



















































Phase contrast micrograph of cells
of Vibrio M.B. 22 (magnification
approximately 1200x).


Figure 1.




















































Electron micrograph of Vibrio M.B. 22.
The cells were mounted on collodion
on copper grids and shadow casted with
chromium. Electron microscopy was
performed by Mr. T. Carlisle, Physics
Department, University of Florida
(magnification approximately 35,000x).


Figure 2.












conditions of the environment from which the organisms were isolated.

Although there was no growth of the bacterium H.B. 22 on solid media

incubated at 4 C, if the inoculated media were removed from this

temperature after several weeks and incubated at 30 C, colonies

developed within 24 hr.

The bacterium was examined for certain characteristics

which were considered to be of taxonomic significance (Bergey's

Manual, 1957; Spencer, 1955). These characteristics included the

type of flagellation, the cell morphology, the ability to reduce

the oxidase reagent, the type of metabolism of carbohydrates, and

the sensitivity to certain antibiotics.

The bacterium was a monotrichous, polarly flagellated rod.

The type of flagellation was indicated using the electron microscope.

Pratt (unpublished) demonstrated the same result using a staining

technique (Leifson, 1951). Cells were straight to curved rods,

0.6 by 1.9 microns (Figure 1). The curvature was slight and was

seen most often in wet mounts made from agar cultures which were 2

to 3 days old, Large coccoid bodies were observed in preparations

made from agar cultures of a week or more in age. Thesignificance

of these bodies was noted by Liston (personal communication) who

observed them in cultures of vibrios. No internal structures were

observed with phase-contrast optics. A cell wall and a flagellum

were evident in an electron micrograph (Figure 2).

Other physiological properties of the bacterium were












also examined (Table 2). The bacterium rapidly reduced tetra-methyl-

p-phenylene-diamine oxidasee reagent) with the formation of a deep

blue spot on the filter paper. Parallel tests of species of

Pseudomonas and Escherichia coli were positive and negative, respec-

tively. Shewan (personal communication) has shown that among motile

rods those with polar flagella gave a positive oxidase test. This

included organisms of the following genera: Pseudomonas, Xanthomonas,

Aeromonas, and Vibrio.

The bacterium reduced nitrate and formed indol but neither

trait is of determinative value on the generic level, although very

few species of Pseudomonas are known to produce indol. The bacterium

was capable of anaerobic growth and produced an extracellular enzyme

(or enzymes) that hydrolyzed starch. It was not capable of attacking

gelatin or cellulose during one month of incubation. The latter

test aids in differentiating Cellvibrio which attacks cellulose

from Vibrio which does not.

The Hugh and Leifson test (1953) was used for assessing the

fermentative versus oxidative abilities of the bacterium with several

substrates. The test was considered to be of taxonomic importance

in separating species of Pseudomonas, Aeromonas, and Vibrio, Species

of Pseudomonas have an oxidative metabolism'while species of Aeromonas

and Vibrio ferment carbohydrates. The marine bacterium M.B. 22

fermented a number of carbohydrates (Table 3). The fermentation

was indicated by an acid reaction in the closed tubes. No gas was











Table 2

PHYSIOLOGICAL CHARACTERISTICS OF BIBRIO M4B. 22


Physiological Properties Result of Test
Examined


Motility +

Oxidase +

Nitrate Reduction +

Indol Formation +

Coccoid Bodies Formed +

Hydrogen Sulfide Production +

Starch Hydrolysis +

Anaerobic Growth +

Gelatin Liquefaction

Lipid Hydrolysis

Cellulose Decomposition


+ = positive reaction; possesses property.

- = negative reaction; does not possess property.












Table 3

FERMENTATION OF CARBOHYDRATES BY VIBRIO M.B. 22


Carbohydrate Result (72 hr)


Open Tube Closed Tube


Glucose A A

Galactose A A

Dextrin A A

Glycerol A* A*

Maltose A A

Mannitol A A

Mannose A A

Salicin A* A*

Sucrose A A

Sorbitol A A

Lactose A* A*

Arabinose -

Raffinose

Rhamnose

Xylose


A = acid produced

= no fermentation.

*Acid reaction developed slowly but was positive by 72 hr.







38


produced. It is this latter point which differentiates the anaero-

genic Vibrio species from the aerogenic Aeromonas species. With

sugars such as glucose and sucrose the acid reaction was evident in

7 to 10 hr. However, the fermentation of lactose required 72 hr. In

the media containing those carbohydrates which were used slowly or

were not used at all the indicator dye was turned colorless after

several weeks.

The fermentation of lactose involved an adaptation to this

substrate. Subsequent to 72 hr incubation after inoculation, the

glucose medium was turbid throughout; in contrast, in the lactose

medium discrete fingers of growth emanating from the original stab

permeated the medium and the acid reaction was limited to localized

areas near the growth. The growth from one of these areas was picked

and reinoculated into fresh lactose agar in a closed tube. An acid

reaction was produced in the medium within 24 hr. Serial passage

through lactose agar gave a culture which would ferment the sugar in

about 10 hr. The adaptation to lactose fermentation was considered

to be a distinctive characteristic of the bacterium.

Several antibiotics were suggested by Shicl:in et al. (1954)

for the differentiation of Pseudomonas and Aeromonas species on the

one hand from Vibrio species on the other. The antibiotics were

terramycin, penicillin, the pteridine compound 0-129, and dimethyl

phenanthroline (DMP). The vibriostatic agent 0-129 was developed by

Collier et al. (1950) for therapy against Vibrio comma. The mechanism

of its inhibition was not known but it was considered to be a specific











inhibitor of vibrios. The DMP was reported to be bacteriostatic to

some pseudomonads and other Gram negative bacteria (Shewan, personal

communication). Saturated solutions of the 0-129 and the DMP were

used. The vibriostatic agent 0-129 and the DMP inhibited the growth

of the bacterium M.B. 22 (Table 4). The zone of inhibition around

each of the compounds was large and well defined. Neither peni-

cillin nor terramycin inhibited growth under the conditions used in

the test. However, it was found by Spencer (1955) that sea water

inhibited the action of terramycin.

The bacterium was a Gram negative, monotrichous rod. The

flagellum was polar and the cells were slightly curved. The organism

fermented carbohydrates, produced coccoid bodies in old cultures and

was sensitive to the vibriostatic agent o-129. These properties were

similar to those attributed to species of the genus Vibrio. Accordingly,

the marine organism M.B.


Spheroplasts of Vibrio M.B. 22

The addition of appropriate amounts of penicillin to cucuLte

of Vibrio M.B. 22 growing in an ASW medium containing trypticase resulted

in the disappearance of normal rods and the formation of spherical cells

in about 3 hr. The transformation occurred in the absence of an addi-

tional osmotic stabilizer such as sucrose and indicated the spheres

to be approximately in osmotic equilibrium with the growth medium.

The stability of spheres in the growth medium might have been antici-

pated since sea water has a relatively high osmotic pressure and contains












Table 4

SENSITIVITY OF VIBRIO M.B. 22 TO ANTIBIOTICS


Penicillin Terramycin Vibriostatic DP**,
(2.5 units) (10 ug) Compound 0/129*


+ +


+ = sensitive.

= insensitive.

*2, 4-diamino-6, 7-isopropylpteridine.

**2, 9-dimethyl-l, 10-phenanthroline.

Solid medium of trypticase (1.0%) -ASW agar (2.0%) flooded
with broth culture (12 hr in age) and allowed to dry. Penicillin
and terramycin added as drug tablets and liquid 0-129 and DMP added
to agar wells. Inhibition of growth determined after 24 hr incuba-
tion at 30 C.











magnesium ions, both of which were reported as necessary to stabi-

lize penicillin-induced spheres of other Gram negative bacteria

(Lederberg, 1956; Gebicki and James, 1960). The formation and proper-

ties of penicillin-induced spheres of Vibrio M.B. 22 were studied to

establish the conditions essential to their formation, the numbers

produced, and their stability.

The growth of Vibrio M.B. 22 was inhibited in the presence

of 1.0 to 100 units of penicillin per ml (Table 5). Whereas 2.5 units

of penicillin per ml on solid media were insufficient to inhibit

growth, in liquid cultures 1.0 unit caused an inhibition. The inhi-

bition was evident 2 hr subsequent to the addition of penicillin,

but inhibition was not complete since growth continued at a reduced

rate for 8 hr. In the presence of 10 or 100 units of penicillin

per ml the turbidity increase of cultures, over the interval tested,

was about one-half that of the increase in cultures without peni-

cillin; over the same interval the turbidity increase in cultures

with 1.0 unit of penicillin per ml was two-thirds that of cultures

without penicillin. The degree of turbidity increase of cultures

in the presence of 1.0 unit of the antibiotic per ml was indicative

of a partial inhibition.

Penicillin treated cultures were examined microscopically

using phase-contrast illumination. The bacterial cells growing in

the presence of penicillin underwent a series of morphological

changes (Table 6) which indicated that the effect of the antibiotic

was reflected in an inhibition in the process of cell division.












Table 5

GROWTH OF VIBRIO M.B. 22 IN GRADED PENICILLIN
CONCENTRATIONS


Penicillin Optical Density
(units/ml)

Age (hr)



0 2 4 6 10

0.0 0.05 0.14 0.39 0.70 0.82

0.0 0.05 0.14 0.39 0.70 0.82

1.0 0.06 0.15 0.34 0.52 0.56

1.0 0.05 0.14 0.33 0.52 0.56

10.0 0.05 0.14 0.32 0.45 0.47

10.0 0.05 0.15 0.33 0.47 0.50

100.0 0.05 0.14 0.33 0.45 0.50

100.0 0.05 0.15 0.34 0.47 0.52


Cultures: 20 ml 0.1% trypticase-ASW broth containing
Vibrio M.B. 22 in 250 ml Erlenmeyer flasks. Incubated on shaker
at 30 C. Turbidity estimated in "Spectronic 20" at 500 mu.
Penicillin added immediately after 2 hr reading.











Table 6

MORPHOLOGICAL FORMS OBSERVED FOLLOWING THE ADDITION OF
PENICILLIN TO GROWING CULTURES OF VIBRIO M.B. 22


Time Penicillin
(hr) (units per ml)


1 100 1,000


1 motile rods motile spheres spheres


2 elongated* spheres crescents
rods crescents**


3 elongated crescents crescents
rods


*Approximately ten times normal length.

As seen in Figure 1.

Vibrio M.B. 22 cultured in 0.1% trypticase-ASW broth for
2 hr at 30 C with shaking. Subsequent to penicillin addition
samples were taken at the designated times; wet mount preparations
were examined microscopically under phase-contrast.










Bacteria incubated for several hr in media with 1.0 unit of penicillin

,per ml were greatly elongated, not unlike the appearance of cotton

fibers, and revealed no tendency to swell. The bacteria growing in

the presence of 100 or 1,000 units of the antibiotic per ml were

converted from normal rods to spheres in a process which involved a

series of morphological alterations and which occurred over a period

of 2 to 3 hrs. A swelling of the rod, either centrally or sub-

terminally, first appeared in about 30 min and became progressively

larger with time. The rod eventually disappeared leaving a spherical

body. The sphere enlarged with time and revealed, with phase-con-

trast optics, a cortex-like area of phase dense material, a trans-

parent body, and a dark periphery (Figure 3). The spheres were

similar to those described by Lederberg (1956) as protoplasts, by

Lark (1958) as crescents, and by others (IIurwitz, et al., 1958;

Gebicki and James, 1960) as spheroplasts. The mature spheres (or

crescents in Lark's nomenclature) were non-motile and revealed no

tendency to clump. Cultures of spheres demonstrated metabolic

activity and were capable of reducing tetrazolium and producing indol

from tryptophan (Tables 17 and 19). Some 20 to 30 per cent of the

spheres were able to engender colonies on a penicillin-free medium

formulated of artificial sea water and trypticase (Table 15). The

spheres lysed immediately when placed in distilled water and were

incapable of forming colonies on agar media following a brief ex-

posure in distilled water (Table 12). The term spheroplasts was

suggested by McQuillen (1960) to describe spherical forms which are





















































Phase contrast micrograph of spheroplasts
of Vibrio M.B. 22 (magnification approx-
imately 1,200x).


Figure 3.











osmotically fragile and are induced by the action of penicillin on

growing bacterial cells. On the basis of this description the

term spheroplasts was used to designate the fragile, spherical

forms of Vibrio M.B. 22.

The number of spheroplasts produced by the action of peni-

cillin from a population of growing cells was estimated by direct

counts of cells, preceding and subsequent to the addition of the

antibiotic. The normal rods of Vibrio M.B. 22 growing in trypti-

case ASW broth were converted quantitatively into spheres in the

presence of 100 units per ml of penicillin in about 2 to 3 hr

(Table 7). The entire population of rods apparently was converted

to spheres. The evidence supported the assumption that each rod

was converted to a single sphere, an interpretation which has also

been made in the case of other Gram negative bacteria (Lederberg,

1956; Gebicki and James, 1960). The spheroplasts were stable and

did not increase in numbers over a period of at least 8 hr, indicating

that the spheres did not multiply and that they were approximately

in osmotic equilibrium with the medium. The optical densities of

the cultures with and without penicillin increased over the period of

the examination. Over the interval from the time of the addition of

penicillin to the final reading, the turbidity increase .of cultures

containing penicillin was one-half the increase of cultures without

penicillin. The effect of penicillin on turbidity was evident 2 hr

subsequent to the addition of the antibiotic. The greatest increment

of change in turbidity in cultures containing penicillin occurred











Table 7

THE RELATION OF SPHEROPLASTS FORMED PER CELL AND THE
GROWTH OF SPHEROPLASTS


Time With Penicillin Without Penicillin


OD Number OD Number


x 107/ml x 107/mi

0 -- 0.04 10.4

1 --- 0.08 14.5

2 0.20 -* 0.20 21.9

3 0.31 20.0 0.32

4 0.39 20.3 0.54

5 0.46 23.8 0.70

6 0.48 0.78

7 ,0.51 20.4 0.85 149.0

10 0.54 22.2 0.90 187.0


Cultures: 20 ml 0.1% trypticase-ASW broth containing Vibrio
M.B. 22 in 250 ml Erlenmeyer flasks. Incubated on shaker at 30 C.
Penicillin to give 100 units per ml added to one of duplicate flasks
after 2 hr cultivation. Samples taken for counts and OD estimated,
with "Spectronic 20" at 500 mu, at designated times.











immediately after the addition of the antibiotic; this increase in

turbidity was exactly paralleled in the cultures without penicillin.

In this regard the effect of the antibiotic was immediately evident as

an inhibition of cellular multiplication. However, the effect on the

assimilatory activities of the cells, as indicated by mass, i.e. optical

density, was possibly not a direct consequence of the antibiotic since

the mass increased with time. The decreasing rate of turbidity change

was probably due both to a diminishing rate in the size increase of

spheroplasts and to their failure to multiply. In comparison the

cultures without penicillin revealed the greatest increment of turbidity

increase between the third and fourth hr of incubation and there was

a steadily decreasing rate of change in turbidity thereafter. The

decrease in this case was probably a consequence of the limitations of

oxygen and nutrients, and of crowding# It was noted that in the cultures

without penicillin an approximate ten-fold increase in optical density

was paralleled by a ten-fold increase in cell numbers. The results of

the experiment indicated that the rod-shaped cells of Vibrio M.B. 22

growing in the presence of suitable amounts of penicillin were converted,

one for one, to spheroplasts and that the spheroplasts were capable of

growth but not multiplication,

The turbidity increase of penicillin-treated cultures was con-

sidered to be an indication of the growth of spheroplasts. This view

was substantiated by an experiment which demonstrated that the optical

density increase of penicillin-treated cultures was directly related











to the amount of nutrient provided to the culture (Table 8). The rela-

tion between the optical density of cultures and the quantity of avail-

able nutrient was determined by cultivating Vibrio M.B. 22 in several

different amounts of nutrient. The method used was as follows. The

bacterium was inoculated into several media, all containing ASW and

penicillin, but each containing a different concentration of trypti-

case. The concentrations used were 0.02, 0.06 and 0.1 per cent tryp-

ticase. A duplicate of each medium without penicillin was also inocu-

lated. A concentrated inoculum was employed to insure that the nutrient

would be rapidly assimilated. The optical density of the inoculated

cultures was determined periodically until it ceased to increase and

then the total number of spheroplasts per ml was estimated by a micro-

scopic count.

Subsequent to incubation for 7 hro, the optical density attained

by each culture was dependent on the concentration of trypticase avail-

able. In penicillin-treated cultures a greater optical density was

attained in 0.1 per cent than in 0.02 per cent trypticase. However,

although optical density was dependent on trypticase concentration the

number of spheroplasts formed in 0.02 and 0.1 per cent trypticase was

approximately the same. This observation was interpreted as an indi-

cation that spheroplasts were capable of growth subsequent to their

formation. The optical density of penicillin-treated cultures was less

than that of the corresponding cultures without penicillin, which

demonstrated again the inhibitory action of the antibiotic. In peni-

cillin-treated cultures the greatest increment in optical density











Table 8

GROWTH OF SPHEROPLASTS IN RESPONSE TO 1NUTFEIIT
CONCENTRATION


Trypticase Penicillin OD Number
(%) ____(7 hr
culture)
Age (hr)


0 1 2 4 7


x 10 /ml

0.02 + 0.22 0.34 0.37 0.37 0.37 37.1

0.06 + 0.22 0.38 0.45 0.52 0.52

0.10 + 0.24 0.41 0.51 0.60 0.62 31.4

0.02 0.22 0.36 0.41 0.44 0.44

0.06 0.21 0.38 0.52 0.61 0.61

0.10 0.22 0.41 0.61 0.82 0.82


Vibrio M.B. 22 inoculated into ASW broth containing the
designated trypticase concentration and 100 units per ml penicillin
when appropriate. Cultures incubated on shaker at 30 C. OD estimated
with "Spectronic 20" at 500 mu. Samples taken at end of 7 hr and
spheroplasts counted with phase-contrast microscopy.












occurred during the first hour of incubation and was paralleled by a

similar increase in cultures without penicillin. Microscopic examina-

tion of the culture containing penicillin and 0.02 per cent trypti-

case which had been incubated for one hr revealed only motile spheres.

The changes in the size of spheres during incubation was not determined

quantitatively but microscopic examination indicated that they did

increase slightly in size. In a separate experiment it was determined

that cells growing in 1.0 per cent trypticase were 0.6 by 1.9 microns

in average size. In the presence of penicillin (100 units per ml)

these rods were converted after 5 hr incubation into spheres whose

average diameter was 5.3 microns. In this regard Lederberg and

St. Clair (1958) have reported that penicillin-induced spheres of

Escherichia coli increased in both size and mass upon incubation in

nutrient.

The stability of spheroplasts of Vibrio M.B. 22 subjected to

various treatments was examined. The number of spheroplasts per ml

did not decrease when washed twice in artificial sea water (Table 9).

The number also did not decrease when preparations were incubated for

12 hr at 4 C; the average number of spheroplasts per ml (suspended

in artificial sea water) was 460+67 x 10 before incubation at 4 C

and 490+34 x 106 subsequent to incubation. The addition of 0.05 ml

of formalin per ml to spheroplast suspensions did not cause a decrease

in the number of these forms. Spheroplasts washed and suspended in

artificial sea water were stable in number for a period of 170 min

(Table 10),







52



Table 9

EFFECT OF WASHING SPHEROPLASTS


Trial Number of Spheroplasts
(per ml)


Unwashed Washed (2x)


x 108 x 108

1 20.6 18.4

2 22.5 17.9

3 21.6 21.0

4 18.6 18.5

5 20.9 19.9

6 -- 16.6

7 -- 19.8


x = 20.8


x 18.9


s.d. = + 1.45


s.d. = + 1.47


Vibrio H.B. 22 cultured in 1.0% trypticase-ASW broth for
8 hr was diluted in fresh medium (1.0% trypticase-ASW) containing
penicillin (final concentration: 100 units per ml). Culture rein-
cubated on shaker at 30 C for 5 hr. Number of spheroplasts counted
before and after washing in ASW.











Table 10

STABILITY OF SPHEROPLASTS IN ARTIFICIAL SEA WATER


Time OD Number per ml Number per ml
(min) (average)


x 106

0 0.30 180; 182; 196; 170 182

10 0.30

20 0.30

30 0.30 176; 228; 204; 198 202

170 0.30 198; 160; 176; 202 184


Subsequent to incubation for 3 hr in medium containing penicil-
lin (100 units per ml) spheroplasts were harvested by centrifugation,
washed twice with ASW and suspended in ASW. OD was measured with the
"Spectronic 20" at 500 mu. Spheroplasts were counted in a Thoma
chamber.











The Osmotic Lysis of Cells and Spheroplasts of
Vibrio M.B. 22

Cells and spheroplasts of Vibrio M.B. 22 underwent rapid lysis

when suspended in distilled water. The lysis of the organism was indi-

cated by a turbidity decrease in the distilled water suspension, by

the loss of certain enzymatic activities, by the leakage into the

suspending fluid of ultraviolet-adsorbing material, and by the loss

of the ability of the organism to engender growth on the surface of an

optimum medium. These effects were not observed when cells (or sphero-

plasts) were suspended in artificial sea water or in 0.5 M sodium

chloride. The lysis of cells and spheroplasts of Vibrio M.B. 22

suspended in graded concentrations of sodium chloride was studied to

establish the minimum salt concentration required to protect the

organism against lysis.

The lysis of cells and spheroplasts exposed to graded concen-

trations of sodium chloride was estimated by several methods which in

effect were indirect estimates of cell damage. Direct evidence for

lysis was demonstrated by the leakage, from cells and from sphero-

plasts, of ultraviolet-adsorbing material and by the leakage, from

cells, of protein.

The susceptibility to lysis in distilled water of a marine



This term was applied to the normal cells which were rod-
shaped.

2
Spheroplasts designated the penicillin-induced cells which
were spherical shaped.












and a non-marine bacterium was compared. Cells of Vibrio M.B. 22

and Pseudomonas aeruginosa were suspended in distilled water and the

effect on turbidity and the ability to reduce 2, 3, 5-triphenyltetra-

zolium chloride (TTC) determined (Table 11). Both organisms were

grown in ASW containing trypticase (1.0 per cent); they were washed

twice in ASW and suspended in 0.3 M sodium chloride and in distilled

water. The optical density of suspensions of both species was less

in distilled water than in 0.3 M sodium chloride, but the difference

was greater for Vibrio M.B. 22. The marine bacterium reduced TTC to

a greater extent in 0.3 M sodium chloride than did the non-marine

bacterium, but did not reduce TTC when suspended .in distilled water.

However, the non-marine species revealed no decrease in TTC reduction

in distilled water over that in 0.3 M sodium chloride. The distilled-

water suspension of the marine bacterium, but not the non-marine bac-

terium, was viscous and foamy. Although both organisms were grown in

a solution high in salts only the marine bacterium revealed evidence

of lysis in distilled water. The decrease in the optical density accom-

panying the suspension of Pseudomonas aeruginosa in distilled water was

considered to be a result of the swelling of the organisms, analogous

to the swelling and shrinking of Gram negative bacteria suspended in

various concentrations of sodium chloride (Mager et al., 1956).

Evidence of lysis of cells and spheroplasts of Vibrio M.B. 22

was revealed by demonstrating the presence of ultraviolet-adsorbing

material in the supernatants obtained from suspensions of the organism











Table 11

EFFECT ON VIBRIO M.B. 22 AND PSEUDOMONAS AERUGINOSA OF
SUSPENSION IN DISTILLED WATER


Culture Suspending OD Formazan
Fluid Produced
(ug)



Vibrio M.B. 22 0.3 M NaCI 0.85 62.0


DW 0.54 0


Pa. aeruginosa 0.3 M NaCi 0.95 5.0


DW* 0.84 6.1



*Distilled water.

Bacteria incubated for 12 hr in trypticase (1.0%)-ASW broth.
Cells harvested and washed twice. OD measured with "Spectronic 20"
at 500 mu. Tetrazolium reduction was determined as described in the
text except that reduction was under aerobic conditions.











in dilute salt solutions (Table 12). Cells (and spheroplasts) were

suspended in graded concentrations of sodium chloride, the particulate

matter was sedimented by high speed centrifugation, and the optical

density of the supernatants was determined at a wave length of 260 mu.

An increase in the optical density, over that of supernatants from

suspensions in ASW, was observed in supernatants of both cells and

spheroplasts suspended in either 0.1 M sodium chloride or in distilled

water. There was a greater absorption in supernatants from sphero-

plasts suspended in 0.1 M sodium chloride and distilled water than in

supernatants from cell suspensions of the same concentrations of salt.

In this regard it should be noted that cell and spheroplast preparations

were adjusted so that sea water suspensions of each gave approximately

the same optical density when measured at a wave length of 500 mu.

The results indicated that from preparations of similar cell mass,

spheroplasts released more ultraviolet-absorbing material than did

normal cells. Protein was also detected from supernatants of cell

suspensions in dilute salt solutions (Table 13),

The lysis of cells and spheroplasts when exposed to graded

concentrations of sodium chloride was estimated by the following

methods: colony counts, optical density, the reduction of 2, 3, 5-

triphenyltetrazolium chloride (TTC), indol production from tryptophan,

and microscopic counts. The methods were indirect estimates of lysis

except for the method of direct counts of surviving organisms.

Lysis estimated by the method of colony counts

The estimation of osmotic damage to cells of Vibrio M.B. 22

suspended in graded concentrations of sodium chloride was determined












Table 12

LYSIS OF CELLS AND SPHEROPLASTS OF VIBRIO M.B. 22
ESTIMATED BY OPTICAL DENSITY AND VIABILITY


Organism Suspension Supernatant Colonies
Suspended OD OD per ml
in NaC1 (500-mu) (260 mu)
(M)


x 106

Cells

0.0 0.41 0.170 (-10)*
0.1 0.48 0.100 19
0.3 0.53 0.055 1,340
0.5 0.56 0.060 1,150
ASW 0.56 0.070 1,250

Spheroplasts

0.0 0.10 0.880 (- 10)*
0.1 0.49 0.340 **
0.3 0.56 0.075 155
0.5 0.60 0.085 206
ASW 0.59 0.085 175


*No colonies observed.

**Between 107 and 105.

Aseptically 0.1 ml of concentrated cell or spheroplast suspen-
sion was placed in 10.0 ml of each of the indicated salt solutions.
All were incubated 15 min at 37 C in a water bath. Turbidity was
determined with the "Spectronic 20" at 500 mu. Aliquots of each
suspension were diluted, where necessary, in broth (1.0% trypticase
in ASW) and suitable dilutions were inoculated onto a solid medium
(1.0% trypticase and 2.0% agar in ASW). Particulate matter in each
suspension was sedimented by centrifugation at 12,000 RCF. Super-
natants were examined for OD at 260 mu with a Beckman DU.












Table 13

LEAKAGE OF PROTEIN FROM VIBRIO M.B. 22


Cells Suspended in NaCI Protein
(M) (ug per ml)


0.0


25.0


13.0


0.05


0.10


0.15


0.20


0.30


5.6


4.6


4.6


4.6


3.0


ASW


Twice washed cells of a culture (12 hr in age) suspended
in the appropriate salt solutions for 10 min at 37 C. Particu-
late matter immediately sedimented by centrifugation at 12,000 RCF.
Supernatants cleared by repeated centrifugation when necessary.
Protein in the supernatants determined as described in the text
by a colorimeter method. OD of color indicative of protein was
measured with "Spectronic 20" at 725 mu. The supernatants analyzed
were taken from cell suspensions containing 0.8 mg dry wt bac-
terial cells per ml.











>y several methods. One method involved an estimation of the number

of organisms capable of engendering colony formation on the surface

of solid media (1.0 per cent trypticase in ASW agar) following exposure

to various concentrations of sodium chloride. Cells were suspended

in sodium chloride solutions, aliquots were taken and suitable

dilutions of each aliquot were inoculated onto the surface of the

solid medium. Following incubation colonies were counted. The results

revealed that few organisms formed colonies after exposure to 0.1 M

sodium chloride or to distilled water (Table 12). The examination

of similar suspensions of spheroplasts revealed a similar decrease

in colony counts (Table 12). Viable organisms capable of colony

formation were not detected from cell or spheroplast suspensions

in distilled water. The results were indicative of osmotic damage but

further investigation revealed a difficulty in using the method of

colony counts as an estimation of lysis. Cell suspensions in artifi-

cial sea water were examined and the total number of bacteria (deter-

mined by microscopic counts) were compared with the number of colony-

forming bacteria. The number of bacteria capable of engendering

colonies on the agar medium was considerably less than the total

number of cells present (Table 14). It was possible that of the

total population of cells a large proportion was non-viable. How-

ever, this view was not consistent with the observation that cell

populations were converted quantitatively to spheroplasts. Although

viability has been used to estimate lysis of halophilic bacteria











Table 14

RELATION OF COLONY COUNT TO DIRECT COUNT OF VIBRIO M.B. 22



Trial OD Direct Count Colonies Viable/Total
(per ml) (per ml) %



x 106 x 106


1 0.33 530 197 37


2 0.28 373 240 65


E. coli -- 255 184 72



Cultures: 20 ml 1.0% trypticase-ASW broth containing
Vibrio M.B. 22 in 250 ml Erlenmeyer flasks incubated several hr on
shaker at 30 C. Samples taken, dilutions made in 1.0% trypticase-
ASW broth, and suitable dilutions plated in 1.0% trypticase-ASW
agar. Direct counts made in a Thoma chamber. Colony counts made
after 48 hr incubation. OD estimated with "Spectronic 20" at
500 mu.











(Christian and Ingram, 1959b), the method was not satisfactory with

Vibrio M4B. 22. A similar low !'plating efficiency" was observed with

spheroplasts (Table 15):. A ratio of viable to total cells of about

50 per cent was reported for spheroplasts of E. coli (Lederberg and

St. Clair, 1958). The ratio obtained with spheroplasts of Vibrio

M.B. 22 was somewhat lower.


Lysis estimated by turbidity measurements

Another method used for estimation of cell lysis involved

measuring the turbidity of cell suspensions in graded concentrations

of sodium chloride. Cell suspensions, each containing the same quantity

of cells, were prepared in various concentrations of sodium chloride

and the turbidity of each suspension was determined (Table 16). There

was a slight decrease in turbidity over the range of salt concentra-

tions from 0.3 H to 0.08 1 with a precipitous decrease in solutions

less than 0*08 M in sodium chloride, This pattern of turbidity decrease

was also observed for spheroplast suspensions (Table 17)4 The sphero-

plasts, however, revealed a greater decrease in dilute salt solutions

than did cells, This perhaps indicated a greater release of cell

material by spheroplasts than by cells. The difficulty in establishing

the lytic threshold based on turbidity measurements came in deciding

in which concentration of salt a precipitous decrease occurred. In

the case of both cells and spheroplasts the lytic threshold was

considered to be over the range of salt concentrations from 0.08

to 0.06 M.











Table 15

RELATION OF COLONY COUNT TO DIRECT COUNT OF SPHEROPLASTS
OF VIBRIO M.B. 22


Time After Spheroplasts Colonies Viable/Total
Penicillin (per ml) (per ml) (%)
Addition
(hr)



x 106 x 106


4 473 132 28


6 505 121 26


9.5 543 112 21



Cultures: 20 ml 1.0% trypticase-ASW broth in 250 ml Erlenmeyer
flasks with 100 units penicillin per ml added. Samples diluted in 1.0%
trypticase-ASW agar. Direct counts made in a Thoma chamber. Colony
counts made after 48 hr incubation.











Table 16

LYSIS OF VIBRIO M.B. 22 IN GRADED CONCENTRATIONS OF HAC1
AS ESTIMATED BY INDOL FORMATION



Cells Suspended Residual Indol per ml Residual
in MaCI Turbidity Cell Suspension Activity
(M) (%) (ug) (%)


(1) (2)

0.0 31 0.0 0.0 0.0

0.02 30 0.0 0.0 0.0

0.04 44 1.1 1.1 10.7

0.06 61 2.9 3.3 30.3

0.08 84 6.2 6.2 60.7

0.10 89 8.4 8.4 82.1

0.14 91 9.5 9.9 94.6

0.16 90 9.9 9.5 94.6

0.20 94 9.5 10.2 96.5

0.30 97 9.9 10.6 100.0

ASW 00 9.1 9.5


% Residual Turbidity = OD x 100.
OD of ASW


% Residual


Activity activity x 100.
activity in 0.3 M NaC1


Cells incubated 5 min at 37 C in the designated salt con-
centrations. Samples taken and indol determined as described in the
text. OD of cells in ASW was 0.64.











Table 17

LYSIS OF SPHEROPLASTS IN GRADED CONCENTRATIONS OF NaCi
AS ESTIMATED BY INDOL FORMATION


Spheroplasts Residual Indol per ml Residual
Suspended Turbidity Spheroplast Activity
in NaCl (%) Suspension (%)
(M) (ug)


0.0


0.02

0.04

0.06

0.08

0.10

0.14

0.16

0.20

0.30


ASW


(1)

0.0

0.0

0.7

2.6

6.9

9.1

10.6

11.7

12.0

12.8

13.5


0.0

0.0

0.7

2.6

6.9

9.1

11.0

11.3


12.8

13.9


0.0

0.0

5.7

20.0

54.3

71.4

84.3

90.1

94.3

100.0


% Residual Turbidity =


% Residual Activity =


OD x 100.
OD of ASW

activity
activity in 0.3 M NaCl


Spheroplasts incubated 5 min at 37 C in the designated salt
concentrations. Samples taken and indol determined as described in
the text. OD of spheroplasts in ASW was 0.62,











Analysis for enzymatic activity was considered as a method

for estimating osmotic damage to cells or spheroplasts suspended in

graded concentrations of sodium chloride. Two systems were examined;

the production of indol from tryptophan and the reduction of 2,3,5-

triphenyltetrazolium chloride (TTC). It was previously established

by Pratt and Happold (1960) that the indol method could be used to

estimate cell lysis of Vibrio M.B. 22.


Lysis estimated by indol production

The production of indol by cells of Vibrio M.B. 22, incubated

in graded salt solutions, was determined. Indol formation was revealed

to be a function of the salt concentration in which cells were

suspended (Table 16). There was a gradual decrease in the amount of

indol formed as cell suspensions were made progressively more dilute

in sodium chloride. In solutions containing less than 0.1 M sodium

chloride a precipitous decrease was observed. In solutions containing

0.02 M sodium chloride, or distilled water, indol was not produced.

In solutions containing 0.14 M sodium chloride, or more, cells were

capable of maximum activity. The concentration of salt required to

support the activity of the organism was considered to be at least

0.1 M sodium chloride.

The activity of spheroplasts, suspended in various salt

solutions, in producing indol was similar to that of cells; the

pattern of indol decrease was also similar (Table 17). It should be

noted again that suspensions of spheroplasts and cells were adjusted

to give approximately the same optical density (at a wave length of











OU mu) in ASW. The formation of indol by spheroplasts in ASW was

slightly higher than for cells in ASW. For spheroplasts there was

a precipitous decrease in indol formation in solutions less than

0.1 M in sodium chloride. The lytic threshold was considered to

occur over the range of salt concentration from 0.1 M to 0.08 M

in sodium chloride.


Lysis estimated by TTC reduction

Lysis of cells and spheroplasts was also estimated by the

ability to reduce TTC. Cells rapidly reduced TTC in suspensions

containing 0.3 M sodium chloride (Table 18). However, as cells

were placed in salt solutions made progressively more dilute there

was a concomitant inhibition of TTC reduction. It appeared that at

least 0.14 M sodium chloride was necessary to protect the activity

of the organism. The reduction of TTC by spheroplasts was also a

function of the sodium chloride concentration (Table 19). Sphero-

plast suspensions, however, demonstrated a somewhat different

pattern of inactivation. With spheroplast suspensions the greatest

inhibition was in solutions less than 0.08 M in sodium chloride.

Both cells and spheroplast suspensions in ASW revealed an

inhibition of TTC reduction, The nature of this inhibition was not

determined. The reduction of TTC was compared to the production of

indol. Both cells and spheroplasts were active in producing indol

in 0.04 M sodium chloride but neither reduced tetrazolium in this

concentration. This possibly could be a reflection of a greater

sensitivity of the TTC-reducing system since the reduction of TTC













LYSIS OF VIBRIO M.B. 22
AS ESTIMATED BY


Table 18

IN GRADED CONCENTRATIONS OF NaCl
TETRAZOLIUM REDUCTION


Cells Suspended Residual Formazan per ml Residual
in NaCl Turbidity Cell Suspension Activity
(M) (%) (ug) (%)


(1) (2)
0.0 31 0.0 0.0 0.0

0.02 30 0.0 0.0 0.0

0.04 44 0.0 0.0 0.0

0.06 61 4.8 4.8 5.0

0.08 84 39.6 40.8 41.7

0.10 89 62.4 61.2 64.0

0.14 91 91.2 96.0 98.5

0.16 90 96.0 -- 99.5

0.20 94 97.9 -- 102.0

0.30 97 95.0 97.9 100.0

'AS 100 51.6 48.1 --


OD
% Residual Turbidity OD f x 100.
OD of ASW
% Residual Activity = activity
activity in 0.3 M NaCl


x 100.


Cells suspended in the designated salt concentrations for
5 min at 37 C. TTC reduction was determined as indicated in text.
OD of cells in ASW was 0.64.











Table 19

LYSIS OF SPHEPOPLASTS IN GRADED CONCENTRATIONS OF NaCI AS
ESTIMATED BY TETRAZOLIUM REDUCTION



Spheroplasts Residual Formazan per ml Residual
Suspended Turbidity Spheroplast Suspension Activity
in NaCI (%) (ug) (%)
(M)

(1) (2)

0.0 11 0.0 0.0 0.0

0.02 8 -

0.04 15 0.0 0.0 0.0

0,06 55 27.4 -- 28.0

0.08 79 72.0 77.8 76.5

0.10 89 85.0 77.8 83.1

0.14 84 80.6 83.5 83.9

0.16 94 86.4 83.5 86.8

0.20 90 85.0 89.3 89.1

0.30 94 97.9 -- 100.0

ASW 100 73.4 74.9 --


% Residual Turbidity =- ---Q 100,
OD of ASW


% Residual Activity =


activity
activity in 0*3 M NaCI


Spheroplasts suspended in the designated salt concentra-
tions for 5 min at 37 C. TTC reduction was determined as indicated
in text. OD of spheroplasts in ASW was 0.62.


x 100.











was believed to involve a multicomponent, multienzyme system (Nachlas

et al., 1960). In comparison with the enzyme system responsible for

indol production, the TTC-reducing system may be more readily sub-

ject to osmotic damage. On the other hand it was determined that there

was an inhibition of TTC reduction in suspensions dilute in cell

numbers (Appendix A). This result could explain the inhibition of

TTC reduction of cell suspensions in 0.04 M sodium chloride.

The two enzymatic methods employed for estimating lysis could

not be used to determine the number of cells surviving in the various

salt concentrations since in either case the enzyme activity was not

a linear function of the concentration of cells (Appendices A and B).

The estimation of osmotic damage to cells by the enzymatic methods

was, in effect, an indirect method. To overcome this difficulty a

more direct means of estimating lysis was developed. The method was

based on the ability of unlysed but not lysed bacteria to form sphero-

plasts in the presence of penicillin.


Development of a direct method for estimating lysis

Cells of Vibrio M.B. 22 were lysed when suspended in dilute

salt solutions and in distilled water, as evidenced by the leakage

of cell constituents. The cells were not completely fragmented in

the process and revealed, with phase microscopy, little change in

morphology, although cells in distilled water were markedly less

dense to phase light than cells in ASW. The lysis of cells was

therefore difficult to determine by direct observation since lysed

and unlysed cells could not easily be differentiated. However, this











difficulty was overcome by converting unlysed bacteria to sphero-

plasts which were easily differentiated from the lysed bacillary

forms. Since it was demonstrated that spheroplasts develop one

for one from the rod-shaped cells and do not multiply, then the

number of spheroplasts formed should be an estimate. of the unlysed

bacteria.

Cells were suspended in graded concentrations of sodium

chloride and after a period of incubation (20 min) to allow for

lysis an aliquot from each suspension was transferred to sea water

broth containing penicillin (1.0 per cent trypticase and 100 units

per ml of penicillin in ASW). Following incubation for 5 hr unlysed

cells had developed into spheroplasts which could be differentiated

from bacillary forms and counted microscopically. The ability of

cells to form spheroplasts was a function of the sodium chloride

concentration to which cells were exposed (Table 20). As the sodium

chloride concentration was lowered from 0.3 M to 0.03 M there was a

progressive increase in lysis and no unlysed cells (i.e., no sphero-

plasts) were observed from suspensions in 0.02 M sodium chloride or

in distilled water. In solutions greater than 0.08 M in sodium

chloride the majority of cells were protected from lysis.

Spheroplasts, like cells, upon lysis left much debris and

intact spheroplasts could not easily be differentiated from ruptured

spheroplasts. However, since spheroplasts had been demonstrated to

grow on incubation, this was used as a means to estimate lysis.

Spheroplasts were suspended in graded concentrations of sodium











Table 20

LYSIS OF VIBRIO M.B. 22 AS ESTIMATED BY DIRECT COUNT
OF SPHEROPLASTS FORMED


Cells Suspended Average Number of Lysis
in NaCI Spheroplasts (%)
(M) Formed


0

0.0

0.02

0.03

0.04

0.05

0.06

0.08

0.10

0.16

0.30


ASW


% Lysis =


x 107/ml


(<105)

0.65


>99

>99

>99


20.5

84.0

117.0

135.0

135.0

173.0

175.0


Spheroplast Numbers
Spheroplast Numbers in ASW


x 100.


Cells suspended in designated salt concentrations for
20 min at 37 C. Microscopic counts determined as indicated in
text. OD cells in ASW was 0.60.











chloride and allowed time in which to lyse (20 min). Aliquots were

taken from each solution and transferred to fresh sea water broth

containing penicillin. After 5 hr incubation intact spheroplasts

had increased in size. The intact spheroplasts could be differen-

tiated from ghosts and counted directly under phase-contrast illum-

ination in a Thoma chamber.

Spheroplasts, like cells, were subject to lysis in dilute

salt solutions (Table 21). In salt solutions less than 0.1 M in

sodium chloride there was a large degree of lysis while in solutions

above 0.1 M the majority of spheroplasts were protected from lysis.

A point should be made concerning the methodology used in

the direct estimation of lysis. Aliquots were taken from the cell

suspensions in the various salt concentrations and placed in ASW

medium containing penicillin. This procedure diluted the sea water

and in each case the dilution was one part of aliquot to four parts

of sea water medium. The aliquot from distilled water suspension,

for example, caused a dilution of all constituents of the sea water

medium. The possibility was considered that spheroplast development

was inhibited in slightly diluted sea water media. However, experi-

ment revealed that spheroplasts could be induced in liquid media

containing 0.075 M sodium chloride, 0.01 M magnesium chloride,

0.001 M potassium chloride and 0.1 per cent trypticase. The medium

containing trypticase and ASW even when diluted with an aliquot from

distilled water suspensions was suitable for the development of

spheroplasts.










Table 21

LYSIS OF SPHEROPLASTS AS ESTIMATED BY DIRECT COUNT


Spheroplasts Average Number of Lysis
Suspended in Spheroplasts (%)
NaCl
(M)


x 107/ml

0.0 (<105) >99

0.02 (<105) >99

0.04 0.38 >99

0.06 8.4 82

0.08 12.0 75

0.10 36.0 25

0.12 37.0 23

0.16 44.0 8

0.30 48.0 0

AS *' 48.0 0


% Lysis =


Spheroplast Numbers x 100.
Spheroplast Numbers in ASW


Spheroplasts suspended in designated salt concentrations
for 20 min at 37 C. Microscopic counts determined as indicated
in text. OD spheroplasts in ASW was 0.86.














DISCUSSION


The present study was an exploration of the nature of the

osmotic fragility of a marine bacterium. Although emphasis was

placed on the osmotic fragility of the bacterium other properties

were examined in order.to allow a more thorough understanding of

the organism in terms of its biological capabilities and its rela-

tionship to other microbial species.

The classification of marine bacteria is of importance for

several reasons; it provides information that allows comparative

studies with other bacteria, both marine and non-marine; it aids

in the demonstration of inter-relationships among the bacteria; it

adds to the knowledge of the types of bacteria found in the sea;

and it facilitates communication. One method of classification is

that typified by Bergey's Manual of Determinative Bacteriology (1957),

a system which has evolved from methods used for classifying higher

organisms. .In this system taxa are established partly upon the basis

of selected characteristics which are considered to represent

natural relationships among bacteria. This system was followed in

classifying the marine bacterium used in this study.

The marine bacterium, M.B. 22, belonged to the order

Pseudomonadales, a group encompassing straight or curved rods which

are motile by means of polar flagella. Within the order, division may











be made on the basis of cell morphology: straight rods belonging to the

family Pseudomonadaceae and curved rods to Spirillaceae. The marine

bacterium, M.B. 22, presented a paradox since the cells were pleomorphic

and existed as straight rods or curved rods. If considered as straight

rods, the bacterium belonged to the genus Pseudomonas, and if considered

as curved rods, to the genus Vibrio. The division of bacteria based on

cell curvature is subjective, and Haynes and Burkholder (Bergey's Manual,

page 90) noted that "The borderline between the straight rods found in

Pseudomonas and the curved rods found in Vibrio is not sharp; occasionally

curved rods nay occur in species that normally are composed of straight

rods, this variation sometimes being dependent on the medium used. h How-

ever, the examination of other characteristics resolved the difficulty.

The marine bacterium demonstrated an anaerogenic fermentation of glucose

and was sensitive to the vibriostatic agent 0-129. Both properties were

considered to be characteristic of bacteria of the genus Vibrio. Bacteria

of this genus are defined as curved rods which are motile by means of

polar flagella. They are heterotrophic, asporogenous, Gram negative,

aerobic (facultative), and grow rapidly on the surface of culture media.

They are found in salt and fresh water. The properties of the marine

bacterium were consistent with these criteria. It was considered to be

a vibrio, and accordingly, was designated Vibrio M.B. 22.

The role of the rigid cell wall in protecting Vibrio M.B. 22

against osmotic lysis was examined by comparing the osmotic fragilities

of normal cells and cells which had been inhibited in the formation of











a rigid cell wall. To this end, spheroplasts of Vibrio M.B. 22 were

induced by penicillin in order to obtain cells lacking the component of

the cell wall responsible for rigidity. The effect of penicillin on

the chemical composition of the walls of Vibrio M.B. 22 was not deter-

mined; rather, the effect was assumed to be analogous to the effect of

penicillin on the cell walls of other Gram negative bacteria. Spheres

have been induced, by the action of penicillin, from Escherichia coli

(Lederberg, 1956), Aerobacter aerogenes (Gebicki and James, 1960),

Alcaligenet faecalis (Lark, 1958) and Salmonella gallinarum (Salton

and Shafa, 1958). The penicillin-induced spheres, but not the normal

rods, of these organisms were reported to be susceptible to osmotic shock.

The observations have implicated the cell wall as one site of the action

of penicillin. In the case of E. coli the effect of penicillin was

believed to involve an inhibition of the incorporation of a mucocomplex

into the cell walls of the bacteria (Park and Strominger, 1957; Weidel

et al,, 1960), The mucocomplex, composed of amino acids and amino

sugars, in conjunction with other material, has been suggested as the

component responsible for the strength of the cell wall (Weidel et al.,

1960). Cells inhibited in the incorporation of the mucocomplex were

believed to possess cell walls incapable of confining the bacterium to

rod shape and incapable of withstanding osmotic shock. The penicillin-

induced spheres of Vibrio M.B. 22 indicated a loss of cell wall function

since rods were converted to spheres which were susceptible to osmotic

lysis. The spheres were considered to have lost the rigid component

of the cell wall and to represent (in analogy to the penicillin-induced

spheres of E. coli) a form of the bacterium induced to osmotic sensitivity.











Stable spheroplasts of Vibrio 1M.B. 22 were induced from cells

growing in an optimum medium without adding an additional osmotic

stabilizer such as sucrose. Stability in the trypticase medium prepared

with sea water indicated the spheroplasts to be in approximate osmotic

equilibrium with the medium. The stability of spheroplasts in media

relatively dilute in.salts (0.075 M sodium chloride, 0.01 M magnesium

chloride and 0.001 M potassium chloride) seemed to substantiate Christian

and Ingram's (1959a) suggestion that the internal osmotic pressure of

halophilic and non-halophilic bacteria reflects the osmotic pressure of

the growth medium. The rods of Vibrio M.B. 22 were converted, one for

one, to spheroplasts which were capable of growth but not multiplication.

The observation was indicative of the nature of the action of penicillin

as an inhibition of cell division; that this was a consequence of the

inhibition of the formation of a complete cell wall appeared likely.

The spheroplasts of Vibrio M.B. 22 demonstrated metabolic activity;

cultures were capable of reducing tetrazolium and producing indol. The

enzymatic activities as well as viability, although evident in sea water

suspensions, were damaged when cells were exposed to distilled water.

In sea water suspensions the spheroplasts were stable and no damage was

revealed by washing of the spheroplasts or subsequent to incubation

at 4 C.

Suspension of Vibrio M.B. 22 in distilled water caused a

decrease in turbidity and in viability. The suspensions were viscous

and became foamy when shaken. Evidence was presented by Johnson and

Harvey (1938) and by Johnson et al. (1943) that the effects observed











subsequent to placing marine bacteria in distilled water were sympto-

matic of osmotic cytolysis. The lysis of Vibrio M.B. 22 was evidenced

by the demonstration that ultraviolet-adsorbing material and protein

were released from the cells upon their exposure to distilled water.

Pratt and Happold (1960) earlier had shown that the organism underwent

cytolysis when cells were placed in distilled water. By the method

of plasmoptysis cell-free extracts were prepared which formed indol

in the presence of tryptophan, pyridoxal phosphate and potassium

phosphate. In the present study the osmotic stress which the bacterium

would stand was determined by estimating the minimum sodium chloride

concentration necessary to protect cells against lysis. Sodium chloride,

the principal salt in sea water, was selected since, when present in

suitable concentration, it has been found to prevent cytolysis and

to maintain the viability of marine bacteria (Riley, 1955). The lysis

of the bacterium was estimated by several types of analyses for the

activity of cells suspended in graded sodium chloride concentrations.

The protection afforded the viability of cells of Vibrio M.B. 22

was positively correlated with the concentration of sodium chloride in

the solutions in which cells were placed. Osmotic damage resulting in

the inability of cells to engender colony formation occurred in solu-

tions containing 0.1 M sodium chloride or distilled water. However, the

estimation of viability by colony counts was not considered to give an

accurate measure of the osmotic damage to cells because the ratio of

colony counts to microscopic counts made from cell suspensions in sea

water was low. The low "plating efficiency" was an indication that











the estimation of lysis in dilute salt solutions by colony counts could

involve other than osmotic damage. The method of colony counts has

been used to estimate the lysis of halophilic bacteria (Christian and

Ingram, 1959b) and marine bacteria (Johnson and Harvey, 1938). Failure

to consider the ratio of colony counts to total counts might explain

some of the inconsistencies in their results.

Turbidity of suspensions of Vibrio M.B. 22 was directly corre-

lated with the salt concentration of the suspending fluid; turbidity

decreased as the salt concentration was made progressively more dilute,

It was considered that turbidity decrease might involve two factors:

swelling of cells and cytolysis. Both would result in a decrease in

light scattering (Mitchell and Mayle, 1956). However, it was believed

that swelling alone was not responsible since the magnitude of decrease

in turbidity was greater than that expected if lysis was not involved

(Mitchell and Moyle, 1956), and lysis was indicated by the viscous

nature of suspensions in dilute salt solutions.

Two enzymatic activities of Vibrio M.B. 22, tetrazolium reduc-

tion and indol formation, were dependent on the concentration of salt

in which the cells were suspended, Both enzymatic reactions were

inhibited in dilute salt solutions. The inhibition of the reactions

was considered as a reflection of osmotic damage to the cells. How-

ever, it should be noted that the enzymatic and turbidity methods

were indirect estimates of cell lysis.

A direct method for estimating cell lysis was developed based

on the ability of unlysed but not lysed bacteria to form spheroplasts.

This method of microscopic observation of spheroplasts formed from











unlysed cells demonstrated the osmotic susceptibility of Vibrio

M.B. 22 in dilute salt solutions. The results revealed an increase

in lysis of the bacterium as the salt concentration of the suspend-

ing fluid was made progressively more dilute. A pertinent observa-

tion that can be made from the data of this study was that in large

populations of Vibrio M.B. 22 there exists a variation in the suscep-

tibility of cells to osmotic lysis and some individual cells survived

for a time even in 0.03 M sodium chloride.

The minimum salt concentration required to protect Vibrio

M.B. 22 was estimated, by the several different methods, to be

between 0.10 M and 0.06 M1 sodium chloride, roughly equivalent to

osmotic pressures of 4.6 to 2.8 atmospheres. Exposure for a short

time to solutions of lower osmotic pressure resulted in rapid lysis.

Either or both of two factors might account for the suscep-

tibility of Vibrio M.B. 22 to cytolysis: a high internal osmotic

pressure or a cell wall of insufficient strength to withstand a

relatively high osmotic stress. Lysis could reflect in part a

high internal osmotic pressure of the marine bacterium, and the

difference in lysis between marine and non-marine bacteria could

possibly reflect a difference in internal osmotic pressure. In this

regard the marine bacterium, Achromobacter fisher, was believed by

Mitchell and Moyle (1956) to have an internal osmotic pressure of

about 20 atmospheres while that for E. coli is considered to be 2 to

3 atmospheres (Mitchell and Moyle, 1956). The internal osmotic

pressure of E. coli grown in concentrated salt solutions is not known;

however, Christian and Ingram (1959a) have suggested that the internal










salt concentration of halophiles and non-halophiles (including

E, coli) is directly related to the salt concentration of the medium.

It is conceivable that non-marine bacteria may develop a high

internal osmotic pressure if grown in media containing a high salt

concentration. However, cells of E. coli adapted to a medium con-

taining 7.0 per cent (1.2 M) sodium chloride were not lysed by trans-

fer to a medium prepared with distilled water (Doudoroff, 1950). After

subculturing E. coli three times in the saline medium a subculture was

made in fresh salt-free medium. There was no lag in the initiation

of growth and no death of the bacteria following the transfer of the

saline cultures to fresh water media. Comparison of this observation

with the observations of Vibrio I.B. 22 indicates a different response

of the two organisms in salt-free media even though both were grown

in media relatively high in salt. Pseudomonas aeruginosa, a non-

marine bacterium, grown in sea water media was apparently not lysed

upon transfer to distilled water.

Salton (1956) has suggested that halophilic bacteria and lum-

inous bacteria (marine species) lyse in hypotonic solutions as a result

of their possessing weak cell walls. A similar implication of the

cell wall has been made by Brown (1960) based on the demonstration

of a lower amount of amino sugar in the cell walls of a marine bac-

terium than that reported to be in the walls of several non-marine

species. In the present study the osmotic fragilities of normal cells

and spheroplasts were compared in an attempt to determine the protec-

tion afforded the cell against osmotic lysis by the rigid component

of the cell wall.












The comparison of the lysis of cells and spheroplasts of Vibrio

M.B. 22 was based on the results obtained with the different methods

of estimating lysis. The comparison was made by computing the slopes

of linear lysis between successive sodium chloride concentrations.

The results obtained were indicative of the degree of lysis over the

increment of sodium chloride concentration examined (Table 22). The

estimation of osmotic damage by the inhibition of indol formation

revealed similar patterns of inactivation for normal cells and sphero-

plasts with maximum slope over the increment of 0.06 M to 0.08 M

sodium chloride. For both cells and spheroplasts the salt concen-

tration which supported 50 per cent activity was only slightly less

than 0.08 M sodium chloride (Tables 16 and 17). The comparison of

the fragilities of cells and spheroplasts based on the method of

inhibition of TTC reduction again gave a maximum slope over the

increment of 0.06 to 0.08 M sodium chloride. However, the sphero-

plasts appeared to be more active than cells in slightly lower salt

concentrations (Tables 18 and 19). This might indicate that indi-

vidually spheroplasts were more active in reducing TTC than were

cells. Osmotic cytolysis as estimated by turbidity decreases gave a

maximum slope for cells over the range 0.06 M to 0.08 M and for sphero-

plasts 0.04 M to 0.06 M sodium chloride. This result might be mis-

leading since the salt concentration supporting 50 per cent residual

turbidity was slightly less than 0.06 M sodium chloride for both cells

and spheroplasts (Tables 16 and 17). The difference in maximum slopes

perhaps indicated a greater release of cellular material by spheroplasts

than by cells. The patterns of lysis as estimated by microscopic









Table 22


COMPARISON OF THE LYTIC PATTERNS OF CELLS AND SPHEROPLASTS OF VIBRIO M.B. 22


Method NaC1 (M)


0.0-0.02 0.02-0.04 0.04-0.06 0.06-0.08 0.08-0.10 0.10-0.30


Indol Cells 0.0 5.4 9.8 15.2 10.7 1.0
Spheroplasts 0.0 2.9 7.2 17.2 8.6 1.4

TTC Cells 0.0 0.0 2.5 18.4 11.2 1.8
Reduced Spheroplasts 0.0 0.0 14.0 24.3 3.3 0.9

Turbidity Cells -0.5 7.0 8.5 11.5 2.5 0.6
Spheroplasts -1.5 3.5 20.0 12.0 5.0 0.6

Micro- Cells 0.0 2.0 22.0 9.5 5.0 1.2
scopic Spheroplasts 0.0 0.0 9.0 3.5 25.0 1.3


Values in the table represent the linear slopes over the indicated increments of sodium
chloride concentration. The slope equals the ratio of inactivation of cell function (I.e.,
decrease in enzyme activity, microscopic count, or turbidity) to increment of salt concentration.
The values for inactivation were considered as fractions of the maximum activity.


0C
*p-











count revealed a maximum slope for cells over the range of salt concen-

trations from 0.04 M to 0.06 M and for spheroplasts over the increment

from 0.08 M to 0.10 M sodium chloride.

The estimation of osmotic damage by reduction in indol forma-

tion, in ability to reduce TTC, and in turbidity revealed no clear

difference in the fragility of cells as opposed to that of spheroplasts

although the lytic patterns were somewhat different depending on the

method used. However, estimation of osmotic lysis by microscopic

counts indicated spheroplasts to be more fragile than cells. A con-

sideration of the salt concentration necessary to protect 50 per cent

of the cells as opposed to 50 per cent of the spheroplasts indicated

a difference of 0.03 M sodium chloride or approximately 1.4 atmospheres

osmotic pressure. The considerable difference might be an indication of

the protection offered the cell by the rigid cell wall. There was also

some indication by the indol method that spheroplasts were slightly

more sensitive than cells in salt concentrations over 0.1 M. It would

seem likely that the rigid cell wall which maintained the rod-shape of

the bacterium would also offer some protection against osmotic lysis.

However, the difference in fragility of cells and spheroplasts of the

marine bacterium should be compared with the difference in fragility

of cells and spheroplasts of non-marine bacteria. Cells of Aerobacter

aerogenes, for example, are not known to be damaged by cultivation in

media dilute in salts (i.e., in the absence of osmotic stabilizers),

whereas spheroplasts of the organism have been shown to be subject

to lysis in solutions of osmotic pressure below 5 atmospheres (Gebicki











and James, 1960). In fact, osmotic cytolysis is one criterion for

distinguishing spheroplasts from normal cells. The difference in the

lysis of cells and spheroplasts of Vibrio M.B. 22 was considerably less

than the difference in lysis of these two forms of non-marine bacteria.

The results indicated that the rigid penicillin-sensitive com-

ponent of the cell wall of Vibrio M.B. 22 offered less than effective

protection to the organism against osmotic lysis. This was believed

to support the suggestions of others (Salton, 1956; Brown, 1960) that

the cell walls of marine and halophilic bacteria are weak. This sug-

gests that the marine bacterium may not require a rigid cell wall to

maintain its cellular integrity in its natural environment. However,

many non-marine bacteria will survive in solutions of relatively low

osmotic pressures and it is under these conditions that the rigid cell

wall of bacteria has a selective advantage, making the organism rela-

tively free of its environment.















SUMMARY


The osmotic fragility of a bacterial species isolated from

coastal sea water was studied. The bacterium grew rapidly in a

nutrient medium prepared with artificial sea water but failed to

grow when cells were transferred to a nutrient medium prepared with

distilled water. The organism was characterized by osmotic cytolysis

as evidenced by the leakage of ultraviolet-adsorbing material and

protein when cells were placed in distilled water or dilute salt

solutions. The bacterium was identified as a vibrio, and was desig-

nated Vibrio M.B. 22.

The osmotic fragility of normal cells and spheroplasts was

estimated by several methods and the results of the lysis of the two

forms were compared in an attempt to estimate the role of the rigid

cell wall in protecting the organism against osmotic lysis. Sphero-

plasts were formed when cells were exposed to 100 units of penicillin.

per ml in nutrient sea water broth. Spheroplasts were stable without

provision of additional osmotic components, were formed one for one

from the cells, and were capable of growth but not multiplication.

The degree of lysis which occurred when cells or spheroplasts

were suspended in graded concentrations of salt was estimated by

several methods: reduction in colony counts, in turbidity, in ability

to reduce tetrazolium, and in ability to produce indol from trypto-

phane. These methods were considered to be indirect estimates of lysis.

87












Consequently, a new method was devised which estimated directly the

osmotic fragility of cells and spheroplasts. The direct method in-

volved the microscopic enumeration of the proportion of cells capable

of spheroplast formation after cytolytic exposure, and of the pro-

portion of spheroplasts capable of growth after such treatment.

Evidence of the validity of this technique was adduced.

Cells of Vibrio M.B. 22 were protected from lysis in 0.06 to

0.08 M sodium chloride solutions, while spheroplasts required 0.08

to 0.10 M, as measured by the direct technique. These values were

not materially different from those obtained by the indirect methods,

although the latter generally gave similar values for cells and

spheroplasts.

It was concluded that the osmotic fragility of cells and

spheroplasts of Vibrio M.B. 22 were similar, although the small dif-

ference might reflect some significant physical attribute. As com-

pared with values reported for cells and spheroplasts of other Gram

negative bacteria, the difference was minor. The results were inter-

preted as an indication that the rigid penicillin-sensitive component

of the cell wall offers little protection against osmotic lysis in

cells of the marine bacterium, Vibrio M.B. 22.





































APPENDICES















APPENDIX A: FACTORS CONCERNED WITH TETRAZOLIUM REDUCTION
BY VIBRIO M.B. 22


Dense suspensions of cells of Vibrio M.B. 22 rapidly reduced

the colorless 2,3,5-triphenyltetrazolium chloride (TTC) to the red

formazan, and the reaction was used to estimate the effect of

suspending cells in various concentrations of sodium chloride.

The examination had as its basis the supposition that cells lysing

in dilute salt solutions would lose the ability to reduce TTC.

Before beginning the investigation of lysis the reduction of TTC by

Vibrio M.B. 22 was examined in detail. Two points were considered:

the conditions necessary for maximum TTC reduction and TTC reduction

as a function of cell concentration. It was found that under anaerobic

conditions in the presence of glucose and the appropriate concentra-

tion of TTC, maximum reduction was obtained. Also, TTC reduction was

not a linear function of the concentration of cells since dilute cell

suspensions were inhibited in TTC reduction.

In order to facilitate the determination of formazan produced

by the TTC reduction, a standard curve was prepared relating ug of

formazan to the optical density of the color of each concentration

(Table 23). The light adsorption by formazan was maximum at a wave

length of 485 mu with the "Spectronic 20." Formazan was prepared

by the reduction of TTC with hyposulfite. A small amount of the

reducing agent was added to a concentrated aqueous solution of TTC

90











Table 23

STANDARD CURVE: OPTICAL DENSITY OF GRADED CONCENTRATIONS
OF FORMAZAN


Prepared Formazan* Commercial Formazan


ug per ml OD ug per ml OD


Trial Trial

1 2 1 2

12.0 12.0 0.75 0.75 12.0 0.66

9.6 9.6 0.60 0.60 9.6 0.50

7.2 7.2 0.45 0.45 7.2 0.37

4.8 4.8 0.30 0.30 4.8 0.24

2.4 2.4 0.15 0.15 2.4 0.11

-- 1.2 -- 0.075 --

0.0 0.0 0.0 0.0 0.0 0.0


*Prepared by the reduction of 2.3,5-triphenyltetrazolium
chloride.

OD/0.0625 = ug formazan per ml.

A weighed amount of formazan was dissolved in an n-propanol-
water mixture. A series of solutions containing different amounts of
formazan were prepared by dilution. Diluent used was 2 parts water to
4 parts n-propanol. OD was determined with the "Spectronic 20" at
485 mu.











and the insoluble formazan precipitated upon formation. The fonnazan

was immediately sedimented by centrifugation and separated from the

supernatant. In a stepwise manner involving the isolation of the

formazan a sufficient amount of material was gathered. Care was taken

not to discolor the formazan by over-reduction. The formazan was

dried in vacuo. Over the range investigated the optical density was

a linear function of the formazan concentration. A commercial prepara-

tion of formazan (Nutritional Biochemicals Corporation) was also

investigated and was found to give a curve with less slope than the

formazan prepared as described above. The relationship obtained with

the synthesized formazan was used throughout this study.

TTC reduction by cells of Vibrio M.B. 22 in 0.4 M sodium

chloride under anaerobic, as opposed to aerobic, conditions was inves-

tigated. Anaerobic conditions were obtained, as described in

METHODS, by incubating the reaction mixture under nitrogen. A greater

degree of reduction was obtained anaerobically than aerobically at

each cell concentration and this was especially notable in the more

dilute solutions (Table 24). The concentration of TTC also affected

the degree of reduction (Table 25). Maximum reduction by cells in

0.4 M sodium chloride was obtained with concentrations of from 200 to

400 ug. In the less concentrated solutions the factor limiting the

reduction may have been the amount of TTC present. The reduction was

also inhibited in the solutions more concentrated in TTC. The nature

of this inhibition was not determined.











Table 24

ANAEROBIC VERSUS AEROBIC REDUCTION OF TETRAZOLIUI4 BY
VIBRIO M.B. 22


Cell Suspension Formazan Produced
(mg dry wt of cells) (ug)


Anaerobic Aerobic


Trial Trial

1 2 1 2

0.0 0.0 0.0 0.0 0.0

0.14 6.6 6.0 0.0 0.0

0.24 11.4 12.6 0.0 0.0

0.33 24.0 24.0 0.0 0.0

0.44 33.6 33.6 12.4 12.4

0.54 79.2 86.4 28.8 --


Each reaction mixture contained: 2.0 ml cell suspension (dry
wt indicated in table), 0.2 ml potassium phosphate buffer (final
concentration 0.01 M, pH 7.0), 200 ug TTC and 200 mumoles glucose.
Total volume 2.2 ml. Reaction time for anaerobic system 15 min, for
aerobic system 25 min. Incubation temperature 37 C. Reaction stopped
with 4.0 ml of n-propanol. Final volume 6.2 ml.












Table 25

TETRAZOLIUM REDUCTION AS A FUNCTION OF TETRAZOLIUM CONCENTRATION


Tetrazolium
(ug)


Formazan Produced
(ug)


Trial


0.0

16.2

36.6

64.2

113.0

115.0

49.8

36.6


200

400

800


1600


2

0.0

16.2

35.4

76.2

119.2

100.0

48.0

34.2


The reaction system was anaerobic. Each reaction mixture
contained: 2.0 ml cell suspension (0.54 mg dry wt of cells), 0.2 ml
potassium phosphate buffer (final concentration 0.01 M, pH 7.0),
250 mumoles glucose and TTC (as shown in table). Total volume 2.2 ml.
Incubation was 15 min at 37 C. Reaction stopped with 4.0 ml of
n-propanol.


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