ABSTRACT
We have shown that the growth, starvation and population heterogeneity of Salmonella typhimurium and its isogenic nuoG and cydA mutants can be monitored by flow cytometry. Bacterial cells were analysed unstained, and after staining with rhodamine 123, propidium iodide and acridine orange. In unstained cultures it was possible to distinguish flagellated and non-flagellated cells. nuoG and cydA mutants were less stained with rhodamine confirming their defects in generating membrane potential. Increase in propidium iodide staining associated with reduced membrane integrity was seen between day 4 and 14 in all the strains. Acridine orange staining showed that there was retarded development in stationary phase in nuoG and cydA mutants. Furthermore, up to day 28, a small portion of cells showed high RNA and DNA levels. To determine whether these cells represent a sub-population better adapted for long term survival, we measured the growth of the population by both OD values and viable counts. Because the OD values increased throughout the whole study in both wild-type and mutant strains, while the viable counts gradually decreased, we propose that even in very old cultures there must be a population of cells undergoing replication.
Subject(s)
Cytochromes/genetics , Electron Transport Chain Complex Proteins , Escherichia coli Proteins , Flow Cytometry , Mutation , NADH, NADPH Oxidoreductases/genetics , Oxidoreductases/genetics , Salmonella typhimurium/physiology , Acridine Orange , Coloring Agents , Cytochrome b Group , Cytochromes/metabolism , DNA Transposable Elements , Electron Transport Complex I , Fluorescent Dyes , Genes, Bacterial , NADH, NADPH Oxidoreductases/metabolism , Oxidoreductases/metabolism , Oxygen Consumption , Phenotype , Propidium , Rhodamine 123 , Salmonella typhimurium/genetics , Salmonella typhimurium/growth & developmentABSTRACT
A simple and universal protocol for the rapid detection of Salmonella spp. in various samples using nested polymerase chain reaction (PCR) was developed. The protocol takes advantage of the rapid purification and concentration of Salmonella by centrifugation onto a layer of 60% sucrose solution. DNA was released by treatment with proteinase K and Triton X-100. Even without pre-enrichment, the detection limit for the nested PCR was 10(5) CFU g-1 of faeces. After pre-enrichment, it was possible to detect Salmonella in faeces where their original concentration was approximately 10(2) CFU g-1 of faeces and in meat samples, it was possible to detect Salmonella in samples where their original concentration was less than 10 cells g-1 of minced meat.
