Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Applied Microbiology logoLink to Applied Microbiology
. 1972 Aug;24(2):179–183. doi: 10.1128/am.24.2.179-183.1972

Fluorometric Determination of Deoxyribonucleic Acid in Bacteria with Ethidium Bromide

J A Donkersloot 1, S A Robrish 1, M I Krichevsky 1
PMCID: PMC380577  PMID: 4561101

Abstract

A simple, sensitive, and rapid method is presented for the determination of deoxyribonucleic acid (DNA) in both gram-positive and gram-negative bacteria. It is based upon the fluorometric determination of DNA with ethidium bromide after alkaline digestion of the bacteria to hydrolyze the interfering ribonucleic acid. The assay takes less than 2 hr. Its sensitivity is at least 0.2 μg of DNA in a final solution of 4 ml and it uses commonly available filter or double monochromator fluorometers. Judicious choice of light source and filters allows an additional 10-fold increase in sensitivity with a filter fluorometer. Turbidity caused by bacteria or insoluble polysaccharides does not interfere with the fluorescence measurements. There was no significant difference between the results obtained with this method and those obtained with the indole and diphenylamine methods when these assays were applied to Escherichia coli and sucrose- or glucose-grown Streptococcus mutans. The method was also tested by determining the specific growth rate of E. coli. This new procedure should be especially useful for the determination of bacterial DNA in dilute suspensions and for the estimation of bacterial growth or DNA replication where more conventional methods are not applicable or sensitive enough.

Full text

PDF
179

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. CERIOTTI G. A microchemical determination of desoxyribonucleic acid. J Biol Chem. 1952 Sep;198(1):297–303. [PubMed] [Google Scholar]
  2. DOWNING M., SCHWEIGERT B. S. Rôle of vitamin B12 in nucleic acid metabolism. III. Preparation of C14 uniformly labeled pyrimidine deoxynucleosides. J Biol Chem. 1956 Jun;220(2):513–520. [PubMed] [Google Scholar]
  3. Hinegardner R. T. An improved fluorometric assay for DNA. Anal Biochem. 1971 Jan;39(1):197–201. doi: 10.1016/0003-2697(71)90476-3. [DOI] [PubMed] [Google Scholar]
  4. KISSANE J. M., ROBINS E. The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system. J Biol Chem. 1958 Jul;233(1):184–188. [PubMed] [Google Scholar]
  5. Koch A. L. Turbidity measurements of bacterial cultures in some available commercial instruments. Anal Biochem. 1970 Nov;38(1):252–259. doi: 10.1016/0003-2697(70)90174-0. [DOI] [PubMed] [Google Scholar]
  6. Le Pecq J. B., Paoletti C. A new fluorometric method for RNA and DNA determination. Anal Biochem. 1966 Oct;17(1):100–107. doi: 10.1016/0003-2697(66)90012-1. [DOI] [PubMed] [Google Scholar]
  7. LePecq J. B., Paoletti C. A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization. J Mol Biol. 1967 Jul 14;27(1):87–106. doi: 10.1016/0022-2836(67)90353-1. [DOI] [PubMed] [Google Scholar]
  8. Munro H. N. The determination of nucleic acids. Methods Biochem Anal. 1966;14:113–176. doi: 10.1002/9780470110324.ch5. [DOI] [PubMed] [Google Scholar]
  9. ROBERTS D., FRIEDKIN M. The fluorometric determination of thymine in deoxyribonucleic acid and derivatives. J Biol Chem. 1958 Aug;233(2):483–487. [PubMed] [Google Scholar]
  10. Robertson F. W., Tait K. Optimal conditions for the fluorometric determination of DNA. Anal Biochem. 1971 Jun;41(2):477–481. doi: 10.1016/0003-2697(71)90168-0. [DOI] [PubMed] [Google Scholar]
  11. SHERRATT H. S. A., THOMAS A. J. The nucleic acid fractions of a strain of Streptococcus faecalis. J Gen Microbiol. 1953 Apr;8(2):217–223. doi: 10.1099/00221287-8-2-217. [DOI] [PubMed] [Google Scholar]
  12. Switzer B. R., Summer G. K. A modified fluorometric micromethod for DNA. Clin Chim Acta. 1971 Apr;32(2):203–206. doi: 10.1016/0009-8981(71)90333-0. [DOI] [PubMed] [Google Scholar]
  13. Tanzer J. M., Wood W. I., Krichevsky M. I. Linear growth kinetics of plaque-forming streptococci in the presence of sucrose. J Gen Microbiol. 1969 Sep;58(1):125–133. doi: 10.1099/00221287-58-1-125. [DOI] [PubMed] [Google Scholar]
  14. WEBB J. M., LEVY H. B. New developments in the chemical determination of nucleic acids. Methods Biochem Anal. 1958;6:1–30. doi: 10.1002/9780470110225.ch1. [DOI] [PubMed] [Google Scholar]

Articles from Applied Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES