skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A radiometric assay for bacterial growth detection and quantitative antibiotic testing

Abstract

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

Authors:
; ;
Publication Date:
Research Org.:
Univ. of Iowa, Iowa City, IA
OSTI Identifier:
6842318
Report Number(s):
CONF-840619-
Journal ID: CODEN: JNMEA
Resource Type:
Conference
Resource Relation:
Journal Name: J. Nucl. Med.; (United States); Journal Volume: 25:5; Conference: 31. annual meeting of the Society of Nuclear Medicine, Los Angeles, CA, USA, 5 Jun 1984
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ANTIBIOTICS; RADIOMETRIC ANALYSIS; SENSITIVITY; ESCHERICHIA COLI; GROWTH; CARBON 14; CARBON DIOXIDE; DOSE-RESPONSE RELATIONSHIPS; GRAPHS; HYDROCHLORIC ACID; INOCULATION; SODIUM HYDROXIDES; TRACER TECHNIQUES; TURBIDITY; ALKALI METAL COMPOUNDS; ANTI-INFECTIVE AGENTS; BACTERIA; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CARBON COMPOUNDS; CARBON ISOTOPES; CARBON OXIDES; CHALCOGENIDES; CHEMICAL ANALYSIS; DRUGS; EVEN-EVEN NUCLEI; HYDROGEN COMPOUNDS; HYDROXIDES; INORGANIC ACIDS; ISOTOPE APPLICATIONS; ISOTOPES; LIGHT NUCLEI; MICROORGANISMS; NUCLEI; OXIDES; OXYGEN COMPOUNDS; QUANTITATIVE CHEMICAL ANALYSIS; RADIOISOTOPES; SODIUM COMPOUNDS; YEARS LIVING RADIOISOTOPES 550701* -- Microbiology-- Tracer Techniques

Citation Formats

Boonkitticharoen, V., Kirchner, P.T., and Ehrhardt, J.C. A radiometric assay for bacterial growth detection and quantitative antibiotic testing. United States: N. p., 1984. Web.
Boonkitticharoen, V., Kirchner, P.T., & Ehrhardt, J.C. A radiometric assay for bacterial growth detection and quantitative antibiotic testing. United States.
Boonkitticharoen, V., Kirchner, P.T., and Ehrhardt, J.C. 1984. "A radiometric assay for bacterial growth detection and quantitative antibiotic testing". United States. doi:.
@article{osti_6842318,
title = {A radiometric assay for bacterial growth detection and quantitative antibiotic testing},
author = {Boonkitticharoen, V. and Kirchner, P.T. and Ehrhardt, J.C.},
abstractNote = {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 which 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.},
doi = {},
journal = {J. Nucl. Med.; (United States)},
number = ,
volume = 25:5,
place = {United States},
year = 1984,
month = 1
}

Conference:
Other availability
Please see Document Availability for additional information on obtaining the full-text document. Library patrons may search WorldCat to identify libraries that hold this conference proceeding.

Save / Share:
  • 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 valuesmore » 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.« less
  • A quantitative technique for the measurement of /sup 14/CO/sub 2/ released from a bacterial culture was evaluated. The technique uses liquid scintillation counting to record /sup 14/CO/sub 2/ accumulation on a fluor-impregnated filter paper within a double-chambered scintillation vial that also houses the bacterial growth medium. We have successfully identified and corrected the major causes for a variably low detection efficiency, and also established the optimum mixture of reagents for the detection system. Incorporation of Triton X-100 into the scintillation fluid used for the detector reduced the variability between identical assays in a single batch from 50% to 5%, and,more » in conjunction with an increase in the scintillator concentration, raised the counting efficiency from 30% to 70-88%. The response of the improved detector is linear over a wide range of count-rates. Another significant modification was the interchange of growth and detector chambers. Overall, a 40-fold increase in count-rate during the exponential phase of bacterial growth was obtained by improving /sup 14/CO/sub 2/ detection efficiency, increasing the rate of /sup 14/CO/sub 2/ transfer from liquid to gas phases and enlarging the growth supporting capacity of the detector system. The minimum detection time for bacterial growth was shortened and the exponential phase of bacterial proliferation was lengthened by at least 2 hr. High counting efficiency, precision, and linearity make the improved detector a sensitive and reliable tool for radiometry of bacterial growth and metabolism.« less
  • The two-compartment radioassay for microbial kinetics based on continuous measurement of the {sup 14}CO{sub 2} released by bacterial metabolism of 14C-labeled substrate offers a valuable approach to testing the potency of antimicrobial drugs. By using a previously validated radioassay with gram-positive and gram-negative bacteria, a group of protein synthesis inhibitors was evaluated for their effect on microbial growth kinetics. All tested drugs induced changes in both the slopes and intercepts of the growth curves. An exponential growth model was applied to quantify the drug effect on the processes of bacterial {sup 14}CO{sub 2} liberation and cell generation. The response wasmore » measured in terms of a generation rate constant. A linear dependence of the generation rate constant on the dose of spectinomycin was observed with Escherichia coli. Sigmoidal-shaped curves were found in the assays of chloramphenicol and tetracycline. The implications of dose-response curves are discussed on the basis of the receptor site concept for drug action. The assay sensitivities for chloramphenicol and tetracycline were similar to those obtained by the cell counting method, but the sensitivity of the radioassay was at least 10 times greater for spectinomycin.« less
  • A /sup 14/C-radiorespirometric assay was used to show the sensitivity of fixed-film (sessile), catheter-associated and free-living (planktonic) cells of Pseudomonas aeruginosa to varying concentrations (100 micrograms/mL to 1000 micrograms/mL) tobramycin sulfate. This strain of P. aeruginosa has an MIC of 0.6 microgram/ml and an MBC of 50 micrograms/mL when tested by conventional methods. When /sup 14/C-glutamic acid was used as a substrate in this radiorespirometric assay, it could be completed in less than one hour and planktonic samples showed a significant reduction in mineralization activity (evolution of /sup 14/CO/sub 2/) within eight hours of the antibiotic challenge. These changes inmore » respiratory activity appeared to be dose and time dependent. Within 18 hr. at 1000 micrograms/mL, there was no significant residual respiratory activity in planktonic samples. Some residual respiratory activity was detected, however, in samples exposed to 100 micrograms/mL for 36 hours. The mineralization activity of sessile catheter-associated bacteria was unaffected by four hr. and eight hr. exposures to 1000 micrograms/mL of the antibiotic. A significant reduction in respiratory activity was recorded in catheter samples exposed for 18 hr. or more at each concentration examined. Unlike the planktonic samples, however, the antibiotic challenge failed to eradicate the metabolic activity of the attached bacteria. Antibiotic stressed, catheter-associated bacteria transferred to a post-exposure enrichment broth showed a limited ability to re-establish respiratory activity. This apparent recovery was limited to antibiotic exposures less than 24 hr. and was not observed in planktonic samples. The radioisotopic assay is a non-culture method which can be used to assess the antibiotic sensitivity of both planktonic bacteria and in situ biofilm populations.« less
  • Spherules of Coccidioides immitis grew readily after inoculation in vented trypticase soy broth, biphasic brain heart infusion media, and aerobic tryptic soy broth bottles used in a radiometric system (BACTEC). However, visible growth was not accompanied by a significant radiometric growth index. Growth of C. immitis can be visually detected in routine bacterial blood culture media while the radiometric growth index remains negative.