- Email: [email protected]
An improved method for fixing amoebae for electron microscopy
252
Experimental
AN
IMPROVED
METHOD
FOR D.
K.
FOR
ELECTRON
BHOWMICK
and
Zentral-Laboratorium Institut
K.
FIXING
40, 252-263
(1965)
AMOEBAE
MICROSCOPY1 E. WOHLFARTH-BOTTERMANN
fiir angewandte cbermikroskopie der Universitiit Bonn, Western Received
Cell Research
February
am Zoologischen Germany
22, 1965
ONE of the most important
facets of cytology is the relation of structure to function, that is, the attempt to show how a certain structure is related to a particular function. But, as a prerequisite for explaining the function, the morphology of the respective cell should be unraveled. Several current theories of amoeboid movement postulate the existence of cytoplasmic diversity within amoebae [ 1, 61. It has rarely been questioned that protoplasmic streaming is causally related to the structure of the protoplasm. Electronmicroscopic investigations on amoeboid moving cells and slime molds revealed the importance of the groundplasm for the generation of the motive force of amoeboid movement [19, 201. The groundplasm as the contractile substrate could be demonstrated morphologically. The investigation of the structural differences of amoebae has met, at the because there is no chemical initial stage, with rather serious difficulties, substance or mixture known, which might serve as a satisfactory fixing agent for the giant amoebae. Lehmann [9] has pointed out this problem. From the extensive literature on this subject, hardly a single work can be mentioned where the cytoplasm is satisfactorily represented. In almost all electronmimatrix of groundplasm is crographs of amoebae, a more or less continuous absent. This suggests an inadequate fixation or embedding technique. An [ 18 ], of Hyuloexception are the figures, shown by Wohlfarth-Rottermann discus simplex, and in certain cases of ruptured amoebae as shown by Wolpert, Thompson, and O’Neill [al] and Nachmias [ll]. More recently, Komnick and Wohlfarth-Bottermann [S] succeeded in revealing a continuous matrix of groundplasm by pretreating the cells with enzyme solutions, thus making the highly impermeable plasmalemma of the amoeba more permeable for the fixing agent. 1 Support knowledged.
from
the
Kultusministerium
Experimental
Cell Research
40
des Landes
Nordrhein-Westfalen
is gratefully
ac-
Fixation
253
of amoebae for electron microscopy
This communication is a further report of our experiments to find improved fixation methods for an analysis of the structural disparity of amoeba cytoplasm. The criterion for a satisfactory fixation was mainly the preservation of the groundplasm within the cells.
METHODS
Cultivation
of
A. proteus
and Chaos chaos
For this study Chaos chaos (strain Copenhagen) and Amoeba proteus (strain Princeton) have been used. They are grown in mass cultures fed on Tetrahymena in Chalkley inorganic salt solution: 480 mg NaCl, 24 mg NaHCO,, 24 mg KCl, 32 mg CaCl, + 2H,O and 10 mg Ca (H,PO,), +H,O were dissolved in 100 ml distilled water and the solution was made up to a volume of 6000 ml with distilled water of pH 6.3. This solution was always kept in a dark amber glass bottle. The amoebae were kept in petri dishes with lids. The diameters of the dishes were 7 cm and the height of about 5 cm. The level of Chalkley solution in the container was about 2 cm over the bottom. For the fast production of amoebae in great numbers [12], they received a steady diet of Tetrahymena every third day. Cultivation of Tetrahymena.---The decoction of dried lettuce (supplied by Difco Laboratories, Detroit, U.S.A.) was mainly used for the cultivation of Tetrahymena, 200 mg of dried lettuce were boiled in 100 ml of distilled water for 15 min. A pinch of CaCO, was always added to make the decoction alkaline. It was filtered and cooled to room temperature. Tetrahymena gained from the stock culture were inoculated in the above medium. On third day after inoculation, a mass production of Tetrahymena was observed. Amoebae were fed with Tetrahymena, washed repeatedly in Chalkley solution. Both cultures were renewed by inoculation into new vessels approximately every third day. Amoebae grown at 22°C on this diet can be expected to be more uniform than in mass cultures or in nature.
OBSERVATIONS
The fixation of amoebae was made in mixtures of osmium tetroxide and potassium dichromate in different concentrations of gradually increasing strength. Preliminary observations had revealed the following facts: immediately after immersing A. proteus of Chaos chaos into 2 per cent osmium fixing solution, one can observe that cytoplasmic streaming does not stop instantaneously but continues for several seconds. Higher concentrations of osmium tetroxide prevent this effect and stop cytoplasmic streaming immediately when the fixative was poured over the cells. With the idea in mind, that higher concentrations of osmium tetroxide may cause an acceleration of fixation, we attempted, through a complementary series of experiments, to overcome the obstacle incurred by the highly impermeable plasmalemma Experimental
Cell Research
40
254
D. K. Bhowmick
and K. E. Wohlfarth-Bottermann
of the giant amoebae. We injected the fixing agent with the aid of a micromanipulator into the amoeba. Each fixing solution was adjusted with potassium hydroxide and the pH of the solution was maintained to 7.2. The fixation was always performed at room temp. (22”C-26°C). The solubility of osmium tetroxide controls the ultimate concentration range of this substance in fixation media. The following solubilities of osmium tetroxide in 100 ccm mater are presented for reference: Room
temp.
6.5 g (cf. manufacturer, IV. C. Heraeus GmbH, Hanau, Germany) 7.24 g (21 9.5 g+O.l g (approx.) (own experiments)
25°C 30°C
Table I gives the concentrations of osmium tetroxide and potassium dichromate as percentage w/v which have been used during the course of these investigations. The criterion for the quality of fixation is the appearan; of the groundplasm in the electron micrographs. Osmium tetroxide was always dissolved in an aqueous solution of 4.5 per cent potassium dichromate, adjusted by KOH to pH 7.2. The fixing agents were either directly poured over amoeba or injected into the amoeba with the help of a Leitz-micromanipulator (see below). The method which we used for fixing amoebae by submersion was as follows: A number of amoebae were transferred to a shallow petri dish with a They are allowed to move until they minimum level of Chalkley solution. presented a normal appearance. The fluid was slowly taken out by means of a glass pipette to the extent that the amoebae remained just moist and normally crawling. About 2-3 ccm aqueous solution of osmium tetroxide (of different concentration as required) was quickly poured over the amoebae. After fixation, the fixation medium was removed and the cells dehydrated and embedded in Vestopal \V using the normal methods [7, 141. An unsatisfactory fixation of amoebae (see Fig. 1 a) obtained by the conventional method (2 per cent 0~0,) is indicated by the disappearance of groundplasm. The cytoplasm shows alveolar structure but the spaces between the membranous structures are more or less empty in the electronFig. 1 u.PChaos chaos fixed in 2 per cent buffered osmium tetroxide submersion. Stained with phosphotungstic acid and many1 acetate. cytoplasm and the lack of ground plasm. x 22,500. Fig. 1 b.-Chaos chaos fixed with the same solution as in Fig. the cell by a micropipette with the help of a micromanipulator. can be observed. x 19,000. Experimental
Cell
Research
40
and potassium dichromate Note the alveolar structure
1 a but the fixative A few remnants
by of
was injected into of ground plasm
Fixation
of amoebae for electron microscopy
Experimental
255
Cell
Research
40
256
D. K. Bhowmick
and K. E. Wohlfarth-Bottermann
micrograph. This appearance is a characteristic hint for artifacts ced with other cell-types and also amoebae. But as the highly fixatives were applied on to amoeba, an obvious change of the obtained (Figs. 3b, 4). This structural appearance of the rather comparable to the figures shown by Wohlfarth-Bottermann TABLE
Ser. no. of experiments
oso, in y0 2 2 2.5 2.5 3.5 4.0 4.5 4.5
9.5
240 (Chaos chaos) 244 (C. chaos) 11 (C. chaos) I2 (A. proteus) I3 (C. chaos) 14 (A. proteus) I5 (C. chaos) 15, (A. profeus)
9.5 9.5 9.5 9.5 9.5 9.5 9.5
- , No groundplasm + and + + , a continuous micrographs.
present, matrix
only membrane of groundplasm
as experienconcentrated pictures was cytoplasm is [IS] in
I.
PH
No. of amoeba fixed
7.1 7.2 7.0 7.3 7.4 7.2 7.2 7.2
6 11 6 7 7 9 11 9
Duration fixation in min
of Quality fixation
90
-
60 40 60
-
20 10 8
AI I + ~..
8
structures; &, a sparse and membrane structures
of
++
groundplasm present; present in all electron-
the amoeba Hyalodiscus and agrees so far with the theoretical conception of cytoplasmic structures of amoeba. By conception of cytoplasmic structures we mean an explicit gradual structural differentiation of cytoplasm into the groundplasm and cytoplasmic membranous components. As an instrument for the injection of the fixation medium, the Leitz micromanipulator with two needleholders, a microsyringe and a stereomicroscope of 12-30 initial magnification was used. Instead of a general type of moist chamber we used depression slides. This allows the amoeba to move on a leveled substratum. Micromanipulator units were mounted at a level a bit higher than that of the steady non-moveable mechanical stage of the stereomicroscope, so that the microneedles may diagonally enter into the depression of the slide. Fig. 2a.-A chaos. Note
mixture of 6.5 per cent 0~0, the network of plasmafilaments
and 2 per cent K,Cr,O, in parallel arrangement.
was microinjected x 24,500.
into
Fig. 2b.-Amoeba (Chaos chaos) injected as in Fig. 2 b. A mass localisation of thicker filaments be observed. Fixation: a mixture of 7.5 per cent 0~0, and 2 per cent K,Cr,O,. x 29,000. Experimental
Cell
Research
40
Chaos can
Fixation
257
of amoebae for electron microscopy
Experimental
Cell Research
40
258
D. K. Bhowmick
and K. E. Wohlfarth-Bottermann
The micropipettes were filled with fixative by capillary action and the tips were plugged with a drop of inert paraffin oil. An amoeba was transferred to the depression slide and was allowed to move normally. Then the microneedle, filled with fixative was suddenly inserted into the cytoplasm. The insertion of the micropipette stopped the streaming and movement of the amoeba, but a short time after the insertion of the pipette, the cell resumed normal locomotion. Then the plunger of the microsyringe was slowly pushed and the fixative streamed into the amoeba. The cells which were fixed by microinjection, were immersed immediately after the injection for 5 min with the same fixing solution. It was observed, even when the fixing agent was introduced into the cell, that the preservation of the groundplasm was unsatisfactory (Fig. 1 b). The amoebae shown in Figs. 2a and 2 h were injected with 6.5 per cent and 7.5 per cent 0~0, respectively. In these amoebae in the vicinity of the introduced pipette, a hbrillar differentiation of the cytoplasm was observed. A large zone of filaments was always found in that part of the cell, where the micropipette had been inserted. These structures are comparable wih the “Iibrillar differentiations”, revealed by Nachmias [l l] when she ruptured the cell membrane before fixation. When a high concentration of aqueous solution of 0~0, was brought by injection into the cell (Fig. 3a), the fixation was not so satisfactory as that obtained by simply pouring the same fixative over the cells (Figs. 3 b and 4). DISCUSSION
Figs. 1 a and 1 b show that the conventional “standard” 2 per cent osmium tetroxide fixation fails to fix satisfactorily the amoeba protoplasm, even when the fixative is directly microinjected into the cell: In both cases, the groundplasm is not revealed. Various explanations may be offered for this failure. In the living cell, the plasmalemma of Amoeba proteus and Chaos chaos is remarkably impermeable to water or any aqueous solution [3, 161. It can be assumed that the plasmalemma is also impermeable to osmium tetroxide. The cause may be the fringed mucous layer of the plasmalemma demonstrated by Schneider and Wohlfarth-Bottermann [15] and Brandt and Pappas Fig. 3a.-A mixture of 9.5 per cent 0~0, and amoeba (Chaos chaos). Note the meagre presence x 19,000. Fig. 3 b.-Amoeba remarked dense x 19,000. Experimental
4.5 per cent K,Cr,O, was microinjected into the of groundplasm and the rupture of mitochondria.
proteus fixed in the same solution as in Fig. 3 a, but appearance of the cytoplasm can be noted because
Cell Research
40
by submersion ground plasm
of the cell. A is preserved.
Fixation
of amoebae for electron microscopy
Eqerimental
259
Cell
Research
40
260
Fig.
D. K. Bhowmick
4.-Chaos
Experimental
chaos fixed Cell Research
in the 40
and K. E. Wohlfarth-Bottermann
same
solution
and
in same
way
as in Fig.
36.
x 29,000.
Fixation
of amoebae for electron
261
microscopy
[5]. More recently Komnick and Wohlfarth-Bottermann [8] presented evidence for this assumption. Although osmium tetroxide normally causes instant cessation of streaming in cytoplasm, often it has been noted in this laboratory as well as in others (K. D. Allen, personal communication) that amoeba cytoplasm continues to move for a certain time when the cells are covered with 1 or 2 per cent osmium tetroxide solution. This may result from a delay in the entry of a slowly-penetrating fixative like osmium tetroxide [13]. Another reason for the disappearance of the groundplasm may be, as Bahr [2] and Wigglesworth [4] suggested, that by prolonged treatment \vith 0~0, much of the cell protoplasm dissolves. And this reaction is presumably shown in Fig. 3a. The cause for this peculiar reaction may be understood, if the suggestion is accepted that a short treatment with osmium tetroxide causes an apparent gelation of protoplasm. But this gelation is temporary only. On further treatment with osmium tetroxide the gel becomes soluble again and is washed out during the dehydrating processes. To avoid these disadvantages, that is, on one side the washing out effect and on the other side the slow penetrating rate of this fixative, Fick’s law of diflusion and concentration can be considered. The law suggests that the acceleration of diffusion is directly proportional to the concentration (i.e. molar value) of a solution. Naturally, the molar value of the super saturated aqueous solution of GO,, which has been used in this investigation, lies high above the molarity “standard” 0~0, fixative. High concentrations of osof the conventional mium tetroxide in water can successfully fix the amoeba, sufficiently well to preserve the groundplasm (Figs. 3 b and 4). From Table I it could be deduced that the longer the duration of fixation, the more unsatisfactory is its quality. Fixation for periods longer than lo-20 min gives no satisfactory results, even when high concentrations of osmium tetroxide are applied. The fixation by injection of the fixing solution into the cell also gives similar results. Due to the impermeable nature of a plasmalemma, perhaps the osmium cannot be washed out as quickly as it is thought, and thus the duration of fixation and the nature of fixation is comparable with that of amoeba fixed directly for a longer period of time. However, it was curious to note that at the immediate vicinity of puncture, in all amoebae fixed by microinjection, a fibrillar organisation of the cytoplasm was observed consisting of a parallel arrangement of many plasmafilaments. Investigations on slime molds have shown that these plasmafilaments are a differentiation of the Experimental
Cell Research
40
D. K. Bhowmick
262
and K. E. Wohlfarth-Bottermann
groundplasm [19, 201. Their appearance is an indication of contraction processes of the cytoplasm. This interpretation is supported by the results of Nachmias [ 111, who got the same structures when she ruptured the cell membrane of the amoebae with needles. With great certainty, this procedure results in a heavy contraction process of the cytoplasm. Injecting the fixative with the micropipettes as in our experiments is a similar mechanical injury and so it is not astonishing to find the same structures as a response of the cytoplasm. In evaluating our experiments to find an improved fixation method for Amoeba proteus and Chaos chaos, apparently the highly concentrated solution of osmium tetroxide (9.5 per cent) in mixture with K,Cr,O, (4.5 per cent), buffered at a pH nearly 7.0 make it possible to reveal the ground plasm of these amoebae (fixing time S-10 min). Further staining of the objects with heavy metal solutions [17] enhances the contrast and contributes a distinct revelation of the delicate elements of the groundplasm. It remains to be seen whether it will be possible with this method to reveal a cytoplasmic diversity in normally moving amoebae, especially to compare the structure of the hyaline cap and the uroid, which are supposed to be involved in the locomotion processes according to the different modern contraction theories. SUMMARY
Mixtures of osmium tetroxide and potassium dichromate at different concentrations were tried on Amoeba proteus and Chaos chaos as fixatives. The fixatives were either introduced into the cell with a micromanipulator or poured directly on the amoeba. The microinjection resulted in a distinct fibrillar organisation of the cytoplasm as a reaction to the mechanical injury. It was found that a high concentration of GO,, applied for S-10 min by pouring over the cell, is a more satisfactory fixative for amoeba and permits observation of the groundplasm of these cells. This is a prerequisite for the analysis of cytoplasmic diversity [4] causally related to the mechanism of amoeboid movement. REFERENCES 1. ALLEN, R. D., The Cell, Vol. 2. Academic Press, New York and London 1961. 2. BAHR, G. F., Exptl Cell Res. 7, 457 (1954). 3. BELDA, W. H., Salesianum 38, 17 (1943). 4. BRANDT, P. W., J. Cell Bid. 15, 55 (1962). 5. BRANDT, P. W. and PAPPAS, G. D., J. Biophys. Biochem. Cytol. 8, 675 (1960). 6. GOLDACRE, R. J. and LORCH, I. J., Nature 166, 497 (1950). 7. KELLENBERGER, E., SCHWAB, W. et A. RYTER, Exprrienfia (Basel) 12, 421 (1956).
Experimental
Cell
Research
40
Fixation 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
of amoebne for electron microscopy
263
KOWNICK, H. and WOHLF~RTH-BOTTER~XA~N, K. E., Z. Zellforsch. In press. (1965.) LEHMANN, F. G., Ergeb. Biol. 21, 88 (1959). LBVTRUP, S. and P&ON, A., Corkpt. ‘Rend: Trau. Lab. Carlsberg 28, 1 (1951). NACHMIAS, V. T., J. Cell Biot. 23, 183 (1964). PRESCOTT, D. M., Compt. Rend. Trau. Lab. Carlsberg 30, 1 (1956). ROMEIS, B., in OLDENBOURG, R. (ed.), Mikroskopische Technik, Miinchen, 1948. RYTER, A., and KELLESBERGER, E., J. lJ/traslruct. Res. 2, 200-214 (1958). SCHNEIDER, L. and WOHLFARTH-BOTTERMANX, K. E., Protoplasma 51, 377 (1959). WIGGLESWORTH, \‘. B., Proc. Rog. Sot. (London) 147, 185 (1957). WOHLFARTH-BOTTERMAXN, I<. E., iVaturwiss. 12b, 164 (1957). ~ Protoplasma 52, 58 (1960). ~ Primitive Motile Systems in Cell Biology. S. 79. Academic Press, New York, 1964a. -Intern. Reu. Cytol. 16, 61 (1964b). WOLPERT, L., THOXPSON, C. M. and O’NEILL, C. H., Primitive Motile Systems in Cell Biology. 173. Academic Press, New York, 1964.
Experimental
Cell
Research
40
Experimental
AN
IMPROVED
METHOD
FOR D.
K.
FOR
ELECTRON
BHOWMICK
and
Zentral-Laboratorium Institut
K.
FIXING
40, 252-263
(1965)
AMOEBAE
MICROSCOPY1 E. WOHLFARTH-BOTTERMANN
fiir angewandte cbermikroskopie der Universitiit Bonn, Western Received
Cell Research
February
am Zoologischen Germany
22, 1965
ONE of the most important
facets of cytology is the relation of structure to function, that is, the attempt to show how a certain structure is related to a particular function. But, as a prerequisite for explaining the function, the morphology of the respective cell should be unraveled. Several current theories of amoeboid movement postulate the existence of cytoplasmic diversity within amoebae [ 1, 61. It has rarely been questioned that protoplasmic streaming is causally related to the structure of the protoplasm. Electronmicroscopic investigations on amoeboid moving cells and slime molds revealed the importance of the groundplasm for the generation of the motive force of amoeboid movement [19, 201. The groundplasm as the contractile substrate could be demonstrated morphologically. The investigation of the structural differences of amoebae has met, at the because there is no chemical initial stage, with rather serious difficulties, substance or mixture known, which might serve as a satisfactory fixing agent for the giant amoebae. Lehmann [9] has pointed out this problem. From the extensive literature on this subject, hardly a single work can be mentioned where the cytoplasm is satisfactorily represented. In almost all electronmimatrix of groundplasm is crographs of amoebae, a more or less continuous absent. This suggests an inadequate fixation or embedding technique. An [ 18 ], of Hyuloexception are the figures, shown by Wohlfarth-Rottermann discus simplex, and in certain cases of ruptured amoebae as shown by Wolpert, Thompson, and O’Neill [al] and Nachmias [ll]. More recently, Komnick and Wohlfarth-Bottermann [S] succeeded in revealing a continuous matrix of groundplasm by pretreating the cells with enzyme solutions, thus making the highly impermeable plasmalemma of the amoeba more permeable for the fixing agent. 1 Support knowledged.
from
the
Kultusministerium
Experimental
Cell Research
40
des Landes
Nordrhein-Westfalen
is gratefully
ac-
Fixation
253
of amoebae for electron microscopy
This communication is a further report of our experiments to find improved fixation methods for an analysis of the structural disparity of amoeba cytoplasm. The criterion for a satisfactory fixation was mainly the preservation of the groundplasm within the cells.
METHODS
Cultivation
of
A. proteus
and Chaos chaos
For this study Chaos chaos (strain Copenhagen) and Amoeba proteus (strain Princeton) have been used. They are grown in mass cultures fed on Tetrahymena in Chalkley inorganic salt solution: 480 mg NaCl, 24 mg NaHCO,, 24 mg KCl, 32 mg CaCl, + 2H,O and 10 mg Ca (H,PO,), +H,O were dissolved in 100 ml distilled water and the solution was made up to a volume of 6000 ml with distilled water of pH 6.3. This solution was always kept in a dark amber glass bottle. The amoebae were kept in petri dishes with lids. The diameters of the dishes were 7 cm and the height of about 5 cm. The level of Chalkley solution in the container was about 2 cm over the bottom. For the fast production of amoebae in great numbers [12], they received a steady diet of Tetrahymena every third day. Cultivation of Tetrahymena.---The decoction of dried lettuce (supplied by Difco Laboratories, Detroit, U.S.A.) was mainly used for the cultivation of Tetrahymena, 200 mg of dried lettuce were boiled in 100 ml of distilled water for 15 min. A pinch of CaCO, was always added to make the decoction alkaline. It was filtered and cooled to room temperature. Tetrahymena gained from the stock culture were inoculated in the above medium. On third day after inoculation, a mass production of Tetrahymena was observed. Amoebae were fed with Tetrahymena, washed repeatedly in Chalkley solution. Both cultures were renewed by inoculation into new vessels approximately every third day. Amoebae grown at 22°C on this diet can be expected to be more uniform than in mass cultures or in nature.
OBSERVATIONS
The fixation of amoebae was made in mixtures of osmium tetroxide and potassium dichromate in different concentrations of gradually increasing strength. Preliminary observations had revealed the following facts: immediately after immersing A. proteus of Chaos chaos into 2 per cent osmium fixing solution, one can observe that cytoplasmic streaming does not stop instantaneously but continues for several seconds. Higher concentrations of osmium tetroxide prevent this effect and stop cytoplasmic streaming immediately when the fixative was poured over the cells. With the idea in mind, that higher concentrations of osmium tetroxide may cause an acceleration of fixation, we attempted, through a complementary series of experiments, to overcome the obstacle incurred by the highly impermeable plasmalemma Experimental
Cell Research
40
254
D. K. Bhowmick
and K. E. Wohlfarth-Bottermann
of the giant amoebae. We injected the fixing agent with the aid of a micromanipulator into the amoeba. Each fixing solution was adjusted with potassium hydroxide and the pH of the solution was maintained to 7.2. The fixation was always performed at room temp. (22”C-26°C). The solubility of osmium tetroxide controls the ultimate concentration range of this substance in fixation media. The following solubilities of osmium tetroxide in 100 ccm mater are presented for reference: Room
temp.
6.5 g (cf. manufacturer, IV. C. Heraeus GmbH, Hanau, Germany) 7.24 g (21 9.5 g+O.l g (approx.) (own experiments)
25°C 30°C
Table I gives the concentrations of osmium tetroxide and potassium dichromate as percentage w/v which have been used during the course of these investigations. The criterion for the quality of fixation is the appearan; of the groundplasm in the electron micrographs. Osmium tetroxide was always dissolved in an aqueous solution of 4.5 per cent potassium dichromate, adjusted by KOH to pH 7.2. The fixing agents were either directly poured over amoeba or injected into the amoeba with the help of a Leitz-micromanipulator (see below). The method which we used for fixing amoebae by submersion was as follows: A number of amoebae were transferred to a shallow petri dish with a They are allowed to move until they minimum level of Chalkley solution. presented a normal appearance. The fluid was slowly taken out by means of a glass pipette to the extent that the amoebae remained just moist and normally crawling. About 2-3 ccm aqueous solution of osmium tetroxide (of different concentration as required) was quickly poured over the amoebae. After fixation, the fixation medium was removed and the cells dehydrated and embedded in Vestopal \V using the normal methods [7, 141. An unsatisfactory fixation of amoebae (see Fig. 1 a) obtained by the conventional method (2 per cent 0~0,) is indicated by the disappearance of groundplasm. The cytoplasm shows alveolar structure but the spaces between the membranous structures are more or less empty in the electronFig. 1 u.PChaos chaos fixed in 2 per cent buffered osmium tetroxide submersion. Stained with phosphotungstic acid and many1 acetate. cytoplasm and the lack of ground plasm. x 22,500. Fig. 1 b.-Chaos chaos fixed with the same solution as in Fig. the cell by a micropipette with the help of a micromanipulator. can be observed. x 19,000. Experimental
Cell
Research
40
and potassium dichromate Note the alveolar structure
1 a but the fixative A few remnants
by of
was injected into of ground plasm
Fixation
of amoebae for electron microscopy
Experimental
255
Cell
Research
40
256
D. K. Bhowmick
and K. E. Wohlfarth-Bottermann
micrograph. This appearance is a characteristic hint for artifacts ced with other cell-types and also amoebae. But as the highly fixatives were applied on to amoeba, an obvious change of the obtained (Figs. 3b, 4). This structural appearance of the rather comparable to the figures shown by Wohlfarth-Bottermann TABLE
Ser. no. of experiments
oso, in y0 2 2 2.5 2.5 3.5 4.0 4.5 4.5
9.5
240 (Chaos chaos) 244 (C. chaos) 11 (C. chaos) I2 (A. proteus) I3 (C. chaos) 14 (A. proteus) I5 (C. chaos) 15, (A. profeus)
9.5 9.5 9.5 9.5 9.5 9.5 9.5
- , No groundplasm + and + + , a continuous micrographs.
present, matrix
only membrane of groundplasm
as experienconcentrated pictures was cytoplasm is [IS] in
I.
PH
No. of amoeba fixed
7.1 7.2 7.0 7.3 7.4 7.2 7.2 7.2
6 11 6 7 7 9 11 9
Duration fixation in min
of Quality fixation
90
-
60 40 60
-
20 10 8
AI I + ~..
8
structures; &, a sparse and membrane structures
of
++
groundplasm present; present in all electron-
the amoeba Hyalodiscus and agrees so far with the theoretical conception of cytoplasmic structures of amoeba. By conception of cytoplasmic structures we mean an explicit gradual structural differentiation of cytoplasm into the groundplasm and cytoplasmic membranous components. As an instrument for the injection of the fixation medium, the Leitz micromanipulator with two needleholders, a microsyringe and a stereomicroscope of 12-30 initial magnification was used. Instead of a general type of moist chamber we used depression slides. This allows the amoeba to move on a leveled substratum. Micromanipulator units were mounted at a level a bit higher than that of the steady non-moveable mechanical stage of the stereomicroscope, so that the microneedles may diagonally enter into the depression of the slide. Fig. 2a.-A chaos. Note
mixture of 6.5 per cent 0~0, the network of plasmafilaments
and 2 per cent K,Cr,O, in parallel arrangement.
was microinjected x 24,500.
into
Fig. 2b.-Amoeba (Chaos chaos) injected as in Fig. 2 b. A mass localisation of thicker filaments be observed. Fixation: a mixture of 7.5 per cent 0~0, and 2 per cent K,Cr,O,. x 29,000. Experimental
Cell
Research
40
Chaos can
Fixation
257
of amoebae for electron microscopy
Experimental
Cell Research
40
258
D. K. Bhowmick
and K. E. Wohlfarth-Bottermann
The micropipettes were filled with fixative by capillary action and the tips were plugged with a drop of inert paraffin oil. An amoeba was transferred to the depression slide and was allowed to move normally. Then the microneedle, filled with fixative was suddenly inserted into the cytoplasm. The insertion of the micropipette stopped the streaming and movement of the amoeba, but a short time after the insertion of the pipette, the cell resumed normal locomotion. Then the plunger of the microsyringe was slowly pushed and the fixative streamed into the amoeba. The cells which were fixed by microinjection, were immersed immediately after the injection for 5 min with the same fixing solution. It was observed, even when the fixing agent was introduced into the cell, that the preservation of the groundplasm was unsatisfactory (Fig. 1 b). The amoebae shown in Figs. 2a and 2 h were injected with 6.5 per cent and 7.5 per cent 0~0, respectively. In these amoebae in the vicinity of the introduced pipette, a hbrillar differentiation of the cytoplasm was observed. A large zone of filaments was always found in that part of the cell, where the micropipette had been inserted. These structures are comparable wih the “Iibrillar differentiations”, revealed by Nachmias [l l] when she ruptured the cell membrane before fixation. When a high concentration of aqueous solution of 0~0, was brought by injection into the cell (Fig. 3a), the fixation was not so satisfactory as that obtained by simply pouring the same fixative over the cells (Figs. 3 b and 4). DISCUSSION
Figs. 1 a and 1 b show that the conventional “standard” 2 per cent osmium tetroxide fixation fails to fix satisfactorily the amoeba protoplasm, even when the fixative is directly microinjected into the cell: In both cases, the groundplasm is not revealed. Various explanations may be offered for this failure. In the living cell, the plasmalemma of Amoeba proteus and Chaos chaos is remarkably impermeable to water or any aqueous solution [3, 161. It can be assumed that the plasmalemma is also impermeable to osmium tetroxide. The cause may be the fringed mucous layer of the plasmalemma demonstrated by Schneider and Wohlfarth-Bottermann [15] and Brandt and Pappas Fig. 3a.-A mixture of 9.5 per cent 0~0, and amoeba (Chaos chaos). Note the meagre presence x 19,000. Fig. 3 b.-Amoeba remarked dense x 19,000. Experimental
4.5 per cent K,Cr,O, was microinjected into the of groundplasm and the rupture of mitochondria.
proteus fixed in the same solution as in Fig. 3 a, but appearance of the cytoplasm can be noted because
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by submersion ground plasm
of the cell. A is preserved.
Fixation
of amoebae for electron microscopy
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260
Fig.
D. K. Bhowmick
4.-Chaos
Experimental
chaos fixed Cell Research
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and K. E. Wohlfarth-Bottermann
same
solution
and
in same
way
as in Fig.
36.
x 29,000.
Fixation
of amoebae for electron
261
microscopy
[5]. More recently Komnick and Wohlfarth-Bottermann [8] presented evidence for this assumption. Although osmium tetroxide normally causes instant cessation of streaming in cytoplasm, often it has been noted in this laboratory as well as in others (K. D. Allen, personal communication) that amoeba cytoplasm continues to move for a certain time when the cells are covered with 1 or 2 per cent osmium tetroxide solution. This may result from a delay in the entry of a slowly-penetrating fixative like osmium tetroxide [13]. Another reason for the disappearance of the groundplasm may be, as Bahr [2] and Wigglesworth [4] suggested, that by prolonged treatment \vith 0~0, much of the cell protoplasm dissolves. And this reaction is presumably shown in Fig. 3a. The cause for this peculiar reaction may be understood, if the suggestion is accepted that a short treatment with osmium tetroxide causes an apparent gelation of protoplasm. But this gelation is temporary only. On further treatment with osmium tetroxide the gel becomes soluble again and is washed out during the dehydrating processes. To avoid these disadvantages, that is, on one side the washing out effect and on the other side the slow penetrating rate of this fixative, Fick’s law of diflusion and concentration can be considered. The law suggests that the acceleration of diffusion is directly proportional to the concentration (i.e. molar value) of a solution. Naturally, the molar value of the super saturated aqueous solution of GO,, which has been used in this investigation, lies high above the molarity “standard” 0~0, fixative. High concentrations of osof the conventional mium tetroxide in water can successfully fix the amoeba, sufficiently well to preserve the groundplasm (Figs. 3 b and 4). From Table I it could be deduced that the longer the duration of fixation, the more unsatisfactory is its quality. Fixation for periods longer than lo-20 min gives no satisfactory results, even when high concentrations of osmium tetroxide are applied. The fixation by injection of the fixing solution into the cell also gives similar results. Due to the impermeable nature of a plasmalemma, perhaps the osmium cannot be washed out as quickly as it is thought, and thus the duration of fixation and the nature of fixation is comparable with that of amoeba fixed directly for a longer period of time. However, it was curious to note that at the immediate vicinity of puncture, in all amoebae fixed by microinjection, a fibrillar organisation of the cytoplasm was observed consisting of a parallel arrangement of many plasmafilaments. Investigations on slime molds have shown that these plasmafilaments are a differentiation of the Experimental
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D. K. Bhowmick
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and K. E. Wohlfarth-Bottermann
groundplasm [19, 201. Their appearance is an indication of contraction processes of the cytoplasm. This interpretation is supported by the results of Nachmias [ 111, who got the same structures when she ruptured the cell membrane of the amoebae with needles. With great certainty, this procedure results in a heavy contraction process of the cytoplasm. Injecting the fixative with the micropipettes as in our experiments is a similar mechanical injury and so it is not astonishing to find the same structures as a response of the cytoplasm. In evaluating our experiments to find an improved fixation method for Amoeba proteus and Chaos chaos, apparently the highly concentrated solution of osmium tetroxide (9.5 per cent) in mixture with K,Cr,O, (4.5 per cent), buffered at a pH nearly 7.0 make it possible to reveal the ground plasm of these amoebae (fixing time S-10 min). Further staining of the objects with heavy metal solutions [17] enhances the contrast and contributes a distinct revelation of the delicate elements of the groundplasm. It remains to be seen whether it will be possible with this method to reveal a cytoplasmic diversity in normally moving amoebae, especially to compare the structure of the hyaline cap and the uroid, which are supposed to be involved in the locomotion processes according to the different modern contraction theories. SUMMARY
Mixtures of osmium tetroxide and potassium dichromate at different concentrations were tried on Amoeba proteus and Chaos chaos as fixatives. The fixatives were either introduced into the cell with a micromanipulator or poured directly on the amoeba. The microinjection resulted in a distinct fibrillar organisation of the cytoplasm as a reaction to the mechanical injury. It was found that a high concentration of GO,, applied for S-10 min by pouring over the cell, is a more satisfactory fixative for amoeba and permits observation of the groundplasm of these cells. This is a prerequisite for the analysis of cytoplasmic diversity [4] causally related to the mechanism of amoeboid movement. REFERENCES 1. ALLEN, R. D., The Cell, Vol. 2. Academic Press, New York and London 1961. 2. BAHR, G. F., Exptl Cell Res. 7, 457 (1954). 3. BELDA, W. H., Salesianum 38, 17 (1943). 4. BRANDT, P. W., J. Cell Bid. 15, 55 (1962). 5. BRANDT, P. W. and PAPPAS, G. D., J. Biophys. Biochem. Cytol. 8, 675 (1960). 6. GOLDACRE, R. J. and LORCH, I. J., Nature 166, 497 (1950). 7. KELLENBERGER, E., SCHWAB, W. et A. RYTER, Exprrienfia (Basel) 12, 421 (1956).
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Fixation 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
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KOWNICK, H. and WOHLF~RTH-BOTTER~XA~N, K. E., Z. Zellforsch. In press. (1965.) LEHMANN, F. G., Ergeb. Biol. 21, 88 (1959). LBVTRUP, S. and P&ON, A., Corkpt. ‘Rend: Trau. Lab. Carlsberg 28, 1 (1951). NACHMIAS, V. T., J. Cell Biot. 23, 183 (1964). PRESCOTT, D. M., Compt. Rend. Trau. Lab. Carlsberg 30, 1 (1956). ROMEIS, B., in OLDENBOURG, R. (ed.), Mikroskopische Technik, Miinchen, 1948. RYTER, A., and KELLESBERGER, E., J. lJ/traslruct. Res. 2, 200-214 (1958). SCHNEIDER, L. and WOHLFARTH-BOTTERMANX, K. E., Protoplasma 51, 377 (1959). WIGGLESWORTH, \‘. B., Proc. Rog. Sot. (London) 147, 185 (1957). WOHLFARTH-BOTTERMAXN, I<. E., iVaturwiss. 12b, 164 (1957). ~ Protoplasma 52, 58 (1960). ~ Primitive Motile Systems in Cell Biology. S. 79. Academic Press, New York, 1964a. -Intern. Reu. Cytol. 16, 61 (1964b). WOLPERT, L., THOXPSON, C. M. and O’NEILL, C. H., Primitive Motile Systems in Cell Biology. 173. Academic Press, New York, 1964.
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