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A cold method for preparing dried reference cultures
Notes and Brief Articles
603
form takes up considerable space (all at the bottom of the condenser) and could cause plugging. Vacuum blockage by condensates, and leaks due to badly fitted joints are the main causes of failure with this system. REFERENCE
DAVIES, D . A. L. ( 1962) . The preservation of larger fungi by freeze-drying. Trans. Br. mycol. Soc. 45, 424-428.
Forest Research Laboratory, Maple, Ontario 44
L. D. TAYLOR, EXPLANATION OF PLATE
Apparatus for freeze-drying fleshy fungi.
A
COLD METHOD FOR PREPARING DRIED REFERENCE CULTURES
The Commonwealth Mycological Institute's method (Anon, 1960) for drying reference cultures, when tried here on locally made hardboard, was found to be unsatisfactory because the cultures either cracked and curled badly or else stuck so tightly that they could not be stripped off. Probably this is due to a diffe rence in the surface of the hardboard used. Pollack (196 7) has given a modification designed to overcome these difficulties but her method, like that of the C.M.I., suffers from the disadvantage of requiring agar to be melted every time a culture needs to be dried. I have found that commercial PYA glue can replace the melted agar: it requires no heating and is always ready for immediate application. There are a number of important points in using this method. (I) It is essential to use a hard type of PVA glue such as that used in woodworking. PYA glues are based on polyvinyl acetate; they are variously formulated but that which I use is known as an 'externally plasticized' type. Bookbinding formulations of PVA glue are often used for mounting herbarium specimens of higher plants but one brand tried was too elastic for preparing dried cultures. (2) The 'Epiglass' PYA used needed diluting approximately 3:2 with water, other brands may need different dilution. The diluting can be done in bulk, but the container needs to be shaken each time before use. (3) A 'Perspex' acrylic plastic sheet is used in place of the hardboard. I t is essential that this be lightly smeared with petroleum jelly (e.g. 'Vaseline') each time before use. (4) The PYA is best applied to the greased surface in numerous separate droplets. (5) A label can be incorporated in the PVA giving a permanent record of the culture number, medium, age and date dried down. It is laid face down on top of the PVA droplets and other PVA droplets are placed on top of it b efore applying the agar culture. Unlike Pollack I have found it unnecessary to partially pre-dry the cultures before removing them from their Petri dishes. I use media Trans. Br, mycol. Soc. 51 (3 & 4) , 1968. Printed in Great Britain
604
Transactions British Mycological Society
with 1·5 % agar and rarely find any difficulty in removing a culture in its entirety from its Petri dish. When laid on the PVA, the cultures may remain uncovered overnight at 20-25 "C in a refrigerated incubator with ice forming on the evaporator. This rapidly dries the cultures and they are completely dry and ready to remove from the plastic plate within 24 h. They can be easily stripped off by inserting a scalpel under the edge of the dried PVA film. Dried cultures produced by this very quick cold method are flexible and are much more tough and durable than those made on melted agar. I am told that it is not likely that the PVA will become brittle with age. I would like to thank Consolidated Chemicals Ltd, Auckland, New Zealand, for advice regarding the formulation and properties of the glue used. REFERENCES
ANON (1960). Herb. I.M.I. Handbook, Commonwealth Mycological Institute. Kew: 103 pp. POLLACK, F. G. (1967). A simple method for preparing dried reference cultures. Mycologia 59,541-544. G. F. LAUNDON,
Horticultural Research Laboratory, Levin, New Zealand
SEPARATION OF ENDOGONE SPORES FROM ORGANIC SOIL DEBRIS BY DIFFERENTIAL SEDIMENTATION ON GELATIN COLUMNS
That Endogone spores are widespread in many soils of N. America, Great Britain, Australia, New Zealand and Africa (Gerdemann & Nicolson, 1963; Mosse & Bowen, 1968b, Redhead priv. comm.) was not fully realized till the technique of wet-sieving was applied to their recovery. Originally used by nematologists to collect eelworms, the technique was modified by Gerdemann (1955) to separate an organic fraction including Endogone spores from the heavier soil particles, by washing the soil through a series of sieves with decreasing pore diameter. The resulting sievings contain Endogone spores mixed with a much larger volume of organic debris, which makes quantitative records or the collection oflarge numbers of spores very laborious. Ohms (1957) attempted to improve spore separation by centrifuging the organic fraction on a sucrose gradient, but we found that the 50 % sucrose needed to separate spores from organic debris damaged the spores. This note describes an alternative technique of sedimentation on layered gelatin columns, which have smaller osmotic pressure than 50 % sucrose. Preparation of gelatin columns. Columns were prepared in glass tubes, 30 em long and 37 mm internal diam, tightly stoppered at one end. Gelatin (Hopkins and Williams, Bacteriological Grade) was dissolved in distilled water to give 20, 15 and 5 % (wjv) solutions. The specific gravities of these solutions were 1·05, 1·04 and I ·02 respectively at 32°C. Liquid 20 % gelatin at about 30° was poured into the glass tubes to a depth of 5 em (approx. 54 ml) and allowed to solidify at 4°. A 5 em Trans. Br. mycol. Soc. 51: (3 & 4), 1968. Printed in Great Britain
603
form takes up considerable space (all at the bottom of the condenser) and could cause plugging. Vacuum blockage by condensates, and leaks due to badly fitted joints are the main causes of failure with this system. REFERENCE
DAVIES, D . A. L. ( 1962) . The preservation of larger fungi by freeze-drying. Trans. Br. mycol. Soc. 45, 424-428.
Forest Research Laboratory, Maple, Ontario 44
L. D. TAYLOR, EXPLANATION OF PLATE
Apparatus for freeze-drying fleshy fungi.
A
COLD METHOD FOR PREPARING DRIED REFERENCE CULTURES
The Commonwealth Mycological Institute's method (Anon, 1960) for drying reference cultures, when tried here on locally made hardboard, was found to be unsatisfactory because the cultures either cracked and curled badly or else stuck so tightly that they could not be stripped off. Probably this is due to a diffe rence in the surface of the hardboard used. Pollack (196 7) has given a modification designed to overcome these difficulties but her method, like that of the C.M.I., suffers from the disadvantage of requiring agar to be melted every time a culture needs to be dried. I have found that commercial PYA glue can replace the melted agar: it requires no heating and is always ready for immediate application. There are a number of important points in using this method. (I) It is essential to use a hard type of PVA glue such as that used in woodworking. PYA glues are based on polyvinyl acetate; they are variously formulated but that which I use is known as an 'externally plasticized' type. Bookbinding formulations of PVA glue are often used for mounting herbarium specimens of higher plants but one brand tried was too elastic for preparing dried cultures. (2) The 'Epiglass' PYA used needed diluting approximately 3:2 with water, other brands may need different dilution. The diluting can be done in bulk, but the container needs to be shaken each time before use. (3) A 'Perspex' acrylic plastic sheet is used in place of the hardboard. I t is essential that this be lightly smeared with petroleum jelly (e.g. 'Vaseline') each time before use. (4) The PYA is best applied to the greased surface in numerous separate droplets. (5) A label can be incorporated in the PVA giving a permanent record of the culture number, medium, age and date dried down. It is laid face down on top of the PVA droplets and other PVA droplets are placed on top of it b efore applying the agar culture. Unlike Pollack I have found it unnecessary to partially pre-dry the cultures before removing them from their Petri dishes. I use media Trans. Br, mycol. Soc. 51 (3 & 4) , 1968. Printed in Great Britain
604
Transactions British Mycological Society
with 1·5 % agar and rarely find any difficulty in removing a culture in its entirety from its Petri dish. When laid on the PVA, the cultures may remain uncovered overnight at 20-25 "C in a refrigerated incubator with ice forming on the evaporator. This rapidly dries the cultures and they are completely dry and ready to remove from the plastic plate within 24 h. They can be easily stripped off by inserting a scalpel under the edge of the dried PVA film. Dried cultures produced by this very quick cold method are flexible and are much more tough and durable than those made on melted agar. I am told that it is not likely that the PVA will become brittle with age. I would like to thank Consolidated Chemicals Ltd, Auckland, New Zealand, for advice regarding the formulation and properties of the glue used. REFERENCES
ANON (1960). Herb. I.M.I. Handbook, Commonwealth Mycological Institute. Kew: 103 pp. POLLACK, F. G. (1967). A simple method for preparing dried reference cultures. Mycologia 59,541-544. G. F. LAUNDON,
Horticultural Research Laboratory, Levin, New Zealand
SEPARATION OF ENDOGONE SPORES FROM ORGANIC SOIL DEBRIS BY DIFFERENTIAL SEDIMENTATION ON GELATIN COLUMNS
That Endogone spores are widespread in many soils of N. America, Great Britain, Australia, New Zealand and Africa (Gerdemann & Nicolson, 1963; Mosse & Bowen, 1968b, Redhead priv. comm.) was not fully realized till the technique of wet-sieving was applied to their recovery. Originally used by nematologists to collect eelworms, the technique was modified by Gerdemann (1955) to separate an organic fraction including Endogone spores from the heavier soil particles, by washing the soil through a series of sieves with decreasing pore diameter. The resulting sievings contain Endogone spores mixed with a much larger volume of organic debris, which makes quantitative records or the collection oflarge numbers of spores very laborious. Ohms (1957) attempted to improve spore separation by centrifuging the organic fraction on a sucrose gradient, but we found that the 50 % sucrose needed to separate spores from organic debris damaged the spores. This note describes an alternative technique of sedimentation on layered gelatin columns, which have smaller osmotic pressure than 50 % sucrose. Preparation of gelatin columns. Columns were prepared in glass tubes, 30 em long and 37 mm internal diam, tightly stoppered at one end. Gelatin (Hopkins and Williams, Bacteriological Grade) was dissolved in distilled water to give 20, 15 and 5 % (wjv) solutions. The specific gravities of these solutions were 1·05, 1·04 and I ·02 respectively at 32°C. Liquid 20 % gelatin at about 30° was poured into the glass tubes to a depth of 5 em (approx. 54 ml) and allowed to solidify at 4°. A 5 em Trans. Br. mycol. Soc. 51: (3 & 4), 1968. Printed in Great Britain