- Email: [email protected]
[5] Culturing and handling of Chlamydomonas reinhardti
144
MICROBIOLOGICAL TECHNIQUES
[5]
[5] Culturing and Handling of Chlamydomonas reinhardti
By G~ZA D/~NES The unicellular green algae of the genus Chlamydomonas provide an excellent system for studying many problems of biochemistry and genetics. Chlamydomonasreinhardti, one of the best-known species of the genus, is a heterothallic isogamous alga: there are two mating types, referred to as plus (ot) and minus (9) which look alike. The vegetative cells are ovoid in shape, and their dimensions are 8 × 15 tz. The cells grown in a liquid medium are motile and swim with the aid of a pair of anterior flagella. Cells grown on solid media are nonmotile because of lack of flagella. The cell contains only one chloroplast. The cytology, sexual cycle, and genetics of Chlamydomonas have been described in detail. 1,2 For most biochemical experiments studying enzymes or regulation of cell metabolism, vegetatively growing cells or cultures are quite suitable. This article deals only with the description of media, techniques of maintaining stock cultures, and preparation of large-scale cultures for the detection or purification of some enzymes. For the sake of reproducibility it is advisable to use strains from the Culture Collection. During the last five years we have used the plus and minus mating-type strains Nos. 89 and 90, obtained from the Culture Collection of Algae, Department of Botany, Indiana University, Bloomington, Indiana. We could not detect any difference between the plus and minus mating-type strains with respect to mechanism and regulation of amino acid biosynthetic pathways.
Maintenance of Stock Cultures Sterile stock cultures are grown on agar slants. For maintaining the strains on agar slants Medium I is used.l'3 One liter of the 10-fold concentrated stock solution (10 × I) contains sodium citrate • 2 H20, 5.0 g; NH4NOz, 3.0 g; FeClz • 6 H20, 0.1 g; CaC12, 0.4 g; MgSO4 • 7 H~O, 3.0 g; KH2PO4, 1.0 g; KeHPO4, 1.0 g. The chemicals are dissolved separately, and the solutions obtained are mixed and diluted to a final volume of 1 liter. The pH of the slightly yellow stock solution is about 6.8. The solution is stored at room temperature in a glass-stoppered container with 10 ml of chloroform. IR. Sager and S. Granick,J. (;en. Physiol. 37, 729 (1954). 2R. P. Levine and W. T. Ebersold, Ann. Rev. Microbiol. 14, 197 (1960). hR. Sager and S. Granick, Ann. N. Y. Acad. Sci. 56, 831 (1953).
[5]
CULTURING OV Chlamydomonas reinhardti
145
M e d i u m I is s u p p l e m e n t e d with trace metals. O n e liter o f h u n d r e d fold c o n c e n t r a t e d trace metal solution contains: HzBO3, 100 mg; ZnSO4 • 7 H20, 100 mg; MnSO4 • 4 H20, 40 mg; COC12 • 6 H 2 0 , 20 mg; CuSO4 • 7 HzO, 4 mg. T h e trace metal stock solution is stored in a glasss t o p p e r e d container at r o o m t e m p e r a t u r e and is stable for months. For p r e p a r a t i o n o f solid m e d i u m , 100 ml o f 10 X I and 40 ml o f trace metal stock solution are diluted with distilled water to make a final v o l u m e o f 1000 ml, and 15.0 g o f Bacto-agar (Difco) is dissolved in the m e d i u m by sterilization at 120 ° for 30 minutes. Aliquots o f the sterile m e d i u m obtained are distributed either in sterile tubes or in sterile petri dishes for p r e p a r a t i o n o f slants or plates. T h e cells are g r o w n on the agar slants at 22-25 ° u n d e r diurnally intermittent illumination o f 15 hours light and 9 hours darkness. T h e y are illuminated by fluorescent tubes (3 tubes, T u n g s r a m F 7, daylight, 60 W, m o u n t e d on a white plastic-covered b o a r d o f 40 cm x 158 cm). During the first 3 days the freshly inoculated slants are placed in a Plexiglas box and aerated with air containing 5% CO.,. T h e box is placed at a distance o f 40 cm f r o m the light source. After this period, the cultures are placed in a storage box (metal with a Plexiglas window) which is at a distance o f 150 cm f r o m the same light source. O r d i n a r y laboratory air contains e n o u g h CO2 to maintain the cultures u n d e r these conditions. T o p r e v e n t desiccation o f the agar slants, o p e n dishes containing distilled water are placed on the bottom of" the storage box. T h e cultures are t r a n s f e r r e d onto fresh agar slants every 10 weeks. T h e same m e t h o d is also used tor maintaining a u x o t r o p h i c mutants. In these cases the m e d i u m also contains the necessary growth factors, such as amino acid, vitamins, or o t h e r c o m p o u n d s . Cultures on the agar slants die r a t h e r quickly in the dark at r e f r i g e r a t o r temperature. M e d i u m I contains citrate as chelating agent. Chlamydomonas c a n n o t use this c o m p o u n d as a carbon source, but some o t h e r microorganisms may use it. For this reason, standard sterile bacteriological techniques are r e q u i r e d because o f the d a n g e r o f contamination. Chlamydomonas (an "acetate" flagellate) can use acetate and some o t h e r fatty acids as a c a r b o n source but c a n n o t utilize carbohydrates. 4 M e d i u m II contains acetate as the carbon source ~,3 and the 10-fold c o n c e n t r a t e d stock solution (10 × I I) contains: sodium citrate.2 HzO, 0.50 g; NH4NO3, 0.30 g; FeC13.6 H20, 0.01 g; CaCI2, 0.04 g; MgSO4.7 H20, 0.30 g; sodium acetate, 2.00 g; NaH2PO4"H20, 3.67 g; K2HPO4, 1.15 g. M e d i u m II, like M e d i u m I, is s u p p l e m e n t e d with trace metals, and the m e t h o d o f p r e p a r a t i o n o f solid M e d i u m II is the same as that 4S. H. Humer and L. Provasoli, in "Biochemistry and Physiology of Protozoa" (A. Lwoff, ed.), p. 27. Academic Press, New York, 1951.
146
MICROBIOLOGICAL TECHNIQUES
[5]
described previously for Medium I. The cells can grow on this medium either in dim light or in darkness. Medium II is especially useful for maintaining mutants damaged in their photosynthetic system.
Laboratory-Scale Liquid Cultures Medium I without agar can be used for the preparation of liquid medium. Since the medium does not contain any carbon source, CO2 is required; 500 ml of the medium is sterilized in culture vessels at 120 ° for 30 minutes. The vessels are 40 cm-long tubes, and their internal diameter is 5 cm. At the bottom of the vessels there is an air inlet tube. Before use the vessels are equipped with air filters. T h e filters are glass tubes (1.5 cm × 30 cm) with rubber tubing on both ends to fit the gas inlet tube of the vessels and the outlet tubes of the pressure air valves. The filter tubes are filled with cotton, wrapped in aluminum foil, and sterilized separately from the vessels at 120 ° for 30 minutes. T h e culture vessels equipped with filters are inoculated with cells (either from agar slants or from a previous liquid culture) and placed in a light thermostat. The light thermostat consists of a water bath and a light source. The water bath (25 × 50 × 130 cm; stainless steel frame with glass windows opposite to each other) is filled with distilled water and equipped with a cooling (or a heating) device and a mixing system to maintain the temperature at 25 ° or any other temperature required. The light source is a set of fluorescent tubes (4 tubes, Tungsram, F 7 daylight, 40 W) mounted on a white plastic-covered board (50 × 125 cm). The cultures are aerated with air (at 10-20 liters per hour). The ordinary laboratory air usually contains enough CO~ to fulfill the CO2 requirement of the growing cells; if it does not, an air-CO2 mixture (5% CO2 in air) is used. Sometimes a strict control of the temperature of the cultures is not necessary and they can be placed on a laboratory desk and illuminated with the light source used for the light thermostat. To obtain uniform illumination it is advisable to slide a sheet of opal glass between the light source and the culture vessels. It is also possible to use Medium II without agar as a liquid medium. The method of preparation is the same as that of Medium I. Wild-type cells growing in Medium II are largely agglutinated, and the determination of the cell number by photometric methods is sometimes difficult. The cultures are grown with slight illumination or in darkness and aerated with air (10-20 liters per hour). Since this medium contains both citrate and acetate, the cultures are easily contaminated under nonsterile conditions.
[5]
CULTURING or Chlamydomonas reinhardti
147
Large-Scale Fermentation Procedure To obtain enough cells for the preparation and purification of enzymes, nonsterile procedure is used. Medium V containing ethylenediaminetetraacetate (EDTA) as a chelating agent is convenient for this purpose, s One liter of 50-fold concentrated stock solution of medium V contains KHePO4, 18.0 g; KzHPO4, 36.0 g; NH4NOn, 15.0 g. The stock solution is sterilized at 120° for 30 minutes. Medium V is supplemented with trace metals; 1 liter of the hundredfold concentrated trace metal solution contains ethylenediaminetetraacetic acid, 5.00 g; potassium hydroxide, 2.90 g; H3BO3, 1.14 g; CaClz.2 H20, 1.00 g; MgSO4-7 H20, 2.00 g; ZnSO4-7 HeO, 2.20 g; MnSO4"4 H20, 0.58 g; FeSO4"7 HeO, 0.49 g; COC12"6 HeO, 0.16 g; CUSO4"5 HeO, 0.16 g; NazMoOa'2 HzO, 0.14 g. The EDTA, boric acid, and potassium hydroxide are dissolved together, but the other chemicals are added to the solution separately in the order listed and completely dissolved before the next is added. The trace metal solution is stored at 2° in darkness and is stable for 7-10 days. For preparation of Medium V, 500 ml of stock solution and 250 ml of trace metal solution are diluted with distilled water to make a final volume of 25 liters in the Plexiglas culture vessel. The size of the vessel (external) is 106 × 57 × 6.7 cm; wall thickness is 0.8 cm. Four or six containers are used for the mass production of the algae, each containing 25 liters of medium. The vessels are placed on both sides of the light source. The light source consists of a set of fluorescent tubes (10 tubes, Tungsram, F 7, daylight, 20 W), mounted on a stainless steel frame. The vessels are inoculated with 2 liters of liquid culture of Chlamydomonas and aerated by air containing 5% COs (250 liters per hour per vessel). The amount of air-CO2 mixture bubbling through the culture is regulated individually for each vessel by a needle valve. To obtain a sterile dust-free and oil droplet-free gas mixture, the gas is filtered through an asbestos filter ("Seitz" EKS, Seitz-Werke, Kreuznach, Germany). The optimum temperature for the cell growth is 25 ° , but we have found that the yield of cells is about the same between 22 ° and 27 °. The cells are harvested after 48 hours by means of a Sharples centrifuge. The yield is 100-120 g of wet cells per 150 liters of medium. The cells are washed with 0.02 M KCI by centrifugation and either used immediately for the preparation of a cell-free extract or stored at--30 ° before use. The culture (2 liters) remaining in the vessels is diluted with 25 liters of fresh medium and a new growth period is continued 5M.Staub and G. D6nes,Biochim.Biophys.Acta 128, 82 (1966).
148
MICROBIOLOGICAL TECHNIQUES
[5]
for 2 days. Since the only organic ingredient of Medium V is EDTA which is not metabolized by most microorganisms, this method of nonsterile fermentation is usually free of contamination. If contamination occurs, this is mostly because of bad growth conditions causing death of the algae and growth of the contaminating microorganisms on the dying algal cells.
Preparation of Cell-Free Extract Three different methods are used for preparation of cell-free extract from Chlamydomonas.The first one is the disintegration of the cells by sonic treatment. This procedure is useful to obtain 5-100 ml of extract for the detection or partial purification of enzymes. The principle of this procedure for Chlamydomonasis the same as for bacteria. The second method is grinding the algae with solid CO2. This procedure is used for preparation of a large amount of extract for purification of allosteric enzymes or of enzymes that are easily inactivated. 5,6 The third method involves two steps: the first is the preparation of acetone-dry powder from the freshly prepared or stored algae cells; and the second is extraction of the powder with buffer. 7 The details of the last two methods are described elsewhere in this volume. 8'9 6A. Farago and G. D6nes, Biochim. Biophys. Acta 136, 6 (1967). 7I. SiJdi and (,, D6nes, Acta Biochim. Biophys. Acad. Sci. Hung. 2, 279 (1967). 8(;. l)enes, this w)lume [28] and [29]. 9(;. D~nes, this volume [30].
MICROBIOLOGICAL TECHNIQUES
[5]
[5] Culturing and Handling of Chlamydomonas reinhardti
By G~ZA D/~NES The unicellular green algae of the genus Chlamydomonas provide an excellent system for studying many problems of biochemistry and genetics. Chlamydomonasreinhardti, one of the best-known species of the genus, is a heterothallic isogamous alga: there are two mating types, referred to as plus (ot) and minus (9) which look alike. The vegetative cells are ovoid in shape, and their dimensions are 8 × 15 tz. The cells grown in a liquid medium are motile and swim with the aid of a pair of anterior flagella. Cells grown on solid media are nonmotile because of lack of flagella. The cell contains only one chloroplast. The cytology, sexual cycle, and genetics of Chlamydomonas have been described in detail. 1,2 For most biochemical experiments studying enzymes or regulation of cell metabolism, vegetatively growing cells or cultures are quite suitable. This article deals only with the description of media, techniques of maintaining stock cultures, and preparation of large-scale cultures for the detection or purification of some enzymes. For the sake of reproducibility it is advisable to use strains from the Culture Collection. During the last five years we have used the plus and minus mating-type strains Nos. 89 and 90, obtained from the Culture Collection of Algae, Department of Botany, Indiana University, Bloomington, Indiana. We could not detect any difference between the plus and minus mating-type strains with respect to mechanism and regulation of amino acid biosynthetic pathways.
Maintenance of Stock Cultures Sterile stock cultures are grown on agar slants. For maintaining the strains on agar slants Medium I is used.l'3 One liter of the 10-fold concentrated stock solution (10 × I) contains sodium citrate • 2 H20, 5.0 g; NH4NOz, 3.0 g; FeClz • 6 H20, 0.1 g; CaC12, 0.4 g; MgSO4 • 7 H~O, 3.0 g; KH2PO4, 1.0 g; KeHPO4, 1.0 g. The chemicals are dissolved separately, and the solutions obtained are mixed and diluted to a final volume of 1 liter. The pH of the slightly yellow stock solution is about 6.8. The solution is stored at room temperature in a glass-stoppered container with 10 ml of chloroform. IR. Sager and S. Granick,J. (;en. Physiol. 37, 729 (1954). 2R. P. Levine and W. T. Ebersold, Ann. Rev. Microbiol. 14, 197 (1960). hR. Sager and S. Granick, Ann. N. Y. Acad. Sci. 56, 831 (1953).
[5]
CULTURING OV Chlamydomonas reinhardti
145
M e d i u m I is s u p p l e m e n t e d with trace metals. O n e liter o f h u n d r e d fold c o n c e n t r a t e d trace metal solution contains: HzBO3, 100 mg; ZnSO4 • 7 H20, 100 mg; MnSO4 • 4 H20, 40 mg; COC12 • 6 H 2 0 , 20 mg; CuSO4 • 7 HzO, 4 mg. T h e trace metal stock solution is stored in a glasss t o p p e r e d container at r o o m t e m p e r a t u r e and is stable for months. For p r e p a r a t i o n o f solid m e d i u m , 100 ml o f 10 X I and 40 ml o f trace metal stock solution are diluted with distilled water to make a final v o l u m e o f 1000 ml, and 15.0 g o f Bacto-agar (Difco) is dissolved in the m e d i u m by sterilization at 120 ° for 30 minutes. Aliquots o f the sterile m e d i u m obtained are distributed either in sterile tubes or in sterile petri dishes for p r e p a r a t i o n o f slants or plates. T h e cells are g r o w n on the agar slants at 22-25 ° u n d e r diurnally intermittent illumination o f 15 hours light and 9 hours darkness. T h e y are illuminated by fluorescent tubes (3 tubes, T u n g s r a m F 7, daylight, 60 W, m o u n t e d on a white plastic-covered b o a r d o f 40 cm x 158 cm). During the first 3 days the freshly inoculated slants are placed in a Plexiglas box and aerated with air containing 5% CO.,. T h e box is placed at a distance o f 40 cm f r o m the light source. After this period, the cultures are placed in a storage box (metal with a Plexiglas window) which is at a distance o f 150 cm f r o m the same light source. O r d i n a r y laboratory air contains e n o u g h CO2 to maintain the cultures u n d e r these conditions. T o p r e v e n t desiccation o f the agar slants, o p e n dishes containing distilled water are placed on the bottom of" the storage box. T h e cultures are t r a n s f e r r e d onto fresh agar slants every 10 weeks. T h e same m e t h o d is also used tor maintaining a u x o t r o p h i c mutants. In these cases the m e d i u m also contains the necessary growth factors, such as amino acid, vitamins, or o t h e r c o m p o u n d s . Cultures on the agar slants die r a t h e r quickly in the dark at r e f r i g e r a t o r temperature. M e d i u m I contains citrate as chelating agent. Chlamydomonas c a n n o t use this c o m p o u n d as a carbon source, but some o t h e r microorganisms may use it. For this reason, standard sterile bacteriological techniques are r e q u i r e d because o f the d a n g e r o f contamination. Chlamydomonas (an "acetate" flagellate) can use acetate and some o t h e r fatty acids as a c a r b o n source but c a n n o t utilize carbohydrates. 4 M e d i u m II contains acetate as the carbon source ~,3 and the 10-fold c o n c e n t r a t e d stock solution (10 × I I) contains: sodium citrate.2 HzO, 0.50 g; NH4NO3, 0.30 g; FeC13.6 H20, 0.01 g; CaCI2, 0.04 g; MgSO4.7 H20, 0.30 g; sodium acetate, 2.00 g; NaH2PO4"H20, 3.67 g; K2HPO4, 1.15 g. M e d i u m II, like M e d i u m I, is s u p p l e m e n t e d with trace metals, and the m e t h o d o f p r e p a r a t i o n o f solid M e d i u m II is the same as that 4S. H. Humer and L. Provasoli, in "Biochemistry and Physiology of Protozoa" (A. Lwoff, ed.), p. 27. Academic Press, New York, 1951.
146
MICROBIOLOGICAL TECHNIQUES
[5]
described previously for Medium I. The cells can grow on this medium either in dim light or in darkness. Medium II is especially useful for maintaining mutants damaged in their photosynthetic system.
Laboratory-Scale Liquid Cultures Medium I without agar can be used for the preparation of liquid medium. Since the medium does not contain any carbon source, CO2 is required; 500 ml of the medium is sterilized in culture vessels at 120 ° for 30 minutes. The vessels are 40 cm-long tubes, and their internal diameter is 5 cm. At the bottom of the vessels there is an air inlet tube. Before use the vessels are equipped with air filters. T h e filters are glass tubes (1.5 cm × 30 cm) with rubber tubing on both ends to fit the gas inlet tube of the vessels and the outlet tubes of the pressure air valves. The filter tubes are filled with cotton, wrapped in aluminum foil, and sterilized separately from the vessels at 120 ° for 30 minutes. T h e culture vessels equipped with filters are inoculated with cells (either from agar slants or from a previous liquid culture) and placed in a light thermostat. The light thermostat consists of a water bath and a light source. The water bath (25 × 50 × 130 cm; stainless steel frame with glass windows opposite to each other) is filled with distilled water and equipped with a cooling (or a heating) device and a mixing system to maintain the temperature at 25 ° or any other temperature required. The light source is a set of fluorescent tubes (4 tubes, Tungsram, F 7 daylight, 40 W) mounted on a white plastic-covered board (50 × 125 cm). The cultures are aerated with air (at 10-20 liters per hour). The ordinary laboratory air usually contains enough CO~ to fulfill the CO2 requirement of the growing cells; if it does not, an air-CO2 mixture (5% CO2 in air) is used. Sometimes a strict control of the temperature of the cultures is not necessary and they can be placed on a laboratory desk and illuminated with the light source used for the light thermostat. To obtain uniform illumination it is advisable to slide a sheet of opal glass between the light source and the culture vessels. It is also possible to use Medium II without agar as a liquid medium. The method of preparation is the same as that of Medium I. Wild-type cells growing in Medium II are largely agglutinated, and the determination of the cell number by photometric methods is sometimes difficult. The cultures are grown with slight illumination or in darkness and aerated with air (10-20 liters per hour). Since this medium contains both citrate and acetate, the cultures are easily contaminated under nonsterile conditions.
[5]
CULTURING or Chlamydomonas reinhardti
147
Large-Scale Fermentation Procedure To obtain enough cells for the preparation and purification of enzymes, nonsterile procedure is used. Medium V containing ethylenediaminetetraacetate (EDTA) as a chelating agent is convenient for this purpose, s One liter of 50-fold concentrated stock solution of medium V contains KHePO4, 18.0 g; KzHPO4, 36.0 g; NH4NOn, 15.0 g. The stock solution is sterilized at 120° for 30 minutes. Medium V is supplemented with trace metals; 1 liter of the hundredfold concentrated trace metal solution contains ethylenediaminetetraacetic acid, 5.00 g; potassium hydroxide, 2.90 g; H3BO3, 1.14 g; CaClz.2 H20, 1.00 g; MgSO4-7 H20, 2.00 g; ZnSO4-7 HeO, 2.20 g; MnSO4"4 H20, 0.58 g; FeSO4"7 HeO, 0.49 g; COC12"6 HeO, 0.16 g; CUSO4"5 HeO, 0.16 g; NazMoOa'2 HzO, 0.14 g. The EDTA, boric acid, and potassium hydroxide are dissolved together, but the other chemicals are added to the solution separately in the order listed and completely dissolved before the next is added. The trace metal solution is stored at 2° in darkness and is stable for 7-10 days. For preparation of Medium V, 500 ml of stock solution and 250 ml of trace metal solution are diluted with distilled water to make a final volume of 25 liters in the Plexiglas culture vessel. The size of the vessel (external) is 106 × 57 × 6.7 cm; wall thickness is 0.8 cm. Four or six containers are used for the mass production of the algae, each containing 25 liters of medium. The vessels are placed on both sides of the light source. The light source consists of a set of fluorescent tubes (10 tubes, Tungsram, F 7, daylight, 20 W), mounted on a stainless steel frame. The vessels are inoculated with 2 liters of liquid culture of Chlamydomonas and aerated by air containing 5% COs (250 liters per hour per vessel). The amount of air-CO2 mixture bubbling through the culture is regulated individually for each vessel by a needle valve. To obtain a sterile dust-free and oil droplet-free gas mixture, the gas is filtered through an asbestos filter ("Seitz" EKS, Seitz-Werke, Kreuznach, Germany). The optimum temperature for the cell growth is 25 ° , but we have found that the yield of cells is about the same between 22 ° and 27 °. The cells are harvested after 48 hours by means of a Sharples centrifuge. The yield is 100-120 g of wet cells per 150 liters of medium. The cells are washed with 0.02 M KCI by centrifugation and either used immediately for the preparation of a cell-free extract or stored at--30 ° before use. The culture (2 liters) remaining in the vessels is diluted with 25 liters of fresh medium and a new growth period is continued 5M.Staub and G. D6nes,Biochim.Biophys.Acta 128, 82 (1966).
148
MICROBIOLOGICAL TECHNIQUES
[5]
for 2 days. Since the only organic ingredient of Medium V is EDTA which is not metabolized by most microorganisms, this method of nonsterile fermentation is usually free of contamination. If contamination occurs, this is mostly because of bad growth conditions causing death of the algae and growth of the contaminating microorganisms on the dying algal cells.
Preparation of Cell-Free Extract Three different methods are used for preparation of cell-free extract from Chlamydomonas.The first one is the disintegration of the cells by sonic treatment. This procedure is useful to obtain 5-100 ml of extract for the detection or partial purification of enzymes. The principle of this procedure for Chlamydomonasis the same as for bacteria. The second method is grinding the algae with solid CO2. This procedure is used for preparation of a large amount of extract for purification of allosteric enzymes or of enzymes that are easily inactivated. 5,6 The third method involves two steps: the first is the preparation of acetone-dry powder from the freshly prepared or stored algae cells; and the second is extraction of the powder with buffer. 7 The details of the last two methods are described elsewhere in this volume. 8'9 6A. Farago and G. D6nes, Biochim. Biophys. Acta 136, 6 (1967). 7I. SiJdi and (,, D6nes, Acta Biochim. Biophys. Acad. Sci. Hung. 2, 279 (1967). 8(;. l)enes, this w)lume [28] and [29]. 9(;. D~nes, this volume [30].