isolation media (i.e. they were isolated from nature 

 in media containing a mixture of known water-solu- 

 ble vitamins) . 



It is remarkable that all the species which 

 have photosynthetic pigments and the related color- 

 less forms require only three vitamins; in order of 

 decreasing incidence, vitamin Bj2, thiamine, and 

 biotin (Table I). Only the phagotrophic flagellates 

 (e.g. Peranema ) seem to need other vitamins and 

 "building blocks". 



Problems in Designing Artificial Media 



It is not generally realized that the classic 

 media for algae (Knop, Beijerinck, Detmer) have 

 been designed before 1900. No pH is mentioned, 

 nor the type of phosphate (mono-, di-, tri-basic) , 

 and, often, if the salts are anhydrous or not. Un- 

 fortunately even the more recent media (up to 19 20 

 and in some cases later) have the same defect. It 

 is not surprising therefore that there exist different 

 interpretations and modifications of these formulas. 

 These solutions were apparently meant to be em- 

 ployed at their natural pH (i.e. pH 5 for Knop, 6.2 

 for the Detmer and 7.2 for the Beijerinck); they pre- 

 cipitate in the more alkaline pHs, yet they have 

 been used successfully even at different pHs. 

 These solutions are in general too concentrated and 

 it was soon found that dilutions of 1:2 to 1:20 per- 

 mitted growth of many more species . Most Chloro- 

 phyceae and many diatoms grow in these media . 



In most algal media, especially the old ones, 

 the only trace metal added is iron. This does not 

 mean that the other trace metals are not needed: it 

 is well known that algae need Mn , Zn , Co, Cu, V, 

 Mo. It only reflects the degree of impurity of the 

 major mineral constituents of the medium. It is 

 worth noting that the industrial methods of purifica- 

 tion of the "chemically pure" salts have undergone 

 many changes since Knop's time resulting in an 

 ever-changing sets of impurities . Besides, with 

 the advent of plastic containers in industry we may 

 expect increasingly pure salts and therefore possi- 

 ble nutritional deficiencies. New deficiencies 

 (and perhaps toxicities) will develop when the nor- 

 mal glassware of the laboratory will be substituted 

 by plastics with consequent absence of the impuri- 

 ties leaching from glass. 



The addition of trace metal-chelate mixtures 

 of media offers the possibility of minimizing the 

 impurities (toxic or favorable) as well as furnishing 

 a metal pool of available trace metals. Therefore 

 chelated media should be more reproducible and 

 withstand fluctuations in impurities of the chemi- 

 cally pure components of media . A further advan- 

 tage is that chelators help in preventing precipita- 

 tion - one of the main goals for reproducibility. 

 A number of chelated media have been developed 

 for a few species of fresh-water algae: Euglena 



(Hutner et al . , 195 6); Ochromonas malhamensis 

 (Hutner et al . , 195 7); O. danlca (Aaronson and 

 Baker, 1959). These media were designed for 

 maximal growth and rapid cell division. To obtain 

 this goal, the media have been enriched, often up 

 to the limits of osmotic tolerance, with as many 

 preformed key metabolites as possible, to spare the 

 organism most of the work of synthesis. Since the 

 success of these media depends upon their exploit- 

 ing all the externally accessible synthetic path- 

 ways (i.e. permeability) and any useful potential 

 and tolerance of the organism in question, they be- 

 come so highly tailored that seldom are they suit- 

 able for other organisms. Only a few chelated 

 fresh-water media are of more general, though still 

 restricted, use, as the Kratz-Myers (1955) medium 

 for Anacystis nidulans , Anabaena variabilis , and 

 Nostoc muscorum , which is also good for other 

 blue-green algae (Phormidium autumnale , Synecho - 

 coccus cedorum , Anabaena cylindrica) , and the 

 medium for Chlamydomonas (Hutner and Provasoli , 

 1951) which serves for its colorless counterpart 

 Polytoma (Cirillo, 1957) and a few other colorless 

 flagellates (Chilomonas , etc.). Perhaps the flexi- 

 bility of these media is due to their being designed 

 for photoautotrophic nutrition, while the others are 

 for heterotrophic nutrition and for eurybionts . Good 

 heterotrophic media contain several amino acids 

 and often other building blocks, some with strong 

 chelating abilities, therefore the trace-metal mix- 

 tures developed for these media are too high in 

 metals for the photoautotrophs . 



The prerequisites for reproducible and ver- 

 satile media for photoautotrophic algae can now be 

 specified: 



a) total-solids concentrations. 



b) concentrations of major elements to suit 

 the prevalent ions required . 



c^) adequate sources of N, and growth fac- 

 tors . 



d) sources of P and avoidance of precipi- 

 tates in alkaline pHs . 



e) pH buffering 



f) trace-metal buffering 



As stated in section I, the best chances to fulfill 

 a) and b) , at least for devising media for isolation 

 and moderate growth, are to mimic as closely as 

 possible the conditions of the natural waters in 

 which the organisms normally bloom. Natural con- 

 ditions in respect to carbonates are hard to repro- 

 duce because carbonates, during sterilization dis- 

 integrate releasing CO2 with resultant alkaliniza- 

 tion and precipitation of the medium. Filter-steri- 

 lized carbonates or CO2 can be added aseptically. 

 Fortunately, carbonates can be substitutes by other 

 anions such as Gl, SO4, PO4 , and NO3 . It is op- 

 portune to introduce as little as possible of chlo- 

 rides and sulphates by employing as much nitrate 

 as is compatible with the organism. Therefore the 

 prevalent ions, often Ga , can be introduced as 



87 



