[7] Synchronized cultures: diatoms

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SYNCHRONOUS CULTURES: DIATOMS

85

[7] S y n c h r o n i z e d C u l t u r e s : D i a t o m s B y W. M. DAaLEY and B. E. VOLCANI

In order to carry out an extensive investigation on the role of silicon in metabolism and shell formation in diatoms, it was necessary to devise methods for obtaining synchronized cultures that could be used for a wide variety of biochemical studies, and that would provide large amounts of cell material, at a high degree of synchrony. Two systems for inducing synchrony were developed: (a) silicon-starvation, which results in synchronized formation of the wall, and (b) a light-dark regime which produces a complete cycle of synchronized growth and division. These synchronies have been used for studies on changes in DNA, RNA, protein, carbohydrate, lipid, and pigment during the life cycle1 and wall formation, 2,3 as well as for studies on changes of nucleoside triphosphates,4,~ photosynthesis, and respiration 6 during wall formation only. Navicula pelliculosa Culture and Media. The strain of N. pelliculosa used in both synchronies is listed as No. 668 in the Culture Collection of Algae at the Indiana University, Bloomington, Indiana. Recently cloned cultures, necessary for best results, are obtained by plating on a freshwater Tryptone medium (FWT) solidified with 1.5% agar composed of Ca(NO3)2.4H20, 0.1 g; K2HPO4"3H20, 0.0135 g; MgSO4.7H20, 0.025 g; Na~SiO3.9H20, 0.1 g; Na2C03, 0.02 g; trace elements, 1 ml; Bacto-Tryptone peptone (Difco) 1 g, in 1 liter of distilled water; pH (not adjusted) 8.3. The mixture of the trace elements7 consists of HsB03, 0.568 g; ZnC12, 0.624 g; CuC12.H20, 0.268 g; Na2MoO4.2H20, 0.252 g; COC12.6H20, 0.42 g; MnC12.4H20, 0.36 g; FeSO4.7H20, 2.50 g, sodium tartrate.2 H20, 1.76 g in 1 liter of glass-distilled water. After 7-10 days growth at 20 °, and exposure to a fluorescent light intensity of 5000 lux, colonies varying in size from small, compact and

1W. M. Darley, C. W. Sullivan, and B. E. Volcani,in preparation. 2j. Coombs, W. M. Darley, O. Holm-Hansen, and B. E. ¥olcani, Plant Physiol. 42, 1601 (1967). F. P. Healey, J. Coombs, and B. E. Volcani, Arch. Mikrobiol. 59, 131 (1967). 4j. Coombs, P. J. Halicki, O. Itolm-Ha~sen, and B. E. Volcani, Exp. Cell Res. 47, 302 (1967). ~J. Coombs, P. J. Halicki, O. Holm-Hansen, and B. E. Volcaai, Exp. Cell Res. 47, 315 (1967). J. Coombs, C. Spanis, and B. E. Volcani, Plant Physiol. 42, 1607 (1967). 7p. R. Burkholder and L. G. Nickell, Bot. Gaz. 110, 426 (1949).

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ISOLATION AND CULTURE TECHNIQUES

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smooth margined, to large and diffuse margined, can be distinguished. A colony of intermediate size is isolated into 8 ml of FWT liquid medium in 12.5 X 2 cm screw-cap tubes. After growth for about 2 weeks under the above conditions, the cultures are placed in dim light; they are transferred once every 2 months. It may be necessary to reclone the cultures periodically to improve the synchrony. Cell Number. The cell samples (about 5 ml) are preserved with a drop of Lugol's solution (6 g of KI, 4 g of Is in 100 ml of H20). To separate clumped cells effectively without causing significant cell breakage, samples are hand-homogenized by plunging at least 20 times with a Teflon pestle in a Potter-Elvehjem tissue grinder. In the light-dark (L-D) synchrony, the homogenized suspension is subjected to mild sonication for 15 minutes in an ultrasonic cleaner (model System Forty, Ultrasonic Industries) and is again hand-homogenized. Then 0.5 ml is pipetted into 49.5 ml of Millipore-filtered 0.85% NaC1 solution; the cell number is determined with a Coulter Model B electronic counter equipped with a 100 t~ pore aperture tube and set at amplification 1/4, aperture current 1/4, upper threshold 100, and lower threshold 10. The settings should be established for each instrument.

Silicon-Starvation Synchrony Principle. An exponential culture is grown in the light in a medium containing a limited amount of silicon, but an excess of other essential nutrients. At the period of silicon starvation, increase in cell number stops and initiation of cell wall occurs in most of the cells. Upon the addition of silicon, wall formation is completed with a resulting synchronous increase in cell number2 ,s,~ Inoculum. Cultures of 50 ml of FWT medium in 125-ml Erlenmeyer flasks inoculated with 5 ml are grown on a reciprocal shaker (120 strokes/minute) for 3 days at 18°-20 ° under continuous illumination at 5000 lux with "cool white" and "warm white" fluorescent lamps. After two further subculturings under the same conditions, the inoculum consists of cells at a stationary stage of growth (about 6 X 108 cells/ml). Approximately 400 ml of culture are required for inoculation. Culture Vessel. A 4-liter polycarbonate bottle (specially manufactured by Nalge Co. Inc., Rochester, New York) is adapted with three openings for a polypropylene aeration tube, a sampling tube, and the 8j. C. Lewin, B. E. Reimann, W. F. Busby, and B. E. Volcani, in "Cell Synchrony-Studies in Biosynthetic Regulation" (I. L. Cameron and G. M. Padilla, eds.), p. 169. Academic Press, New York, 1966. ~W. F. Busby and J. Lewin,J. Phycol. 3, 127 (1967).

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87

Fio. 1. Vessels for silicon-starvation (left) and light-dark (right) synchronization cultures. 1, Aeration tube (5 mm, i.d.) ; 2, sampling tube (5 mm, i.d.) ; 3, inoculation port; 4, glass tubing plugged with cotton for suction of sample; 5, replaceable glass cap for aseptically removing sample; 6, magnetic bar, Teflon coated, octagonal surfaces, 5 cm long; 7, magnetic stirrer, solid state; 8, inlet for sterile dilution medium; 9, resin flask cover; 10, Teflon washer; 11, bank of fluorescent lamps. introduction of inoeulum (Fig. 1). The bottle is placed between two banks of 40-W fluorescent lamps, each bank containing two "cool white" and two "warm white" lamps, equally distributed; this provides a light intensity of 17,000 lux midway between the two banks. The temperature is maintained at 21 ° ± 1 ° ; the culture is aerated at the rate of 10 liters/ minute and stirred with a magnetic stirrer (a solid state speed control stirrer is used since it does not generate heat). Synchronization Procedure. Three liters of F W T medium to which is added 9 ml of a sterile antifoam agent [polypropylene glycol (P-2000) : water 2:1000 by volume], and 5 ml of a 5% filter-sterilized solution of thiosulfate are inoculated to an initial cell density of about 6.5 X 105 cells/ml. After 36 hours of growth, by which time silicon is depleted from the medium and cell division has ceased, the culture is starved for 14 hours. T h e synehrony is initiated by adding a solution of neutralized

88

ISOLATION AND CULTURE TECHNIQUES

[7]

Si Cell uptake seperotion Stationory 14 hr

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Fie. 2. Course of silicon-starvation synchrony of Navicula pelliculosa, and depletion of silicon from the medium [J. Coombs, P. J. Halicki, 0. ttolm-Hansen, and B. E. Volcani, Exp. Cell Res. 47, 315 (1967)]. Silicon-starvation synchrony

Light-dark synchrony

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Fie. 3. Schematic representation of division cycle of Navicula pelIiculosa. Left: Light-dark synchrony (W. M. Darley, C. W. Sullivan, and B. E. Volcani, in preparation). Right: Silicon-starvation synchrony [J. Coombs, P. J. Ha]icki, O. Holmttansen, and B. E. Volcani, Exp. Cell Res. 47, 315 (1967)]. n, nucleus; ch, chloroplast; pl, plasmalemma; cw, cell wall.

[7]

SYNCHRONOUS CULTURES: DIATOMS

89

sodium silicate to a concentration of 17 ~g/ml silicon, together with L-methionine and L-cysteine, to concentrations of 10-5 and 10-4 M, respectively. If the pH has risen above 8 during the starvation period, it is adjusted to pH 7.5 by the addition of sterile HC1. Properties o] the Synchrony. Cell separation ceases once the medium is depleted of silicon (36 hours). However, cellular development continues until cytokinesis has taken place and the deposition of new walls has begun. Thus, after 14 hours the cell population consists of biprotoplastic cells, i.e., two daughter protoplasts, each surrounded by a new plasmalemma and separated by intercellular space, contained within the parent frustule (Fig. 3). When silicon is resupplied, a 3-4 hour period of rapid silicon uptake, during which new walls are completed, is followed by a 3-4 hour period of cell separation, at which time 80-95% of the cells divide (Figs. 2 and 3).

Light-Dark Synchrony Principle. An exponential culture is placed in the dark for 24 hours. At the end of this period, cell division has ceased and the culture consists for the most part of small, young cells. The culture is then exposed to a repetitive light-dark cycle such that the light period provides enough energy for one division cycle and the dark period allows completion of the division cycle. Inoculum. Prior to inoculation of the synchrony vessel, the F W T culture is subcultured at least twice in a defined freshwater glycylglycine medium (FWG); this consists of the medium described above in which Tryptone is replaced by glycylglycine 0.66 g/liter, and which is supplemented with vitamin BI~, l~g; biotin, 2 ~g; and thiamine.HC1, 0.5 mg/ liter. The pH is adjusted to 8.2 with N a 0 H . Fifty-milliliter cultures in 125-ml Erlenmeyer flasks are grown at 20 °, and illuminated with fluorescent lamps (5000 lux), on a reciprocal shaker (120 strokes/minute) for 24 hours to a cell count of 4 to 5 X l0 s cells/ml. Culture Vessel and Chamber. The culture vessel consists of a 4-liter Pyrex reaction kettle (14 cm in diameter and 39 cm in height). The glass cover, resting on a Teflon gasket, has four openings through which an aeration tube, a sampling tube, and an inlet for fresh medium enter the vessel through cotton plugs; the fourth opening is used for introduction of the inoculum (Fig. 1). The synchrony is carried out in the glycylglycine medium to which is added sodium lactate (FWGL), 2 g/liter (6.6 ml of a 30% sodium lactate sterilized separately and added at inoculation). All media are autoclaved at least 24 hours prior to use. Medium to be used for dilution of the synchrony is aerated for 12 hours prior to use at the temperature

90

ISOLATION AND CULTURE TECHNIQUES

[7]

of the synchrony, and transferred to the synchrony vessel through silicone rubber tubing. The sychronization is maintained in a lightproof chamber (50 cm wide X 100 cm long X 75 cm high) provided with fans for ventilation. Illumination of the culture vessel is the same as that for the siliconstarvation synchrony, and the culture is aerated at the rate of 10 liters/ minute. The light regime is controlled by a time-switch electric clock. Synchronization Procedure. To initiate the synchrony, 3 liters of FWGL medium in the synchrony vessel are inoculated to a final concentration of 1.7 to 2.0 X 104 cells/ml. A 24-hour culture in FWG medium in the late exponential phase of growth (4 to 5 X 106 cells/ml) is used for inoculum. The synchrony vessel is placed in the synchrony chamber in the light for 32-36 hours; then the lights are turned off. After 24 hours of starvation in the dark, cell number is 0.5 to 0.75 X 106 cells/ml. Cells are then placed in a L-D regime of 5 hours light and 7 hours dark (one cycle) for as many cycles as desired. The cell number is approximately doubled during each of the first 3 L-D cycles, and reaches 4 to 6 X 106 cells/ml at the end of the third cycle (Fig. 4). The culture is diluted by half with fresh medium at the beginning of i

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[7]

SYNCHRONOUS CULTURES: DIATOMS

91

the fourth cycle and every cycle thereafter, resulting in 2 to 3 )< 106 cells/ml at the beginning of each cycle. Occasionally the culture is diluted slightly at the beginning of the third cycle if the cell number exceeds 3 X 10~ cells/ml. To dilute the culture, 1.5 liters of culture is removed by suction through the sample tube into a large bottle, and 1.5 liters of medium is aseptically added from the reservoir of fresh medium. Continuous-Light Synchrony. When studying light-dark synchronized cells, it is possible to avoid abnormal shifts in metabolism resulting from the light-dark changes, simply by leaving the already L-D synchronized culture in continuous light until cell division is completed. Dilution is carried out in the usual manner after the period of cell division. The light-dark synchronized culture retains a reasonable degree of synchrony for at least two successive cycles in continuous light. Properties o] the Synchrony. Cell separation occurs only during the dark period. During the first 3-4 cycles, however, the length of the separation burst steadily decreases from 6-7 hours during the first cycle to 4-5 hours during cycle 3. By cycle 5, however, the culture has reached a consistent degree of synchrony. On the average, the separation burst lasts for 3 hours (range 2.0-4.0), with the midpoint occurring between hour 7 and 10 of the L-D cycle; 75-100% of the cells divide at each burst, for an average of 88%. Experiments are therefore confined to the fifth and successive cycles. The culture can be maintained for a~ many as 19 cycles without loss of synchronization. The sequence of events occurring both in the light-dark cycle and in the second cycle in continuous light of a synchronized culture consists of: cell growth culminating in mitosis and cytokinesis, followed by silieic acid uptake, wall formation, and separation of complete daughter cells. The division cycle is shown in Fig. 3.

Cyllndrotheca fusfformis Light-Dark Synchrony

Principle. The synchronizing principle for C. ]usiJormis is the same as that for the L-D synchrony of N. pelliculosa. However, in contrast to N. pelliculosa which requires a repetitive L-D cycle, C. ]usi]ormis requires only a single period of dark starvation followed by illumination at high intensity to synchronize cell separation. 4,8 Culture and Media. Axenic cultures of Cylindrotheca ]usi]orm~s (S. Watson's strain 13) are maintained and cloned on an enriched seawater medium (ESW) composed of NAN03, 0.25 g; K~HPO4.3H20, 0.027 g; Na2SiO~.9H~O, 0.1 g; trace elements, 1 ml; vitamin B12, 1 ~g; thiamine.HC1, 0.5 mg; Tryptone, 1 g, in 1 liter filtered seawater. In this synchrony it is essential to use a recently isolated clone for

92

ISOL&TION AND CULTURE TECHNIQUES

[7]

inoculum. The clone is obtained by plating on ESW medium solidified with 1.5% agar. Single colonies are transferred to test tubes containing 5 ml of ESW. The synchrony is carried out in a medium prepared from commercial synthetic seawater (Utility Chemical Co., Paterson, New Jersey) of the following composition: NaC1, 27.5 g; MgC]¢.6H20, 5.38 g; MgS04"7 H20, 6.77 g; KC1, 0.722 g; NaHCO~, 0.20 g; SrC12.6H20, 19.7 mg; MnSO,.H20, 3.95 mg; Na2MoO4"2H20, 0.987 mg; Na2HPO4.7H20, 3.29 mg; LiCl, 0.987 mg; CaCl2, 1.375 g; KI, 0.095 mg; KBr, 0.0285 mg; A12(SO4)3, 0.475 mg; COS04, 0.0526 mg; RbC1, 0.157 rag; CuSO,.5H20, 0.488 rag; ZnSO4.TH20, 0.101 mg; and calcium gluconate, 0.659 mg. To 40 g of the above is added: NAN03, 0.16 g; Na2SiO3.9H20, 0.1 g; trace elements, 1 ml; 2 Na-EDTA (ethylenediamine tetraacetie acid, disodium), 0.012 g; thiamine hydrochloride, 0.5 mg; Tryptone, 1 g in 1 liter of glass distilled water. Before autoclaving, the pH is adjusted with NaOH to 8.0. 3 0 0 0 LUX

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[7]

SYNCHRONOUS CULTURES: DIATOMS

93

Cell Number. Samples are preserved and handled as in N. pelliculosa. Before counting, one drop of 5 N HC1 is added to the suspension to dissolve any precipitated salts. Cells are counted in the light microscope with a Levy counting chamber, or in the Coulter counter set at amplification 1/4, aperture current 0.354, upper threshold 100, and lower threshold 10. Culture Vessel. The culture vessel consists of a 2-liter Erlenmeyer flask adapted with an aeration and sampling assembly (as for N. pelliculosa) which enters through a cotton plug at the top. The flask is kept in a thermostatically controlled water bath at 25 °. Illumination is provided either by two "warm-white" and two "cool-white" fluorescent lamps, providing about 3000 lux at the center of the flask, or by an iodine lamp ("Quartzline," General Electric Co.) which provides 20,000 lux. Air is bubbled through the culture at 6 liters/minute. Inoculum. A 7-10-day-old clone is inoculated (2 ml) into 50 ml of ESW medium in a 125-ml Erlenmeyer flask and grown for 3 days on an illuminated reciprocal shaker as for N. pelliculosa. This culture is inoculated (5 ml) into identical medium; when it reaches 9 to 10 X 10~ cells/ ml (approximately 24 hours) it is used for inoculum; 40-50 ml of culture is required for inoculation. Synchronization Procedure. Artificial seawater medium, 1.5 liters, in the synchrony vessel is inoculated to a final cell density of 4 X 104 cells/ml. After incubation at 3000 lux for about 20 hours, the culture

e

FIa. 6. Schematic representation of division cycle of Cylindrotheca ]usiIormis. n, nucleus; ch, chloroplast; pl, plasmalemma, cw, cell wall [J. Coombs, P. J. ttalicki, O. Holm-Hansen, and B. E. Volcani, Exp. Cell Res. 47, 302 (1967)].

94

ISOnATION AND CULTURE TECHNIQUES

[7]

reaches a cell number of 1.2 X 10~ cells/ml; the flask is then placed in the dark by wrapping the synchrony vessel with aluminum foil. After 24 hours of darkness, the culture is exposed to the high intensity iodine lamp and at the same time filter-sterilized solutions of L-cysteine, Lmethionine, and sodium lactate are added to final concentrations of 10-4, 10-°, and 2 X 10- ~ M, respectively. The culture is left in the light for 10-12 hours for the completion of the synchronized division. Properties o] the Synchrony. The time course of increase in cell number during the entire synchrony procedure is shown in Fig. 5a. When the exponential culture is placed in the dark, cell division continues for about 10 hours, resulting in an 80% increase in cell number. When the culture is reexposed to light (20,000 lux) there is a 7-hour lag during which the cells increase in size and carbon content and take up silicic acid (Fig. 5b); cell separation then takes place during a 2-2.5 hour period. Cell number increases by 92% on the average. A schematic representation of the division cycle is shown in Fig. 6. As with Navicula pelliculosa, a sequence of events that may be grouped into 3 phases can be distinguished: (1) increase in carbon mass; (2) nuclear division, cytokinesis, silicon uptake, and cell wall formation; (3) cell separation (see phases 4, 5, and 6 respectively, in Fig. 5). Light-Dark Synchrony of Other Diatoms Light-dark regimes have been used to obtain synchronized cell division in four other species. Comparative data on seven L-D synchronies are presented in the table. The varying degrees of synchronization and the applicability to biochemical research vary with the organism and reflect, to some extent, the purposes for which the synchronies were devised. The comparatively low cell concentrations in the last four synchronies are due to the fact that laboratory conditions were designed to simulate those of the natural environment. Two different methods have been used to synchronize Ditylum brightwellii. In the first, 1° an 8:16 hour L-D regime produced a comparatively high degree of synchronization; this synchrony was used for morphological and metabolic studies. In another set of experiments 11 with D. brightwellii and Nitzschia turgidula using a variety of cycles, growth rate was measured as a function of light intensity. By interpolating from these growth curves, combinations of photoperiod and light intensity were chosen that gave a growth rate of one division per day. It was ~°R. W. Eppley, R. W. Holmes, and E. Paasche, Arch. Mikrobiol. 56, 305 (1967). 11E. Paasche, Physiol. Plant. 21, 66 (1968).

[7]

SYNCHRONOUS

CULTURES:

DIATOMS

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ISOL)~TION AND CULTURE TECHNIQUES

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found that short photoperiods of bright light yielded the highest degree of synchrony. The synchrony developed for Skeletonema costatum, a familiar experimental species, was only slightly better than exponential growth.

[8]

Tissue

Culture:

Plant

B y W. M. LAETSCH

The first true tissue cultures were apparently green, 1 but the use of such plant material for investigating photosynthesis and/or chloroplast development was essentially ignored until recent years. 2,a This is unfortunate, because tissue cultures offer the prospect of providing the desirable experimental features of algal cultures while possessing the unique developmental patterns of higher plants. The delineation of some of the advantages of this system will perhaps indicate some of the ways in which the following methods can be used. Techniques for handling cultured tissues are very similar to those for microorganisms, and the physical environment can be controlled in a common manner. The long-term control of temperature and illumination is, therefore, much more precise than is presently possible for either seedlings or mature plants. It is notoriously difficult, for example, to expose intact plants or detached organs to very high light intensities fo ~ an extended time period. This problem is minimized with cultured cells or tissues. The same is true for quantitative work involving light quality. Since most work on the development of higher plant chloroplasts centers on the light-induced etioplast to chloroplast transformation, the value of a well-defined illumination system cannot be underestimated. The control of the chemical environment is also susceptible to far greater precision than is possible with either whole plants or plant parts. Sterile conditions open the way to a variety of experiments, and the absence of a cuticle in cultured tissues lessens the problems of absorption of exogenous chemicals which so often plague those working with higher plant tissue. A defined substrate for cultured tissues offers opportunities which too often have been ignored. This permits the isolation of events in chloroplast development from general cell responses such as replication and growth. " is next to impossible to regulate the chemical imputs of R. J. Gautheret, C. R. Acad. Sci. 198, 2195 (1934). W. M. Laetsch and D. A. Stetler, Amer. J. Bot. 52, 798 (1965). 3L. Bergma~ and C. Berger, Planta 69, 58 (1966).