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Accelerated aging tests for stability of freeze-dried BCG (Bacillus Calmette-Guerin) vaccine
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room temperature on a variety of media, and will normally give survival values of O.l-2% depending on the medium in which it is suspended when subjected to a wide range of freezing and thawing procedures. Pseudomonas F 8 is well protected by both extracellular protective agents (dextran) and intracellular protective agents (dimethyl sulfoxide) and thus appears to be well suited for cryobiological studies. Supplies of this bacterium are available for distribution to interested investigators. (Supported by Canadian Medical Research Council Grant MA 4055.) 41. Cryobiology of the Relict form Kakabekia barghoorniana Siegel. B. Z. SIEGEL* AND S. M. SIEGEL (Departments of Microbiology and Botany, University of Hawaii, Honolulu, Hawaii 96822). Kakabekia barghoorniana Siegel is the sole living member of a microbial genus known otherwise only as a microfossil from the Precambrian Gunflint Chert of Ontario. It was first discovered in topsoil from Wales incubated in ammonia waterglucose media. These umbrella-shaped forms 5-30 pm in diameter were subsequently sought in soil samples from tropical to Arctic sites. They were found in Alaska and Iceland at several sites. Study of these forms has been impeded by failure to establish pure cultures, but some aspects of cytomorphology and cytochemistry have been examined (Proc. Nat. dcad. Sci. U.S.A. 67, 1005 1010, 1970; and NASA TM X-62,009 Section 8.11, pp. 117-121, 1971). Biogeographic correlations revealed that all sites for Kakabekia lie north of the 15” C July summer isotherm. This suggested cryobiotic capabilities and we now report that Kakabekia (a) can be grown readily at 4°C and even at -7°C in ammonia and other alkaline media, and (b) is found naturally in Hawaii only at elevations of 3000 m or more on Mount Haleakala and Mauna Kea. (Supported in part by National Aeronautics and Space Administration Research Grant NGR-012-001-042.) 42. Accelerated Aging Tests for Stability of Freeze-Dried BCG (Bacillus Calmette-Guerin) Vaccine. R. G. CRISPEN (Institution for Tuberculosis Prevention, University of Illinois at the Medical Center, Chicago, Illinois, 60612). Rapid aging studies have been developed previously for various biological products that have been preserved by freeze-drying. Preliminary work was carried out to determine if such tests could be used to determine the longevity under suitable conditions of freeze-dried BCG vaccine. With suitable modifications, the experimental procedure was adopted to conform with the requirements of
ABSTRACTS
BCG. Experiments indicate that thermal degradation causes a first-order kinetic loss in viability of the organisms. The thermal degradation constant can be determined for any temperature from an Arrhenius plot and used to predict the length of time required to lose a predetermined amount of viability. Based on this prediction, the storage period of a vaccine can be determined for various conditions of storage with a reasonable degree of accuracy. 43. Nondestructive Analysis of Freeze-Dried Mycobacteria (BCG) in Sealed Ampules by Nuclear Magnetic Resonance. R. G. CRISPEN (Institution for Tuberculosis Prevention, University of Illinois at the Medical Center, 1853 West Polk Street, Chicago, Illinois 60612). High resolution proton nuclear magnetic resonance spectra of glass sealed 5-ml ampules have been taken. The signal intensity measured under a set of standard conditions was seen to vary with the amount of water present in the ampules. Thus it appears that it is possible to obtain accurate analysis of residual moisture in the sealed ampule nondestructively by NMR. Ten ampules prepared in as an identical manner as possible were analyzed by this technique. Residual moisture as obtained by proton (‘H) NMR was seen to vary within narrow limits in all but one sample. In the anomalous sample the recorded signal intensity indicated that a large amount of residual water was still present. Thus, the NMR technique offers the possibility of surveying large numbers of ampules in order to detect those which do not meet certain specifications 44. Hibernation Pattern, Body Temperature, and Posture in Marmota monax. J. A. PANBRA AND F. ALBERT* (Department of Biology, THOMAS Georgetown University, Washington, D. C. 20007). Fifteen Marmota monax, trapped in Maryland, were observed frequently during natural hibernation at an ambient temperature of 46°C. Body temperatures were monitored continuously from two marmots in which thermocouples had been chronically implanted in the intraperitoneal fat associated with the reproductive tract (IFR) and under the skin and connective tissue on the right side of the thorax (ST). During entrance into states of hibernation, the marmots assumed a curled upright posture. During hibernation, however, they were observed more often in other postures: somewhat curled on their sides or lying on their backs. During hibernation, therefore, the body surface area exposed to the cold apparently was not minimized; body temperatures were us-
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room temperature on a variety of media, and will normally give survival values of O.l-2% depending on the medium in which it is suspended when subjected to a wide range of freezing and thawing procedures. Pseudomonas F 8 is well protected by both extracellular protective agents (dextran) and intracellular protective agents (dimethyl sulfoxide) and thus appears to be well suited for cryobiological studies. Supplies of this bacterium are available for distribution to interested investigators. (Supported by Canadian Medical Research Council Grant MA 4055.) 41. Cryobiology of the Relict form Kakabekia barghoorniana Siegel. B. Z. SIEGEL* AND S. M. SIEGEL (Departments of Microbiology and Botany, University of Hawaii, Honolulu, Hawaii 96822). Kakabekia barghoorniana Siegel is the sole living member of a microbial genus known otherwise only as a microfossil from the Precambrian Gunflint Chert of Ontario. It was first discovered in topsoil from Wales incubated in ammonia waterglucose media. These umbrella-shaped forms 5-30 pm in diameter were subsequently sought in soil samples from tropical to Arctic sites. They were found in Alaska and Iceland at several sites. Study of these forms has been impeded by failure to establish pure cultures, but some aspects of cytomorphology and cytochemistry have been examined (Proc. Nat. dcad. Sci. U.S.A. 67, 1005 1010, 1970; and NASA TM X-62,009 Section 8.11, pp. 117-121, 1971). Biogeographic correlations revealed that all sites for Kakabekia lie north of the 15” C July summer isotherm. This suggested cryobiotic capabilities and we now report that Kakabekia (a) can be grown readily at 4°C and even at -7°C in ammonia and other alkaline media, and (b) is found naturally in Hawaii only at elevations of 3000 m or more on Mount Haleakala and Mauna Kea. (Supported in part by National Aeronautics and Space Administration Research Grant NGR-012-001-042.) 42. Accelerated Aging Tests for Stability of Freeze-Dried BCG (Bacillus Calmette-Guerin) Vaccine. R. G. CRISPEN (Institution for Tuberculosis Prevention, University of Illinois at the Medical Center, Chicago, Illinois, 60612). Rapid aging studies have been developed previously for various biological products that have been preserved by freeze-drying. Preliminary work was carried out to determine if such tests could be used to determine the longevity under suitable conditions of freeze-dried BCG vaccine. With suitable modifications, the experimental procedure was adopted to conform with the requirements of
ABSTRACTS
BCG. Experiments indicate that thermal degradation causes a first-order kinetic loss in viability of the organisms. The thermal degradation constant can be determined for any temperature from an Arrhenius plot and used to predict the length of time required to lose a predetermined amount of viability. Based on this prediction, the storage period of a vaccine can be determined for various conditions of storage with a reasonable degree of accuracy. 43. Nondestructive Analysis of Freeze-Dried Mycobacteria (BCG) in Sealed Ampules by Nuclear Magnetic Resonance. R. G. CRISPEN (Institution for Tuberculosis Prevention, University of Illinois at the Medical Center, 1853 West Polk Street, Chicago, Illinois 60612). High resolution proton nuclear magnetic resonance spectra of glass sealed 5-ml ampules have been taken. The signal intensity measured under a set of standard conditions was seen to vary with the amount of water present in the ampules. Thus it appears that it is possible to obtain accurate analysis of residual moisture in the sealed ampule nondestructively by NMR. Ten ampules prepared in as an identical manner as possible were analyzed by this technique. Residual moisture as obtained by proton (‘H) NMR was seen to vary within narrow limits in all but one sample. In the anomalous sample the recorded signal intensity indicated that a large amount of residual water was still present. Thus, the NMR technique offers the possibility of surveying large numbers of ampules in order to detect those which do not meet certain specifications 44. Hibernation Pattern, Body Temperature, and Posture in Marmota monax. J. A. PANBRA AND F. ALBERT* (Department of Biology, THOMAS Georgetown University, Washington, D. C. 20007). Fifteen Marmota monax, trapped in Maryland, were observed frequently during natural hibernation at an ambient temperature of 46°C. Body temperatures were monitored continuously from two marmots in which thermocouples had been chronically implanted in the intraperitoneal fat associated with the reproductive tract (IFR) and under the skin and connective tissue on the right side of the thorax (ST). During entrance into states of hibernation, the marmots assumed a curled upright posture. During hibernation, however, they were observed more often in other postures: somewhat curled on their sides or lying on their backs. During hibernation, therefore, the body surface area exposed to the cold apparently was not minimized; body temperatures were us-