Tuatara: Volume 11, Issue 1, March 1963
A Culture Method for Marine Diatoms and Flagellates
A Culture Method for Marine Diatoms and Flagellates
There is a Considerable literature on the culture of the smaller marine algae (diatoms and flagellates). Pringsheim's (1946) book contains much valuable information, including the earlier references. Butcher (1959) includes later references on the culture and taxonomy and Lewin (1959) summarises isolation methods. As pointed out by Butcher (1959), all known culture methods have failed to promote the growth of many of the smaller marine algae; including many of the commonest North Sea diatoms. Some diatoms and flagellates have, however, been grown in culture in various laboratories for many years, without expensive equipment or complicated media. The four species whose culture is described here fall into this category, and it seems likely that because subcultures of these species were readily available from well-established cultures, workers such as Spencer (1954), Kain and Fogg (1958), and McLachlan (1960) were stimulated to study optimum growth requirements of these species. Such studies may lead to the development of culture methods that can be applied successfully to some of the more difficult species. The medium and growth conditions described below are probably not optimum for any of the four species concerned, but have been found satisfactory for maintaining these and fifteen other species of diatoms and flagellates in culture at this laboratory for several years.
The isolation of a species from the sea and its purification into a unialgal laboratory culture is time-consuming, and it is usually preferable to obtain an inoculum from an existing culture. Fortunately, several laboratories have been building up collections for many years and these are regularly subcultured so that they remain in a healthy growing condition. Three of the cultures described here were obtained originally from the Plymouth Laboratory through the courtesy of Dr Mary Parke. The fourth (Nannochloris atomus Butcher) was isolated from Sydney inshore page 21 waters. This was done by towing a small phytoplankton net behind a launch, filtering off and discarding the zooplankton from the rest of the sample, and enriching the latter with the Erdschreiber medium described later. After a week or so such samples often produce a bloom of one or several species of diatoms and/or flagellates. They are then subcultured for several months, the objective being to make the subculture when the desired species is most abundant and the undesired least. Lewin (1959) has outlined the alternative methods that can be used for isolation, and also for making the cultures bacteria-free. The latter is difficult and is necessary only in special circumstances.
Fig. 1. Left: Three normal cells of the small diatom Phaeodactylum tricornutum Bohlin. The pair on the right are separating after division. Length approx. 30μ. Centre : Single motile cells of the green flagellate Dunaliella tertiolecta Butcher. Length (excl. flagella) approx. 11μ. (After Butcher, 1959.) Right: Older motile stage of the golden-yellow flagellate Iseochrysis galbana Parke, length (excl. flagella) aprox. 6μ; and mature cyst (diam. approx. 6μ) containing 16 developing motile stages. (After Parke, 1949.)
The small diatom Phaeodactylum tricornutum Bohlin (Fig. 1) is probably better known under its old name Nitzschia closterium (Ehrenb.) W.Sm. forma minutissima Allen and Nelson. It was isolated about 1910 at the Plymouth Laboratory, where it has been maintained in culture ever since and has been used extensively for feeding marine invertebrate larvae. The reproduction and growth requirements have been described by Wilson (1946) and Spencer (1954) respectively. The green flagellate Dunaliella tertiolecta Butcher was isolated from Oslo Fjord, and entered the Plymouth Laboratory collection about 1928 under the name ‘Chlamydomonas III’…. Its anatomy and growth requirements have been described by Butcher (1959) and McLachlan (1960) respectively. The second page 22 green flagellate Nannochloris atomus is very small (1-2μ) and has not be figured, since little detail can be seen under the light microscope. Its anatomy has been described briefly by Butcher (1952). The golden-yellow flagellate Isochrysis galbana Parke was isolated at Port Erin in 1940 for feeding oyster larvae and was considered by Walne (1956) to be thoroughly satisfactory for this purpose. Its anatomy and life history were described by Parke (1949), and its growth requirements by Kain and Fogg (1958).
Preparation of Media
The Erdschreiber medium used is based on a formula kindly supplied by Dr. Mary Parke, and is similar to that used at the Plymouth Laboratory. It consists of a freshwater extract of garden soil which is enriched with nitrate and phosphate and added to sea-water just before inoculation with the desired algal species. Although the soil extract fraction is indeterminate and variable, Erdschreiber forms the basis of many satisfactory culture solutions and is probably the most easily prepared medium in use. The simplest method of preparing the soil extract is to boil 2 1. of rich garden soil and 2 1. of tap-water in an enamel bucket for 15 min., and then filter overnight in a series of large funnels containing paper hand towels or coarse filter paper (e.g. Eaton-Dikeman No. 541). Alternatively, if an autoclave is available, the soil and tap-water can be autoclaved at 15 lb./sq.in. for 3 hr. before filtering. The colour of the resulting soil extract varies with the type of soil used, and the treatment (boiling or autoclaving). Boiled soil extract is often yellow-amber in colour and the nitrate and phosphate can be added to it without dilution. The soil used in this laboratory is black and sandy and the autoclaved extract is dark brown: it is diluted with about 6 parts of tap-water until it becomes yellow-amber. Each batch is colour matched by eye against a standard to reduce variation between batches. Some workers advocate distilled water for all dilutions, but this seems unnecessary in view of the indeterminate and variable composition of the soil extract. Our tap-water comes through copper pipes from the mains but does not seem to be detrimental to the cultures.
This soil extract is enriched by adding, per 50 ml., 0.02 g. sodium nitrate and 0.03 g. sodium dihydrogen phosphate (NaH2PO4 · 12H2O). Butcher (1959) uses 0.1 g. and 0.05 g. respectively of the corresponding potassium salts. This enriched soil extract is poured into bottles or flasks, heated to 100° C., plugged with cotton wool, and allowed to cool to 20-30° C. before the nitrate and phosphate are added. The alternative autoclave method is to add the nitrate and phosphate, plug, and autoclave at 5 lb./sq.in. for 1 hr. Enriched soil extract can be prepared in bulk since it lasts page 23 several months without deterioration. The working medium is prepared by adding 50 ml. of enriched soil extract to 950 ml. of sea-water. Contaminant algal species are removed from the latter by fine filtering (e.g. Whatman No. 40 filter paper), pasteurisation (heating to 70°C. and allowing to cool) or sterilisation (autoclaving at 5 lb./sq.in. for 2 hr.).
Routine subcultures are usually made every 7-10 days to keep the cultures in a healthy, growing condition. The pipettes used to transfer the inocula from flasks are 25 cm. lengths of 4 mm. (internal diameter) glass tubing drawn out to give a 1-2 mm. opening at one end. It is important to use a fresh sterile pipette for each species to avoid cross-contamination. Pipettes that have been wrapped in tissue paper and incubated at 150°C. in a drying oven for 1 hr. are satisfactory, but well-boiled pipettes can be used. All flasks used need to be well rinsed in boiling water beforehand to destroy traces of previous cultures that might lead to cross-contamination. 250 ml. flasks containing about 200 ml. of culture are normally used. The enriched soil extract and sea-water are mixed in the proportions of 1 in 20 respectively, pasteurised by bringing to 70°C. in a suitable container, and poured into the flasks. The latter are plugged and next day, when they have cooled to room temperature, an inoculum of about 10 ml. is added from a previous culture. In the autoclave method the flasks are filled with the enriched soil extract and sea-water mixture, plugged, and autoclaved at 5 lb./sq.in. for 2 hr. before cooling and inoculating.
Larger cultures can be obtained readily from these stock cultures as required. Four, 10 or 25 l. glass containers are suitable and the same procedure is followed, but it is more convenient to use fine filtered sea-water (Whatman No. 40 filter paper) rather than pasteurised or sterilised. Usually, the larger the inoculum the more rapid the growth. Aeration is needed for cultures of these volumes to keep the algae in suspension and possibly to assist in supplying carbon dioxide.
In addition to the nutrients contained in the Erdschreiber, the cultures require light, carbon dioxide, and a suitable temperature range. Many workers stand their cultures near a window where they receive daylight, but not direct sunlight. In this laboratory cultures are placed on shelves parallel to. and 10-20 cm. distant from, horizontal fluorescent lamps (40 W. ‘Osram White’). No special arrangements are made to provide the 200 ml. stock cultures with carbon dioxide. The 4 l. and larger cultures are aerated to prevent the algae sinking to the bottom of the containers and, since air page 24 contains about 0.03 per cent carbon dioxide by volume, this may prevent a shortage of the latter. Temperatures of 16-20°C. are usually advocated for diatoms and flagellates. Initially, cultures were kept close to this range by using a thermostat-controlled heater in winter, and a waterbath in summer. The lower limit could be controlled accurately, but the upper one was dependent on the temperature of the sea-water circulating in the waterbath and rose to 23°C. during much of the summer. This system worked for the few species then in culture, but as more species came into culture it became cumbersome and was replaced by a thermostat-controlled air conditioner. The latter maintains the temperature in the culture room at 18-22°C. and the cultures have grown steadily in this range for several years. Temperatures above 25°C. may be detrimental for continued growth of Phaeodactylum. Spencer (1954) suggested that increase in temperature above 25°C could be expected to lead to a rapid increase in the mean generation time of this species, followed by a complete cessation of growth resulting from denaturation of proteins and disorganisation of the cell. Wisely and Purday (1961) obtained poor growth of Phaeodactylum at 25-28°C.
Cultures of the four species described briefly earlier were made following the above method, but not using an autoclave. Aerated half-gallon glass jars were used as containers at 20 ± 2°C. Densities were estimated every few days using a Petrov Hauser bacteria counter (Phaeodactylum, Nannochloris) or a Neubauer haemocytometer (Dunaliella, Isochrysis). Since only duplicate counts were made (and then averaged) the counting errors were large, but, as shown in Fig. 2, substantial growth occurred. ..Isochrysis reached a peak concentration of about 4 million cells/ml. in 14 days and then declined. Phaeodactylum increased in density for 50 days to reach a concentration of about 11 million cells/ml. Nannochloris was similar, but reached a peak concentration of about 62 million cells/ml. Dunaliella reached a concentration of about 7 million cells/ml. around the thirty-sixth day and maintained this until about the fifty-fourth day.
Butcher, R. W., 1952. Contributions to our knowledge of the smaller marine algae. J. Mar. biol. Ass. U.K. 31: 175-191.
Butcher, R. W., 1959. An introductory account of the smaller algae of British coastal waters. Part I. Introduction and Chlorophyceae. Fish. Invest., Lond. Ser. IV: 1-74.
Kain, J. M., and Fogg, G. E., 1958. Studies on the growth of marine phytoplankton. II Isochrysis galbana Parke. J. Mar. biol. Ass. U.K. 37: 781-8.
Lewin, R. A., 1959. The isolation of algae. Rev. algol. no. 3: 182-197.
McLachlan, J., 1960. The culture of Dunaliella tertiolecta Butcher — a euryhaline organism. Can. J. Microbiol. 6: 367-79.page 25 page 26
Parke, Mary, 1949. Studies on marine flagellates. J. Mar. biol. Ass. U.K. 28: 255-86.
Pringsheim, E. G., 1946. Pure Cultures of Algae. Cambridge.
Spencer, C. P., 1954. Studies on the culture of a marine diatom. J. Mar. biol. Ass. U.K. 33: 265-90.
Walne, P. R., 1956. Experimental rearing of the larvae of Ostrea edulis L. in the laboratory. Fish. Invest., Lond. Ser. II: 1-23.
Wilson, D. P., 1946. The triradiate and other forms of Nitzschia closterium (Ehrenberg) Wm. Smith, forma minutissima of Allen and Nelson. J. Mar. biol. Ass. U.K. 26: 235-70.