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Chapter 3 Axenic Culture of Acetabularia in a Synthetic Medium
Chapter 3 Axenic Czlltzue of Acetdbdurid in d Synthetic Medizm DAVID C . SHEPHARD Department of Anatomy. Case Western Reserve University. Clevehnd. Ohio
I . Introduction
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I1. Technical Aspects . A . The Organism .
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B. Culture Conditions . . . . C. Culture Vessels and Apparatus . D . Antibiotics and Bacteriocidal Agents E . Sterility Testing . . . . F. The Synthetic Medium . . . Procedures . . . . . . A Rationale . . . . . . B . Cyst Decontamination . . . C. Reproduction and Early Growth . D . The Main Growth Phase . . E . Enucleation and Enucleate Growth F. Maturation . . . . . G. Evaluatiton . . . . . References . . . . . . . Note Added in Proof . . . .
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I. Introduction The giant unicellular alga Acetabularia and its relatives combine a number of unusual features that make them desirable experimental organisms. In the following partial list of these features there is at least one that will appeal to almost every cell biologist. (The references given are to recent papers or reviews.) 1. Enucleation by surgical mtans is simple. and subsequent growth of the anucleate segments is prolonged (Hammerling et aL. 1958) and 49
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DAVID C . SHEPHARD
accompanied by nucleic acid and protein synthesis (Gibor, 1967; Schweiger et al., 1967; Brachet et al., 1955; Keck and Clauss, 1958; and Shephard, 1966). 2. Intra- and interspecies nuclear grafts can be made readily (Richter, 1962). 3. They are single cells with a distinct morphogenesis that can be manipulated by light (Clauss, 1968)) chemical means (Zetsche, 1965; devitry, 1963), and surgery ( Hammerling, 1963). 4. They grow exponentially a billionfold without nuclear division ( Shephard, 1965). 5. At a given stage ten or more rapid cycles of caryokinesis occur ( Schulze, 1939). 6. They have well-defined circadian rhythm ( Vanden Driessche, 1966). 7. They respond to at least one plant hormone (Thimann and Beth, 1959) . 8. Enucleate protoplasts can be made from them (Gibor, 1965). 9. Chloroplasts that carry out complete photosynthesis at intact cell rates can be isolated from them in high yield (Shephard et al., 1968). 10. They reproduce sexually every generation ( Schulze, 1939). 11. Since they can be kept growing indefinitely by repeated stalk amputation ( Hammerling et al., 1958) or low light intensities (Beth, 1953), they are appropriate single cells for aging studies. Opposed to these advantages there is one major disadvantage of these organisms-slow growth: 4-8 days for mass doubling. Two other factors augment the problem of slow growth and have made Acetabularia difficult to culture and unsuitable for many types of experiments. These factors are not inherent, however; they are the lack of a suitable defined culture medium and the lack of a reliable means for completely eliminating Contaminating organisms in cultures. The usual Erd-Schreiber medium varies greatly in its ability to support growth, depending on thc source of seawater and the source, age, and treatment of the earth extract (Provasoli et al., 1957). Erd-Schreiber must be considered an undefined medium, especially in the presence of contaminating microorganisms. The usual cultures of Acetahtclaria are subject to loss from “green infections” and show highly variable growth rates, depending on the vagaries of Erd-Schreiber medium and the particular types of bacteria or molds that are present. Moreover, the presence of any contaminating organisms is a rnajor impediment to detailed physiological or biochemical studies. Thus, many laboratories attempting to culture Acetabularia have found it to be a disappointment. The purpose of this paper is to present means for eliminating contaminating organisms and culturing Acetabularia axenically in a com-
3. Acetabularia
CULTURE
51
pletely defined medium for at least the period of time necessary for most experimental needs. No attempt will be made to review the many details of the organism and the techniques for handling it. The excellent papers of Keck ( 1964), Lateur ( 1963), and others (Beth, 1953; Terborgh and Thimann, 1964; and Gibor and Izawa, 1963) should be consulted for this information.
11. Technical Aspects
A. The Organism These methods were developed using Acetabularia mediterranea. The strain used, and the one that many laboratories have, comes originally from Hhmerling's laboratory. It was collected about 1930 and is undoubtedly a highly selected strain by now. Little is known of the possible differences between strains, but different species require variations in technique ( Keck, 1964). Some differences in medium requirements, growth rates, and susceptibilities to antibiotics may exist in other strains and species; however Acicularia schenckii has been successfully cultured using these methods.
B. Culture Conditions The cultures are maintained at a constant temperature between 22' and 24'C. This can often be accomplished quite safely by rapid circulation of air, in an air-conditioned room, through the incubator to carry away the heat from the lamps. Growth rates increase at higher temperatures to about 30°C,but abnormal growth is often noticed above 25°C. Fluorescent illumination is used at an intensity of about 250 footcandles (6 to 8 x lo3 ergs/cm2/second), using a mixture of deluxe warm white ( WWX) and daylight ( D ) bulbs. Higher intensities are appropriate at higher temperatures. A 12-hour on, 12-hour off light cycle is convenient and commonly used. Acetabularia has a circadian rhythm, and there is a potential problem in comparing experiments carried out at different times of day (Hellebust et al., 1967). The cells become completely asynchronous after a week in the dark ( Vanden Driessche, 1966). For some experimental purposes it might be useful to introduce asynchronous cells into a continuous light regimen with the intensity reduced to about 100 footcandles; however, we have had poor success with long-term cultures
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DAVID C. SHEPHAFiD
of Acetabulariu in continuous light. Considerable data are available on the effects of light intensity, color, and cycle on Acetabularia (Beth, 1953; Terborgh, 1965; Terborgh and Thimann, 1964).
C.
Culture Vessels and Apparatus
Vessels. Roux flasks ( 1 liter) make excellent culture vessels for several reasons, including space saving in the incubator and Iarge surface area for illumination and gas exchange. They should be dry-heat sterilized with the necks wrapped in foil. After the cells and 200 ml of medium are added, the flasks are cotton stoppered, or better, the mouth and neck are covered with two layers of autoclave paper to facilitate subsequent sterile transfers. The maximum cell densities in such cultures are determined by CO, diffusion (Allen and Arnon, 1955). Two grams of fresh weight of cells is the limit that these flasks will support, and 1 gm should not be exceeded if maximum growth rates are desired. Aerating the flasks might increase their capacity, but it adds severe difficulties to sterile culture. Moreover, the medium may not support the extra mass. If they are aerated, the CO, concentration must be less than 0.5%. Smaller amounts of cells (to 0.1 gm) can be maintained conveniently in screw-cap BOD bottles on their sides. Nursery Bottles. Figure 1 diagrams the container we have developed for the reproductive phase of the life cycle. It is a 250-ml widemouth specimen jar with a Bakelite screw cap. A l-inch hole punched in the cap is covered with cotton and paper. A cone of 270 mesh bolting "silk" (nylon) suspended from the lid with platinum wire so that it reaches almost to the bottom is added to those bottles which will receive the cysts. The top of the bottle is wrapped in autoclave paper and the whole assembly autoclaved. Washing Funnels. These are used with various meshes of bolting silk or membrane filters for various procedures ( a Gelman I-inch vacuum filter funnel is suitable). Bolting silk of 74 mesh will pass cysts and retain cells or large debris; 270-mesh nylon will retain cysts and pass contaminating organisms. A large-pore membrane filter (such as Millipore type NC) is used for washing very small cells that tend to become entangled in the nylon. For cyst washing the funnel is mounted in the sterile hood immediately above a magnetic stirrer. Teflon-covered magnetic stirring bars (1.5 x 8 mm) may be used in these funnels. Larger bars tend to be excessively damaging. The entire apparatus must be autoclaved or gas sterilized. lnverted Diwecting Microscope. This arrangement is shown in Fig. 1. It provides the only means of examining the cells in detail while they are
3. Acetabularia
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CULTURE
.Cyst cone Plote glass
rnl
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Nursery bottle
I
3
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1
Inverted dissecting microscope
25 gauge plo tinum
glass
1
/
\
Hook
10 cm Swimming tube
Light
c
FIG. 1. Special apparatus. (See Section II,C for description.)
in R o w flasks or nursery bottles. Optically poor glass surfaces can be masked simply by immersing them in a puddle of water on the glass stage. The construction is quite simple. Miscellaneous A p r a t u s . The swimming tube is used at times to obtain sterility by forcing the phototactic gametes to swim through sterile medium (Keck, 1964). A platinum hook is the simplest means for handling and selecting larger cells, which can be hooked by the rhizoids or caps. It is made from platinum wire (25 gauge) and is sterilized by flaming. Very small cells are counted or manipulated using finer wire or a hair loop.
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DAVID C. SHEPHMUI
D. Antibiotics and Bacteriocidal Agents Table I presents much of our experience in using chemical agents to obtain sterility in Acetabularia cultures. Many agents are more toxic to Acetabularia than to contaminants. The acceptable antibiotics are very useful, but must be used carefully; otherwise, mutation and selection may make them useless when they are most needed. Only bacteriocidal antibiotics are used, and their effectiveness should be ascertained beforehand. Other treatments and agents are used whenever possible. There are three somewhat different sets of circumstances under which all of these substances are used. The first is when dealing with heavy, mixed contamination of the cysts. The object is to eliminate most species and decimate the number of survivors. Sodium lauryl sulfate and formaldehyde are used for this. The details of the procedure are given in Section II1,B. The second set of circumstances is the temporary control of a light, accidental contamination of large cells or as a precaution after an experimental manipulation of the cells. While reestablishing sterility may be desirable, the 2 weeks necessary to accomplish and confirm it is unreasonably long at this stage of the life cycle. The object of this type of treatment is to eliminate as many bacteria as possibIe with a quick and simple treatment. It should be difficult to detect bacteria for a week or two afterwards, and the cells may be used for many experimental purposes. A 5-minute treatment with 0.1%formaldehyde will suffice with sturdy cells or an overnight treatment in 5 pglml sodium omadine is a reasonable alternative. Any treatment should be followed by washes in sterile medium. The third and most important circumstance is the complete eliniination of the few species of contaminants remaining either after the initial treatment of the cysts or found in young cells while there is time to confirm the results. This is the time to use the antibiotics. Such contamination is detected as colonies on agar plates (Section 11,E). These colonies should be collected and their sensitivities to the antibiotics determined before treatment is attempted. We use a filter disc technique (Bailey and Scott, 1966) and make up the discs with the antibiotic stocks on hand. The antibiotics in Table I fall into two categories, bacterial cell wall inhibitors and miscellaneous metabolic inhibitors. The first group is very valuable because of its low toxicity to Acetabularia, even at exorbitant concentrations. (There is little point in using most antibiotics at more than 100 pg/ml.) Antibiotics in the second category are unpredictable in their effects on Acetabularia. We have tested relatively few members
3. Acetabularia ANTIBIOTICS AND
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CUL~RE
TABLE I BACTERIOCIDES FOR
USE WITH
Acetabularia
Maximum tolerated concentrations and timesD
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Effects noticed on hetabidaria*
Bacterial cell wall inhibiting antibiotics Ampicilin Carbenicillin Cephalo thin Cy closerine Penicillin G Vancomycin
1 mg/ml, indefinite 1 mg/ml, indefinite
100 pg/ml, indefinite 100 pg/ml, indefinite 1 mg/ml, indefinitec 1 mg/ml, indefinite
Other antibiotics Amphotericin B (Fungizone) Cephaloridine Gentamicin snlfate Kanamy cin Lincomycin Neomycin Sodium colistimet,hate Streptomycin
1 pg/ml, indefinite” 100 p g / m l , indefinite 1 mg/ml, indefinite 100 pg/ml, indefinite 1 mg/ml, indefinitec 100 pg/ml, 4 days 50 pg/ml, 2 days 1 nig/ml, indefinite
Bacterion’des Formaldehyde (dilute formalin) Furadantin Sodium omadine (Olin Mathieson) Sodium lauryl sulfate
0.1%. 5 minutes (cells) 2%, 5 minutes (cysts) 1 mg/ml, indefinite 5 pg/ml, 2 days l%,12 hours (cysts)
T S N S
s
Agents found to be excessively toxic to Acetabulariad ~~
~
Antibiotics Aureomycin Mycostatin Novobiocin Polymixin
~
______
~~
Bacteriocides Argyrol (Crooks-Barnes) Copper sulfate Domiphen bromide Hexylresorcinol
Hydrogen peroxide Phenol Sodium hypochlorite Zephrin chloride
a Concentrations and times may be far in excess of those needed for bacteriocidal activity. Indefinite times imply a 10-day or longer test,, a period probably longer than the life expectancy of most of these agents. b (N) No toxic effects noted; (S) some toxicity noted a t higher coilcentrations (or longer times); (T) some toxicity noted a t useful times and concentrations. c Compound so unstable that treatment time is almost meaningless. d These agents kill Acetabularia cells and cysts a t times and concentrations necessary to kill other microorganisms.
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DAVID C. SHEPHARD
of this group, and even those listed in the table must be used with care. Most inhibit Acetabularia cells, but any effect at the times and concentrations given are apparently reversible. Still, their use should be limited to cysts if possible. Of the bacteriocidal agents, Furadantin and perhaps sodium omadine fit more appropriately in this second category. After the antibiotic sensitivities of the contaminants have been determined, two or more effective antibiotics are selected for each species to be killed (wall-active antibiotics are not usually mixed with other types). Treatments of cells or cysts with antibiotics should always be carried out in the dark for two reasons. First, many of the antibiotics are destroyed fairly rapidly by light and, second, the Acetabularia is photosynthetically inactive in the dark and is much less sensitive to antibiotic effects. Some substrate (usually 0.05% glucose) is added simultaneously with the antibiotics, especially wall-active ones, to insure that the bacteria will be growing slowly. Since spore-forming organisms are likely contaminants of the cysts following the preliminary decontamination, the antibiotic treatment must be long enough for complete excystment. Threeday treatments are usually adequate; however, observation of the times of colony appearance on the plates will help determine suitable treatments. The life expectancies of some antibiotics in alkaline solutions may be less than 3 days. Cyst treatments are often more effective if carried out in distilled water adjusted to a pH suitable for the antibiotics being used. We have not tried continuous culture in antibiotics partly because few of them are sufficiently stable and because many of them are slightly inhibitory to Acetabuluria. There is also the possibility of developing highly resistant bacterial strains; therefore, continuous treatment is not recommended. However, if necessary, the cell-wall antibiotics, streptomycin, neomycin, and kanamycin, may be added to the culture medium at 50 pg/ml, and arnphotericin B (for fungi) may be added at 0.25 pg/ml. Chloramphenicol at 5-10 g / m l is an effective growth inhibitor for many bacteria, but appears to have little effect on Acetabukzriu. One last point should be emphasized. If reasonable treatments do not result in sterility, it is best, if possible, to discard the culture.
E. Sterility Testing We have found that many of the contaminating organisms grow on the surface of Acetabularia; thus, plating or testing the medium may give a negative result, whereas plating the cells or cysts (with 0.5 ml of medium) will reveal contamination. We plate on tryptone glucose yeast extract agar made up in water (see Section 11,F). Unfortunately, some
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contaminants of these cultures are heat sensitive and very slow growing. We keep the plates in the same incubator as the cells. To find colonies, 48 hours is necessary, and it may take several days to reveal all species. A plate should be kept a minimum of 5-7 days. The delay is not serious at the two most crucial stages for sterility testing-cysts and very small cells. These can be held in the dark until the plate is read. If contamination is found, treatment can be given or the culture discarded. Routine platings of the medium supply and cell stocks are required.
F. The Synthetic Medium The requirements of a suitable medium are: (1) that it can be autoclaved; ( 2 ) that it is chemically defined; ( 3 ) that it supports normal cell growth; and ( 4 ) that all cell nutrients must be in adequate supply to support cell growth from the negligible wet weight of the inoculum to the maximum weight that the CO, diffusion rate will permit under the standard culture conditions. The ASP-6 medium of Provasoli and associates (1957) was found to satisfy the first three requirements. Modifications of this medium which meet the last requirement are given in Table 11. Growth is normal in this medium and as rapid as any reported, 4 4 days for mass doubling until cap initiation, and if the inoculation size is appropriate, no medium change is ever necessary. The major salt solution given in the table can probably be replaced by artificial seawater supplemented with nitrate, pH adjusted to 7.8. The other components are more important. Bicarbonate is added in an amount appropriate for the pH of the medium and to replace that lost during autoclaving. Additional bicarbonate added initially or at intervals during culture seems to be detrimental. Thiamin HC1 and vitamin B,, are the only vitamins tested that have a dramatic effect on growth rate. They constitute the minimum supplementation of any medium. Other vitamins seem to be only slightly beneficial, but have not been completely retested under axenic conditions. The micronutrient solution determines the number of cells that the medium will support in long-term culture. It, too, should be added to any other medium used. We have not found a dramatic effect of added auxin (naphthalene acetic acid for stability), but auxin and other plant hormones have not been adequately tested; amino acids and other organics, some of which might be beneficial to cell growth rates, have not been tested either. The cells will not modify the pH of the medium appreciably until they become overcrowded, and they tolerate a fairly wide range of pH. The pH selected (7.8) is a compromise between lower values favorable
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DAVID C. SHEPHARD
TABLE I1 A SYNTHETIC MEDIUMFOR Acetabularia
Major salts NaCl MgSOd. 7 H20 CaC12 2 Hz0 Tris KC1 NaN03 KzHP04 Micronutrient sa1t.s NaZEDTA ZnSOr. 7 HzO NazMoOd. 2 HzO FeC4 . 6 HzO MnClz.4 HzO CoCli. 6 Hz0 C U S O ~5. HzO Bicarbonate NaHC03 Vitamins Thiamine HC1 p-Aminobenzoate Ca-Pan tothenate Vitamin Blz
Amountfliter
Instructions
24 gm 12 gm 1 gm 1 gm 0.75 gm 40 mg 1 mg
The phosphate is added last; then the pH is adjust,ed to 7.8 with 1N HC1 (about 5 ml/liter) ;finally the solution is autoclaved
12 mg 2 mg
This solution is made up separately and added to the major salt solution before autoelaving
1 mg 0 . 5 mg 0.2 mg 2 rg 2 rg
100 mg 300 r g 20 PP 10 Pg 4 rg
These solutions are made up separately arid added to the salt solution through a sterilizing membrane filter after the autoelaving
to autoclaving without precipitation and higher ones that increase the bicarbonate concentration. It seems likely that bicarbonate rather than carbon dioxide is the substrate for photosynthesis in marine algae ( Thomas and Tregunna, 1967). Perhaps the greatest problem with the medium is handling it to maintain sterility. We autoclave carboys of medium (16 liters) for 1 hour with the siphon and all tubing in place. The bicarbonate and vitamins are added subsequently through a rubber injection port using a syringe equipped with a membrane filter. Since subsequent contamination of the medium can occasionally occur and is catastrophic, the culture bottles should be filled by pumping the medium through an easily replaceable, terminal sterilizing filter. Vacuum filtration is easier but will degas and decarbonate the medium. The medium should be plated each time it is used for setting up stock cultures and more frequently if terminal sterilization is not used; however, it should be remembered that it will be at least 48 hours before the plate can be read.
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111. Procedures A. Rationale Figure 2 is a diagram of the life cycle of Acetabularia from the point of view of culture technique. The dormant cyst stage offers the best possibility for obtaining complete sterility. It is the most resistant stage to
FIG.2. The life cycle of Acetabularia in culture. The subdivisions of the life cycle represent changes in technique rather than natural phases of growth. These subdivisions are described in the text. The main growth phase shows the cells diagrammatically, including: the rhizoid, a rootlike organ containing the nucleus; the cylindrical stalk, which increases in diameter as well as in length; the whorls-hairlike branches that are formed at the apex and shed after the tip has grown several millimeters past their point of attachment; and the disc-shaped cap, which is initiated in 2.5 to 3-cm cells, whereupon stalk elongation ceases and all further growth takes place by the increase in cap diameter. The times (weeks) and fresh weights indicated are those observed under the culture conditions described here. They are mean values. The variability in the population will increase with the age of the culture, and the times become almost meaningless after the formation of small caps. The period in axenic culture is determined by the size of the cells needed experimentally.
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DAVID C. SHEPHARD
chemical and mechanical treatments, and it can be held indefinitely until sterility is obtained and confirmed. Since problems often arise in breaking cyst dormancy on schedule, it is best to accumulate and decontaminate large numbers of them at once. Then the occasional massive reproductions, if they can be grown to dark storage size without contamination, will provide enough cells to keep the incubator full for many months. The main growth phase is initiated 6y inoculating an appropriate number of dark storage cells into the culture flasks and placing them in the incubator. Maintaining sterility for long periods in the absence of antibiotics requires considerable care. The culture schedule is set up to grow large numbers of sterile cells only to the size required by the experiments planned. Small numbers of cells which become contaminated accidentally or are not used experimentally are saved for reproduction. They should be handled with care to insure that no algal contamination occurs. Tne cysts will almost certainly need to be decontaminated before reproduction. Assuming that the cysts can be freed from all contaminants, that the cells will grow normally in synthetic medium, and that a suitable sterile change hood and other facilities are available for culture and experimental procedures, the following requirements still should be met if routine axenic culture is to be successful: 1. Handling subsequent to cyst decontamination must be minimal. 2. Handling at stages when it is unreasonable to test for sterility afterward and treat to eliminate any contaminants found must be avoided. 3. Time in sterile culture must be kept to the minimum necessary. These points form the basis of the procedures described below.
B. Cyst Decontaininatioii The cysts must be totally freed of all contaminating organisms before attempting axenic culture. Cysts are more resistant to mechanical, 0smotic, and chemical treatments than cells. Furthermore, they can be stalled in the dark for long periods of time while sterility is being checked. The procedure for decontaminating them takes advantage of these facts. From the mature caps in dark storage, about fifty perfectly normal ones are selected. This represents about 5 10' cysts. The cysts are released from the cell walls of the caps with a loose fitting Tenbroeck tissue grinder (clearance should be close to 0.01 inches). The resulting slurry is filtered through 74-mesh bolting silk, which will retain the wall fragments and large debris. The cysts should be washed several times with fresh medium in a 25 x 200-mm test tube. If the tube is shaken, the re-
x
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sulting foam will tend to float debris and dead cysts. Living cysts will settle to the bottom of the tube in about 5 minutes, and the supernatant can be aspirated off. The cysts are next transferred to a sterile washing funnel with a filter of 270-mesh bolting silk and drained. A sterile magnetic stirring bar is placed in the funnel, and the latter is filled with sterile medium and drained several times. The stirrer is run constantly during this process, and all medium and other solutions used should be absolutely sterile. Terminal filtration is recommended. After the rinses the funnel is filled with 1%sodium lauryl sulfate (SLS) in salt medium and the cysts are stirred in several complete changes of this mixture for a total period of 10 minutes. (They will survive several hours of this treatment.) The SLS is then washed out with several changes of sterile medium. The funnel is next filled to overflowing with 2%formaldehyde (5%formalin) in sterile medium. The cysts are stirred in this solution for 5 minutes. The formaldehyde is washed out with at least six fullfunnel rinses of sterile medium over a 30-minute period. This treatment should kill or wash away practically all contaminants; however, there are likely to be a few spores or highly resistant organisms trapped in debris or perhaps in dead cysts. To achieve complete sterility it will be necessary to plate the contaminants and determine a treatment to eliminate them. The cysts are suspended in about 10 ml of medium in the funnel and 0.5 ml of this suspension is plated; the balance is transferred to a sterile vial and glucose is added. A sample of the medium that has been used for washing should be plated at the same time. The plates are placed in the incubator and checked daily for a week. If no contaminants appear on the plates, the cysts are probably clean; however, the medium in the vial of cysts should be plated again. If this plate is negative, the cysts can be considered sterile. If mold colonies appear on the plates, the cysts are immediately returned to the washing funnel and given a formaldehyde treatment. If this is done before the mold can sporulate it will usually be completely effective. A fresh plate should be made of the cysts, and they should be returned to a sterile vial with 0.05%glucose in the dark. A representative of each type of bacterial colony that appears on the plates should be isolated and its antibiotic sensitivities determined (see Section 11,D). Then a treatment is designed that uses at least two antibiotics that are effective against each species of contaminant. It is necessary to consider the known antagonisms and synergisms of the drugs, and it may be necessary to give sequential treatments. In general the treatments should be carried out in darkness and in distilled water (perhaps with glucose added) at a pH suitable to the antibiotics being
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DAVID C. SHEPHARD
used. Each treatment should last 2-3 days. Finally the cysts are washed, a sample plated, and the rest returned to sterile medium with glucose in the dark. If the first plate is blank after a week another is made of the cysts plus glucose medium. If this plate reveals no contamination it is virtually certain that the cysts are axenic. This procedure is somewhat time consuming; however, the final cysts represent a potential lo6 cells, and the procedure will need to be carried out only once or twice a year.
C. Reproduction and Early Growth Most published pocedures for Acetabularia calI for various manipulations during these culture phases. Since handling is not conducive to maintaining sterility and many of the gametes and small cells may be lost in transfers, the procedure we have developed keeps handling to an absolute minimum. Considerable difficulty in breaking cyst dormancy should be anticipated. No treatments that we have tried have consistently improved the stimulation of gamete emission provided by cycled light under the standard conditions of the incubator. In these conditions, gametes will usually appear sometime during the first week or two and then at odd intervals for several months afterwards. Only a small percentage of the cysts will be involved each time, but since each of the thousands of cysts will produce about 100 gametes, yields of lo5 gametes are often obtained and represent a potential kilogram of cells. The procedure is as follows: The decontaminated cysts from fifty caps are placed in the bolting silk cone of a nursery bottle (Fig. 1) with 50 ml of sterile medium to cover them. The bottle, which is placed at the front of an incubator shelf, must be checked daily about 5 hours after the lights come on. When gamete emission occurs, the phototactic gametes will be seen as a green line at the air-water interface at the lightest (rear) side of the bottle. After waiting several hours to insure that gamete emission is complete the lid with the attached cone of cysts is transferred to a fresh bottle for future emissions, and a sterile lid (without the cone) is placed on the bottle with the gametes. The bottom twothirds of this bottle is wrapped in black paper and the bottle returned to the incubator. Late in the day the newly formed zygotes will become negatively phototactic, swim to the bottom, and attach to the glass. If the bottle is not masked with paper, they will all gather at the darkest point and grow into dense clumps of attached cells which are useless for many experimental procedures. The paper may be removed the following day. The technique described by Keck (1964) utilizing the phototactic
3. Acetabularia
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swimming of the gametes to decontaminate them is a useful one, but we find that it does not insure sterility. Species of very motile pseudomonads will be found in such cultures unless they have been killed by some other means beforehand. This technique, however, may be the only reasonablz means for eliminating very difficult spore-forming bacteria, The swimming tube shown in Fig. 1 is used for this. It is filled with sterile medium, and the gametes are added to the bulb with a pipette. If the other end is illuminated with a microscope lamp, the gametes will swim in that direction at a rate of about 1 cm/minute. When a good green spot has appeared, it is collected with a sterile Pasteur pipette and transferred to a nursery bottle ( a plate should be made). We have observed a considerable loss of gamete number and viability during this procedure, perhaps due to the shock of two pipettings into fresh medium. The zygotes are left in the nursery bottle for the early growth phase. They will grow to a length of 2-3 mm in a month. After about the third week it is desirable to count them. The cells are freed from the glass by the gentle use of a sterile magnetic stirrer or pipette. While the bottle is being stirred, a 1-ml sample is removed with a pipette ( 1 mm bore). The number of cells in this sample is determined by counting them with a dissecting microscope or judicious use of a counting chamber. The desired number of cells is 10,000 per bottle. Excess cells are pipetted to new bottles, and medium is added to bring all bottles to 100 ml (or the cell count to 100/ml). The bottles are returned to the incubator until the cells reach 2-3 mm in length. At this time the bottles are placed in a dark cabinet at room temperature until needed. If they are to be stored many months, an exposure to several days of light every three months is recommended ( Keck, 1964 ) . If suitable sterile precautions have been taken in handling these cells, there is little chance of contamination. If it does occur, it mny be difficult to eliminate.
D. The Main Growth Phase During this phase the dark storage cells are inoculated into Roux flasks and grown to the size desired for experimental purposes. Since it is difficult to anticipate the need for cells a month in advance, we plan a weekly inoculation schedule that will keep culture facilities filled to capacity, assuming that the bottles will be removed from the incubator when the cells reach the desired size. Since there is no reason to manipulate the cells between the time of inoculation and the time they reach experimental size, the number of cells to be inoculated into each bottle is determined by the final size desired. The flasks will support normal
64
DAVID C. SHEPHARD
growth to 1 gm of cells; that is 1000 cells to 1 cm, 100 to cap initiation, perhaps 50 to large caps or 25 to full maturity (see Fig. 2). If very large cells are desired, a subculture or two along the way will, of course, allow more efficient use of incubator space, but at the risk of contamination. Before the inoculation is made it is necessary to demonstrate the absence of contamination in the dark storage bottle. About a milliliter of cells and medium from a bottle should be plated at least a week before its first anticipated use. If it is sterile, it should be handled very carefully for it will provide the weekly inoculum for a month or two (another plate should be made each time that it is used, however). If the cells are not sterile, they should be discarded; but if it is necessary, there is a reasonable chance that they can be decontaminated, although they are much less . resistant than the cysts. An antibiotic sensitivity test and treatment as outlined in Section II,B must be used. The cells will not withstand SLS, and small cells are often killed by even mild formalin treatments. If a washing funnel is used with cells of this size, a large pore membrane filter (e.g., hlillipore type NC) should be substituted for the bolting silk. After washing the cells are plated. Assuming that the cell density in a sterile dark storage bottle has been determined, the inoculation is made simply by pipetting the appropriate volume from the stirred bottle into each Roux flask. The flasks should already contain their 200 ml of sterile medium. An excess number of cells may be needed, depending on previous experience with the cell viability in the dark storage bottle. Viability may diminish slowly with time and precipitously with decontamination. After inoculation the flasks are capped and placed in the incubator. Little additional attention is needed, but it is well to record their length and condition at weekly intervals. If the temperature and light intensity of the incubator are not uniform, rotating the positions of the flasks daily will help insure the uniformity of the cells in all flasks. The growth of these cells during the main growth phase is approximately exponential, as measured by wet mass or, more dramatically, when the reduced weight of individual cells is followed with a Cartesian diver balance (Holter et al., 1956), as shown in Fig. 3. There is a change in growth rate at the time of cap initiation, and there may be other changes in very early and very late stages. The exponential growth period of cells between 10 and 25 mm in length is probably the most useful stage for work of a chemical or physiological nature. The mass doubling time during this period is about 4 days under the culture conditions described here. If large cells are contaminated accidentally or during an experimental manipulation, a 5-minute treatment in 0.1%formaldehyde or overnight in
3. Acetabularia
65
CULTURE
P
r P
initiation
Normol cells Jj I I I I 1 1
I
v Enucleoie cells
I I I 1 1111 I I I 1 1 1 , I I ,I 1 I I t I t t I t
IILLLLLLUIIV!IJLIlL~ILILL.IIU
O o-Iv s_
FIG. 3. Single-cell growth curves. These measurements were made with a Cartesian diver balance and therefore represent dry weight growth curves. Their exponential character is obvious, the change in rate occurring at the time of cap initiation. All cells were about 8 mm at the beginning of the measurements. The normal cells went on to form 7-mm caps. While the enucleates grew for only 2-4 weeks and did not form caps, their growth rates are comparable to those of the normal cells. These cells were not axenic and were grown in Erd-Schreiber medium.
5 pg/ml sodium omadine will suffice to make surviving bacteria difficult
to detect for a week or two. It is usually not reasonable to put the cells in the dark while antibiotic sensitivities are determined and treatments given. Dark storage is not kind to larger cells, but they can be stalled by light intensities of about 25 footcandles. However, after any such treatment the cells should be given a week to 10 days in the incubator before they can be considered normal again.
66
DAVID C. SHEPHARD
Once the scheduled gram of cells per flask is reached, the growth rate will decrease and the cells may begin to deteriorate. If there is no foreseeable experimental need for them, the largest should be selected for reproduction and the rest discarded.
E. Enucleation and Enucleate Growth The internal pressure in AcetabuZuriu is appreciable. If the cells are enucleated by simply amputating the rhizoid, a large amount of cytoplasm is immediately forced out, and more continues to be lost until the cut end seals itself. A stalk fragment may lose more than 50% of its cytoplasm before it heals. Since the growth rate observed after enucleation will be proportional to the remaining cytoplasm, negative or nil growth rates are likely to be obtained. To avoid this problem we tie the stalk off with fine nylon and then amputate the rhizoid just basal to the knot. Turgor is maintained, and no cytoplasmic loss occurs from the severed stalk. The rhizoids are usually kept because their regener at'ion serves as proof that the nucleus was contained in them rather than in the stalk. The practice of dividing the cell into an anucleate apical segment and a nucleate basal one which serves as a control appears ideal in theory, and it is true that the apical portion is not an entire cell lacking only the nucleus. However, much of the usefulness of AcetabuZuria resides in the fact that the apical segment is a good approximation of an enucleate cell; thus, it seems more reasonable to consider it as such and compare its growth with that .of an intact cell of equivalent size rather than with the regenerating stump of a cell. Figure 3 shows that the growth rates of intact and enucleate cells are entirely comparable. The length of time that enucleates will grow is variable from cell to cell and group to group (Shephard, 1965). Careful selection of healthy cells to be enucleated, axenic culture and defined media reduce the variability but do not eliminate it. Mean data from groups of enucleates often show an approximately linear rate of growth, apparently because the exponential growth rate is masked by a similar rate of decline in the number of cells growing at all. Exponential growth has been observed to continue for periods of a month or more in enucleate Acetabularia ( Shephard, 1965).
F. Maturation Since the reproductive potential of one Acetabularia cell is about 10,000 new cells, only a small percentage of the cells need to be carried
3. Acetabularia
CULTURE
67
on to maturity. Five percent will provide an enormous safety margin if they are selected late enough in the growth phase to insure that they are healthy and normal. For reproduction the cells must be maintained for several months beyond the period of their experimental usefulness. It is extremely dficult to maintain sterility in such cultures, especially since manual selection of cells and several medium changes may be necessary. Figure 2 shows the times and cell weights involved; however, these tend to be minimum times, many cells taking considerably longer to reach maturity. When cells with full-sized caps are seen in a culture being saved for reproduction, they are selected and transferred to fresh medium (50 cells per flask). Nuclear division in these cells is indicated by loss of color in the stalk as the cytoplasm migrates into the cap. Once this process is complete and the cap is full of uniform round cysts, another selection is in order. The cyst-containing cells are collected from several bottles and placed together in the incubator for another 2 weeks to insure complete maturation. After this all caps which still appear perfectly normal are placed in dark storage until they are needed. There is a tendency for completely mature caps to detach from the stalk. Generally, a period of a month in the dark will facilitate breaking cyst dormancy. Longer storage may result in a gradual diminution of viability, but viable gametes can be obtained even after several years. An occasional medium change is desirable during long periods of dark storage,
G. Evaluation The improved methods for the culture of Acetabularia presented here offer potential success in the performance of a variety of biochemical and physiological studies on Acetabularia which have been impossible with contaminated cultures and an undefined medium. Moreover, the defined conditions should result in more reproducibility of experimental results than has been experienced in the past. However, even cultured as described, Acetabularia still lacks several requirements of a good laboratory organism. The variability in growth rates is not completely eliminated by standard conditions and may reflect a high degree of genetic variability. Careful selection for some standard growth pattern may be required before growth rates become predictable. The growth rate is still too slow and the generation time too long. It would seem that a mass doubling time of 1 day could be achieved by truly optimum culture conditions and medium. This would reduce the generation time to a month and the main growth phase to a week. The problem of breaking cyst dormancy is
68
DAVID C. SHEPHARD
twofold. Not only is it a nuisance to obtain reproduction but it is also practically impossible to self- or crossbreed individual cells. A hormone or shock treatment that will induce gamete production efficiently and quickly is necessary if Acetabularia is to realize its potential as a laboratory organism. The procedures described, however, make it possible to obtain and maintain reasonable quantities of healthy and sterile Acetabularia with a minimum of effort and space. ACKNOWLEDGMENTS A number of people have assisted in the development of the mediums and procedures: Marilyn Nagare, Mary Cammock, Patricia Roche, Florence Dark, and Yona Victor. The assistance of Fenton Moore in developing the antibiotic screening technique has been invaluable. This work was supported by a grant from the National Science Foundation (GB-5210) and by general research support awards from Case Western Reserve University. I would like to thank the Schering Corp. of Bloomfield, New Jersey for the sample of Gentamicin-sulfate, and Chas. Pfizer & Co., Groton, Connecticut, for the sample of Carbenicillin.
REFERENCES Allen, M. B., and Arnon, D. I. (1955). Pknt Physioz. 30, 366. Bailey, W. R., and Scott, E. G. (1966). “Diagnostic Microbiology,” 2nd Ed. Mosby, St. Louis, Missouri. Beth, K. (1953). 2. Naturforsch. 8b, 334. Brachet, J,, Chantrenne, H., and Vanderhaeghe, F. ( 1955). Biochim. Biophys. Actu 18, 544. Clauss, H. (1968). Protoplasniu 65, 49. deVitry, F. (1963). Protoplasma 55, 313. Gibor, A. (1965). €‘roc. Natl. Acad. Sci. U.S. 54, 1527. Gibor, A. (1967). In “Biochemistry of the Chloroplasts” ( W . T. Goodwin, ecl.), Vol. 11, pp. 321328. Academic Press, New York. Gihor, A,, and Izawa, M. (1963). PTOC.Natl. Acad. Sci. U. S. 50, 1164. Hammerling, J. (1963). Ann. Reu. Plant Physiol. 14, 65. Hhmerling, J., Clauss, H., Keck, K., Richter, G., and Wertz, G. (1958). Exptl. CeZl Res. Strppl. 6, 210. Hellebust, J. A., Terborgh, J., and McLeod, G. C. (1967). Biol. Bull. 133, 670. Holter, H., Linderstr@m-Lang,K., and Zeuthen, E. (1955). In “Physical Techniques in Biological Research (G. Oster and A. W. Pollister, eds.), Vol. 111, pp. 577-625. Academic Press, New York. Keck, K. ( 1964). In “Methods in Cell Physiology” (D. Prescott, ed.), Vol. I, pp. 189213. Academic Press, New York. Keck, K., and Clauss, H. (1958). Botan. Gaz. 120, 43. Lateur, L. (1963). Revue Algol. 1, 26. Provasoli, L., McLaughlin, J.-J. A., and Droop, M. R. (1957). Arch. Microbiol. 25, 392. Richter, G. (1962). In “Physiology and Biochemistry of Algae” (R. A. Lewin, ed.), pp. 633-652. Academic Press, New York.
3. Acetabuluria
CULTURE
69
Schulze, K. L. (1939). Arch. Protistenk. 92, 179. Schweiger, H. G., Dillard, W. L., Gibor, A., and Berger, S. (1967). Protoplasm 64, 1. Shephard, D. C. ( 1965). Ex$. Cell Res. 37, 93. Shephard, D. C. ( 1966). Biochim. Biophys. Acta 108, 635. Shephard, D. C., Levin, W., and Bidwell, R. G. S. (1968). Biochem. Biophys. Res. C m m u n . 32, 413. Terborgh, J. (1965). Nature 207, 1360. Terborgh, J., and Thimann, K. V. (1964). Planta 63, 83. Thimann, K. V., and Beth, K. (1959). Nature 183,946. Thomas, E., and Tregunna, E. B. (1967). Can. J . Botany 45, 411. Vanden Driessche, T. (1966). Biochim. Biophys. Acta 126, 456. Zetsche, K. (1965).Planta 64, 119. NOTE ADDEDIN PROOF In June 1969, an International Symposium on Acetabularia was held in Brussels. The proceedings provide a valuable source d information on various aspects of Acetabularia and are published in “The Biology of Acetabularia” (J. Brachet and S. Bonotto, ds., Academic Press, New York, 1970).
I . Introduction
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I1. Technical Aspects . A . The Organism .
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B. Culture Conditions . . . . C. Culture Vessels and Apparatus . D . Antibiotics and Bacteriocidal Agents E . Sterility Testing . . . . F. The Synthetic Medium . . . Procedures . . . . . . A Rationale . . . . . . B . Cyst Decontamination . . . C. Reproduction and Early Growth . D . The Main Growth Phase . . E . Enucleation and Enucleate Growth F. Maturation . . . . . G. Evaluatiton . . . . . References . . . . . . . Note Added in Proof . . . .
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49 51 51 51 52 54 56 57 59 59 60
62 63 66 66 67 68 69
I. Introduction The giant unicellular alga Acetabularia and its relatives combine a number of unusual features that make them desirable experimental organisms. In the following partial list of these features there is at least one that will appeal to almost every cell biologist. (The references given are to recent papers or reviews.) 1. Enucleation by surgical mtans is simple. and subsequent growth of the anucleate segments is prolonged (Hammerling et aL. 1958) and 49
50
DAVID C . SHEPHARD
accompanied by nucleic acid and protein synthesis (Gibor, 1967; Schweiger et al., 1967; Brachet et al., 1955; Keck and Clauss, 1958; and Shephard, 1966). 2. Intra- and interspecies nuclear grafts can be made readily (Richter, 1962). 3. They are single cells with a distinct morphogenesis that can be manipulated by light (Clauss, 1968)) chemical means (Zetsche, 1965; devitry, 1963), and surgery ( Hammerling, 1963). 4. They grow exponentially a billionfold without nuclear division ( Shephard, 1965). 5. At a given stage ten or more rapid cycles of caryokinesis occur ( Schulze, 1939). 6. They have well-defined circadian rhythm ( Vanden Driessche, 1966). 7. They respond to at least one plant hormone (Thimann and Beth, 1959) . 8. Enucleate protoplasts can be made from them (Gibor, 1965). 9. Chloroplasts that carry out complete photosynthesis at intact cell rates can be isolated from them in high yield (Shephard et al., 1968). 10. They reproduce sexually every generation ( Schulze, 1939). 11. Since they can be kept growing indefinitely by repeated stalk amputation ( Hammerling et al., 1958) or low light intensities (Beth, 1953), they are appropriate single cells for aging studies. Opposed to these advantages there is one major disadvantage of these organisms-slow growth: 4-8 days for mass doubling. Two other factors augment the problem of slow growth and have made Acetabularia difficult to culture and unsuitable for many types of experiments. These factors are not inherent, however; they are the lack of a suitable defined culture medium and the lack of a reliable means for completely eliminating Contaminating organisms in cultures. The usual Erd-Schreiber medium varies greatly in its ability to support growth, depending on thc source of seawater and the source, age, and treatment of the earth extract (Provasoli et al., 1957). Erd-Schreiber must be considered an undefined medium, especially in the presence of contaminating microorganisms. The usual cultures of Acetahtclaria are subject to loss from “green infections” and show highly variable growth rates, depending on the vagaries of Erd-Schreiber medium and the particular types of bacteria or molds that are present. Moreover, the presence of any contaminating organisms is a rnajor impediment to detailed physiological or biochemical studies. Thus, many laboratories attempting to culture Acetabularia have found it to be a disappointment. The purpose of this paper is to present means for eliminating contaminating organisms and culturing Acetabularia axenically in a com-
3. Acetabularia
CULTURE
51
pletely defined medium for at least the period of time necessary for most experimental needs. No attempt will be made to review the many details of the organism and the techniques for handling it. The excellent papers of Keck ( 1964), Lateur ( 1963), and others (Beth, 1953; Terborgh and Thimann, 1964; and Gibor and Izawa, 1963) should be consulted for this information.
11. Technical Aspects
A. The Organism These methods were developed using Acetabularia mediterranea. The strain used, and the one that many laboratories have, comes originally from Hhmerling's laboratory. It was collected about 1930 and is undoubtedly a highly selected strain by now. Little is known of the possible differences between strains, but different species require variations in technique ( Keck, 1964). Some differences in medium requirements, growth rates, and susceptibilities to antibiotics may exist in other strains and species; however Acicularia schenckii has been successfully cultured using these methods.
B. Culture Conditions The cultures are maintained at a constant temperature between 22' and 24'C. This can often be accomplished quite safely by rapid circulation of air, in an air-conditioned room, through the incubator to carry away the heat from the lamps. Growth rates increase at higher temperatures to about 30°C,but abnormal growth is often noticed above 25°C. Fluorescent illumination is used at an intensity of about 250 footcandles (6 to 8 x lo3 ergs/cm2/second), using a mixture of deluxe warm white ( WWX) and daylight ( D ) bulbs. Higher intensities are appropriate at higher temperatures. A 12-hour on, 12-hour off light cycle is convenient and commonly used. Acetabularia has a circadian rhythm, and there is a potential problem in comparing experiments carried out at different times of day (Hellebust et al., 1967). The cells become completely asynchronous after a week in the dark ( Vanden Driessche, 1966). For some experimental purposes it might be useful to introduce asynchronous cells into a continuous light regimen with the intensity reduced to about 100 footcandles; however, we have had poor success with long-term cultures
52
DAVID C. SHEPHAFiD
of Acetabulariu in continuous light. Considerable data are available on the effects of light intensity, color, and cycle on Acetabularia (Beth, 1953; Terborgh, 1965; Terborgh and Thimann, 1964).
C.
Culture Vessels and Apparatus
Vessels. Roux flasks ( 1 liter) make excellent culture vessels for several reasons, including space saving in the incubator and Iarge surface area for illumination and gas exchange. They should be dry-heat sterilized with the necks wrapped in foil. After the cells and 200 ml of medium are added, the flasks are cotton stoppered, or better, the mouth and neck are covered with two layers of autoclave paper to facilitate subsequent sterile transfers. The maximum cell densities in such cultures are determined by CO, diffusion (Allen and Arnon, 1955). Two grams of fresh weight of cells is the limit that these flasks will support, and 1 gm should not be exceeded if maximum growth rates are desired. Aerating the flasks might increase their capacity, but it adds severe difficulties to sterile culture. Moreover, the medium may not support the extra mass. If they are aerated, the CO, concentration must be less than 0.5%. Smaller amounts of cells (to 0.1 gm) can be maintained conveniently in screw-cap BOD bottles on their sides. Nursery Bottles. Figure 1 diagrams the container we have developed for the reproductive phase of the life cycle. It is a 250-ml widemouth specimen jar with a Bakelite screw cap. A l-inch hole punched in the cap is covered with cotton and paper. A cone of 270 mesh bolting "silk" (nylon) suspended from the lid with platinum wire so that it reaches almost to the bottom is added to those bottles which will receive the cysts. The top of the bottle is wrapped in autoclave paper and the whole assembly autoclaved. Washing Funnels. These are used with various meshes of bolting silk or membrane filters for various procedures ( a Gelman I-inch vacuum filter funnel is suitable). Bolting silk of 74 mesh will pass cysts and retain cells or large debris; 270-mesh nylon will retain cysts and pass contaminating organisms. A large-pore membrane filter (such as Millipore type NC) is used for washing very small cells that tend to become entangled in the nylon. For cyst washing the funnel is mounted in the sterile hood immediately above a magnetic stirrer. Teflon-covered magnetic stirring bars (1.5 x 8 mm) may be used in these funnels. Larger bars tend to be excessively damaging. The entire apparatus must be autoclaved or gas sterilized. lnverted Diwecting Microscope. This arrangement is shown in Fig. 1. It provides the only means of examining the cells in detail while they are
3. Acetabularia
53
CULTURE
.Cyst cone Plote glass
rnl
-
Nursery bottle
I
3
i
1
Inverted dissecting microscope
25 gauge plo tinum
glass
1
/
\
Hook
10 cm Swimming tube
Light
c
FIG. 1. Special apparatus. (See Section II,C for description.)
in R o w flasks or nursery bottles. Optically poor glass surfaces can be masked simply by immersing them in a puddle of water on the glass stage. The construction is quite simple. Miscellaneous A p r a t u s . The swimming tube is used at times to obtain sterility by forcing the phototactic gametes to swim through sterile medium (Keck, 1964). A platinum hook is the simplest means for handling and selecting larger cells, which can be hooked by the rhizoids or caps. It is made from platinum wire (25 gauge) and is sterilized by flaming. Very small cells are counted or manipulated using finer wire or a hair loop.
54
DAVID C. SHEPHMUI
D. Antibiotics and Bacteriocidal Agents Table I presents much of our experience in using chemical agents to obtain sterility in Acetabularia cultures. Many agents are more toxic to Acetabularia than to contaminants. The acceptable antibiotics are very useful, but must be used carefully; otherwise, mutation and selection may make them useless when they are most needed. Only bacteriocidal antibiotics are used, and their effectiveness should be ascertained beforehand. Other treatments and agents are used whenever possible. There are three somewhat different sets of circumstances under which all of these substances are used. The first is when dealing with heavy, mixed contamination of the cysts. The object is to eliminate most species and decimate the number of survivors. Sodium lauryl sulfate and formaldehyde are used for this. The details of the procedure are given in Section II1,B. The second set of circumstances is the temporary control of a light, accidental contamination of large cells or as a precaution after an experimental manipulation of the cells. While reestablishing sterility may be desirable, the 2 weeks necessary to accomplish and confirm it is unreasonably long at this stage of the life cycle. The object of this type of treatment is to eliminate as many bacteria as possibIe with a quick and simple treatment. It should be difficult to detect bacteria for a week or two afterwards, and the cells may be used for many experimental purposes. A 5-minute treatment with 0.1%formaldehyde will suffice with sturdy cells or an overnight treatment in 5 pglml sodium omadine is a reasonable alternative. Any treatment should be followed by washes in sterile medium. The third and most important circumstance is the complete eliniination of the few species of contaminants remaining either after the initial treatment of the cysts or found in young cells while there is time to confirm the results. This is the time to use the antibiotics. Such contamination is detected as colonies on agar plates (Section 11,E). These colonies should be collected and their sensitivities to the antibiotics determined before treatment is attempted. We use a filter disc technique (Bailey and Scott, 1966) and make up the discs with the antibiotic stocks on hand. The antibiotics in Table I fall into two categories, bacterial cell wall inhibitors and miscellaneous metabolic inhibitors. The first group is very valuable because of its low toxicity to Acetabularia, even at exorbitant concentrations. (There is little point in using most antibiotics at more than 100 pg/ml.) Antibiotics in the second category are unpredictable in their effects on Acetabularia. We have tested relatively few members
3. Acetabularia ANTIBIOTICS AND
55
CUL~RE
TABLE I BACTERIOCIDES FOR
USE WITH
Acetabularia
Maximum tolerated concentrations and timesD
-
Effects noticed on hetabidaria*
Bacterial cell wall inhibiting antibiotics Ampicilin Carbenicillin Cephalo thin Cy closerine Penicillin G Vancomycin
1 mg/ml, indefinite 1 mg/ml, indefinite
100 pg/ml, indefinite 100 pg/ml, indefinite 1 mg/ml, indefinitec 1 mg/ml, indefinite
Other antibiotics Amphotericin B (Fungizone) Cephaloridine Gentamicin snlfate Kanamy cin Lincomycin Neomycin Sodium colistimet,hate Streptomycin
1 pg/ml, indefinite” 100 p g / m l , indefinite 1 mg/ml, indefinite 100 pg/ml, indefinite 1 mg/ml, indefinitec 100 pg/ml, 4 days 50 pg/ml, 2 days 1 nig/ml, indefinite
Bacterion’des Formaldehyde (dilute formalin) Furadantin Sodium omadine (Olin Mathieson) Sodium lauryl sulfate
0.1%. 5 minutes (cells) 2%, 5 minutes (cysts) 1 mg/ml, indefinite 5 pg/ml, 2 days l%,12 hours (cysts)
T S N S
s
Agents found to be excessively toxic to Acetabulariad ~~
~
Antibiotics Aureomycin Mycostatin Novobiocin Polymixin
~
______
~~
Bacteriocides Argyrol (Crooks-Barnes) Copper sulfate Domiphen bromide Hexylresorcinol
Hydrogen peroxide Phenol Sodium hypochlorite Zephrin chloride
a Concentrations and times may be far in excess of those needed for bacteriocidal activity. Indefinite times imply a 10-day or longer test,, a period probably longer than the life expectancy of most of these agents. b (N) No toxic effects noted; (S) some toxicity noted a t higher coilcentrations (or longer times); (T) some toxicity noted a t useful times and concentrations. c Compound so unstable that treatment time is almost meaningless. d These agents kill Acetabularia cells and cysts a t times and concentrations necessary to kill other microorganisms.
56
DAVID C. SHEPHARD
of this group, and even those listed in the table must be used with care. Most inhibit Acetabularia cells, but any effect at the times and concentrations given are apparently reversible. Still, their use should be limited to cysts if possible. Of the bacteriocidal agents, Furadantin and perhaps sodium omadine fit more appropriately in this second category. After the antibiotic sensitivities of the contaminants have been determined, two or more effective antibiotics are selected for each species to be killed (wall-active antibiotics are not usually mixed with other types). Treatments of cells or cysts with antibiotics should always be carried out in the dark for two reasons. First, many of the antibiotics are destroyed fairly rapidly by light and, second, the Acetabularia is photosynthetically inactive in the dark and is much less sensitive to antibiotic effects. Some substrate (usually 0.05% glucose) is added simultaneously with the antibiotics, especially wall-active ones, to insure that the bacteria will be growing slowly. Since spore-forming organisms are likely contaminants of the cysts following the preliminary decontamination, the antibiotic treatment must be long enough for complete excystment. Threeday treatments are usually adequate; however, observation of the times of colony appearance on the plates will help determine suitable treatments. The life expectancies of some antibiotics in alkaline solutions may be less than 3 days. Cyst treatments are often more effective if carried out in distilled water adjusted to a pH suitable for the antibiotics being used. We have not tried continuous culture in antibiotics partly because few of them are sufficiently stable and because many of them are slightly inhibitory to Acetabuluria. There is also the possibility of developing highly resistant bacterial strains; therefore, continuous treatment is not recommended. However, if necessary, the cell-wall antibiotics, streptomycin, neomycin, and kanamycin, may be added to the culture medium at 50 pg/ml, and arnphotericin B (for fungi) may be added at 0.25 pg/ml. Chloramphenicol at 5-10 g / m l is an effective growth inhibitor for many bacteria, but appears to have little effect on Acetabukzriu. One last point should be emphasized. If reasonable treatments do not result in sterility, it is best, if possible, to discard the culture.
E. Sterility Testing We have found that many of the contaminating organisms grow on the surface of Acetabularia; thus, plating or testing the medium may give a negative result, whereas plating the cells or cysts (with 0.5 ml of medium) will reveal contamination. We plate on tryptone glucose yeast extract agar made up in water (see Section 11,F). Unfortunately, some
3. Acetabularia
CULTURE
57
contaminants of these cultures are heat sensitive and very slow growing. We keep the plates in the same incubator as the cells. To find colonies, 48 hours is necessary, and it may take several days to reveal all species. A plate should be kept a minimum of 5-7 days. The delay is not serious at the two most crucial stages for sterility testing-cysts and very small cells. These can be held in the dark until the plate is read. If contamination is found, treatment can be given or the culture discarded. Routine platings of the medium supply and cell stocks are required.
F. The Synthetic Medium The requirements of a suitable medium are: (1) that it can be autoclaved; ( 2 ) that it is chemically defined; ( 3 ) that it supports normal cell growth; and ( 4 ) that all cell nutrients must be in adequate supply to support cell growth from the negligible wet weight of the inoculum to the maximum weight that the CO, diffusion rate will permit under the standard culture conditions. The ASP-6 medium of Provasoli and associates (1957) was found to satisfy the first three requirements. Modifications of this medium which meet the last requirement are given in Table 11. Growth is normal in this medium and as rapid as any reported, 4 4 days for mass doubling until cap initiation, and if the inoculation size is appropriate, no medium change is ever necessary. The major salt solution given in the table can probably be replaced by artificial seawater supplemented with nitrate, pH adjusted to 7.8. The other components are more important. Bicarbonate is added in an amount appropriate for the pH of the medium and to replace that lost during autoclaving. Additional bicarbonate added initially or at intervals during culture seems to be detrimental. Thiamin HC1 and vitamin B,, are the only vitamins tested that have a dramatic effect on growth rate. They constitute the minimum supplementation of any medium. Other vitamins seem to be only slightly beneficial, but have not been completely retested under axenic conditions. The micronutrient solution determines the number of cells that the medium will support in long-term culture. It, too, should be added to any other medium used. We have not found a dramatic effect of added auxin (naphthalene acetic acid for stability), but auxin and other plant hormones have not been adequately tested; amino acids and other organics, some of which might be beneficial to cell growth rates, have not been tested either. The cells will not modify the pH of the medium appreciably until they become overcrowded, and they tolerate a fairly wide range of pH. The pH selected (7.8) is a compromise between lower values favorable
58
DAVID C. SHEPHARD
TABLE I1 A SYNTHETIC MEDIUMFOR Acetabularia
Major salts NaCl MgSOd. 7 H20 CaC12 2 Hz0 Tris KC1 NaN03 KzHP04 Micronutrient sa1t.s NaZEDTA ZnSOr. 7 HzO NazMoOd. 2 HzO FeC4 . 6 HzO MnClz.4 HzO CoCli. 6 Hz0 C U S O ~5. HzO Bicarbonate NaHC03 Vitamins Thiamine HC1 p-Aminobenzoate Ca-Pan tothenate Vitamin Blz
Amountfliter
Instructions
24 gm 12 gm 1 gm 1 gm 0.75 gm 40 mg 1 mg
The phosphate is added last; then the pH is adjust,ed to 7.8 with 1N HC1 (about 5 ml/liter) ;finally the solution is autoclaved
12 mg 2 mg
This solution is made up separately and added to the major salt solution before autoelaving
1 mg 0 . 5 mg 0.2 mg 2 rg 2 rg
100 mg 300 r g 20 PP 10 Pg 4 rg
These solutions are made up separately arid added to the salt solution through a sterilizing membrane filter after the autoelaving
to autoclaving without precipitation and higher ones that increase the bicarbonate concentration. It seems likely that bicarbonate rather than carbon dioxide is the substrate for photosynthesis in marine algae ( Thomas and Tregunna, 1967). Perhaps the greatest problem with the medium is handling it to maintain sterility. We autoclave carboys of medium (16 liters) for 1 hour with the siphon and all tubing in place. The bicarbonate and vitamins are added subsequently through a rubber injection port using a syringe equipped with a membrane filter. Since subsequent contamination of the medium can occasionally occur and is catastrophic, the culture bottles should be filled by pumping the medium through an easily replaceable, terminal sterilizing filter. Vacuum filtration is easier but will degas and decarbonate the medium. The medium should be plated each time it is used for setting up stock cultures and more frequently if terminal sterilization is not used; however, it should be remembered that it will be at least 48 hours before the plate can be read.
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111. Procedures A. Rationale Figure 2 is a diagram of the life cycle of Acetabularia from the point of view of culture technique. The dormant cyst stage offers the best possibility for obtaining complete sterility. It is the most resistant stage to
FIG.2. The life cycle of Acetabularia in culture. The subdivisions of the life cycle represent changes in technique rather than natural phases of growth. These subdivisions are described in the text. The main growth phase shows the cells diagrammatically, including: the rhizoid, a rootlike organ containing the nucleus; the cylindrical stalk, which increases in diameter as well as in length; the whorls-hairlike branches that are formed at the apex and shed after the tip has grown several millimeters past their point of attachment; and the disc-shaped cap, which is initiated in 2.5 to 3-cm cells, whereupon stalk elongation ceases and all further growth takes place by the increase in cap diameter. The times (weeks) and fresh weights indicated are those observed under the culture conditions described here. They are mean values. The variability in the population will increase with the age of the culture, and the times become almost meaningless after the formation of small caps. The period in axenic culture is determined by the size of the cells needed experimentally.
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chemical and mechanical treatments, and it can be held indefinitely until sterility is obtained and confirmed. Since problems often arise in breaking cyst dormancy on schedule, it is best to accumulate and decontaminate large numbers of them at once. Then the occasional massive reproductions, if they can be grown to dark storage size without contamination, will provide enough cells to keep the incubator full for many months. The main growth phase is initiated 6y inoculating an appropriate number of dark storage cells into the culture flasks and placing them in the incubator. Maintaining sterility for long periods in the absence of antibiotics requires considerable care. The culture schedule is set up to grow large numbers of sterile cells only to the size required by the experiments planned. Small numbers of cells which become contaminated accidentally or are not used experimentally are saved for reproduction. They should be handled with care to insure that no algal contamination occurs. Tne cysts will almost certainly need to be decontaminated before reproduction. Assuming that the cysts can be freed from all contaminants, that the cells will grow normally in synthetic medium, and that a suitable sterile change hood and other facilities are available for culture and experimental procedures, the following requirements still should be met if routine axenic culture is to be successful: 1. Handling subsequent to cyst decontamination must be minimal. 2. Handling at stages when it is unreasonable to test for sterility afterward and treat to eliminate any contaminants found must be avoided. 3. Time in sterile culture must be kept to the minimum necessary. These points form the basis of the procedures described below.
B. Cyst Decontaininatioii The cysts must be totally freed of all contaminating organisms before attempting axenic culture. Cysts are more resistant to mechanical, 0smotic, and chemical treatments than cells. Furthermore, they can be stalled in the dark for long periods of time while sterility is being checked. The procedure for decontaminating them takes advantage of these facts. From the mature caps in dark storage, about fifty perfectly normal ones are selected. This represents about 5 10' cysts. The cysts are released from the cell walls of the caps with a loose fitting Tenbroeck tissue grinder (clearance should be close to 0.01 inches). The resulting slurry is filtered through 74-mesh bolting silk, which will retain the wall fragments and large debris. The cysts should be washed several times with fresh medium in a 25 x 200-mm test tube. If the tube is shaken, the re-
x
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sulting foam will tend to float debris and dead cysts. Living cysts will settle to the bottom of the tube in about 5 minutes, and the supernatant can be aspirated off. The cysts are next transferred to a sterile washing funnel with a filter of 270-mesh bolting silk and drained. A sterile magnetic stirring bar is placed in the funnel, and the latter is filled with sterile medium and drained several times. The stirrer is run constantly during this process, and all medium and other solutions used should be absolutely sterile. Terminal filtration is recommended. After the rinses the funnel is filled with 1%sodium lauryl sulfate (SLS) in salt medium and the cysts are stirred in several complete changes of this mixture for a total period of 10 minutes. (They will survive several hours of this treatment.) The SLS is then washed out with several changes of sterile medium. The funnel is next filled to overflowing with 2%formaldehyde (5%formalin) in sterile medium. The cysts are stirred in this solution for 5 minutes. The formaldehyde is washed out with at least six fullfunnel rinses of sterile medium over a 30-minute period. This treatment should kill or wash away practically all contaminants; however, there are likely to be a few spores or highly resistant organisms trapped in debris or perhaps in dead cysts. To achieve complete sterility it will be necessary to plate the contaminants and determine a treatment to eliminate them. The cysts are suspended in about 10 ml of medium in the funnel and 0.5 ml of this suspension is plated; the balance is transferred to a sterile vial and glucose is added. A sample of the medium that has been used for washing should be plated at the same time. The plates are placed in the incubator and checked daily for a week. If no contaminants appear on the plates, the cysts are probably clean; however, the medium in the vial of cysts should be plated again. If this plate is negative, the cysts can be considered sterile. If mold colonies appear on the plates, the cysts are immediately returned to the washing funnel and given a formaldehyde treatment. If this is done before the mold can sporulate it will usually be completely effective. A fresh plate should be made of the cysts, and they should be returned to a sterile vial with 0.05%glucose in the dark. A representative of each type of bacterial colony that appears on the plates should be isolated and its antibiotic sensitivities determined (see Section 11,D). Then a treatment is designed that uses at least two antibiotics that are effective against each species of contaminant. It is necessary to consider the known antagonisms and synergisms of the drugs, and it may be necessary to give sequential treatments. In general the treatments should be carried out in darkness and in distilled water (perhaps with glucose added) at a pH suitable to the antibiotics being
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used. Each treatment should last 2-3 days. Finally the cysts are washed, a sample plated, and the rest returned to sterile medium with glucose in the dark. If the first plate is blank after a week another is made of the cysts plus glucose medium. If this plate reveals no contamination it is virtually certain that the cysts are axenic. This procedure is somewhat time consuming; however, the final cysts represent a potential lo6 cells, and the procedure will need to be carried out only once or twice a year.
C. Reproduction and Early Growth Most published pocedures for Acetabularia calI for various manipulations during these culture phases. Since handling is not conducive to maintaining sterility and many of the gametes and small cells may be lost in transfers, the procedure we have developed keeps handling to an absolute minimum. Considerable difficulty in breaking cyst dormancy should be anticipated. No treatments that we have tried have consistently improved the stimulation of gamete emission provided by cycled light under the standard conditions of the incubator. In these conditions, gametes will usually appear sometime during the first week or two and then at odd intervals for several months afterwards. Only a small percentage of the cysts will be involved each time, but since each of the thousands of cysts will produce about 100 gametes, yields of lo5 gametes are often obtained and represent a potential kilogram of cells. The procedure is as follows: The decontaminated cysts from fifty caps are placed in the bolting silk cone of a nursery bottle (Fig. 1) with 50 ml of sterile medium to cover them. The bottle, which is placed at the front of an incubator shelf, must be checked daily about 5 hours after the lights come on. When gamete emission occurs, the phototactic gametes will be seen as a green line at the air-water interface at the lightest (rear) side of the bottle. After waiting several hours to insure that gamete emission is complete the lid with the attached cone of cysts is transferred to a fresh bottle for future emissions, and a sterile lid (without the cone) is placed on the bottle with the gametes. The bottom twothirds of this bottle is wrapped in black paper and the bottle returned to the incubator. Late in the day the newly formed zygotes will become negatively phototactic, swim to the bottom, and attach to the glass. If the bottle is not masked with paper, they will all gather at the darkest point and grow into dense clumps of attached cells which are useless for many experimental procedures. The paper may be removed the following day. The technique described by Keck (1964) utilizing the phototactic
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swimming of the gametes to decontaminate them is a useful one, but we find that it does not insure sterility. Species of very motile pseudomonads will be found in such cultures unless they have been killed by some other means beforehand. This technique, however, may be the only reasonablz means for eliminating very difficult spore-forming bacteria, The swimming tube shown in Fig. 1 is used for this. It is filled with sterile medium, and the gametes are added to the bulb with a pipette. If the other end is illuminated with a microscope lamp, the gametes will swim in that direction at a rate of about 1 cm/minute. When a good green spot has appeared, it is collected with a sterile Pasteur pipette and transferred to a nursery bottle ( a plate should be made). We have observed a considerable loss of gamete number and viability during this procedure, perhaps due to the shock of two pipettings into fresh medium. The zygotes are left in the nursery bottle for the early growth phase. They will grow to a length of 2-3 mm in a month. After about the third week it is desirable to count them. The cells are freed from the glass by the gentle use of a sterile magnetic stirrer or pipette. While the bottle is being stirred, a 1-ml sample is removed with a pipette ( 1 mm bore). The number of cells in this sample is determined by counting them with a dissecting microscope or judicious use of a counting chamber. The desired number of cells is 10,000 per bottle. Excess cells are pipetted to new bottles, and medium is added to bring all bottles to 100 ml (or the cell count to 100/ml). The bottles are returned to the incubator until the cells reach 2-3 mm in length. At this time the bottles are placed in a dark cabinet at room temperature until needed. If they are to be stored many months, an exposure to several days of light every three months is recommended ( Keck, 1964 ) . If suitable sterile precautions have been taken in handling these cells, there is little chance of contamination. If it does occur, it mny be difficult to eliminate.
D. The Main Growth Phase During this phase the dark storage cells are inoculated into Roux flasks and grown to the size desired for experimental purposes. Since it is difficult to anticipate the need for cells a month in advance, we plan a weekly inoculation schedule that will keep culture facilities filled to capacity, assuming that the bottles will be removed from the incubator when the cells reach the desired size. Since there is no reason to manipulate the cells between the time of inoculation and the time they reach experimental size, the number of cells to be inoculated into each bottle is determined by the final size desired. The flasks will support normal
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DAVID C. SHEPHARD
growth to 1 gm of cells; that is 1000 cells to 1 cm, 100 to cap initiation, perhaps 50 to large caps or 25 to full maturity (see Fig. 2). If very large cells are desired, a subculture or two along the way will, of course, allow more efficient use of incubator space, but at the risk of contamination. Before the inoculation is made it is necessary to demonstrate the absence of contamination in the dark storage bottle. About a milliliter of cells and medium from a bottle should be plated at least a week before its first anticipated use. If it is sterile, it should be handled very carefully for it will provide the weekly inoculum for a month or two (another plate should be made each time that it is used, however). If the cells are not sterile, they should be discarded; but if it is necessary, there is a reasonable chance that they can be decontaminated, although they are much less . resistant than the cysts. An antibiotic sensitivity test and treatment as outlined in Section II,B must be used. The cells will not withstand SLS, and small cells are often killed by even mild formalin treatments. If a washing funnel is used with cells of this size, a large pore membrane filter (e.g., hlillipore type NC) should be substituted for the bolting silk. After washing the cells are plated. Assuming that the cell density in a sterile dark storage bottle has been determined, the inoculation is made simply by pipetting the appropriate volume from the stirred bottle into each Roux flask. The flasks should already contain their 200 ml of sterile medium. An excess number of cells may be needed, depending on previous experience with the cell viability in the dark storage bottle. Viability may diminish slowly with time and precipitously with decontamination. After inoculation the flasks are capped and placed in the incubator. Little additional attention is needed, but it is well to record their length and condition at weekly intervals. If the temperature and light intensity of the incubator are not uniform, rotating the positions of the flasks daily will help insure the uniformity of the cells in all flasks. The growth of these cells during the main growth phase is approximately exponential, as measured by wet mass or, more dramatically, when the reduced weight of individual cells is followed with a Cartesian diver balance (Holter et al., 1956), as shown in Fig. 3. There is a change in growth rate at the time of cap initiation, and there may be other changes in very early and very late stages. The exponential growth period of cells between 10 and 25 mm in length is probably the most useful stage for work of a chemical or physiological nature. The mass doubling time during this period is about 4 days under the culture conditions described here. If large cells are contaminated accidentally or during an experimental manipulation, a 5-minute treatment in 0.1%formaldehyde or overnight in
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CULTURE
P
r P
initiation
Normol cells Jj I I I I 1 1
I
v Enucleoie cells
I I I 1 1111 I I I 1 1 1 , I I ,I 1 I I t I t t I t
IILLLLLLUIIV!IJLIlL~ILILL.IIU
O o-Iv s_
FIG. 3. Single-cell growth curves. These measurements were made with a Cartesian diver balance and therefore represent dry weight growth curves. Their exponential character is obvious, the change in rate occurring at the time of cap initiation. All cells were about 8 mm at the beginning of the measurements. The normal cells went on to form 7-mm caps. While the enucleates grew for only 2-4 weeks and did not form caps, their growth rates are comparable to those of the normal cells. These cells were not axenic and were grown in Erd-Schreiber medium.
5 pg/ml sodium omadine will suffice to make surviving bacteria difficult
to detect for a week or two. It is usually not reasonable to put the cells in the dark while antibiotic sensitivities are determined and treatments given. Dark storage is not kind to larger cells, but they can be stalled by light intensities of about 25 footcandles. However, after any such treatment the cells should be given a week to 10 days in the incubator before they can be considered normal again.
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Once the scheduled gram of cells per flask is reached, the growth rate will decrease and the cells may begin to deteriorate. If there is no foreseeable experimental need for them, the largest should be selected for reproduction and the rest discarded.
E. Enucleation and Enucleate Growth The internal pressure in AcetabuZuriu is appreciable. If the cells are enucleated by simply amputating the rhizoid, a large amount of cytoplasm is immediately forced out, and more continues to be lost until the cut end seals itself. A stalk fragment may lose more than 50% of its cytoplasm before it heals. Since the growth rate observed after enucleation will be proportional to the remaining cytoplasm, negative or nil growth rates are likely to be obtained. To avoid this problem we tie the stalk off with fine nylon and then amputate the rhizoid just basal to the knot. Turgor is maintained, and no cytoplasmic loss occurs from the severed stalk. The rhizoids are usually kept because their regener at'ion serves as proof that the nucleus was contained in them rather than in the stalk. The practice of dividing the cell into an anucleate apical segment and a nucleate basal one which serves as a control appears ideal in theory, and it is true that the apical portion is not an entire cell lacking only the nucleus. However, much of the usefulness of AcetabuZuria resides in the fact that the apical segment is a good approximation of an enucleate cell; thus, it seems more reasonable to consider it as such and compare its growth with that .of an intact cell of equivalent size rather than with the regenerating stump of a cell. Figure 3 shows that the growth rates of intact and enucleate cells are entirely comparable. The length of time that enucleates will grow is variable from cell to cell and group to group (Shephard, 1965). Careful selection of healthy cells to be enucleated, axenic culture and defined media reduce the variability but do not eliminate it. Mean data from groups of enucleates often show an approximately linear rate of growth, apparently because the exponential growth rate is masked by a similar rate of decline in the number of cells growing at all. Exponential growth has been observed to continue for periods of a month or more in enucleate Acetabularia ( Shephard, 1965).
F. Maturation Since the reproductive potential of one Acetabularia cell is about 10,000 new cells, only a small percentage of the cells need to be carried
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on to maturity. Five percent will provide an enormous safety margin if they are selected late enough in the growth phase to insure that they are healthy and normal. For reproduction the cells must be maintained for several months beyond the period of their experimental usefulness. It is extremely dficult to maintain sterility in such cultures, especially since manual selection of cells and several medium changes may be necessary. Figure 2 shows the times and cell weights involved; however, these tend to be minimum times, many cells taking considerably longer to reach maturity. When cells with full-sized caps are seen in a culture being saved for reproduction, they are selected and transferred to fresh medium (50 cells per flask). Nuclear division in these cells is indicated by loss of color in the stalk as the cytoplasm migrates into the cap. Once this process is complete and the cap is full of uniform round cysts, another selection is in order. The cyst-containing cells are collected from several bottles and placed together in the incubator for another 2 weeks to insure complete maturation. After this all caps which still appear perfectly normal are placed in dark storage until they are needed. There is a tendency for completely mature caps to detach from the stalk. Generally, a period of a month in the dark will facilitate breaking cyst dormancy. Longer storage may result in a gradual diminution of viability, but viable gametes can be obtained even after several years. An occasional medium change is desirable during long periods of dark storage,
G. Evaluation The improved methods for the culture of Acetabularia presented here offer potential success in the performance of a variety of biochemical and physiological studies on Acetabularia which have been impossible with contaminated cultures and an undefined medium. Moreover, the defined conditions should result in more reproducibility of experimental results than has been experienced in the past. However, even cultured as described, Acetabularia still lacks several requirements of a good laboratory organism. The variability in growth rates is not completely eliminated by standard conditions and may reflect a high degree of genetic variability. Careful selection for some standard growth pattern may be required before growth rates become predictable. The growth rate is still too slow and the generation time too long. It would seem that a mass doubling time of 1 day could be achieved by truly optimum culture conditions and medium. This would reduce the generation time to a month and the main growth phase to a week. The problem of breaking cyst dormancy is
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DAVID C. SHEPHARD
twofold. Not only is it a nuisance to obtain reproduction but it is also practically impossible to self- or crossbreed individual cells. A hormone or shock treatment that will induce gamete production efficiently and quickly is necessary if Acetabularia is to realize its potential as a laboratory organism. The procedures described, however, make it possible to obtain and maintain reasonable quantities of healthy and sterile Acetabularia with a minimum of effort and space. ACKNOWLEDGMENTS A number of people have assisted in the development of the mediums and procedures: Marilyn Nagare, Mary Cammock, Patricia Roche, Florence Dark, and Yona Victor. The assistance of Fenton Moore in developing the antibiotic screening technique has been invaluable. This work was supported by a grant from the National Science Foundation (GB-5210) and by general research support awards from Case Western Reserve University. I would like to thank the Schering Corp. of Bloomfield, New Jersey for the sample of Gentamicin-sulfate, and Chas. Pfizer & Co., Groton, Connecticut, for the sample of Carbenicillin.
REFERENCES Allen, M. B., and Arnon, D. I. (1955). Pknt Physioz. 30, 366. Bailey, W. R., and Scott, E. G. (1966). “Diagnostic Microbiology,” 2nd Ed. Mosby, St. Louis, Missouri. Beth, K. (1953). 2. Naturforsch. 8b, 334. Brachet, J,, Chantrenne, H., and Vanderhaeghe, F. ( 1955). Biochim. Biophys. Actu 18, 544. Clauss, H. (1968). Protoplasniu 65, 49. deVitry, F. (1963). Protoplasma 55, 313. Gibor, A. (1965). €‘roc. Natl. Acad. Sci. U.S. 54, 1527. Gibor, A. (1967). In “Biochemistry of the Chloroplasts” ( W . T. Goodwin, ecl.), Vol. 11, pp. 321328. Academic Press, New York. Gihor, A,, and Izawa, M. (1963). PTOC.Natl. Acad. Sci. U. S. 50, 1164. Hammerling, J. (1963). Ann. Reu. Plant Physiol. 14, 65. Hhmerling, J., Clauss, H., Keck, K., Richter, G., and Wertz, G. (1958). Exptl. CeZl Res. Strppl. 6, 210. Hellebust, J. A., Terborgh, J., and McLeod, G. C. (1967). Biol. Bull. 133, 670. Holter, H., Linderstr@m-Lang,K., and Zeuthen, E. (1955). In “Physical Techniques in Biological Research (G. Oster and A. W. Pollister, eds.), Vol. 111, pp. 577-625. Academic Press, New York. Keck, K. ( 1964). In “Methods in Cell Physiology” (D. Prescott, ed.), Vol. I, pp. 189213. Academic Press, New York. Keck, K., and Clauss, H. (1958). Botan. Gaz. 120, 43. Lateur, L. (1963). Revue Algol. 1, 26. Provasoli, L., McLaughlin, J.-J. A., and Droop, M. R. (1957). Arch. Microbiol. 25, 392. Richter, G. (1962). In “Physiology and Biochemistry of Algae” (R. A. Lewin, ed.), pp. 633-652. Academic Press, New York.
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Schulze, K. L. (1939). Arch. Protistenk. 92, 179. Schweiger, H. G., Dillard, W. L., Gibor, A., and Berger, S. (1967). Protoplasm 64, 1. Shephard, D. C. ( 1965). Ex$. Cell Res. 37, 93. Shephard, D. C. ( 1966). Biochim. Biophys. Acta 108, 635. Shephard, D. C., Levin, W., and Bidwell, R. G. S. (1968). Biochem. Biophys. Res. C m m u n . 32, 413. Terborgh, J. (1965). Nature 207, 1360. Terborgh, J., and Thimann, K. V. (1964). Planta 63, 83. Thimann, K. V., and Beth, K. (1959). Nature 183,946. Thomas, E., and Tregunna, E. B. (1967). Can. J . Botany 45, 411. Vanden Driessche, T. (1966). Biochim. Biophys. Acta 126, 456. Zetsche, K. (1965).Planta 64, 119. NOTE ADDEDIN PROOF In June 1969, an International Symposium on Acetabularia was held in Brussels. The proceedings provide a valuable source d information on various aspects of Acetabularia and are published in “The Biology of Acetabularia” (J. Brachet and S. Bonotto, ds., Academic Press, New York, 1970).