The microbial community of any natural aquatic environment, when stained with nucleic acid stains and analyzed by flow cytometry, tend to cluster into two distinct fractions when sideward scatter (SSC) and green fluorescence is taken into account. One cluster shows low SSC as well as low green fluorescence and the other one shows high SSC and fluorescence. Based on these observations these two clusters have been given names LNA and HNA bacteria respectively. The majority of studies carried out in this field have focused on the abundance of these HNA and LNA cells in different environments and their relative activity. The main reason for this is a lack of suitable culture methods. There are many conflicting results about the activity and phylogeny of these LNA and HNA bacteria. HNA bacteria are generally regarded as active part of microbial community where as the LNA are regarded as the inactive part. However few authors regard LNA as active bacteria. The aim of this work was to better understand the underlying factors of this flow cytometry phenomenon. Two major factors namely stain accessibility due to cell membrane permeability and DNA compaction, were investigated for their influence on intensity of fluorescence. It was found that cell membrane permeability does not play a significant role, but DNA compaction may play a major role for low fluorescence intensity of LNA. Study of growth characteristics of an LNA isolate revealed that fluorescence intensity and SSC is influenced by the growth phase of bacteria. The present study pinpoints the limitation of classifying a natural community into low nucleic acid and high nucleic acid content bacteria based on fluorescence intensity observed by flow cytomery. The fluorescence of a bacterium may be influenced by many factors like cell membrane permeability, DNA topology, copy number of chromosomal DNA, physiological state of bacteria etc., which was also reflected in the study of growth characteristic of LNA isolate. So there exists a strong need to investigate the multiple factors which are responsible for the clustering of natural community.