M\Iorton and Engley (1945) broth or agar. Microcolonies of E. coli were grown

Transcription

1 THE "FIXATION" OF ELECTRON\MICROSCOPIC SPECIMENS BY THE ELECTRO'N BEAM1 JAMIES HILLIER, STUART -MUDD, ANDREW G. SMITH,2 AND ERNST H. BEUTNER T'he RCA Laboratories, Princeton, New Jersey, and the Departmnent of Bacterioloyy, School of Mlledicine, University of Pennsylvania, Philadelphia 4, Pennsylvania Received for publication August 4, 1950 The characteristic pattern of contrast between nuclear sites and cytoplasm of Escherichia coli B, and the responsiveness of this pattern to steps of cytological processing, afford a sensitive indicator for possible effects of electron bombardment during electron micrography. The pattern of contrast of nuclear sites and cytoplasm is the more valuable as an indicator because this pattern can be discerned also with the light microscope (Robinow, 1945; Hillier, Mudd, and Smith, 1949), even, under suitable conditions, in the living cells (Tulasne, 1949a,b; Stempen, 1950). The present communication will show that the characteristic contrast pattern is maintained in electron micrographs, even in second and third electron pictures of the same specimen site taken with a cathode-biased electron gun. We shall also show that the electron bombardment used in taking a first picture so "fixes" the specimen that the latter is not visibly altered by chemical procedures which produce characteristic alterations in specimens not previously exposed to electron bombardment. Alteration of the chemical properties of the specimen as a result of electron bombardment has also been encountered in processing thin sections for electron cytological study. This "fixing" action of the electron beam must certainly be borne in mind as the electron microscope is progressively introduced into histochemical research. EXPERIMENTAL METHODS AND RESULTS Escherichia coli, strain B, was used throughout. The nutrient medium was M\Iorton and Engley (1945) broth or agar. Microcolonies of E. coli were grown on collodion membranes overlying the agar (Hillier, Knaysi, and Baker, 1948). Alternatively, when the fragility of the collodion membrane would be an obstacle to taking multiple pictures of the same sites, impression preparations were made on "formvar" membranes as described in the legends for figures 7 and 8. For multiple pictures of the same sites five exposures were made of as many fields of a given specimen. The specimen was then removed from the microscope, subjected to a step in cytological processing (fixation in vapor over OS04 or acetic 1 This research has been aided by a grant to the University of Pennsylvania from the Damon Runyon Fund through the American Cancer Society, recommended by the Committee on Growth of the National Research Council, and also by a grant from the Committee on Therapeutic Research of the Council on Pharmacy and Chemistry of the American imedical Association. 2 Present address: Department of Bacteriology, School of 'Medicine, University of Maryland, Baltimore 1, imaryland. 641

2 642 HILLIER, MUDD, SMITH, AND BEUTNER [VOL. 60 acid, or immersion in N HCI at 60 C), washed in distilled water, dried under an infrared lamp, and returned to the microscope. As many as possible of the five original fields were identified and micrographed. For figures 3, 4, and 5 the specimen was removed a second time from the microscope, processed, and returned to the microscope for a third set of pictures. The RCA model EMU with a biased electron gun wvas used throughout. The electron pictures were taken by Mrs. Jean Minkin tunder the supervision of Downloaded from Figure 1. Microcolonies of E. coli B grown for 2' to 3 hours at 37 C on thin collodion membrane overlying Morton and Engley agar. A and C: Specimens unfixed. B and D: Same specimens fixed 12 minutes in vapor over 2 per cent solution of OS04. Original magnification on all electron microscopic plates was 5750 X. Reproduced at same magnification. Dr. Marshall D. Earle, through the courtesy of the Franklin Institute Laboratories for Research and Development. Multiple pictures of specimens in relation to chemical fixation. The pattern of contrast between nuclei and cytoplasm in young gram-negative bacteria before fixation is usually only rather dimly discernible in pictures taken with ordinary electron lenses. Typical pictures are reproduced as figure 6 in Hillier, Mudd, and Smith (1949) and as figure 1 in Mudd and Smith (1950). The pattern of contrast even in unfixed specimens may be brought out with beautiful clarity, however, in the best pictures taken with the two-component objective lens (Hillier, 1949). Such pictures are reproduced as figures 1 and 2 in Hillier, Mudd, and Smith (1949). on April 11, 2019 by guest

3 1950] FIXATION OF ELECTRON MICROSCOPIC SPECIMENS 643 In figure 1 of the present paper are reproduced electron pictures of two fields before and after OS04 fixation. The patterns of contrast reproduced as figure 1A were exceptionally clear for an unfixed specimen. After pictures had been taken of five fields, the specimen was removed from the microscope, placed in a closed jar over 2 per cent OS04 solution for I' minutes, and returned to the microscope. The fields of which pictures hadl been taken were identified and second pictures taken. Subsequently the tw-o plates wer-e placed side by side and a contact print Downloaded from Figuire 2. E. coli B gronwn 3 hours at 37 C on collodion overlying M. and E. agar. Fixed for 3 minutes in vapor over 2 per cent 0804, then for 3 minutes in vapor over 50 per cent acetic acid solution. X 13,000. of the two contiguouis fields was made w-ith a single exposure (figure 1). The elements of pattern made by the light nuclei against the darker cytoplasm appear to be identical in the first pictures (figure la,c) and in the second pictures of the same fields (figure 1B,D). It is also apparent from these pictures that there was no significant selective absorption of OS04 by structures smaller than the nuclei. Whether or not, the 0S04 w-as absorbed in greater quantities by either the nuclei or the cytoplasm, or whether or not it was absorbed in significant amounts by either cannot be determined from these pictures owing to the obviolus lack of control on exposure and hence on contrast. "Fixation" of th.e specimen by the electron beam. N'either light nor electron on April 11, 2019 by guest

4 -644 HILLIER, MUDD, SMITH, AND BEUTNER [VOL. 60 microscopic pictures have satisfactorily defined the state of the chromatin in the nuclear sites of E. coli. We have tried various chromatin precipitants and nuclear fixatives without definitive results. An electron picture of a microcolony of E. coli fixed in OS04 vapor and then in the vapor over 50 per cent acetic acid is shown in figure 2. In this case the specimen was not introduced into the electron microscope until after fixation both over OS04 and acetic acid. Whether or not chromatin was precipitated within the nuclear sites by the acetic acid vapor we could not tell with certainty. In the hope of removing this uncertainty, pictures of specimens fixed over OS04 were taken, the specimens were removed from the microscope and fixed over acetic acid, and second pictures of the same sites were taken; the specimens were again removed, given additional treatment over acetic acid, and returned to the microscope for a third set of pictures. The results are shown in figures 3 and 4. The elements of the pattern of contrast between nuclei and cytoplasm seem to be identical throughout each series. Had the authors been aware at this time of the power of "fixation" of the electron beam itself, they would not have expected mere exposure to acetic acid vapor to produce visible change in a specimen which had already been exposed to electron bombardment. That the bombardment to which the specimen is subjected in electron micrography has the power to fix the specimens has become clearly apparent from experiments in which specimens are immersed in hydrochloric acid, which normally reverses the contrast pattern (Mudd and Smith, 1950). Specimens that have been micrographed before the acid treatment are not reversed in pattern by the acid. Such sequences are shown in figures 5 and 6. The same elements of pattern are seen in figure 5 in the specimen before chemical fixation, after exposure to OS04 vapor, and after immersion in N HCI. In figure 6 the same elements of pattern appear before and after immersion in N HCl, although the specimen after the hydrochloric acid treatment is shrunken appreciably in comparison to the same specimen before acid treatment. Figure 7 shows the same phenomenon of fixation by the electron beam in a slightly different way. Impression preparations of osmic-acid-fixed microcolonies of E. coli were made on "formvar." Pictures of five fields of one such specimen were made, and the specimen was removed from the electron microscope. This specimen and one which had not been in the electron microscope were immersed together in N HCl at 60 C for 6 minutes, washed, and dried. Figure 7B shows -that the pattern of contrast between nuclear sites and cytoplasm in the specimen not previously subjected to the electron beam was reversed as usual by the HCI treatment (Mudd and Smith, 1950). In the specimen (figure 7A) previously irradiated with electrons the original pattern of contrast was retained despite the HCl treatment. Finally, figures 8, 9, 10, and 11 show the fixation phenomenon in still a different way. Impression preparations were made on "formvar" films on glass slides and the films transferred to mounting screens as described by Mudd, Smith, Hillier, and Beutner (1950). Because examination of one such specimen in the electron microscope showed the presence of some obscuring impurity, all speci-

5 3B :A.... WMINNOm ::.. Figure 3. Cells of E. coli grown for 3 hours on collodion overlying M. and E. agar. A: Specimen fixed 3 minutes in OS04 vapor, B: Same specimen after additional exposure to vapor of 50 per cent acetic acid solution for 3 minutes. C: Same specimen after additional exposure to vapor of 50 per cent acetic acid for 6 minutes. Exposure in making electron picture greatest in C. Figures 3 to 7A, X 10,500 approximately. Figure 4. Cells of E. coli prepared and processed as in figure 3. Exposure in making electron picture greatest in B. 645

6 Downloaded from 6 B Figure 6. A microcolony of E. coli B grown for 2' to 3 hours at 37 C on collodion overlying M. and E. agar. A: Specimen unfixed. B: Same specimen fixed 11 minutes over OS04. C: Same specimen after being in N HCl solution at 56 C for 8 minutes. Figure 6. Microcolony of E. coli B grown for 3 hours on collodion overlying M. and E. agar. A: Specimen fixed 1 minute over OS04. B: Same specimen after being in N HCl solution at CO C for 9 minutes. The bright circles near the centers of these pictures are due to light coming directly from the electron microscope filament. Figure 7A. E. coli B grown 1 hour and 20 minutes on M. and E. agar at 37 C. Fixed 1 minute in vapor over 2 per cent OSO4. Agar cut out and inverted on "formvar" film overlying agar (Mudd, Smith, Hillier, and Beutner, 1950). Upper agar square removed, "formvar" film floated off from lower agar layer on water and picked up on screen. Specimen previously exposed to electron beam in course of taking electron pictures. Immersed in solution of N HCl at 60 C for 6 minutes. 646 on April 11, 2019 by guest

7 *^,:..... : : FIXATION OF ELECTRON MICROSCOPIC SPECIMENS 647 <.. <....s,.,s, 85 a: ::':: :. :. M *10 11.::;.E:.. '. _ Figure 7B. Conditions as in figure 7A. Specimen not previously exposed to electron beam. Immersed in solution of N HCl at 60 C for 6 minutes. This specimen shows usual reversal of constrast by HCl; specimen A "fixed" by electron bombardment, retains original contrast. Figures 7B to 11, X 9,000 approximately. Figure 8. E. coli B grown 1 hours on M. and E. agar at 37 C. Fixed 1 minute over OS04. Impression preparation on "formvar" film on glass microscope slide; film floated off on water and picked up on stainless steel metal screen. Specimen washed in distilled water, electron picture taken; specimen immersed in N HCI solution at 60 C for 6 minutes. Finally picture shown in figure 8 taken. Black line at reader's left is a stainless steel metal wire of specimen screen; parallel line through bacteria marks line of protection from electron bombardment of first picture by metal wire. Figures 9, 10, and 1i. Conditions as in figure 8. mens were washed with distilled water. Five fields of the specimen were micrographed, and the specimen was removed from the microscope and immersed in N HC1 at 60 C for 6 minutes. The specimen was washed in distilled water, dried,

8 648 HILLIER, MUDD, SMITH, AND BEUTNER [VOL. 60 and reintroduced into the electron microscope. These manipulations were found to have shifted the position of the specimen slightly with respect to the stainless steel mesh of the supporting screen. Many fields were found in which the specimen had undergone slight lateral displacement with reference to the wire. Across such fields sharp lines of demarcation were observable parallel to the wires. On the sides of these lines of demarcation adjacent to the wire, the bacterial cells had been cytolyzed by the action of the distilled water and hydrochloric acid. On the side of the line away from the wire the protoplasm of the cells was fixed in an insoluble gel refractory to the action of distilled water and acid. Obviously the lines of demarcation defined areas fixed by electron bombardment from those protected by the wire from this bombardment. It is particularly noteworthy that the lines separating gelation from cytolysis traversed many individual cells. Some additional experiments that throw further light on this phenomenon were carried out by one of us (JH). In the first group of these, collodion membranes approximately 150 A thick were bombarded at an electron intensity of somewhat less than 0.01 amp per cm2 with 50 kv electrons for approximately 2 seconds. This intensity is of the same order as that used in routine exposures, and therefore the total bombardment of the membrane was much less than that given to any field micrographed. Since the arrangement of the instrument used permitted one screen opening at a time to be irradiated, an identifiable pattern of irradiated openings could be traced. Membranes that had been treated in this fashion were tested to determine their resistance to solvents and to heating in a vacuum. The membranes on the irradiated openings were found to be intact after being heated to approximately 600 C and after immersion in all of the solvents tried: amyl acetate, acetone, and ether-alcohol. The unirradiated membranes were all completely destroyed by any of these tests. In passing it should be pointed out that such treatment may have an important practical value for the production of insoluble supporting membranes since the irradiated and tested membranes showed no change in structure when reexamined in the electron microscope. In a second experiment a tissue section, 0.2, thick and consisting of osmiumperfused mouse liver doubly embedded in collodion and paraffin, was irradiated. The section was placed in the microscope without removing any of the embedding material. The irradiation amounted to approximately 3 seconds at an electron intensity of slightly more than amp per cm2. During the irradiation the image was observed at low magnification. The section was relatively opaque so that only gross details were visible. During the bombardment no change in opacity was observed, nor was there any indication that the paraffin was melted. At higher bombardment intensities either or both of these changes can occur and are readily observable. After irradiation the specimen was immersed successively in chloroform, xylol, and amyl acetate and then dried by touching its edge to dry filter paper. Upon re-examination in the electron microscope no change could be detected. When a section that has not been irradiated is given such treatment, no more than a few shreds of embedding material remain.

9 1950] FIXATION OF ELECTRON MICROSCOPIC SPECIMENS 6492 When the bombardment intensity is increased slowly on a section as it is being observed, an increase in transparency is observed until the finer details of the section are easily observable on the screen. Very often this procedure can be continued until the specimen is withstanding the maximum available intensity. If this process is followed photographically, no change in structure is detected. However, changes in the finest details can have occurred since they are not visible from the very beginning of the treatment. One important observation is that in this process the "thinning" does not follow the increase in bombardment immediately. Instead there is a very short interval during which there is no change. This is followed by an observable period during which the thinning of the specimen due to a given increase in intensity is completed. When the bombardment is increased rapidly, a number of different phenomena occur which depend to at least some extent on the nature of the specimen within the bombarded area. Often the embedding paraffin can be seen to melt and flow. More often the thinning occurs very rapidly and proceeds beyond the final point described above, with the result that a large number of small holes appear. The section then appears as a fine network supported on the coarser structures. The section may also appear to explode, split, or tear and to be completely ruined. DISCUSSION Questions of the fidelity of the electron microscopic picture to the specimen in the living state, and of changes brought about in organic materials by electron bombardment, have been touched upon in a number of previous investigations. Desiccation artifacts such as shrinkage of the bacterial protoplast from its cell wall and wrinkling of the cell wall have been encountered by most investigators of bacteria. Such artifacts are not without compensation, however, for they have aided in the clear demonstration of the solid state of the bacterial cell wall, its distinctness from the protoplast and its enveloping plasma membrane, and the relatively greater capacity of the bacterial protoplast as compared to the cell wall to shrink on dehydration. Lembke and Ruska (1940) in a study of tubercle bacilli reported that the short exposures required for pictures were without visible effect on cell structure, but that intensive and prolonged bombardment with electrons could cause the appearance of clear areas within the dark intracellular granules and eventually the destruction of the granules. Ruska (1942) demonstrated the irradiation sensitivity of the polar granules of diphtheria bacilli and of intracellular granules in streptococci and dysentery bacilli. These apparently spheroidal granules after intensive bombardment appeared vacuolated. Ruska also described the particles of coli, typhoid, and dysentery bacteriophages as susceptible to vacuolation by intense electron bombardment, staphylococcus phage as less susceptible. Several other authors have described artifacts arising from electron bombardment wrell in excess of the intensities and durations of exposure required for making ordinary electron pictures. Thus K6nig (1947) showed in successive pictures of the same specimen of charcoal that particles of graphite could be melted and vaporized in a high intensity electron beam. Burton, Sennett, and Ellis (1947)

10 6505 HILLIER, MUDD, SMITH, AND BEUTNER VOL. O{ described the sublimation and migration of crystals under intense irradiation from the cathode-biased gun. Watson (1948) described a variety of artifacts in inorganic specimens and in bacteria subjected to excessive bombardment with the cathode-biased electron gun. Draper and Hodge (1949) have actually utilized very high current densities through the specimen, procurable with the biased gun, for "micro-incineration" of thin fragments of muscle. By this technique they report that they have been able to locate inorganic matter in the myofibrils with a precision of approximately 100 A. Watson (1947) and Hillier (1948) have described contamination of specimens subject to prolonged irradiation by the condensation of vapors present in the electron microscope. Hillier (1948) noted "that the deposit occurs mainly on the area of the substrate simultaneously bombarded by the gas molecules and the electron beam," which "strengthens the suggestion that the surface bombarded by the electron beam takes part in the chemical reaction resulting in the deposit." Konig (1948) claims to have demonstrated that the contaminating material deposited was the carbonaceous residue of hydrocarbon or other organic molecules broken down by the intense ionizing action of the electron beam and to have prepared carbon replicas on various crystals merely by prolonged irradiation in the electron microscope; these replicas were refractory to heating in the vacuum oven but in the presence of air were readily burned to CO2. By analyzing the deposit formed on a silica film by means of the microanalyzer Hillier (1948) also came to the conclusion that the deposit consisted largely of carbon. In more recent unpublished work in the RCA Laboratories, S. G. Ellis has shown that migrating grease and oil films may provide the source of supply of most of the contaminating material. The deposit itself is then merely the accumulation of the decomposition or polymerization products that occur as a result of the bombardment. The phenomenon of contamination, which has been introduced here as a common type of artifact and which can change the dimensions of protruding structures or can reduce the contrast of fine internal details by the superposition of a thick uniform layer, also provides observable evidence that the electron bombardment can induce chemical changes in solid materials. Visible artifacts have been observed also from the use of specimens of excessive thickness. Richards and Anderson (1942) described vacuolization of thick sections of insect cuticle due to evolution of gas, shrinkage, discoloration, and loss of tensile strength on excessive electron irradiation. Bombarded areas were found to be refractory to 5 per cent solutions of NaOH and to have lost their staining affinity for acid fuchsin (Richards and Anderson, 1941). The phenomenon of "fixation" of the specimen that is presented in this paper occurs, however, under conditions suitable for the routine making of electron pictures. The pattern of contrast between nuclear sites and cytoplasm is a faithful representation of the contrast pattern observed in similarly prepared specimens viewed with the light microscope (Hillier, Mudd, and Smith, 1949, figure 3). The pattern of contrast in Os04-fixed specimens examined with the light and electron microscopes is also, to the best of our knowledge and belief, a faith-

11 1950] FIXATION OF ELECTRON MICROSCOPIC SPECIMENS 651 ful representation of the pattern of contrast that may be discerned in the same kind of cells examined with the phase microscope in the living state (Tulasne, 1949a,b; Stempen, 1950). Visible changes do not occur in making successive electron pictures of the same specimen. The specimen that has been subjected to electron micrography even under these routine conditions which do not produce visible structural change is, however, very different chemically from the same specimen before electron micrography. Studies in Germany which were not known to us when performing the present experiments have been brought to our attention by the recent book, Die Uibermikroskopie, by Von Borries (1949). In these studies the phenomenon under discussion has been described. Ruska (1940) and Von Borries and Glaser (1944) reported that under electron bombardment changes in the supporting film appear which, on the one hand, influence its chemical character (for example, make the nitrocellulose insoluble in amyl acetate) and, on the other, cause great stability with reference to temperature. Pictures of diphtheria bacilli were reproduced (Von Borries and Glaser, 1944), in the first of which the bacilli were micrographed with as little irradiation as possible; afterward the preparation was heated for 10 minutes in the vacuum oven at 300 C and a second picture was taken of the same field. Alteration of the specimen and of the supporting film by the heating was scarcely detectable. The authors attributed this fixation of specimen and film to heating by energy absorbed from the electron beam. K6nig (1946) showed that whereas a fresh collodion film was destroyed by heating to 150 to 200 C in the vacuum oven, a similar film after bombardment in the electron microscope with electrons of the usual intensity could withstand 1,000 C. The electron diffraction pattern of such an irradiated film could be interpreted, according to Konig, as that of finely crystalline graphite. K6nig and Winkler (1948) and Winkler and Konig (1949) reported that bacteria, after observation in the electron microscope, no longer stained with the usual aniline dyes. Diphtheria bacilli, in confirmation of Von Borries and Glaser (1944), after electron micrography were heated in vacuo to 350 C, almost without visible alteration. Control bacilli similarly heated were reduced to residues in which only the polar granules retained a semblance of their original appearance. After electron micrography the calcium phosphate residues of the polar granules were refractory to solution in hydrochloric acid, and tellurium crystals in the cytoplasm were refractory to solution in bromine water. These effects were attributed to impermeability of the irradiated bacterial protoplasm to water and ions. In a model experiment a film of copper was laid down between films of collodion. The copper film could be dissolved out through the collodion in a few minutes from unirradiated preparations but not from similar preparations after electron micrography. Konig and Winkler attributed the chemical and thermal refractoriness of irradiated specimens less to heating in the electron beam than to ionization caused by the beam. The ionization effects, they believe, drive off volatile products containing hydrogen, oxygen, and nitrogen, leaving largely carbon rings held together by high bonding energies. Such carbon rings would yield the electron

12 HILIER, MUDDD, SMIT, AND BEUTNER 652 [VOL. 60 diffraction pattern of graphite, which has been demonstrated in collodion films after electron micrography (Konig, 1946; KEnig and Winkler, 1948). The experimental work presented here indicates quite conclusively that even low-level bombardment of solid specimens induces drastic chemical modifications. The changes are all in the direction of reduced chemical activity and appear to occur without modification of any observable structure. However, the observations made under these somewhat different conditions do not support the explanations presented in the literature. For instance, the explanations based on a simple heating of the specimen by the bombardment does not seem correct in view of the experiments with the cut sections when the intensities used were very low and the absence of melting of the paraffin provided an internal indication of the temperature. The possibility that the bombardment might prevent the melting of the paraffin is eliminated by the fact that melting is observed at higher intensities. The present work does not support the suggestion of K6nig and Winkler that an ionization produced by the bombardment results in the driving off of all material except carbon. Carbon probably accounts for no more than half of the mass of the sections used. The change in opacity caused by the removal of half the mass would be considerable, yet no change was observed at the low-level bombardment. Here, as above, higher intensities of bombardment would tend to support Konig and Winkler. However, even when more than half of the mass of the section has been removed by bombardment, there is no indication of discoloration when the section is examined in a light microscope. Discoloration appears only after prolonged intense bombardment and could then be explained by the build-up of contamination. The fact that low-level bombardment can produce a "fixation" of organic specimens without a great increase in temperature and without observable loss of material indicates that the phenomenon is the result of the fundamental interaction between the bombarding electrons and the specimen. While it is probable that the new data presented here are far from sufficient for devising a completely satisfactory explanation, some further deductions can be drawn. The two most characteristic changes observed resulting from the bombardment appear to be the loss of solubility and the increase in the melting or sublimation point. Both of these effects could be explained by an increase in the binding between the molecules of the material. The decreased chemical reactivity that the bombarded material displays could be a manifestation of a radical modification of the molecules themselves. Such changes are not inconceivable when it is realized that a single inelastic collision between an impinging electron and a molecular electron will, on the average, release more than enough energy to break any bond in the molecule. The two or more parts of such a fragmented molecule are highly reactive. They may recombine or they may react with adjacent molecules to form entirely new bonds, their acquired energy then being released as heat. Since, in these experiments, the specimen has always contained a high proportion of very large molecules, those molecules are largely immobilized. Thus it could be expected that the rearrangement of the bonds would bind the

13 1950] FIXATION OF ELECTRON MICROSCOPIC SPECIMENS 653 entire mass of molecules together without causing any large-scale redistribution of mass. On this basis the first effect of bombardment might be considered as a form of polymerization. At higher levels of bombardment there is undoubtedly an increase in temperature, with the result that the more volatile molecular fragments are able to escape. It would be only in the extreme limit of treatment that one would expect the residue to consist only of carbon. CONCLUSIONS The pattern of contrast between nuclear sites and cytoplasm seen in electron micrographs of young cells of Escherichia coli fixed in osmic acid vapor resembles the pattern of contrast seen in similarly prepared cells in light micrographs, and also resembles the pattern seen with the phase contrast microscope in living cells. The contrast pattern appears unchanged in second and third pictures of the same specimen taken in routine manner with no special precautions against electron bombardment. After micrography in the electron microscope, specimens may be subjected, without undergoing change in appearance, to chemical or thermal influences that would profoundly alter the specimen before exposure to electron irradiation. Thus bacterial protoplasm is "fixed" by electron irradiation so that it is refractory to normal hydrochloric acid or to cytolysis by distilled water followed by acid treatment. Collodion films and embedding media are refractory to their usual solvents and to high temperatures. Organic specimens subjected to the electron irradiation of routine micrography are therefore altered in chemical constitution and properties if not in visual appearance. This chemical alteration has been attributed to the high energy which a single electron can impart to a molecule of a specimen. REFERENCES BORRIES, B. VON 1949 Die tlbermikroskopie. Verlag Editio Cantor Aulendorf, Wuirttemberg. Refer to p , BORRIES, B. VON, AND GLASER, W ttber die Temperaturerhohung der Objekte im t)bermikroslkop. Kolloid-Z., 106, BURTON, E. F., SENNETT, R. S., AND ELLIS, S. G Specimen changes due to electron bombardment in the electron microscope. Nature, 160, 56&-566. DRAPER, M. H., AND HODGE, A. J Sub-microscopic localization of minerals in skeletal muscle by internal "microincineration" within the electron microscope. Nature, 163, 576. HILLIER, J On the investigation of specimen contamination in the electron microscope. J. Applied Phys., 19, HILLIER, J Some remarks on the image contrast in electron microscopy and the two-component objective. J. Bact., 57, HILLIER, J., KNAYSI, G., AND BAKER, R. F New preparation techniques for the electron microscopy of bacteria. J. Bact., 56, HILLIER, J., MUDD, S., AND SMITH, A. G Internal structure and nuclei in cells of Escherichia coli as shown by improved electron microscopic techniques. J. Bact., 67,

14 654 6HlLIER, MUDD, SMIT, AND BEUTNER [VOL. 60 SKONIG, H Verinderung organischer Praparate im Elektronenmikroskop. Nachr. d. Akad. Wiss. G5ttingen, Mathem-Physik. K1., K6NIG, H lber das Schmelsen des Kohlenstoffs. Naturwisenschaften, 34, K8NIG, H Die Rolle der Kohle bei elektronenmikroskopischen Abbildungen. Naturwussenschaften, 35, K8NIG, H., AND WINKLER, A VUber Einschltsse in Bakterien und ihre Verfinderung im Elektronenmikroskop. Naturwissenschaften, 35, LEMBEE, A., AND RusKA, H Vergleichende mikroskopische und flbermikroskopische Beobachtungen an den Erregern der Tuberkulose. Klin. Wochschr., 19, MORTON, H. E., AND ENGLEY, F. B., JR The protective action of dysentery bacteriophage in experimental infections in mice. J. Bact., 49, MUDD, S., AND SMITH, A. G Electron and light microscopic studies of bacterial nuclei. I. Adaptation of cytological processing to electron microscopy; bacterial nuclei as vesicular structures. J. Bact., 59, MUDD, S., SNaTH, A. G., HILuIER, J., AND BEuTNER, E. H Electron and light microscopic studies of bacterial nuclei. III. The nuclear sites in metal-shadowed cells of Escherichia coli. J. Bact., 60, RicHAaDs, A. G., JR., AND ANDERSON, T. F Unpublished observations. RIcHAIs, A. G., JR., AND ANDERSON, T. F Electron microscope studies of insect cuticle, with a discussion of the application of electron optics to this problem. J. Morphol. 71, ROBINOW, C. F Nuclear apparatus and cell structure of rod-shaped bacteria. Addendum to R. J. Dubos, The bacterial cell. Harvard Univ. Press, Cambridge, Mas. RusKA, H Bedeutung und Ergebnisse der tjbermikroskopie. Siemens-Z., 20, RusKA, H Morphologische Befunde bei der bakteriophagen Lyse. Arch. Virusforsch., 2, STEzPEN, H Demonstration of the chromatinic bodies of Escherichia coli and Proteus vulgaris with the aid of the phase contrast microscope. J. Bact., 60, TuLAsNz, R. 1949a Sur la cytologie des bact6ries vivantes 6tudi6es gr&ce au microscope & contraste de phase. Compt. rend. soc. biol., 143, TuLAsNE, R. 1949b Donn6es nouvelles sur la cytologie des bact6ries apport6es par la microscopie de contraste. Compt. rend soc. biol., 148, WATSON, J. H. L An effect of electron bombardment on carbon black. J. Applied Phys., 18, 153. WATSON, J. H. L Pseudostructures in electron microscope specimens. J. Applied Phys., 19, WINKLER, A., AND KONIa, H Zur Deutung elektronenoptischer Befunde an Bakterien. Zentr. Bakt. Parasitenk., I, Orig., 153, 9-15.