ON THE CULTURE OF THE DIATOM MELOSIRA SULCATA (EHR.) KUTZING

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1 ON THE CULTURE OF THE DIATOM MELOSIRA SULCATA (EHR.) KUTZING BY V. D. RAMAMURTHY AND K. KRISHNAMOORTI-1Y (U.G.C. Centre for Advanced Study and Research ht Marine Biology, Porto Novo, S. India) Received January 16, 1965 (Communicated by Prof. R. V. Seshaiya, F.A.SC.) INTRODUCTION ALLEN AND NELSON initiated in 1910 the study of marine planktonic diatoms by laboratory cultures. Since then there has been considerable progress in the investigation of diatoms by laboratory cultures. Allen (1914) cultured successfully the diatom Thalassiosira gravida in artificial sea-water. Ketchum (1939) studied the phosphorus and nitrogen absorption in cultures of Nitzschia closterium. Ketchum and Redfield (1949) studied the chemical composition of algae grown in mass cultures, and Goldberg et al. (1951) studied phosphorus utilization in cultures of Asterionella japonica using labelled phosphorus. Chfi (1945) found that in diatom cultures pyrophosphate could not be utilised as a source of phosphorus as effectively as orthophosphate in Phaeocystis poucheti, Skeletonema costatum and Nitzschia closterium. However, he found that phytin could be a good source of orthophosphate for the growth of Skeletonema and Nitzschia. Harvey ( ) discovered that iron and manganese are important elements for healthy diatom growth. He also investigated (1953) the various sources of nitrogen supply and studied synthesis of chlorophyll in cultures of Nitzschia closterium. Enomoto (1961) reared successfully bacteria-free cultures of Melosira nummuloides. Parsons et al. (1961) investigated sugars, amino-acids, nitrogen and silica in eleven species of diatoms grown in mass cultures. More recent studies are by Soli (1963, 1964) and Spencer (1952, 1954) on bacteria-free diatom cultures. There have been some studies also on comparative growth of different diatoms like Nitzschia closterium and Skeletonema costatum (Matudaria, 1939). Gross (1936, 1937) studied the growth of diatoms, dinoflagellates and invertebrate larvae in culture media. Jacobson (1910) demonstrated the effect of the addition of a rich supply of organic substances to cultures of soil algae volvocales, such as Carteria, Chlamydomonas and Sphondylomorum. They showed vigorous ~s 25

2 26 V.D. RAMAMURTHY AND K. KRISHNAMOORTHY development in the presence of protein when the cultures were illuminated, but in the dark they disappeared and Polytoma appeared. The present account describes the successful culturing of Melosira sulcata in the laboratory. This diatom occurs in the estuarine and nearshore waters of Porto-Novo and is abundant during April, July and August. The salinity of the waters during the period ranged from to 34.55~o and the temperature from to C. Z 3 4 Fro. 1. Melosira sulcata (Ehrenberg). 1. Ordinary chain during cell division, Cells enlarged, 1, Before auxospore formation, x 1, After auxospore formation, 1,400. MATERIAL AND METHODS Samples of Melosira were collected from the mouth of the Vellar estuary on The material was cleaned, sobsampled, subcultured and isolated in Foyn's Erdschreiber solution. The Erdschreiber medium wasmade according to the method given by Gross (1936, 1937), by adding Schreiber's medium to soil extract (Plingshein, 1946). The composition of Erdschreiber, was as follows:

3 On the Culture o f the Diatom Melosira sulcata (Ehl.) Kutzing NaNO~ Na~HPO4 Soil extract Sea/estuarine water gm. gin. c.c. c.c. The soil extract was made by boiling for one hour 1 kg. of good garden soil, with one litre of glass distilled water and boiled in an autoclave for three hours. After allowing it to stand for 3 to 4 weeks, the coarse particles settle to the bottom and the fluid becomes transparent and ready for use. To prevent the growth of bacteria and mould, this extract was sterilized again. Repeated decanting and boiling enhance the clarification of the liquid. Before use, the soil extract was poured into another flask and boiled for a short time, and to this the required amounts of filtrate and phosphate were added. The sterilized sea-water was then poured into this. For obtaining large quantities of culture media at short intervals, one or more litres of soil extract with nitrate and phosphate added to it may be kept in stock and the culture medium can be quickly made up with sea-water before use. It is advisable to keep the stock solution in a refrigerator and avoid boiling the extract, once the salts had been added. The seawater used in the present experiment was filtered with Whatman No. 1 paper and then heated to boiling point. 2SO00 299O0 24O0O 2300O '' o00 110oo go00 9'000 8' ! ~g 32 Temperature G Fro, 2, Showsthe growth and ce!ldivi~on of Melosira~ulcata--atdifferent tempcrat~ ~,

4 28 V. D. RAMAMURTHY AND K. KRISHNAMOORTHY Small watch-glasses and small petri dishes were used as glassware. All the glassware, including pipettes, were first cleaned with a mixture of sulphuric acid and bichromate for several hours. They were then thoroughly rinsed with tap water, and sterilised in glass distilled water. The diatoms (Melosira sulcata) were isolated under the low power microscope and squirted into a watch-glass containing 2 ml. of sterile culture medium. These washings were repeated thrice and the diatoms were finally transferred to the petri dishes. The petri dishes containing cultures were incubated at 15 C.-28 C. Subcultures were made every 10 to 15 days and were inoculated mostly with 2-3 cells. Adequate dosages of the antibiotic streptomycin (sodium pencillin) were also added (0.01gm./ 100ml.) to avoid fungal and bacterial infection of the cultme. OBSERVATIONS Melosira sulcata grows normally in the Erdschreiber medium without much difficulty and the culture can be maintained in the laboratory for about five months. The temperature of the medium was from 15 to 28 C., which is tbe tempelature in which the diatcm normally lives, and the ph from 7 to 8.4, generally, one end of the diatcm chain grows attached to the petri dish. When the diatom becomes 1educed in size auxospores are produced. The auxospore is small and slender, but with the addition of 0.01 gm./100 ml. of vitamin B12 to every 100 ml. of the solution the auxospore increased in size and appears healthier. Auxospore formation in enriched sea-water media has been reported by Gross (1937), Harvey (1939; Braarud, 1945) and Tailing (1960). The auxospore formation in Melosira sulcata takes place once in ten days, and is essentially similar to that of other eentrales, described by Fritisch (1956). During the auxospore formation the walls separating two adjacent diatom cells in the chain separate. The protoplast inside each cell becomes enveloped in a thin membrane, the perizonium within which the protoplast undergoes rapid enlargement. The valves and connecting bands are secreted internal to the perizonium, and a new individual larger than the parent one is produced. Usually 2 to 4 auxospores are produced at a time. The cells inclease in number every 2-3 days and in each ch/fin 6 to 8 cells are present. The optimum temperature for the cell division ofmelosira sulcata is 17 C. Figure 2 shows the growth and cell division of Melosira s~lcata at different!~emlperatures,

5 On the Culture of the Diatom Melosira sulcata (Ehr.) Kutzing 29 DISCUSSION Enomoto (1961) studied in Melosira nummuloides in Mastue medium with ph ranged from 7 to 8.4 and temperature 9 to 30 C. He observed that the diatom had the propensity to grow with the end of the chain wall attached to the glass wall. Fritsch (1936) records that in Melosira varians the auxospore formation is comparatively frequent and in Melosira furgensii the spore occupies the part of the epitheca of the parent individual, while the opposite rounded end projects into the hypotheca. In Melosira nummuloide~ the auxospore lies outside the case of the parent, but in Melosira fungensii the auxospore becomes terminal and new cells grow attached to it. The formation of auxospore in Melosira sulcata is similar to that of M. furgensii but unlike that of M. nummuloides. The optimum temperature for the growth and cell division of Melosira suleata in cultures is 17 C., as mentioned befole. At this temperature the diatom grows best (22,000 cells/1.). It has also been found that vitamin B12 promotes rapid growth. As in other centrales cell division and multiplication of the chain occurs every 2 to 3 days and the formation of auxospore once in 10 days. When the size of the auxospore became reduced, addition of vitvmin Biz (0.01 gm./100 ml.) restored them to a healthy state. SUMMARY Cultures of Melosira sulcata were maintained in the laboratory for five months using Erdschreiber medium. The temperature in cultures ranged from C. and the ph from 7 to 8.4. The diatom grows best at about 17 C. (22,000 cells/1.). Cell division and multiplication of the diatom occurs every 2 to 3 days as in other centrales and lhe formation of auxospore once in 10 days. The auxospores, which have become reduced, can be restored to healthy growth by the addition of vitamin B12 (0.01 gm./100ml.). ACKNOWLEDGEMENTS We thank Prof. R.V. Seshaiya, Director of the Marine Biological Station, Porto Novo, for suggesting the problem and for guidance. Our thanks are also due to Prof. T. V. Desikachary, University Botany Laboratory, Madras, for criticism and suggestions. One of us (V. D. R.) is

6 30 V.D. RAMAMURTHY AND K. KRISHNAMOORTHY grateful to the University Grants Commission, New Delhi, for the award of a Junior Research Fellowship. We thank Mr. R. S. Manohardoss for help in preparing the illustrations. REFERENCES Allen, E. J and Nolson, E. VrV. Braarud, T. Chu, S. P. Enomoto, Y. Fritsch, F. E. Goldberg, E. J., Walker, T. J. and Whisenand, A. Gross, F. "On the culture of plankton diatom Thalassiosira gravida (Cleve) in artificial sea-water," J. mar. biol. Ass., U.K., 1914, 10, "On the artificial culture of marine plankton organisms," ibid., 1910, 8, Cited by Lewin, "Note on the technique of making bacteria-free cultures of marine diatoms," ibid., 1946, 26, "On the bacteria-free culture of Melosira nummuloides (dillow)," Bull. Japanese, Soc. Sci. Fish., 1961, 26, The Structure and Reproduction of the Algae, Cambridge University Press, 1956, 1, 791. "Phosphate utilization by diatoms," Biol. Bull. Woods Hole, 1951, 101, "Note on the culture of some marine plankton organisms," J. mar. biol. Ass., U.K., , 21, "The life-history of some planktonic diatoms," Phil. Trans. Roy. Soc., 1937, 228, Harvey, H. W. "The supply of iron to diatoms," ibid., 1937, 22, "Substances controlling the growth of a diatom," ibid., 1939, 23, "Manganese and the growth of phytoplankton," ibid., 1947, 23, Jacobsen, H. C. "Synthesis of organic nitrogen and chlorophyll by Nitzschia closterium, J. mar. biol, Ass. U.K., 31, "Kulturvenuche mit eingen niederen volvocacean," Z. Bot., 1910, 2, 145. Ketchum, B. H. A.C. and Redfield, Lewin, J. C. and GuiUard, R. C, "The absorption of phosphate and nitrate by illuminated cultures of Nitzschia closterium,'" Amer. J. Bot., 1939, 26 (6), "Some physical and chemical characteristics of algae grown in mass culture," J. Cell. Comp. Physiol., 1949, "Diatoms," Ann. Rev. MicrobioL, 1963, 17~ ,

7 On the Culture of the Diatom Melosira sulcata (Ehr.) Kutzing 31 Matudaira, T. Parsons, T. R., Stephen, K. and Strictland, J. D. H. Pringsheim, E.G. Soli, G. Spencer, C. P. Sweeney, B. M. TaRing, J. "The physiological property of sea-water considered from the effect upon the growth of diatoms with special reference to its vertical and seasonal change," Bull. Japanese Soc. Sci. Fish,. 1939, 8, "On the chemical composition of eleven species of marine phytoplankton," J. Fish. Res. Bd., Canada, 1961, Pure Cultures of" Algae, Cambridge University Press, 1946, pp "Axemic cultivation of a pelagic diatom," Symposium on Marin~ Microbiology. (Ed.) C. H. Oppenheimer, 1963, "A system for isolating phytoplankton organisms in unialgal and bacteria-free culture," Limnol. and Oceanogr., 1964, 9 (2), "On the use of antibiotic for isolating bacteria-free cultures of marine phytoplankton organisms," J. mar. biol Ass., U.K., 1952, 31, "Studies on the culture of a marine diatom," ibid., 1953, 31, "Culture of dinoflagellate Gymnodinium in the soil extract," Amer. J. Bot., 1951, 38(9), Cited by Lewin (1963).