Abstract: The invention relates to methods for the determination and detection of nucleic acids sequences in a sample. The nucleic acid may be RNA or DNA or both. The invention also relates to methods for the determination of the presence and species of various microorganisms in a sample. We have also identified a set of oligonucleotide nucleic acid sequences within the rRNAs of Gram-negative organisms that facilitates both the broad identification of Gram-negative organisms as a class when used as a pool, or in combination, for example in a hybridization assay. This set of oligonucleotides may detect sequences that are indicative of the presence of organisms of the broad class of Gram-negative organisms while exhibiting little or no false identification of Gram-positive organisms, and fungi, or other microorganisms. The assay includes concurrent incubation with at least one nucleotide sequence of interest, at least one nucleic acid probe, a fluorosurfactant, and a nuclease.