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The effect of adding trace elements to Czapek-Dox medium
[ 280 ]
THE EFFECT OF ADDING TRACE ELEMENTS TO CZAPEK-DOX MEDIUM By GEORGE SMITH
Department
if Biochemistry, London School if Hygiene and Tropical Medicine
Culture media containing only well-defined, chemically pure substances are widely used for studying fungi. For taxonomic work their chief advantage is their reproducibility by workers in different laboratories, whilst for biochemical studies of all kinds they are obviously more suitable than more complex media made from plant and animal extracts, even though the latter may be essential for maintaining vigour in stock cultures of many species. Probably the most generally used synthetic medium is that devised by Czapek and modified by Dox, containing, per litre, sodium nitrate 2'0 g., potassium chloride 0'5 g., crystalline magnesium sulphate 0'5 g., potassium phosphate (either KH 2P04 or K 2HP04 according to the pH required) 1'0 g., crystalline ferrou s sulphate 0'01 g., and any desirable source of carbon. In this laboratory Czapek-Dox medium, containing 3 % sucrose and 1'5 % agar, as recommended by Thom (1930), has been used for many years in routine examinations of species of Aspergillus and Penicillium, and the same basal medium but with 5 % glucose as source of carbon has been used for numerous biochemical studies. However, during the late war (there is no record ofthe exact date, but, for reasons given below, it was probably th e summer of 1941) the agar medium suddenly changed in character, although, as far as could be ascertained, it was still made up from the same brands of pure chemical s, and from the same batch of agar, a large quantity of which had been purchased prior to the war. It was found that many species of Penicillium began to grow atypically, producing semi-floccose, pale browni sh colonies, with tardy and sparse production of conidia. Sporing usually commenced only at the edges of colonies which had grown to fair size over a period of five or more days. All the constituents of the medium were tested for possible toxic impurities, but nothing was found which could explain the change. Next a batch of medium was made up with tap water and tested alongside the usual medium made with distilled water. There was no appreciable difference between th e two media with any of a large number of species tested. Similarly, additions of yeast extract or corn-steep liquor affected the growth of very few species. It was th en recollect ed that, prior to 1941 , all the distilled wat er used for making culture media came from an old tinned copper still which had been in continuous use since 1929, and that it had frequ ently been reported from various departments of the School that the water contained traces ofcopper. The still was completely destroyed in May 1941 and a more modern type, subsequently installed, produced a distillate in which no trace of copper
Adding Trace Elements to Czapek-Dox Medium. G. Smith 281 could be detected. It was therefore decided to test the effect of adding copper and other trace elements to the ordinary Czapek-Dox medium. (a) Addition of copper andzinc. Four batches of medium were made, one containing no trace elements (except the usual iron), one containing 0'001 % CuS04.5H20, one with 0'001 % ZuS04.7H20, and the fourth with the same percentages of both salts. A number of species of Penicillium, all of which had been found to grow abnormally on the usual medium, were sown simultaneously on all four media. Both copper and zinc, used separately or together, had a marked stimulatory effect on spore production. In general, copper alone was better than zinc alone, and the combination, for a few species, better than either element used singly. (b) Other trace elements. As a preliminary the mixture of trace elements used by Brian, Curtis and Hemming (1946), including iron, copper, zinc, manganese and molybdenum, was tried. There was slight stimulation of some species, but in general the results were much inferior to those obtained with copper and zinc in Exp. I. The concentrations of trace elements in this mixture are apparently too low for maximum effect, iron and zinc being approximately 1/10, and copper 1/70, of the amounts used in Exp. I. (c) Five media were made and tested simultaneously on a number of species of Penicillium. They contained trace elements as follows: (I) Brian's mixture at ten times the recommended concentration; (2) similar but with increased copper; (3) as (2) but with addition of calcium as chloride; (4) copper and zinc alone; (5) none. The actual percentage amounts of salts of trace elements in the four modified media, omitting iron which was present at the same concentration in all five media, were: Copper sulphate Zinc sulphate Manganese sulphate Potassium molybdate Calcium chloride
(I)
(2)
(3)
0' 0 0 0 15 0'001
0'0005 0'001
0'0005 0'001
0'0001 0'0001
0'0001 0'0001
0'0001 0'0001
(4) 0'0005 0'001
0'001
With all the species used cultures on (2), (3) and (4) were almost indistinguishable, and a great improvement on the cultures on ordinary CzapekDox. With some species, but not all, medium (I) was as good as the other trace element media, but in no case was it better. The results indicated that copper and zinc are the essential trace elements and that additions of small amounts of manganese, molybdenum and calcium have little if any effect. (d) In order to find whether copper and zinc, used in the comparatively high concentrations as in medium (4) above, have any adverse effect, this medium was used for the routine subculturing of a collection of about 250 isolates of species of Penicillium. In no case was there any evidence of a toxic effect and with some sixty species there was marked improvement in freedom of spore production. The same medium was then used for growing several hundred species and strains of other genera of moulds. In many cases there was definite stimulation, and only with a few strains of yeasts was there indication of a slight toxic effect.
282
Transactions British Mycological Society
(e) In view of the observed effects of copper and zinc on spore production it was obviously of interest to test the effect of these elements on yields of metabolic products, since it has often been noted in these laboratories that the best yields of characteristic products are associated with rapid and typical growth. Very encouraging results have already been obtained with two species of Penicillium. Citromycetin. This substance, first described by Hetherington and Raistrick (193Ia), is produced by most strains of P. glabrum (Wehmer) Westling. This species was chosen because it had already been found that most "trains grow atypically on ordinary Czapek-Dox and are markedly stimulated by additions of copper and zinc. Citromycetin is readily detected in culture fluids by adding ferric chloride solution, this giving a very intense green colour. With some strains of the mould the ferric chloride reaction, after about two weeks' incubation, is so intense that the solution, contained in an ordinary test-tube, is quite opaque and has to be diluted many times before becoming transparent. The relative amounts of citromycetin produced under different cultural conditions, or by different strains of the mould, may be roughly estimated by comparing the dilutions required to bring the solution to some arbitrary standard depth of colour. Seven strains of P. glabrum were used for the experiment. Each strain was sown in four flasks of ordinary Czapek-Dox solution containing 5 % glucose (hereafter denoted as C-medium) and four flasks of the same medium to which was added 0'0005 % copper sulphate and 0'001 % zinc sulphate (CT-medium). The flasks were of one litre capacity and each contained 350 mI. of medium. All the flasks used for one strain were sown from as nearly as possible identical slopes and were incubated at 240 C. Flasks were harvested after five, eight, eleven and fifteen days' incubation. Growth on CT-medium was much more rapid than on C-medium, as was also the consumption of sugar. For example, after eleven days' incubation residual glucose in the CT-medium varied, for different strains, from 1'3 to 1·6 %, and on C-medium from 3'0 to 3'9 %. On C-medium none of the strains gave, at any stage of incubation, more than a faint reaction for citromycetin. On CT-medium the worst strain gave a ferric chloride colour which was only just transparent, whilst the other six strains gave reactions ten to seventeen times as intense. Citrinin. This yellow pigment was first isolated from P. citrinum Thorn by Hetherington and Raistrick (1931 b). It is also produced in good yield by P. implicatum Biourge (author's unpublished observation). It is readily isolated, as it is precipitated almost completely on acidification of the culture fluid. P. implicatum was sown on C- and CT-media (as described above), thirty flasks of each medium. It had been found previously that the yield of citrinin reaches a maximum when the apparent residual glucose (by polarimeter) has fallen to between o-5 and 1'0 %. The cultures on CT-medium were harvested after nineteen days' incubation, when the apparent residual glucose was 0·66 %. The culture fluid, on acidification, gave 20'0 g. of good quality citrinin. The cultures on C-medium showed considerable variation. Of two flasks tested after nineteen days' incubation one had 1'13 % residual glucose and gave no precipitate on acidification, whilst the other had 1'32 %glucose and gave a fair amount of precipitate.
Adding Trace Elements to Czapek-Dox Medium. G. Smith 283 The cultures were finally harvested after twenty-seven days and gave only 5'9 g. of crude citrinin which was somewhat brownish and of inferior quality to that obtained from the CT-medium. SUMMARY 1. Some moulds, particularly a number of species of Penicillium, grow atypically on Czapek-Dox medium made up with pure chemicals. 2. The medium is satisfactory if copper (o-oooj % CuS04.SH20) and zinc (0'001 % ZnS04.7H20) are added. 3. Addition of copper and zinc stimulates production of citromycetin by P. glabrum and citrinin by P. implicatum.
REFERENCES BRIAN, P. W., CURTIS, P. J. & HEMMING, H. G. (1946). A substance causing abnormal development of fungal hyphae produced by Penicillium Jaru;zewskii Zal. Trans. Brit. mycol. Soc. XXIX, 173-87. HETHERINGTON, A. C. & RAISTRICK, H. (193Ia). On citromycetin, a new yellow colouring matter produced from glucose by species of Citromyces. Phil. Trans. Roy. Soc., Lond., B, ccxx, 20g-44HETHERINGTON, A. C. & RAISTRICK, H. (1931 b). On the production and chemical constitution of a new yellow colouring matter, citrinin, produced from glucose by Penicillium citrinum Thorn. Phil. Trans. Roy. s«, Lond., ccxx, 269-95. THOM, C. (1930). The Penicillia. London: Bailliere, Tindall and Cox.
(Accepted for publication 12 April 1948)
THE EFFECT OF ADDING TRACE ELEMENTS TO CZAPEK-DOX MEDIUM By GEORGE SMITH
Department
if Biochemistry, London School if Hygiene and Tropical Medicine
Culture media containing only well-defined, chemically pure substances are widely used for studying fungi. For taxonomic work their chief advantage is their reproducibility by workers in different laboratories, whilst for biochemical studies of all kinds they are obviously more suitable than more complex media made from plant and animal extracts, even though the latter may be essential for maintaining vigour in stock cultures of many species. Probably the most generally used synthetic medium is that devised by Czapek and modified by Dox, containing, per litre, sodium nitrate 2'0 g., potassium chloride 0'5 g., crystalline magnesium sulphate 0'5 g., potassium phosphate (either KH 2P04 or K 2HP04 according to the pH required) 1'0 g., crystalline ferrou s sulphate 0'01 g., and any desirable source of carbon. In this laboratory Czapek-Dox medium, containing 3 % sucrose and 1'5 % agar, as recommended by Thom (1930), has been used for many years in routine examinations of species of Aspergillus and Penicillium, and the same basal medium but with 5 % glucose as source of carbon has been used for numerous biochemical studies. However, during the late war (there is no record ofthe exact date, but, for reasons given below, it was probably th e summer of 1941) the agar medium suddenly changed in character, although, as far as could be ascertained, it was still made up from the same brands of pure chemical s, and from the same batch of agar, a large quantity of which had been purchased prior to the war. It was found that many species of Penicillium began to grow atypically, producing semi-floccose, pale browni sh colonies, with tardy and sparse production of conidia. Sporing usually commenced only at the edges of colonies which had grown to fair size over a period of five or more days. All the constituents of the medium were tested for possible toxic impurities, but nothing was found which could explain the change. Next a batch of medium was made up with tap water and tested alongside the usual medium made with distilled water. There was no appreciable difference between th e two media with any of a large number of species tested. Similarly, additions of yeast extract or corn-steep liquor affected the growth of very few species. It was th en recollect ed that, prior to 1941 , all the distilled wat er used for making culture media came from an old tinned copper still which had been in continuous use since 1929, and that it had frequ ently been reported from various departments of the School that the water contained traces ofcopper. The still was completely destroyed in May 1941 and a more modern type, subsequently installed, produced a distillate in which no trace of copper
Adding Trace Elements to Czapek-Dox Medium. G. Smith 281 could be detected. It was therefore decided to test the effect of adding copper and other trace elements to the ordinary Czapek-Dox medium. (a) Addition of copper andzinc. Four batches of medium were made, one containing no trace elements (except the usual iron), one containing 0'001 % CuS04.5H20, one with 0'001 % ZuS04.7H20, and the fourth with the same percentages of both salts. A number of species of Penicillium, all of which had been found to grow abnormally on the usual medium, were sown simultaneously on all four media. Both copper and zinc, used separately or together, had a marked stimulatory effect on spore production. In general, copper alone was better than zinc alone, and the combination, for a few species, better than either element used singly. (b) Other trace elements. As a preliminary the mixture of trace elements used by Brian, Curtis and Hemming (1946), including iron, copper, zinc, manganese and molybdenum, was tried. There was slight stimulation of some species, but in general the results were much inferior to those obtained with copper and zinc in Exp. I. The concentrations of trace elements in this mixture are apparently too low for maximum effect, iron and zinc being approximately 1/10, and copper 1/70, of the amounts used in Exp. I. (c) Five media were made and tested simultaneously on a number of species of Penicillium. They contained trace elements as follows: (I) Brian's mixture at ten times the recommended concentration; (2) similar but with increased copper; (3) as (2) but with addition of calcium as chloride; (4) copper and zinc alone; (5) none. The actual percentage amounts of salts of trace elements in the four modified media, omitting iron which was present at the same concentration in all five media, were: Copper sulphate Zinc sulphate Manganese sulphate Potassium molybdate Calcium chloride
(I)
(2)
(3)
0' 0 0 0 15 0'001
0'0005 0'001
0'0005 0'001
0'0001 0'0001
0'0001 0'0001
0'0001 0'0001
(4) 0'0005 0'001
0'001
With all the species used cultures on (2), (3) and (4) were almost indistinguishable, and a great improvement on the cultures on ordinary CzapekDox. With some species, but not all, medium (I) was as good as the other trace element media, but in no case was it better. The results indicated that copper and zinc are the essential trace elements and that additions of small amounts of manganese, molybdenum and calcium have little if any effect. (d) In order to find whether copper and zinc, used in the comparatively high concentrations as in medium (4) above, have any adverse effect, this medium was used for the routine subculturing of a collection of about 250 isolates of species of Penicillium. In no case was there any evidence of a toxic effect and with some sixty species there was marked improvement in freedom of spore production. The same medium was then used for growing several hundred species and strains of other genera of moulds. In many cases there was definite stimulation, and only with a few strains of yeasts was there indication of a slight toxic effect.
282
Transactions British Mycological Society
(e) In view of the observed effects of copper and zinc on spore production it was obviously of interest to test the effect of these elements on yields of metabolic products, since it has often been noted in these laboratories that the best yields of characteristic products are associated with rapid and typical growth. Very encouraging results have already been obtained with two species of Penicillium. Citromycetin. This substance, first described by Hetherington and Raistrick (193Ia), is produced by most strains of P. glabrum (Wehmer) Westling. This species was chosen because it had already been found that most "trains grow atypically on ordinary Czapek-Dox and are markedly stimulated by additions of copper and zinc. Citromycetin is readily detected in culture fluids by adding ferric chloride solution, this giving a very intense green colour. With some strains of the mould the ferric chloride reaction, after about two weeks' incubation, is so intense that the solution, contained in an ordinary test-tube, is quite opaque and has to be diluted many times before becoming transparent. The relative amounts of citromycetin produced under different cultural conditions, or by different strains of the mould, may be roughly estimated by comparing the dilutions required to bring the solution to some arbitrary standard depth of colour. Seven strains of P. glabrum were used for the experiment. Each strain was sown in four flasks of ordinary Czapek-Dox solution containing 5 % glucose (hereafter denoted as C-medium) and four flasks of the same medium to which was added 0'0005 % copper sulphate and 0'001 % zinc sulphate (CT-medium). The flasks were of one litre capacity and each contained 350 mI. of medium. All the flasks used for one strain were sown from as nearly as possible identical slopes and were incubated at 240 C. Flasks were harvested after five, eight, eleven and fifteen days' incubation. Growth on CT-medium was much more rapid than on C-medium, as was also the consumption of sugar. For example, after eleven days' incubation residual glucose in the CT-medium varied, for different strains, from 1'3 to 1·6 %, and on C-medium from 3'0 to 3'9 %. On C-medium none of the strains gave, at any stage of incubation, more than a faint reaction for citromycetin. On CT-medium the worst strain gave a ferric chloride colour which was only just transparent, whilst the other six strains gave reactions ten to seventeen times as intense. Citrinin. This yellow pigment was first isolated from P. citrinum Thorn by Hetherington and Raistrick (1931 b). It is also produced in good yield by P. implicatum Biourge (author's unpublished observation). It is readily isolated, as it is precipitated almost completely on acidification of the culture fluid. P. implicatum was sown on C- and CT-media (as described above), thirty flasks of each medium. It had been found previously that the yield of citrinin reaches a maximum when the apparent residual glucose (by polarimeter) has fallen to between o-5 and 1'0 %. The cultures on CT-medium were harvested after nineteen days' incubation, when the apparent residual glucose was 0·66 %. The culture fluid, on acidification, gave 20'0 g. of good quality citrinin. The cultures on C-medium showed considerable variation. Of two flasks tested after nineteen days' incubation one had 1'13 % residual glucose and gave no precipitate on acidification, whilst the other had 1'32 %glucose and gave a fair amount of precipitate.
Adding Trace Elements to Czapek-Dox Medium. G. Smith 283 The cultures were finally harvested after twenty-seven days and gave only 5'9 g. of crude citrinin which was somewhat brownish and of inferior quality to that obtained from the CT-medium. SUMMARY 1. Some moulds, particularly a number of species of Penicillium, grow atypically on Czapek-Dox medium made up with pure chemicals. 2. The medium is satisfactory if copper (o-oooj % CuS04.SH20) and zinc (0'001 % ZnS04.7H20) are added. 3. Addition of copper and zinc stimulates production of citromycetin by P. glabrum and citrinin by P. implicatum.
REFERENCES BRIAN, P. W., CURTIS, P. J. & HEMMING, H. G. (1946). A substance causing abnormal development of fungal hyphae produced by Penicillium Jaru;zewskii Zal. Trans. Brit. mycol. Soc. XXIX, 173-87. HETHERINGTON, A. C. & RAISTRICK, H. (193Ia). On citromycetin, a new yellow colouring matter produced from glucose by species of Citromyces. Phil. Trans. Roy. Soc., Lond., B, ccxx, 20g-44HETHERINGTON, A. C. & RAISTRICK, H. (1931 b). On the production and chemical constitution of a new yellow colouring matter, citrinin, produced from glucose by Penicillium citrinum Thorn. Phil. Trans. Roy. s«, Lond., ccxx, 269-95. THOM, C. (1930). The Penicillia. London: Bailliere, Tindall and Cox.
(Accepted for publication 12 April 1948)