Automatic quantitative radiometric assay of bacterial metabolism. [/sup 14/C tracer for scintillation counting]
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In a two-compartment scintillation vial, suspensions of bacteria were cultured with 1 ..mu..Ci of (U-/sup 14/C) glucose and the released /sup 14/CO/sub 2/ was measured continuously, cumulatively, and automatically in a liquid-scintillation counter modified to maintain sample temperature at 37/sup 0/C. The metabolism of bacterial populations were followed through their early phase of exponential growth with good precision. The data were obtained conveniently, with use of conventional reagents, glassware, and counting equipment. From analysis of the exponential portion of the curves for cumulative activity vs. time, cell replication rate could be measured precisely in units of time. The resulting values were demonstrably independent of some common experimental variables, including the number of bacteria in the inoculum and counting system sensitivity. Sensitivity of the bacteria to antibiotics was measured to within a few percent by noting the relative prolongation of replication time in the presence of those inhibitors. The digital data from the scintillation counter are susceptible to on- or off-line computer analysis, thus providing the prospect for a totally-automated analytical system. The method shows promise for the mechanized quantitative analysis of bacterial growth, and its inhibition.Keywords:
Liquid Scintillation Counting
Bacterial growth
TRACER
Replication
Quantitative Analysis
Vial
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In a two-compartment scintillation vial, suspensions of bacteria were cultured with 1 muCi of [U-14C] glucose and the released 14C02 was measured continuously, cumulatively, and automatically in a liquid-scintillation counter modified to maintain sample temperature at 37 degrees C. We could follow the metabolism of bacterial populations through their early phase of exponential growth with good precision. The data were obtained conveniently, with use of conventional reagents, glassware, and counting equipment. From analysis of the exponential portion of the curves for cumulative activity vs. time, we could measure cell replication rate precisely in units of time. The resulting values were demonstrably independent of some common experimental variables, including the number of bacteria in the inoculum and counting system sensitivity. Sensitivity of the bacteria to antibiotics was measured to within a few percent by noting the relative prolongation of replication time in the presence of those inhibitors. The digital data from the scintillation counter are susceptible to on- or off-line computer analysis, thus providing the prospect for a totally-automated analytical system. The method shows promise for the mechanized quantitative analysis of bacterial growth, and its inhibition.
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Radiometry
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MTT assay
Formazan
Statistical Analysis
Colorimetry
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Buddemeyer's two-compartment radiometric assay for bacterial growth using respired C-14 carbon dioxide promised major advantages over other available methods, but limitations of the technique have restricted its application. Through a systemic study of relevant physical and chemical factors the authors sought to improve the assay for earlier detection of bacterial growth and to extend its use to measurement of antibiotic drug susceptibility and potency. A 35-fold improvement in count rate response was achieved by a) reversing growth and detector chambers to permit rigorous agitation, b) increasing NaOH quantity and using a supersaturated PPO solution, and c) adding detergent to stabilize NaOH-PPO contact. Bacterial growth may be detected as early as 1/2 hour after inoculation. For rapidly growing bacteria the growth rate constant is defined as the slope of the growth curve (log count rate vs. time). The validity of the growth behavior was verified by measuring growth at several inoculum sizes over 3 orders of magnitude using standard strains of S. aureus and E. coli. The growth rate constant proved to be independent of inoculum size. To test the merit of the system as an antibiotic assay, E. coli were exposed to doses of spectinomycin hydrochloride in the range whichmore » yielded a nonlinear dose-response relation by a turbidity assay. The test, however, showed a linear relation between growth rate constant and antibiotic dose. The results clearly indicate the radiometric growth rate assay to be a rapid, valid and objective assay for bacterial growth and antibiotic sensitivity.« less
Bacterial growth
Spectinomycin
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The objective of the research was to seek a simple and rapid counting method of E.coli.The OD value of E.coli suspension that was identified was determined by spectrophotometer and E.coli number was calculated by direct smear counting method.When the OD value of bacteria suspension diluted by appropriate concentration was in the range of 0.1～1.0,the OD value of bacteria suspension and living bacteria number were in linear relation.Determining E.coli number by using OD value was a simple and rapid bacteria counting method.
Suspension
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