Electron microscope studies of sectioned and non-sectioned bacterial cells

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ELECTRON MICROSCOPE STUDIES OF SECTIONED AND NON-SECTIONED BACTERIAL

CELLS

A Thesis Presented to The Faculty of

the Department of Bacteriology

The University of

Southern California

In Partial Fulfillment of the Requirements for the Degree Master of

Science in Bacteriology

by E. Elizabeth Tamblyn June

1950

UMI Number: EP55019

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.

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l3A

'.5" e

T / S ' s '-

T h is thesis, w r itte n by

....E....El i_zab eth ..Tarablyn............. u n d e r the g u id a n c e o f h ..§ _E. F a c u l t y C o m m itte e , and approved

by a l l its

m em bers,

has

been

presented to a n d accepted by the C o u n c il on G r a d u a te S tu d y a n d R e s ea rch in p a r t i a l f u l f i l l ­ m e n t o f the re q u ire m e n ts f o r the degree o f

...Master...of...Science., in..Bacteriology...

Faculty Committee

Chairman

J , C.'TSH^Wc,

ff

ACKNOWLEDGMENTS The author wishes to express her appreciation to Dr# Richard F. Baker for operating the electron microscope, and for guidance in the thin section technique.

Acknowledg­

ments are also due. Dr. James W. Bartholomew for suggesting the problem, and for his aid in the interpretation of the results.

TABLE OF CONTENTS PAGE INTRODUCTION

..........................................

The need for eruss-sectionsof bacteria

.1

. . . .

1

The approach to the p r o b l e m ................ REVIEW OF THE LITERATURE METHODS AND MATERIALS USED

1

..............................

1

...........................

2

Imbedding and sectioning .......................

2

Reduction of cell opacity.... ...................

5

Starvation method

...........................

5

Effect of age of c e l l s ....................

6

..................

7

Desoxyribonuclease and ribonuclease digestion.

7

Digestion by ribonuclease

R E S U L T S .................................. ..............

8

Bacillus subtilis. not sectioned ..............

8

Normal cells ..................................

8

Digested cells ................................

9

Bacillus subtilis. sectioned,.enzymedigested

.

10

Bacillus subtilis. sectioned, nitrogen-free m e d i u m .....................................

10

* Bacillus subtilis. sectioned, ageexperiment . .

11

Serratia marcescens. not sectioned .............

13

Normal cells ..................................

13

Digested cells ................................

13

Serratia marcescens. sectioned, enzymedigested.

13

iv PAGE Serratia marcescens.sectioned,

ageexperiment

Saccharomyces cerevisiae. notsectioned

.

lb

. . . .

15

Normal cells ..................................

15

Digested cells

15

.........................

Saccharomyces cerevisiae. sectioned, enzyme d i g e s t e d ...................................

15

D I S C U S S I O N ........ '................................... S U M M A R Y .............................................. BIBLIOGRAPHY

16 19

..........................................

21

ILLUSTRATIONS ..........................................

23

LIST OF FIGURES FIGURE

PAGE

1.

Normal B. subtilis. not sectioned, 17500X

2.

B . subtilis. no I sectioned, ribonuclease digested.

23

3.

Normal Vibrio, not sectioned, ^-OOOX

2b

... .

. • . .

23

. . -. .

U-. B. subtilis. not sectioned, ribonuclease and desoxyribonuclease digested, 17500X

2b

5* B. subtilis. sectioned, nitrogen-free medium, 1 7 5 0 0 X ....................................... 6.

Same

7.

B. subtilis. sectioned, nitrogen-free medium,

25

as number 5 ................

1 7 5 0 0 X .......................................

25

26

8.

Same

as number 7 ...................................

26

9.

Same

as number 7 ...................................

27

10.

Same

as number 7 ...................................

27

11.

Same

as number7, 2 0 0 0 0 X ..........................

28

12.

B. subtilis. sectioned, eight hour old cell, 1 7 5 0 0 X ........................................

13.

28

B. subtilis. sectioned, eight hour old cell, 1 7 5 0 0 X .......................................

29

l b . B. subtilis. sectioned, twenty-four hour old cell, 1 7 5 0 0 X ....................................... 15*

29

B. subtilis. sectioned, twenty-four hour old cell, * + 0 0 0 X .......................................

30

vi FIGURE

PAGE

1 6 . B. subtilis. sectioned, twenty-four hour old cell, 1 7 500 X .......................

30

17-

Same as number 1 6 ..................................

31

18.

Same as number 16.

31

. . . ..........................

1 9 . B. subtilis. thirty-six hour old cell, sectioned,

1 7 5 0 0 X .......................................... 20.

B. subtilis. sectioned, thirty-six hour old cell, 1 7 5 0 0 X ............

21.

32

.

S. marcescens. not sectioned, twenty-four hour old cell, 1 7 5 0 0 X ............

22.

2b.

Same as number 23...........

25.

Serratia marcescens. sectioned, ribonuclease digested, 17500X .

...........................

3** 3^

35

S. marcescens. sectioned, ribonuclease and desoxyribonuclease digested, 17500X

27•

S. marcescens. same as number 2 6 .................

28.

S. marcescens. sectioned, eight hour old cells, 17500 X ..........................................

29-

33

S. marcescens. not sectioned, ribonuclease and desoxyribonuclease digested, 17500X

26.

33

S. marcescens. not sectioned, ribonuclease digested, 17500 X ................................

23.

32

35 36

36

S. marcescens. sectioned eight hour old cells, * f 0 0 0 X ..........................................

37

FIGURE

PAGE

30.

Same as number 2 8 .................................

31.

S. marcescens. sectioned twenty-four hour old cells, 17500X.... ................................

32.

Same as number 3 1 ..........

33•

S. marcescens. sectioned, thirty-six hour old 39.

S. marcescens. sectioned, ten day old cells, 1 7 5 0 0 X .......... .

35*

38

38

cell, 1 7 5 0 0 X .................................... 3^-.

37

............................ ..

39

S. marcescens. sectioned, twenty-four hour old cells, dry ice used,1 7 5 0 0 X ......................

,

*+0

36.

Same as figure 3 5 without the dry i c e ...........

*+0

37*

Normal yeast, not sectioned, 1 7 5 0 0 X ............

l+l

38.

Normal yeast, not sectioned, ^ O O O X ..............

*+1

39*

Yeast, not sectioned, ribonucleasedigested, N-000X

*+0.

Same as number 3 9 .................................

*+1.

k2 *+2

Yeast, sectioned, ribonuclease and desoxyribo­ nuclease digested,17500X

.

.....................

1+3

b2.

Same as number H-l..................

*+3

^3.

Same as number *+1.................................

M+

ELECTRON MICROSCOPE STUDIES OF SECTIONED AND NON-SECTIONED BACTERIAL CELLS Bacterial cytologists have, for many years, anticipated a technique which would enable them to cut cross-sections of microorganisms.

Such a technique would make it possible to

confirm the existing ideas on bacterial morphology, and to study the internal structure of the cells.

Since the con­

ventional microtome cuts sections of from one to fifteen mi­ crons, it is of little value to the bacterial cytologist. Recently, Pease and Baker (19^8) introduced a technique by which it became theoretically possible to section bacterial cells. The thin section technique of Pease and Baker (19^8) was later modified by them for the sectioning of bacterial cells, (Baker and Pease 19^9)*

The modified procedure was

used in the work reported here to prepare 0.0? micron sections of bacterial cells for study in the electron microscope. Bacillus subtilis. was chosen to represent the gram-positive organisms, Serraria marcescens. to represent the gram-nega­ tive organisms, and Saccharomyces cerevisiae. to represent the yeast. Schumacher (1926) sectioned and stained frozen yeast cells, noting the change which took place in the gram re­ action.

The intact cells remained gram-positive, while the

sectioned cells became gram-negative. and stained yeast cells.

Kemp (1931) crushed

He noted that the cell walls stained

gram-positive while the inner structure stained gram-negative. Green (193$) succeeded in cutting intact bacterial colonies in sections of from three to five microns.

He noted the

course of sporulation within the colonies of Bacillus species, and Clostridium species.

Laurell (19^9) sectioned bacterial

cells by tearing the cells off an impression slide with a film of beryllium.

In his electron micrographs, he noted

that in sections of Streptococcus faecalis MThe dividing culture appears to contain a round, well-defined body which seems to divide at the same time as the cell.” cut 0.1 micron sections of bacterial cells.

Baker (191+9)

In describing

one of his electron micrographs, he notes, Sufficient in­ ternal detail is visible in both cells to make it clear that the sections are adequately thin for effective use with a 50kv electron microscope.11 As far as the author is aware, nothing further has been published concerning the morphology of the sectioned bacterial cells. METHODS Imbedding and Sectioning.

The microtome used for the

thin sections was a standard Spencer Rotary microtome equip­ ped with the thin section adapter of Pease and Baker (19*+8). The imbedding procedure for the bacteria was that of

3 Baker and Pease (19^9) with certain minor modifications.

It

was as follows: 1.

The cells were grown on a suitable agar plate.

When they had reached the desired age, they were scraped off the plate with a curved glass rod and put directly into a 10 per cent neutral formalin solution in a centrifuge tube. Care was exerted to avoid scraping off any of the agar.

All

of the procedures were carried out in centrifuge tubes, tightly stoppered, until the hardened collodion block could be handled with forceps.

The cells were packed by centri­

fugation between each step in the dehydrating and embedding procedure.

In this way the supernatant fluids could be

poured off from the cells. 2.

Fixed in neutral 10 per cent formalin one to six

3.

30 per cent ethyl alcohol one

hour.

*+.

70 per cent ethyl alcohol two

hours.

5*

95 per cent.ethyl alcohol two

hours.

6.

Absolute ethyl alcohol one hour.

7*

Ether alcohol (50-50 per cent solution) overnight.

8.

3 per cent parlodion in 5 0 -5 0 ether alcohol four

9*

6 per cent parlodion in 5 0 -5 0 ether alcohol four

hours.

1

hours.

hours. 10.

12 per cent parlodion in 50-50 ether alcohol

overnight, 11.

Chloroform one hour in the centrifuge tube.

At

this point the tube was broken, and the semisolid collodion with the cells embedded in it was taken out of the tube and placed in a bottle of chloroform for an additional hour.

The

block was solid after this step, and could be trimmed and handled as a piece of tissue.

The collodion block was then

carried through the following solutions. 12.

Carbol-xylol two hours.

13*

Xylol one hour.

1*+.

Paraffin #1 (M.P. 65C.) one hour.

15.

Paraffin #2 one hour.

16.

Imbed in paraffin #3.

quickly as 17*

The paraffin was cooled as

possible to avoid the formation of large crystals. The doubly imbedded organisms were then mounted

on

sectioning blocks. 18.

Dry ice was placed in the microtome, and on the

block so that the cold vapor glowed over the section and the knife.

This facilitated cutting thin sections. 19.

The cut sections were placed on an ordinary glass

slide with a drop of dioxane placed adjacent to the section. The section was allowed to float onto the dioxane and spread out. 20.

The dioxane was then completely evaporated, and

the sections washed with benzene to remove the paraffin.

21.

The slide with the sections was dipped into a

2 per cent solution of parlodion in amyl acetate. 22.

When the parolodion was dry, the electron micro­

scope 200 mesh screens were placed over the sections.

The

parlodion film was cut, and floated off the slide onto water. The film carrying the screens and sections was picked up on another glass slide.

After drying, the sections were

ready for observation in the electron microscope. Reduction of cell opacity.

Normal cells of Bacillus

subtilis and Saccharomyces cerevisiae taken from twentyfour hour agar plates were found to be almost opaque to the electron beam, even when 0.0? micron sections were observed. The size of sections cut with the same microtome, was deter­ mined by the use of Dow polystyrene latex spheres. and Pease (19^9b).

Baker

Normal cells of a twenty-four hour cul­

ture of Serratia marcescens were less opaque to the beam, but not much internal structure could be observed.

It was

thought that this opacity was due to the high nucleic acid content of the bacterial cells, Knaysi and Baker (19^+7)* Therefore various methods were used in an attempt to render the cells less opaque to the electron beam. 1.

Starvation method.

The first attempt to render

the cells less opaque to the beam was according to the method of Knaysi and Baker (19^7)•

This procedure consisted of

growing the organisms in a nitrogen-free medium, the idea

being to supply a source of carbon, but no source of nitro­ gen.

Endogenous respiration might then occur at the expense

of the nucleic acids.

Variations in age of the culture, time

in the nitrogen-free medium, and incubation temperatures were tried.

The best conditions were found to be, twenty-four

hour old cells, nitrogen-free medium at 37° C. for seventytwo hours.

At the time of fixing, the normally gram-positive

cells were 100 per cent gram-negative.

They appeared to be

somewhat smaller than the normal cells.

The cells were im­

bedded and sectioned in the manner described. was only partially successful.

This treatment

All three of the species of

test organisms were treated in this manner with varying re­ sults.

A total of fourteen blocks were prepared and forty-

five electron micrographs were taken. 2.

Effect of age of cells.

It was thought advisable

to do a study of the effect of age of the cells as to their opacity to the electron beam.

Serratia marcescens and

Bacillus subtilis were grown in nutrient broth for twentyfour hours, then this broth culture was inoculated evenly on nutrient agar plates.

The growth was scraped from the plates

and fixed in formalin after *+-8-16-2^-36-*+8 hours incubation. A ten-day-old culture was also included in this series.

The

regular imbedding procedure was followed on all of the samples.

The electron micrographs showed some variation in

the opacity of the cells.

Sixteen blocks were prepared and

100 electron micrographs were preapred. 3.

Digestion by ribonuclease.

Theoretically, the

enzyme ribonuclease should liberate the ribonucleic acid from the cytoplasm of the cells* to do this.

Therefore an attempt was made

The idea was to reduce the opacity of theceLls,

by ridding them of some of their cytoplasmic nucleic acids. Twenty-four hour cultures of Bacillus subtilis. Serratia marcescens. and Saccharomyces cerevisiae were suspended in distilled water, and heat killed by autoelaving.

The crys­

talline enzyme was dissolved in sterile distilled water to give a final concentration of 0.*+ mgs. per ml. of water. Ten mis. of the bacterial suspensions were placed together with ten mis', of the enzyme solution in centrifuge tubes. The tubes were kept at 55° C.

Samples were removed at

15 minutes, 30 minute, 1 hour, and 2 hour intervals.

At the

end of the indicated time interval, the suspension was cen­ trifuged and the enzyme activity was stopped by fixing the cells in formalin for imbedding.

These cells were sectioned

and observed in the electron microscope.

Six blocks were

prepared and thirty-five electron micrographs taken. }+.

Desoxyribonuclease, and ribonuclease digestion.

This experiment involved the use of both desoxyribonuclease and ribonuclease for the digestion of the cells.

The cells

were prepared as in the previous experiment, and the enzymes made in solution, using *+.0 mgs. of each of them in 100 mis.

.of distilled water.

The experiment was again carried out at

55° C. and samples removed after each hour incubation for three hours.

The two-hour digestion period was shown to be

the best for imbedding.

The cells were fixed and imbedded

for observation in the electron microscope.

Six blocks were

prepared and thirty-five micrographs taken. The usual collodion slide method of preparing bac­ terial cells for observation in the electron microscope was also used.

This made it possible to observe the changes in

the treated cells before they were imbedded for sectioning. Approximately sixty electron micrographs were made of the unsectioned cells. RESULTS The results of each of the experiments to render the cells less opaque to the electron beam will be discussed separately.

The figures shown and discussed are ones selected

from over three hundred electron micrographs.

It was hoped

that these experiments would prove or disprove the theories of Churchman (1927), Gutstein (192*0, and Eisenberg (1909), as to the possible presence of ectoplasm and endoplasm layers in the cytoplasm of the bacterial cells. Bacillus subtilis not sectioned;

plates prepared to

show the apparent three regions of the cells. Figure 1 showed a normal twenty-four hour old cell of

9 Bacillus subtilis.

There was evidence of a cell wall, but no

evidence of an endoplasm, ectoplasm arrangement of the cell protoplasm.

Figure 2 showed a cell of Bacillus subtilis

after digestion with ribonuclease.

This treatment brought

out the cell wall clearly, and showed some evidence of the ectoplasm, endoplasm arrangement of the cell protoplasm. However, in these pictures there was no way of proving that the endoplasm was a real structure, or if it was simply the result of the round shape of the bacteria.

The existence of

the endoplasm was indicated, however, by the lack of this structure in the normal cells, and an analogous structure sharply defined, as seen in the Sprillum picture, Figure 3 ? (recently isolated by Rittenberg and Williams).

Unfixed. •.

cells, mounted for the electron microscope, are dried into a very flat shape.

Thus the observed dark line is probably

not due to the round shape of the bacterial cell. Digestion of the cells with both ribonuclease and desoxyribonuclease was marked by a progressive disappearance of the cell cytoplasm as shown in figure ^f.

There was some

evidence of an endoplasm, ectoplasm arrangement of the cyto­ plasm in a few of the less digested cells. Autoclaved Bacillus subtilis cells undergo autolysis in such a way as to give the suggestion of the double enzyme digestion.

The gram stain reaction was changed, and the

cells were not normal.

10 Bacillus subtilis sectioned enzyme digested*

Twenty-

four hour old cultures of Bacillus subtilis were washed off the slants, heat killed, and digested with ribonuclease, and with ribonuclease and desoxyribonuclease together.

The cells

which were digested with the ribonuclease alone were too opaque in the cross-section to observe structure.

Comparing

these with the unsectioned cells after the same treatment probably indicates that the fixation and imbedding procedure prevents the clear demonstration of the cell wall, ectoplasm, and the endoplasm. 3?he cells which were digested with both desoxyribo­ nuclease and ribonuclease, showed some morphology in the sectioned cells.

Figure 5 showed both the cross-sections and

longitudinal sections of the cells.

These cells showed the

ectoplasm endoplasm arrangement of the cytoplasm.

The cells

marked with an arrow showed an indication of a thin cell wall still intact.

Rather sharp differentiation can be shown be­

tween the endoplasm and the ectoplasm.

Figure 6 showed the

end of a cell with the cell wall, the ectoplasm and the endo­ plasm in evidence.

There was considerable difficulty in

obtaining prints from the negatives to show the three areas as clearly as seen on the negatives. Bacillus subtilis sectioned nitrogen-free medium. This treatment of the cells was designed to get self diges­ tion of the cells by catabolism of nucleic acids by endo-

11 respiration resulting from starvation techniques.

Figures 7,

8, 9, 10, and 11, all show cross-sections of the cells fol­ lowing this treatment.

They all show evident thinness of

’’holes in the center’* of the cells.

For some reason, after

the starvation treatment, the center of the cells seem to fall out.

The absent centers may represent the endoplasm,

and the rings which were left, the ectoplasm.

Thus, we have

some small additional evidence of the ectoplasm endoplasm arrangement of the cytoplasm. Bacillus subtilis sectioned age experiment.

This

experiment was designed to find an age at which the cells might be less opaque to the electron beam, starting with the very young cells, and ending with ten-day-old cultures. Figure 12 shows an eight hour cell of Bacillus subtilis with nice cross-sectioning of the cell with the outer cell wall still intact.

In the majority JDf the cross-

sections, the cell wall is not intact.

In figure 13, there

is an indication of the ectoplasm endoplasm arrangement of the etyoplasm in the cross-sectioned cells.

The cell wall

is not visible. In the twenty-four hour old cross-sectioned Bacillus subtilis. figure l*f shows nice sectioning and electron pene­ tration, but no internal structure.

Figure 15 shows sec­

tioned cells, but again, no internal structure is discernable.

Figure 16 shows cross-sectioning with some indication

12 of internal structure, but no clear definition of the struc­ tures.

Figures 17 and 18 show some hint of ectoplasm endo­

plasm arrangement of the cytoplasm, but since they were cut at 0.1 micron, the results are poor. The thirty-six hour old Bacillus subtilis cells gave essentially the same results as did the twenty-four hour old cells, except for figure 19 in which there is a cell which may have been sectioned longitudinally, (by virtue of the size of the sections).

It is similar to the picture which

Baker and Pease (19*+9) published in Nature.

It shows some

internal structure, and the cell wall is intact. Figure 20 shows cross-sections of two objects with very good internal structure, but the problem is whether or not these are sections through vegetative cells, or through spores showing spore, and spore case.

The latter is the most

probably explanation. The forty-eight hour, and ten-day-old cultures showed nothing that was not shown in the twenty-four hour old cul­ tures. We saw no marked influence of age of the cultures on the opacity of the sectioned cells to the electron beam. Some sectioned cells were transparent, but showed no internal structure, others showed a hint of ectoplasm, endoplasm arrangement. demonstrated.

Occasionally an intact cell wall could be

13 Serratia marcescens, not sectioned, normal and enzyme digested.

Figure 21 shows normal cells of Serratia marcescens.

The cell walls generally are still intact, and there is some internal differentiation, hut not into endoplasm and ecto­ plasm.

Figure 22 shows the cells after they had been digested

with ribonuclease. irregular in shape.

This treatment caused the cells to become There was no clear cut differentiation

of the internal structure.

The cells appeared similar to

their appearance after they had been autoclaved.

Figures 23

and 2V show cells of Serratia marcescens after they had been digested with both ribonuclease and desoxyribonuclease.

This

treatment cause the cells to appear much smaller than the normal cells.

They showed irregular shapes, and there was

some faint evidence of the endoplasm, ectoplasm arrangement of the cytoplasm.

This treatment of the gram-negative cells

did not result in as clear cut differentiation of ectoplasm endoplasm as that shown for the gram-positive cells. Serratia marcescens. sectioned, enzyme digested. Figure 25 shows the cells after they had been digested with ribonuclease.

The cells were somewhat transparent, and had

the "hole in the center*1 appearance.

This same characteris­

tic is again demonstrated in figure 2 6 .

Figure 27 shows a

very transparent cross-section of a cell of Serratia marcescens after digestion with both enzymes.

This picture

shows a granule in the center of the cell, but no internal

lb differentiation of the cytoplasm.

It again is similar to

the picture published by Baker and Pease in Nature (19*+9)Digestion of the cells with ribonuclease and desoxy­ ribonuclease made the ccll3 more transparent, but did not help in differentiation of the internal structure of the cells. Serratia marcescens. sectioned, age experiment.

For

the most part, the very young cells of Serratia marcescens were quite opaque to the electron beam, however in figure 2 8 , one cell shows the ffhole in the center” which has been ob­ served in cells under various conditions.

Figures 29 and 30

are of the eight-hour-old cells, and show various degrees of opacity, some almost transparent, others quite opaque. Figures 31 and 32 show good cross-sections of the twenty-four hour cells, some of which have a suggestion of the endoplasm ectoplasm arrangement of the cytoplasm.

These cells were for

the most part, well penetrated by the electron beam.

Figure

33 is of a thirty-six hour old culture of Serratia marcescens. The cell marked with an arrow has been sectioned longitu­ dinally and shows the presence of the cell wall, and a light center.

The forty-eight hour cultures showed nothing dif­

ferent in the cells.

The ten-day-old cultures showed some

cells with light centers; figure 3^*

The age of the cells

had very little influence on the appearance of these sec­ tioned celLs.

Luck seems to play an important part on how

15 these cells appear after cross-sectioning, with the thinner sections giving the best results, as was shown in figures 35 and 3 6 .

figure 35 was cut using dry ice in the microtome,

figure 36 was cut without dry ice*

The dry ice facilitates

thinner sections, and the cutting seems to be the key to the success of the pictures. Saccharomyces cerevisiae. not sectioned, normal and enzymes digested.

Normal yeast cells showed no differenti­

ation in their internal structure, figures 37 and 3 8 •

Fig­

ures 39 and *+0 are of yeast cells which have been digested with ribonuclease.

The large clear area around the outside

of the cells may represent the gram positive outer layer which has been rendered transparent by the enzyme action. The cells stained gram negative at the time they were pre­ pared for the electron microscope.

After digestion of the

yeast with both ribonuclease and desoxyribonuclease, the cells became more irregularly transparent and the digestion was more general than in the other cells studied.

The cells

were very irregular in shape. Saccharomyces cerevisiae. sectioned, enzyme digested. Normal sectioned yeast cells showed no differentiation within the cytoplasm.

The digestion with ribonuclease did not bring

out in the sectioned cells results comparable to the non­ sectioned cells.

There was no differentiation of the cyto­

plasm, nor could the outside layer of the cells be demonstrated.

16 In the sectioned cells where both ribonuclease and desoxy­ ribonuclease were used for digestion, there was some evidence of the ectoplasm, endoplasm arrangement of the cytoplasm. However, there were areas of disintegration and of opacity. Figure *fl showed a group of yeast cells, one of which showed some internal differentiation.

Figures b2 and *+3 showed

single cells, both of which showed some internal differenti­ ation. DISCUSSION It is now obvious that a method of cross-sectioning for bacterial cells has been achieved.

However, the cross-

sections of 0 . 0 5 micron are so thin that preparation of slides for observation in the light microscope was not achieved.

The 0.1 micron sections can be stained and ob­

served in the light microscope, but it is extremely diffi­ cult to observe morphological details of the cells.

Some

darkfield observations were made, but this possibility was not investigated fully.

With the use of either a phase

microscope, ultraviolet microscope, or a 100 kv electron microscope, these thin sections of bacteria may have great possibilities.

The 0.0? micron sections in the ?0 kv elec­

tron microscope do show some internal structure.

In general

however, the ability of the electron beam to penetrate the sections was very disappointing.

17 Churchman (1927) expounded the theory that grampositive cells consisted of a gram-positive cortex, and a gram-negative medulla.

He believed that the gram-positive

characteristic of the cell resided in the outer cortex, and that the gram-negative cells did not have this cortex.

In

Churchman1s own words !,Positive proof of this explanation must await the evidence furnished by cross-sections of bac­ teria. **

In the present experiments, we found that the cross-

sections of normal cells did not give much confirmation to this theory.

However, the electron micrographs of the enzyme

digested, but unsectioned cells, showed hints of the cell wall and the endoplasm, ectoplasm arrangement of the cyto­ plasm.

The cross-sections gave further evidence of this ar­

rangement, but the imbedding procedures interfered somehow with the sharpness of the differentiation.

Our results dif­

fered from Churchman*s theory in that there is some evidence for this arrangement of the cytoplasm in both the gram-posi­ tive and the gram-negative species studied.

It was shown for

Bacillus subtilis. Serratia marcescens. Saccharomvces cerevisiae. and the Sprillum. of structure.

This seems to be a general type

In the unsectioned electron micrographs, this

structure might be ascribed by some to the roundness of the cells, thus causing an artifact, but in the cross-sections no such conditions exist.

Actually, the dry cells in the

electron microscope are flat, and not round unless the round

18 shape is preserved by proper fixing before electron micro­ scopy. It is known that magnesium ribonucleate is responsible for the gram-positive reaction of the bacterial cells. and Steacy 19^-3).

(Henry

Gram-positive cells can be rendered gram-

negative, and then replated with magnesium ribonucleate to the gram-positive form again.

Normally gram-negative cells

cannot be made gram-positive by this treatment.

This concept

was experimentally confirmed by Bartholomew and Umbreit (1 9 ^ ) in their experiments using ribonuclease to strip off the gram-positive layer.

The gram-negative forms of the nor­

mally gram-positive organisms often appeared smaller than the gram-positive forms in the same preparation.

Therefore,

we decided to use the enzyme ribonuclease to render the .cells gram-negative, and to imbed and cross-section these prepar­ ations. The yeast cells, after treatment with ribonuclease had a thick, almost transparent outer layer.

This can be

interpreted as being Churchman's "cortex" in that the mag­ nesium ribonucleate responsible for the gram-positiveness of the cells had been digested by the enzyme. were gram-negative when fixed.

These cells

Nucleic acids are quite

opaque to the electron beam (Knaysi and Baker, 19*+7)> and here we have demonstrated a layer on the outside of the cell which is quite well penetrated by the beam, after digestion

19 with the enzyme.

This layer is not evident in the normal

gram-positive cells. SUMMARY 1.

Cross-sections of bacterial cells have been

obtained. 2.

Normal cells of Bacillus subtilis cut at 0.05

microns were opaque to the electron beam.

Sections of

Serratia marcescens were much less opaque to the electron beam.

Normal cells of Saccharomyces cerevisiae were opaque

to the electron beam. 3.

Evidence for cell wall, ectoplasm and endoplasm

regions was presented for gram-positive Bacillus subtilis. and Yeast, and for gram-negative Snrillum and Serratia marcescens. b.

The age of the cells had no effect on the electron

beam penetration. 5.

Digestion with ribonuclease made it possible to

demonstrate the layered arrangement of the protoplasm of the gram-positive organisms.

Comparable results were not ob­

tained with the gram-negative organisms. 6.

Digestion of the cells with both ribonuclease

and desoxyribonuclease made the cells less opaque to the electron beam. 7.

The nitrogen-free medium treatment of the cells

20 brought out some evidence of the layered arrangement of the cell protoplasm.

BIBLIOGRAPHY

21

Baker, R. F., and Pease, D. C., “Sectioning of the Bacterial Cell Electron Microscope,” Nature. 163, 282, 19^9a. Baker, R. F., and Pease, D. C., “Improved Sectioning Tech­ nique for Electron Microscopy,” Journal of Applied Physics. 20, ti-80, 19^9b. Bartholomew, J. D . , and Umbreit, W. W . , “Ribonucleic Acid and the Gram Stain,” J ournal of Bacteriology. *+8, 567578, 19bb. Boivin, Andre, “Direct Mutation in Colon Bacillus, by In­ ducing principle of Desoxyribonucleic Nature: Its Meaning for the General Biochemistry of Heredity,” Cold Springs Harbor Symposium on Quantitative Biology. 12, 7-17, 19^7. Churchman, John W . , “The Structure of Bacillus Anthracis and Reversal of the Gram Reaction,” J ournal of Experimental Medicine. **6, 1009-1029, 1927Churchman, John W . , “Gram Structure of Cocci,” Journal of Bacteriology. *fl3-*+29, 1929* Eisenberg, P., “Studien zur Ektoplasmatheorie,” Centralblatt Fur Bakteriologie. A b t , 1. Originale. *+9, *+65, 1909* Green, H. C., “Colony Organization of Certain Bacteria with Reference to Sporulation," Journal of Bacteriology. 35,

261-270, 1938. Gutstein, M . , ”Veber die Farberische Darstellung dis Bakterien Ektoplasmas, Zugleich ein Beitrag Theorie der Gramschen Farbung,” Centralblatt Fur Bakteriologie. A b t . 1. Originale. 93, 233, 192*f. Henry, H . , and Steacy, M . , “Histochemistry of Gram Staining Reaction for Microorganisms,” Nature. 151 (38^1), 6 7 1 ,

19^3 . Kemp, Hardy A., "Gram Reaction in Crushed Yeast,” Stain Technology. 6, 53-56, 1931* Knaysi, B . , Baker, R. F . , and Hillier, J., “A Study with the High-Voltage Electron Microscope of Endospore and Life Cycle of Bacillus Mycoides,” Journal of Bacteriology.

53, 252-237, 19^7.

Knayse, G . , and Baker, R. F . , ’’Demonstration with the Elec­ tron Microscope? of a Nucleus in Bacillus Mycoides Grown in a Nitrogen-free Medium,” J ournal of Bacteri­ ology. 53, 539-553, 19^7. Kunitz, M . , "Crystalline Ribonuclease,” Journal of General Physiology. 2b y 15-32, 19^0. Laurell, A. F. H . , ”A Method of Sectioning Bacteria in Situ for Electron Microscope and Cytochemical Investigations, Nature. 163, 282-283, 19*+9. Pease, D. C., and Baker, R. F . , "Sectioning Technique for Electron Microscope Using a Conventional Microtome," Procedures of the Society for Experimental Biology and Medicine. T?T, 19>+HT Schumacher, Joseph, "Zur Gramschen Farbung," Centralblatt Fur Bakteriologie. Abt. 1. Originale. 9 6 , lO^-llB, 1926 Schumacher, Joseph, "The Ectoplasm of Yeast Cells," Central­ blatt Fur Bakteriologie. Abt. 1. Originale. IOBT 193-207, 1 9 2 8 .

ILLUSTRATIONS

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