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Vol.. XXXIV SEPTEMBER I, Wislsal @ 197r CAROLINA BIOLOGICAL SUPPLY COMPANY Bunr,rNcroN, Nonrn CARoLTNA 27215

ISOLATION AND PURIFICATION OF ALGAE Cultires DANIEL E. JAMES From the Algae Department, Furity and Genera Guaranteed Carolina Biological Supply Coinpany, Burlingtott, North Carolina 27215 The September, 1969, and December l, ment of a few cells, or a piece of a thallus. While looking through a dissecting mi- 1969, issues of Carolina Tips discussed cul- Isolation of algae should proceed with croscope, place a small sample of the Unialgal Cultures are guaranteed to contain only the alga on the label. Please order all ture techniques and media for freshwater the idea that a truly unialgal culture collection containing the organisms in a individual cultures by catalog number genus. and Prices are for cultures shipped at one and marine algae. Here I summarize some should be established as a clone culture. depression of a sterile spot plate, a de- time to one address. Each is suitable culture for a class of 12 to 25 students. of the techniques for isolating and purify- pression slide, or a watch glass. At least 6 Unialgal cultures, each....$3.50; 5 to 9, each..,.93.00; 10 to 19, each....g2.50; 20 or more. each....$2.00 ing algae from mixed populations. Micropipette Washing Technique to 12 other depressions should be filled 15-2260 Golenktnla. YELLOW-GR.EEN ALGAE As each organism reacts differently and with sterile water or media. While watch- 15-22@ Gonium. G. pectorale. must be handled as an individual. the tech- One of the simplest techniques for iso- ing through the dissecting microscope, l5-1710 Anabaena. 15-2890 Botrydlopsls. 1 5-2280 Haemstococcus. purification 15-1715 Anacystis. nldulans. niques for isolation and must lating microscopic forms uses micropi- move the tip of a micropipette over the A. 15-2297 Hyalotheca. l5-2895 Botrydlum. 15-1725 Calothrlx. be adjusted and adapted to meet individual pettes to carry individual cells through a 15-2300 Hydrodic8on. 15-2940 Ophiocytlum. cell. Carefully dip the tip into the me- 15-1755 Cyllndrospennum. 15-2330 Mesotaenium. 15-2970 Tribonema. needs. series of sterile washes. dium, pick up the cell, and expel into Flscherella. M. it 15-1770 kramstai, 15-2993 Vaucherla. V. geminata. 15-1?90 Glaucocystls. G. another depression of sterile water or 15-2345 Mlcruterias. 15-2995 Vaucherla. V. sessills. nostochinearum. Conrcrror medium (Fig. 15 15-2350 Micrcspom. 3). At least 12 to 15-1800 Gloeocapsa, DIATOMS 15-2360 Mougeotla. single cells should be isolated in this 15-1810 Gloeotrlchla. The initial supply of algal material is 15-2380 Netrlum. Lynsbya. 15-3095 Synedra. way and carefully washed 6 to 12 times 15-1830 l5-2400 Oedogonlum. usually from a natural population. FloaG 15-1835 Medsmopedla. in depressions of sterile fluid. A new 15-2415 Pandorina. P. morum. GOLDEN.BROWN ALGAE ing and swimming algae can be collected 15-1847 Nostoc. Balls. each 15-2430 Pedlastrum, plank- micropipette should be used for 15-1865 Osclllatorla. 15-3140 Chrysochronullna. and concentrated efficiently with 15-2435 Percursarla. Marine. washing. 15-1880 Scytonema. Marine. ton nets (Fig. l). Attached and filamentous 15-2445 Pithophora. Splrullna. 15-3145 Coccolithophora. Maritre. After sufficient washings, single celts l5-1900 15-2460 Platydorlna. P. caudqtq. forms can be collected by scraping rocks, 15-1935 Tolypothrlx. 15-3150 Coccolithus. C. hwleyl, should be expelled into each of eight tubes 15-24?5 Plabmonas. leaves, and other large algae. When algal 15-3180 Isochrysls. Marine. 15-2480 Polytoma. of sterile medium. The tubes are placed 15-3200 Ochromonas. specimens are collected directly, it is 15-2490 Protostphon. under lights stimulate growth. GREEN ALGAE necessary to make observations and isola- to algal 15-25 l0 Scenedesmus. DINOFLAGELLATES Cultures started from single cell isolates 15-2525 Splroeura. tions within a very short time, as many of 15-1955 Anklstrodesmus. often take 3 to 6 weeks to attain notable 15-2540 Staumstrum. 15-3240 Amphidinium. Marlne. (and 15-1970 Astrephomene. z{. the more delicate often the more growth. gubernaculilera. 15-2568 StephanoDten. Marine. 15-3250 Glenodinlum. Marine. interesting) genera will begin to disappear 15-2570 Stephanosphaem. 15-3260 Gymnodlnlum. Marine. A variation of this technique can be used 15-1978 Bfastophysa. B. rhi2opus, within a few hours. 15-1980 Bryopsis. Marine. 15-2585 Stichococcus. 15-3290 Peddinium. for isolating filamentous algae. Through 15-2600 Stigeocloniun. A second method of collection utilizes 15-1985 Bulbochaete. FrcuRE 1 Plankton net (65-2160) being used to a procedure similar to making a micro- 15-2CO0 Cartcrla. 15-2620 Trebouia. air-dried soil. Small amounts of mud and collect algae sample. 15-2640 Ulothrix. BROWN ALGAI' pipette, a small microhook can be formed- 15-2015 Chaetomorpha. Marine. sand, collected at the water line, are air 15-2645 Germlings 15-2030 Chlamydmonall Ulva. only. 15-3355 Dictyota. Marine. Either soft glass tubing ( 3 to 4 mm Single filaments of an alga are carefully 15-2655 dried until the soil feels dry and crumbles 15-2071 C. VolYox. V. aurew, 15-3360 Ectocanus. Marine. hooked, isolated, Chlore[a. globator. bore) or disposable pasteur pipettes can and washed. After care- p)'renoidosa. 15-2665 Volvox. V. 15-3420 Sphacelaria. Marine. easily. When small samples of this dried 15-2680 Volwlina. V. steinii, be used to form a micropipette. While ful washing, the algal filament should be 15-2075 Chforella. C, vulgaris, soil are placed in petri dishes, covered with pulled petri 15-2090 Chlorococcum. 15-2690 Zoochlorela. Z, RED ALGA,E clasping the tip of a sterilized pipette with through a dish of very soft parasitica. sterile water or media, and placed under a 15-2105 Clsdophora. Marine. a pair of forceps, hold the glass a agar (0.5 percent to 0.75 percent) several L5-:2695 15-3465 Acrochaetium. Marine. light, many different algae and protozoa in 15-2115 Clostedum. z,yenesa. Agardhlella. small bunsen burner flame until it begins times to help remove any epiphytes. 15-2128 Coleochaete. 15-3480 Marine. will begin to emerge within 24 hours. New 15-3495 Antithamnion. Marine. (Fig. 15-2lzl0 Cosmadum. organisms continue to appear in the dishes to soften 2). In a motion that co- EUGLENOIDS 15-3510 Bangla. Marine. 15-2155 Derbesla. Marine. incides with the removal of the glass from Atomizer Technique 15-3525 Ca||lthamnlon. Marhe. for several days. Adding a sterilized barley 15-2158 Desmldlum. 15-2?20 Astasia. 15-3600 Porphyrldlum. grain or a pea cotyledon to the dishes often the flame, the micropipette tip is stretched l5-2159 D:cbosphaerlum. 15-2725 Astasla. longa. A. 15-3605 Pterocladlr. Marinc. with a smooth, rapid pull. By varying the A fairly simplified technique developed 15-2160 Duna|lella. Marine. 15-2765 Dlstlgma. encourages growth of genera which might 15-3635 Rhodymenla. Marine. 15-2165 Dysmorphococcus. 15-2785 Euglena. E. ac6, otherwise go unnoticed. timing and strength of the pull, the bore by Wiedeman.et al. (1964) can be used 15-3650 Selrospora. Marine. 15-2200 Enteromorpha. Marlne, 15-2800 Euglena. E. gracilis. Algal zygotes, spores, and other resting of the micropipette can be adjusted to to both isolate and purify collections of Eremosphaelr" 15-3655 Spermothamnlon. Marlne. 15-2215 l5-2825 Menoldlum. forms will be viable for a number of years meet the size needed for any microorga- algae. Eight to nine milliliters of algae 15-2230 Eudorlna. E, calllornlca, 15-2845 Phacus. RESIDUAL FLAGELLATES nism. Using forceps, break the from washed (see 15-2235 Eudorlna. E. eleguns. 15-2855 Rhabdomonas. if dried soil samples ar€ stored in sealed tip are by centrifugation Purifi- gentle, 15-2250 Gloeocystk. 15-2870 TlacheloiloDas. 15-3684 Crf,ptomonas, jars or in plastic bags. the micropipette with a steady, cation), and after the final wash, all but outward pull in a straight line with the 2 ml of the supernatant is decanted. A IsoLATroN pipette to make a smooth even tip. Bend- 15-cm long microtube is drawn out fol- ing or crushing the tip produces a jagged lowing the instructions for making a mi- Gan0lina Bi0l0gical $upRlu G0m0anu To assure homogeneity, unialgal cultures edge which is difficult to use. Attach a cropipette. The microtube is inserted in are started from clones. A clone is prop- rubber bulb or piece of soft rubber tubing the bottom of the centrifuge tube and Burfingon, N. C.27215 / Gladstone,0re.9702l agated from a single cell, a single fila- to the top of the pipette. held in place with a cotton stopper. The 34 CAROLINA BIOLOGICAL SUPPLY COMPANY CAROLINA BTOLOGICAL SUPPLY COMPANY 35

several of the techniques used for purify- Agar Plate added to agar media rather than to liquid to assure that it is bacteria-free. The testing ing algal cultures. media. is normally done by inoculating at least six standard bacteriological media. Some UV Light Technique llashing by C entrilugation Technique of the media used for this test are: nutrient broth and agar, yeast-dextrose agar, pro- Bacteria and algae usually can be sepa- UV light can be used as a selective teose peptone agar, malt agar, sodium rated readily by centrifugation and wash- agent. because most algae are more resis- ca- seinate agar, and thioglycollate ing in sterile medium. A sterile, thick- tant than bacteria to the lethal effects of agar. walled, 15-ml centrifuge tube is filled with UV treatment. This technique can be suc- algae from a vigorously growing culture. cessful in purifying algae having gelatinous Funnrnn. RrentNc The material is spun at approximately 2000 matrices which interfere with conventional Brown, R. Malcolm, and Bischoff, Harry W., A rpm for 45 to 90 seconds, the supernatant purifying techniques. A 2750 Angstrom new and useful method for obtaining axenic is decanted, and the algae is resuspended UV light is placed about 25 cm above cul- culture of algae, Phycological News Bull.,1962, in sterile water or medium. This procedure tures on agar or in shallow liquid. The cul- James, Daniel E., Unialgal cultures, Carolina is repeated at least 12 times. tures are irradiated for 8 to 16 minutes. Individual cells are then transferred to Tips, 1969, 32, 33. After the last washing, the liquid is James, Daniel E.. Maintenance and media for decanted and the algae resuspended in fresh medium. marine algae, Carolina Tips, 1969, 32, 45, about 1 ml of sterile liquid. With a sterile Filter Technique Pessoney, George F., Field and Laboratory In- pipette, a few drops are pipetted onto an vestigation of Zygnemataceous Algae, Ph.D. Thesis, University of Texas at Austin, 1968. agar plate (Bristols agar, proteose agar, Millipore@ or membrane filters can be soil-extract agar). The material is streaked Provasoli, L., Effect of plant hormones on Ulva, used to separate filamentous algae and Biol. Bull., 7958, l14, 315. in the form of a pentagon with a flamed (Pessoney, FIcttRB 2 Making a micropipette. bacteria 1968). Filaments are Wiedeman, V. E., Walne, P. L., and Trainor, bacteriological transfer loop. At each cor- cut into small pieces (one to five cells long) F. R., A new technique for obtaining axenic cul- centrifuge tube is fastened to a ring stand but not yet solidified agar containing a ner of the pentagon, the transfer loop is re- Algal and suspended in sterile medium in the tures of algae, Can. J. Bot., 196/., 42,958. and compressed air is directed through a weak nutrient solution. This mixture is flamed and only those cells at the end of bowl of a sterilizing filter outfitted with an small opening (e.g., through a dropper swirled, poured into a petri dish, and al- the last streak are used on the next streak 8 p filter disk. The filter is connected to FrcuRE 4 Atomizer appalatus. OFFPRINT pipette) so that the stream of air crosses lowed to harden. After several days of (Fig. 5). This procedure leaves a decreasing a vacuum, and the filament pieces are kept the top of the microtube extending from growth under the light, colonies are cut number of cells in each streak. After the Filter sterilize and store the stock in a off the filter disk and continually swirled 44-8202 Isolation and Purification of the centrifuge tube. The algal suspension from the agar and inoculated into fresh culture has grown, colonies in the later frozen condition. To use. add I ml of stock by the addition of 2 liters of sterile medi- Algae. Daniel E. James. Illustrated with is drawn the microtube atomized up and medium. streaks will be spaced farther apart and to 100 ml of medium. Transfer algae to the um in a continual flow. The filter disk photographs and drawings. A reprint of the into spray. plate a fine A sterile of agar rexrs: Motile algae will often react will almost always be from single cells. medium, leave for 48 hours, and retransfer is changed several times during this opera- September l, 1971 issue of Carolina Tips. medium passed quickly is through the either positively or negatively to light or After the culture has grown for several to fresh, sterile medium without antibiotics. tion. Each pack contains 30 copies. spray at a distance about 25 (Fig. genera plate of cm electrical current. Also, different days, the agar is examined with a The washed pieces of filament are Per pack, postpaid....$3.00; 12 packs....$30.00 4), recovered and put under the lights. may show their tactic responses at differ- dissecting microscope. Colonies that show Formula II (Provasoli, 1958) poured directly into a flask of cool but After several days growth, single cells or ent rates which allow for differential sep- no sign of bacterial or fungal contamina- unsolidified Bacto nutrient agar. Mix the colonies free of bacteria and fungi are aration. Through repeated isolations, near- tion are transferred with either a micro- Make a stock solution so that 1 ml con- agar and the alga well and pour into sterile UNIALGAL SETS picked up from the plate with a micro- ly pure separations of genera can be pipette or bacteriological transfer loop to tains the following: petri dishes. Leave the alga suspended in Survey Set. Designed pipette and expelled into sterile liquid achieved. fresh agar slants. 15-1520 Unialgal for 12.000 units K Penicillin agar for 2 to 3 days, after which pieces of beginning student. Fifteen representa- medium. osMorrc BALANcE: Some of the hardier Brown and Bischoff (1962) have further the 50 pg Chloramphenicol filament not surrounded by bacteria are algae demonstrate diversity of algae can be cleaned by rapid changes in modified the above techniques to include tive the 50 pg Polymyxin B cut from the agar and inoculated into morphology the Selective Media Technique osmotic concentration. Moving the speci- several treatments of the algal cells with and habit found in all 60 pg Neomycin sterile medium. groups. men from distilled water to a weak salt detergent solutions and with short pulses in major Filter sterilize and use 1.5 stock per The techniques mentioned above for iso- solution and back to distilled water will an ultrasonic water bath (low intensity with ml of Testing lor Purity Per set....$30.00 100 ml of medium. Leave the organism in lating microscopic algae often are not ap- facilitate removal of protozoans and naked a frequency in the range of 90 kc per sec- the antibiotic medium for up to 7 days. After a culture has been carried tbrough 1.5-1540 Marine Algae Set. Ten cultures of plicable to the larger and more complex flagellates. ond) to facilitate algal and bacterial sepa- Either Formula I or Formula II may be a purification technique, it should be tested our selection representing a cross section of marine forms. To simplify isolation of REPRoDUcrrvE srRUcruRES: Resting ration. Prior to the centrifugation and the diversity found among the green, vegetative material, a series of selective spores, cysts, zygotes, tetraspores, and washings, the algal cells are treated with a brown, and red algae. media often useful assist freeing percent is to in other reproductive structures can be iso- synthetic detergent solution or a 5 Per set....$20.00 the alga from some of the worst con- lated and germinated to give rise to pure solution of the nonionic surface active taminants. cultures. agent, "Tween 80." The cells in detergent Select a branch or piece of the alga that solution are given several ultrasonic vibra- SUPPLIES is as clean and as free as possible of tion treatments of from 60 seconds to sev- PuntrtclltoN Seawater. Natural. epiphytes. While viewing through a dis- eral minutes duration depending on the gal....$8.50 secting microscope, brush the piece of After a unialgal culture has been estab- organism. The cells are then washed by 16-4550 4x1 thallus with a camel's-hair brush or cotton lished and is growing well, many types centrifugation as described above, and re- Alga-Gro@ Concentrate. Sterile enrichment to free it of as many epiphytes as possible. of research require that the culture be suspended after each washing with 10 sec- for seawater, or spring water. One tube A microhook is also helpful in removing axenic (bacteria-free). The following are ond bursts of ultrasonic vibration. rnakes I liter. times epiphytes. Wash the thallus several 16-4600 Per 12 tubes....$3.55: 36....$9.60 in sterile seawater and place in medium Antibiotics Technique containing 10 germanium dioxide. AIga-Gro Seawater. Pasteurized medium mglliter Mixes of antibiotics can be successfully dioxide silica marine algae. Germanium is a antagonist used in purifying cultures of algae which for culturing prevent growth qt....$?.00 and will the of diatoms are too large for the centrifuge technique. 16-4810 and other siliceous organisms which are The following two formulas allow for a Alga-Gro Freshwater. Sterile medium for the worst contaminators of marine cul- variation in approach. tures. culturing freshwater algae. Formula I 16-5050 qt.....$6.00 Additional Techniques Make a stock solution containing the fol- 73-4700 Disposable Plastic Filter Unit. Presterilized. Capacity, 115 ml, There are several additional techniques lowing: Each....$1.25; pkg. of 12....$15.00 and variations which can effect some suc- 0.6 g Penicillin "G" (1625 units per cess. mg) 73-6744 Disposable Pasteur Pipet Borosili- AGAR PouR PLATEs: A small inoculum FIGURE 3 Isolating single algal cells with a 1.0 g Streptomycin sulfate cate Class. 7r/z O.D.,9" long. of a raw collection is mixed with cool micropipette. 200 ml Distilled water FrcuRB 5 Decreasing.inoculum method of streaking washed algal cells, Per shelf pack (144)....$4.03; case (720)....$18.10