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Effect of additives and storage conditions on antibody titers of tobacco mosaic and Southern Bean Mosaic virus antisera
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observed that the supernatant fraction had an almost similar effect as the crude hemolymph, indicating that the active component may be a free amino acid pool, but not proteins. EFFECT OF ADDITIVES AND STORAGE CONDITIONS ON ANTIBODY TITERS OF TOBACCO MOSAIC AND SOUTHERN BEAN MOSAIC VIRUS ANTISERA H. E. WATERWORTH, J. W. BLIZZARD, AND R. H. LAWSON Plant Science Research, New Crops Branch, U.S. Pbnt Introduction Station, Glenn Dale, Maryland 20769, American Type Culture Collection, Rockville, Maryland 20852, and Omamgntal Investigations, Plant Industry Station, Beltsville, Maqland 20705 There is no report of a systematic study on the effects of various methods of storing plant virus antisera on reactive antibody titer. As a part of a cooperative program between the U.S. Department of Agriculture, The American Type Culture Collection, and individual plant virologists, antisera to viruses were donated by the latter to the ATCC for distribution to others. In this study antisera to tobacco mosaic virus (TMV) and Southern Bean Mosaic virus (SBMV) were stored using those preservatives, storage temperatures, and methods of handling commonly used by plant virologists. TMV and SBMV antisera were selected because they represent a rod-shaped and spherical virus, respectively. One-half-milliliter aliquots were (1) stored at -70, -20, t4, +26, or f37” C; (2) frozen and thawed 260 times; (3) stored in 5% peptone, 0.02% sodium azide; or an equal volume of glycerine; (4) diluted prior to storage: or (5) freeze-dried. Most of the treatments had little or no effect on reactive antibody titer except: (1) when stored at 37” C the titer of non-freeze-dried TMV antiserum dronped to 0 after 1 year while that of SBMV lost about 75% of its reactivity; (2) freeze-dried TMV antiserum did not drop in reactivity when stored at 37” C while SBMV again lost about 75% of its reactivity; (3) freeze-dried SBMV antiserum stored with peptone maintained all of its reactivity at all storage temperPture.7 while sodium azide was onlv partially effective at 37” C; (4) sodium azide was superior to glycerine in both antisera stored at +26 or +37” C for more than 6 months: (5) non-freeze-drying was superior to freeze-drying TMV antiserum when stored at the lowest four temperatures; and (6) SBMV maintained its reactivity better when stored nreviously diluted I:512 to I:1024 with saline. Consult Ann. Appl.
CONFERENCE Biol. 1973-1974 work. FREEZING
ABSTRACTS for
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OF PARASITIC AND RELATED FREE-LIVING AMOEBAE ROGER G. ZIM:
American Type Culture Collection, Rockville, Ma yland Preliminary studies on the preservation of parasitic and related free-living amoebae indicate that the majority of these organisms can he preserved either by freezing the trophozoites or by drying the encysted stage. Recently two species of soil amoebae, Acanthamoeba and Naegbria, that were thought to he free-living, have been proved to he causative agents of human disease. As a result of this renewed interest in these forms the ATCC has acquired representative cultures isolated from environmental sources as well as from infected hnmans. Free-living isolates derived from soil, swimming pools, lakes, and streams, if grown in the presence of bacteria, readily form cysts that are resistant to drying. We have found that when we suspend these cysts in a sucrose solution they can he dried, sealed, and stored under vacuum for long periods of time. Until now isolates of Naegleria isolated from fatal cases of meningoencephalitis have been propagated on monkey kidney tissue culture. These pathogenic amoebae have proved to be taxonomically similar to Naegleria found free-living in nature. However, pathogenic amoebae grow at a higher temperature and’, if the culture is bacteria-free, do not form cysts. Our studies indicate that there is a difference in the way that various strains of these pathogenic amoebae recover from freezing when DMSO is used as a cryoprotectant. Although few organisms survive freezing, we have found no gross cultural or morphological changes in cultures derived from preserved material. Initial culture growth is slower at first hut gradually becomes comparable to that of unfrozen amoebae after one passage. Amoebae recovering from preservation are still able to cause normal cytopathogenic effects in tissue cultures. Although various methods of freezing axenic cultures of Entamoeha have been reported, they have proved to he quite unreliable and require a large number of organisms. These intestinal parasites of animals fail to form cysts in axenic cultures. Therefore, effective cryobiologic preservation of these organisms must involve the vegetative cell. By using sucrose and DMSO in the freezing suspension we have heen able to improve rrcovery of frozen Entamoeba cultures. The way in which the culture is handled prior to freezing and after thawing also has a marked effect on recovery and subsequent growth of these organisms.
FREEZING
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FREEZE-DRYING
observed that the supernatant fraction had an almost similar effect as the crude hemolymph, indicating that the active component may be a free amino acid pool, but not proteins. EFFECT OF ADDITIVES AND STORAGE CONDITIONS ON ANTIBODY TITERS OF TOBACCO MOSAIC AND SOUTHERN BEAN MOSAIC VIRUS ANTISERA H. E. WATERWORTH, J. W. BLIZZARD, AND R. H. LAWSON Plant Science Research, New Crops Branch, U.S. Pbnt Introduction Station, Glenn Dale, Maryland 20769, American Type Culture Collection, Rockville, Maryland 20852, and Omamgntal Investigations, Plant Industry Station, Beltsville, Maqland 20705 There is no report of a systematic study on the effects of various methods of storing plant virus antisera on reactive antibody titer. As a part of a cooperative program between the U.S. Department of Agriculture, The American Type Culture Collection, and individual plant virologists, antisera to viruses were donated by the latter to the ATCC for distribution to others. In this study antisera to tobacco mosaic virus (TMV) and Southern Bean Mosaic virus (SBMV) were stored using those preservatives, storage temperatures, and methods of handling commonly used by plant virologists. TMV and SBMV antisera were selected because they represent a rod-shaped and spherical virus, respectively. One-half-milliliter aliquots were (1) stored at -70, -20, t4, +26, or f37” C; (2) frozen and thawed 260 times; (3) stored in 5% peptone, 0.02% sodium azide; or an equal volume of glycerine; (4) diluted prior to storage: or (5) freeze-dried. Most of the treatments had little or no effect on reactive antibody titer except: (1) when stored at 37” C the titer of non-freeze-dried TMV antiserum dronped to 0 after 1 year while that of SBMV lost about 75% of its reactivity; (2) freeze-dried TMV antiserum did not drop in reactivity when stored at 37” C while SBMV again lost about 75% of its reactivity; (3) freeze-dried SBMV antiserum stored with peptone maintained all of its reactivity at all storage temperPture.7 while sodium azide was onlv partially effective at 37” C; (4) sodium azide was superior to glycerine in both antisera stored at +26 or +37” C for more than 6 months: (5) non-freeze-drying was superior to freeze-drying TMV antiserum when stored at the lowest four temperatures; and (6) SBMV maintained its reactivity better when stored nreviously diluted I:512 to I:1024 with saline. Consult Ann. Appl.
CONFERENCE Biol. 1973-1974 work. FREEZING
ABSTRACTS for
full-length
account
of this
OF PARASITIC AND RELATED FREE-LIVING AMOEBAE ROGER G. ZIM:
American Type Culture Collection, Rockville, Ma yland Preliminary studies on the preservation of parasitic and related free-living amoebae indicate that the majority of these organisms can he preserved either by freezing the trophozoites or by drying the encysted stage. Recently two species of soil amoebae, Acanthamoeba and Naegbria, that were thought to he free-living, have been proved to he causative agents of human disease. As a result of this renewed interest in these forms the ATCC has acquired representative cultures isolated from environmental sources as well as from infected hnmans. Free-living isolates derived from soil, swimming pools, lakes, and streams, if grown in the presence of bacteria, readily form cysts that are resistant to drying. We have found that when we suspend these cysts in a sucrose solution they can he dried, sealed, and stored under vacuum for long periods of time. Until now isolates of Naegleria isolated from fatal cases of meningoencephalitis have been propagated on monkey kidney tissue culture. These pathogenic amoebae have proved to be taxonomically similar to Naegleria found free-living in nature. However, pathogenic amoebae grow at a higher temperature and’, if the culture is bacteria-free, do not form cysts. Our studies indicate that there is a difference in the way that various strains of these pathogenic amoebae recover from freezing when DMSO is used as a cryoprotectant. Although few organisms survive freezing, we have found no gross cultural or morphological changes in cultures derived from preserved material. Initial culture growth is slower at first hut gradually becomes comparable to that of unfrozen amoebae after one passage. Amoebae recovering from preservation are still able to cause normal cytopathogenic effects in tissue cultures. Although various methods of freezing axenic cultures of Entamoeha have been reported, they have proved to he quite unreliable and require a large number of organisms. These intestinal parasites of animals fail to form cysts in axenic cultures. Therefore, effective cryobiologic preservation of these organisms must involve the vegetative cell. By using sucrose and DMSO in the freezing suspension we have heen able to improve rrcovery of frozen Entamoeba cultures. The way in which the culture is handled prior to freezing and after thawing also has a marked effect on recovery and subsequent growth of these organisms.