Physiological studies on phytoplankton such as nitrogen assimilation requires the use of pure cultures. It is a matter of relative ease to separate the various forms and cultivate them under artificial conditions but difficulty is frequently encountered in freeing the phytoplankton of contaminating bacteria

Long, J. Harold

Zobell, Claude E.

English

Yale University

      P.O. BOX 208118 | NEW HAVEN CT 06520-8118 USA | PEABODY.YALE. EDU   JOURNAL OF MARINE RESEARCH The Journal of Marine Research, one of the oldest journals in American marine science, published important peer-reviewed original research on a broad array of topics in physical, biological, and chemical oceanography vital to the academic oceanographic community in the long and rich tradition of the Sears Foundation for Marine Research at Yale University.  An archive of all issues from 1937 to 2021 (Volume 1–79) are available through EliScholar,  a digital platform for scholarly publishing provided by Yale University Library at  https://elischolar.library.yale.edu/.  Requests for permission to clear rights for use of this content should be directed to the authors, their estates, or other representatives. The Journal of Marine Research has no contact information beyond the affiliations listed in the published articles. We ask that you provide attribution to the Journal of Marine Research.  Yale University provides access to these materials for educational and research purposes only. Copyright or other proprietary rights to content contained in this document may be held by individuals or entities other than, or in addition to, Yale University. You are solely responsible for determining the ownership of the copyright, and for obtaining permission for your intended use. Yale University makes no warranty that your distribution, reproduction, or other use of these materials will not infringe the rights of third parties.  This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. https://creativecommons.org/licenses/by-nc-sa/4.0/   STUDIES ON THE ISOLATION OF BACTERIA-FREE CULTURES OF MARINE PHYTOPLANKTON BY CLAUDE E. ZOBELL AND .J. HAROLD LONG* Scripps Insti tution of Oceanographyt University of California, La Jolla, California Physiological studies on phytoplankton such as nitrogen assimilation re-quires the use of pure cultures. It is a matter of relative ease to separate the various forms and cultivate them under artificial conditions but difficulty is frequently encountered in freeing the phytoplankton of contaminating bacteri a. For the isolation of phytoplankton from the sea All en (1913) and Schrei-ber (1927) recommend the use of mineral enrichment media exposed to li ght which favors the multiplication of photosynthetic organisms but not of saprophytic bacteri a. Pringsheim (1924) gives directions for separating the various species of algae or diatoms from the enrichment cultures either mechanicall y by picking single cell s under a microscope with a loop or micro-pipette, or culturally on solid media. The latter method consists of inoculating agar or sili ca gel media impregnated with mineral salts with a loopful of the enrichment culture and exposing to li ght. If t he inoculum i properly diluted discrete colonies will develop which can be isolated wi.th an inoculating needle, transferred to tubes of media and examined microscop-icall y or culturally for purity. Freedom from other algae or diatoms can usuall y be ascertained by the direct microscopic examination of slide preparations but it is necessary to inoculate nutrient media to prove the absence of bacteri a because the latter may escape visual detection. Due to their tendency to grow associated mth phytoplankton as epiphytes or periphytes (ZoBell , 1937) bacteria will nearl y always be present. The careful examination of a single diatom may rewal the presence of numerous tenaciously-attached bacteria. In order to free the phytoplankton of these contaminants, it may be necessary to sub-cul-ture them several times seriatim on enrichment media. Although the mineral-enrichment media do not provide for the multipli cation of sapro-phytic bacteria, a few bacteria will nearl y ahvays grow at the expense of the organic matter synthesized by the photosynthetic organisms. It is only * Professor of Biology, Christian Coll ege, Columbia, Missouri. Visiting investi-gator at the Scripps Institution during the summer of 1938. t Contribution from the Scripps Institution of Oceanography, New Series No. 41. ( 328 ) 1937-8] JOURNAL OF i\lIARINE RESEARCH 3:29 because the phytoplankton multiply much faster than the bacteria in the enrichment media exposed to light that it is possible eventually to obtain bacteria-free cultures by serial transfers. One's chances for success are enhanced by picking the colonies as soon as they appear before the associated bacteria have had an opportunity to multiply at the expense of the diatoms or algae which die as the cultures become older. In an attempt to simplify and improve the procedure of eliminating the bacteria from pure cultures of phytoplankton several different methods have been examined. Luck, Sheets and Thomas (1931) have summarized the methods of ob-taining bacteria-free cultures of protozoa. It was thought that some of these methods might be applicable to our problems. Washing the organisms in several changes of sterile water is impracticable because it was found that most of the bacteria are firmly attached to the diatoms which were examined. ZoBell and Allen (1933) found that about half of the bacteria in the sea are periphytes which attach so tenaciously to solid surfaces that they resist being washed off with running water. This periphytic characteristic of bacteria also invalidated the use of the cataphoretic method (Chapman, 1929) of freeing the diatoms of the bacteria although in an electrical fi eld most of the unattached bacteria moved to the anode while the diatoms moved to the cathode. While one can attempt to select with the micro-manipulator the single cells which appear to be bacteria-free, many attached bacteria escape detection from the mere microscopic examination of the diatoms. Because of their photosynthetic mode of existence one may expect phyto-plankton to be more resistant to radiations than bacteria. However, we have not been able to find any intensity of wave-lengths between 2760 and 4100 A which were bactericidal but not injurious to the diatoms. Several enriched raw cultures from the sea as well as cultures of Nitzschia closteriurn, N. bilobata, Navicula sp. and Ghlorella sp. have been tried. Shallow layers of water containing the organisms, as well as agar plates seeded with them, were exposed for different time intervals to varying intensities of radiations from a quartz mercury vapor lamp. In no instance did the phytoplankton survive intensities of ultra-violet radiations which were required to kill the bacteria. Rather broad ranges of frequencies were used, however, so it is possible that a selective action may be found if narrow radiation bands are used. Also it is possible that wave-lengths shorter than 2760 A may be more efficacious. According to Duggar (1936) wave-lengths from 1900 to 2950 A are more bactericidal than the longer ultra-violet radiations. Various workers have obtained bacteria-free cultures of protozoa by sterilizing the cysts with heat or chemicals. We find that t~ere are m~ny bacteria in the sea which are more heat-tolerant than the diatoms, which rules out temperature as a means of ridding phytoplankton of contaminating 330 SEARS FO UNDATION [I, 4 bacteria. The observations are summarized in Table I which shows the relative numbers of bacteria and phytoplankton which survived exposure to diff erent temperatures. The heat resistance of the bacteria was tested by plating out on nutrient agar samples of sea water which bad been held at the stated temperatures for ten minutes. The number of colonies which developed from the sample held at 22° C. (room temperature) was arbitrarily taken as 100 per cent and the others were calculated upon a basis of this. The temperature tolerance of" phytoplankton" was determined by heating those mentioned above, including one mixed enriched culture, to the stated temperature for ten minutes and then incubating them to see if they grew. TABL E I P ER CENT OF MARINE BACTERIA AND PHYTOPLANKTON SURVIVING TEN ~lI KUTES' EXPOSURE TO THE STATED TEMPERATURES T emperature in °C 22° 30° 40° 50° 60° 80° Bacteria(seawaterfiora) 100 74. 1 27.6 5 .7 3 .5 1.8 Phytoplankton (five specimens) 100 60 20 0 0 0 In the search for selective bactericidal substances the following ha Ye been tried in various concentrations ranging from that which was high enough to kill the mixed marine bacterial flora to that which was suffi ciently low to be tolerated by the bacteria for ten minutes at room temperature: Toluene, chloroform, sodium carbonate, hydrochloric acid, chloramine T, sodium perborate, potassium chlorate, mercurochrome, boric acid, thymol and certain aniline dyes. With the exception of toluene and chloroform which are not readily soluble in water, critical concentrations of nearly all of the chemicals were found which killed many bacteria and in which the phyto-plankton survived, but some bacteria always survived in concentrations of the chemicals which are lethal for the phytoplankton. In view of the great tolerance of certain kinds of bacteria it seems doubtful if an,r bactericidal agent will be found which will kill all contaminating bacteria without being deleterious to the phytoplankton. The use of the highest concentrations of the germicides tolerated by the diatoms may somewhat enhance one' chances of obtaining bacteria-free cul tures, but the adrnntage of uch treatment are not sufficiently great to warrant the procedure. In some of the foregoing experiments it was noted that bacteri a were attenuated by treatment with sub-lethal concentrations of certain germi-cides. This suggested the possibili ty of finding a selecfo·e bacteriostatic substance which would restri ct the multipli cation of bacteria in concentra-tions in which the phytoplankton would grow. Certain aniline dies were tried, of which there are many as pointed out by Churchman (1923a), which suppress the reproductive mechanism of bacteria without otherwi se injuring protoplasm. 1937-8] JOURNAL OF MARINE RESEAR CH 331 In the preliminary tests concentrations of the dyes ranging from 1 : 1000 to 1 : 100,000 were added to appropriate media for bacteria and diatoms respectively. The nutrient medium for the bacteria consisted of 0.5 per cent Bacto-peptone and 0.3 per cent beef extract in sea water. Tubes containing 10 cc. each were inoculated with a mixed culture of marine bacteria and incubated in the dark. The enrichment medium for the diatoms consisted of aged sea water to which was added 0.10 per cent KN03 and 0.005 per cent each of Na2HP0d2H20, CaCl2.6H20, Na2Si03.9H 20 and FcCl3.6H20. It was dispensed in 10 cc. quantiti es in test tubes, inoculated with a pure bacteria-free culture of N. closteriiirn, and exposed to li ght. The relative amounts of growth, as indicated by turbidity, which occurred after different periods of incubation are recorded in Table II . TABLE II COMPARATIVE MULTIPLI CATION OF MARINE BAC'l'ERIA ( MIX ED CULTURE) AN D DIATOMS (N . closterium) I N MEDIA CONTAINING DIFFERENT CONCE NTRATIONS OF DYE S Growth of bacteria c;fter Growth of diatoms after Dye Concen-/ration 4 days 7 days 14 days 4 days 7 days 14 cloys ------ ---------Crystal violet 1 : 10,000 - - - - - -Crystal violet 1 : 100,000 - + ++ + ++ ++++ Brill i ant green 1 : 10,000 ++ +++ +++ - - -Brill iant green 1 : 100,000 ++ +++-t ++++ + ++ ++ Thionin 1 : 10,000 + ++ ++++ - - -Thionin 1 : 100,000 + + ++++ ++++ - + +++ Basic fushsin 1 : 1000 - - ++ - - -Basic f ushsin 1 : 10,000 + ++ +++ - + ++ Acrifiavine 1 : 10,000 - - - + ++ +++ + Acrifiavine 1 : 100,000 + ++ ++ + ++ ++++ Fl it01 escein 1 : 1000 - + ++ - - -Fluore.~cein l : 10,000 ++ +++ ++++ + + +++ None (cnntrol) ++ ++++ ++++ + ++ ++++ Acriflavine was the only dye in the series which proved to be bacterio-stalic in concentrations which did not interfere greatly with the growth of the diatoms. Crystal violet showed no selecti ve ac~io~, bein~ equal!y inimical to bacteria and diatoms. Brilliant green, th1onm, basic fuchsm and fluorescein were more toxic for diatoms than for bacteria. Acriflavine was more bacteriostatic when the cultures were exposed to the light than when they were incubated in the dark probably due to the photodynamical activation of the dye. 332 SEARS FOUN DATION [I, 4 Additional experiments were performed with acrift avine using concentra-tions of the dye ranging from 1 : 5000 to 1 : 20,000. Table III shows the comparative numbers of bacteria and diatoms which grew. The data on bacteria were obtained by plating samples of raw sea water with nutrient media containing the dye and counting the colonies which developed. The diatom data were obtained by inoculating sea water containing nutrient salts plus the dye with N. closterium and determining the amounts of growth after 12 days incubation in the li ght by centrifuging 5 cc. of the medium in Hopkins tubes. The amount of growth or the number of colonies which developed in the respective media lacking dye was arbitrarily taken as 100 and the others calculated on a percentage basis. Only 13 per cent as many bacteria (mixed microflora indigenous to the sea) developed colonies on medium containing acrifl avine in a concentration of 1 : 10,000 as on simi lar medium lacking the dye. The growth of the diatom, N. closterium, was inhibited very li ttle by this concentration of the dye. Similarly N. bilobata, Navicula sp. and Chlorella sp. grew in the presence of one part of acriflavine in 10,000 of sea-water medium. Several bacteria-free cultures of algae and phytoplankton have been isolated fr om enrichment cultures to which acriflavine had been added. Under these conditions the bacteria associated with the raw cultures multi-plied very littl e or not at all, the dye being more bacteriostatic in the mineral enrichment media than in the organic-matter-rich media in which the results reported in Table III were obtained. The dye was used in the TABL E III COMPARATIVE MULTIPLICATIO N OF BACTERIA AN D DIATOMS I N SEA WATER 11EDIA CONTAINING DIFFERENT CONCENTRATIONS OF ACRIFLAVINE Concentration of Acrifiavine 1 : 5,000 1 : 7,500 1 : 10,000 1 : 15,000 1: 20,000 None Bacteria (sea water flora ) 6 10 13 38 67 100 Diatoms (N. closteriurn) 20 54 89 93 96 100 initi ally inoculated mineral enrichment medium as well a-S in a similar medium solidifi ed with agar upon which the enriched culture '-a-S treaked to obtain pure cultures of the phytoplankton. Most of the colonies picked from the fir st sub-culture on the solid medium proved to be bacteri a-free. With less than ten per cent of the cul tures it has been neces aJ·y to re ort to serial sub-cultures on solid medium to eliminate contaminating bacteri a. Unfortunately there are a few acrifl avine-fast bacteri a from which the phytoplankton can be separated only by repeated diluti on by uccessive 1937-8] JOURNAL OF MARI NE RESEARCH 333 sub-cultures. It is possible that some other selecti ve bacteriostatic sub-stance could be used in combination with acriflavine to inhibit the multi-plication of such acriflavine-fast strains of bacteria. Churchman (1929b) found that a mixture of acriflavine and gentian violet was much more bactericidal than either dye used alone. Additional dyes alone and in combination are being tried in an attempt further to simplif y the purifica-tion of cultures of marine phytoplankton. Although the procedure has not been appli ed to enough phytoplankton nor under enough different conditions to warrant definite conclusions, the results obtained in the foregoing experiments seemed to recommend the use of acriflavine in enrichment media to facilit ate the isolation of bacteria-free cultures of certain diatoms. Whether or not the procedure is generall y applicable to marine phytoplankton can be ascertained only by working on many more cultures under various conditions of culti vation. Experiments along this line are being continued. SUMMARY The methods of isolating bacteria-free cultures of phytoplankton are discussed. Periphytic bacteria which are tenaciously attached to the phytoplankton invalidate washing, cataphoresis or the use of the micromanipulator for freeing the phytoplankton of bacteria. Ultra-violet radiations of wave-lengths between 2760 and 4100 A suffi-cientl y intense to kill contaminating bacteria also kill ed the diatoms which were tested. Several bactericidal substances were tried but they were all lethal for N. clostcriiirn and other diatoms in concentrations which kill ed bacteria. Certain aniline dyes were found to have a selecti ve genesistatic action. Acriflavine in a concentration of 1 : 10,000 inhibits the multipli cation of most marine bacteria without appreciably effecting the reproduction of diatoms. Its use in enrichment media greatly facilitates the isolation of bacteria-free cultures of certain phytoplankton. The authors acknowledge with gratitude the technical assistance rendered by Catharine B. Feltham and Charles C. Teter. 334 SEARS FOUNDATION REFERENCES ALLEN, E. J . [I, 4 l l l l l 1913. On the culture of the plankton diatom, Thalassiosira gravida Cleve, in artificia l sea water. Jour. M ar. Biol. Assoc., 10: 417-439. CHAPMAN, G. H. 1929. E lectrophoretic potential as an aid in identifying strains of the B. coli group. Jour. Bact., 18: 339-342. CHURCHMAN, J . W. 1923a. Genesistatic action of dyes. Proc. Nat. Acad. Sci., 9: 78---84. CHURCHMA N, J . W. 1923b. Bacteriostasi  by mixture of dyes. Jour. Exper. Med., 38: 1-8. DUGGAR, B. M. 1936. Biological effects of radiation. McGraw Hill Book Co., Kew York, Vol. II, pp. 1119- 1150. LUCK, J. M ., SHEETS, G., AN D THOMAS, J . 0. 1931. The role of bacteria in the nutrition of protozoa. Quart. Rev. Biol. , 6: 46-58. PRINGSHEIM, E. G. 1924. Algenkultur. Abderhalden's Handb. d. biol. Arbeitsmethoden. Abt. XI , t. 2: 377-406. S CHREIBER, E. 1927. Die Reincultur von marinen Phytoplankton und deren Bedentung fur die Erforschung der Produktionsfahigheit des iVIeerwassers. TI"iss. :i\Ieeres-untersuchungen, 16: 1-35. ZoBELL, C. E., AND ALLEN, E. C. 1935. The significance of marine bacteria in the fouling of submerged urfaces. Jour. Bact., 29: 239-251. ZoBELL, C. E. 1935. The assimilation of ammonium nitrogen by Nitzschia. closterium and other marine phytoplankton. Proc. Nat. Acad. Sci., 21 : 517-522. ZoBELL, C. E. 1936. Periphytic habits of some marine bacteria. Proc. Soc. Exper. Biol. and Med., 35: 270-273. 

Studies on the isolation of bacteria-free cultures of marine phytoplankton

https://elischolar.library.yale.edu/cgi/viewcontent.cgi?article=1536&context=journal_of_marine_research

Studies on the isolation of bacteria-free cultures of marine phytoplankton

Abstract

Similar works

Full text

Available Versions

Yale University