Rapid method for the differentiation of gram-positive and gram-negative bacteria on membrane filters

Abstract

Microfiltration has become a popular procedure for the concentration and enumeration of bacteria. We developed a rapid and sensitive method for the differentiation of gram-positive and gram-negative bacteria, utilizing a polycarbonate membrane filter, crystal violet, iodine, 95% ethanol, and 6% carbol fuchsin, that can be completed in 60 to 90 s. Gram reactions of 49 species belonging to 30 genera of bacteria were correctly determined by the filter-Gram stain. The sensitivities of the filter-Gram stain and conventional slide-Gram stain were compared by testing dilutions of Escherichia coli, Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenza suspensions in the presence and absence of whole human blood. The filter-Gram stain was approximately 100-fold more sensitive than the slide-Gram stain. The filter-Gram stain detected 2 to 100 bacteria, whereas the slide-Gram stain failed to detect less than 1,000 bacteria. The sensitivities of the methods were not significantly altered by the addition of whole human blood to the dilutions of bacteria tested. The filter-Gram stain could be a useful tool for the examination of body fluids with very low numbers of bacteria. * Corresponding author. MATERIALS AND METHODS Microorganisms. A total of 49 clinical and reference isolates belonging to 30 genera of aerobic, anaerobic, and facultatively anaerobic bacteria (Achromobacter sp., Acinetobacter calcoaceticus subsp. anitratus, Aeromonas hydo

A comparison of three methods for estimating dental plaque removal from individual teeth

“…Filtration through membranes was found to be a useful tnethod of concentrating bacteria from suspension (2,11). The results of this study suggest that large volumes of fluid containing microgratn atnounts of plaque can be quantified by concentration on filter surfaces for air-dried weight measurements.…”

Rapid detection of bacteria in cerebrospinal fluid by immunofluorescence staining on membrane filters

“…Other modifications also improved the FFA procedure. Using 0.2-,um-pore-size polycarbonate membrane filters provided maximum retention of bacteria and a microscopically fiat surface for viewing at x 1,000 magnification (18). Reagents were sterilized by filtration to prevent entrapment of particulates and clogging of membrane filters.…”

“…In the slide Gram stain, crystal violet and iodine were used for staining bacteria, acetonealcohol for decolorizing, and safranin for counterstaining. The same reagents were used in the filter Gram stain except 95% ethanol was used for the decolorizer and 6% carbol fuchsin was used for counterstaining (18). All reagents were obtained from GIBCO Laboratories (Lawrence, Mass.…”

“…Filter Gram stain procedure. The filter Gram stain was performed as previously described (18). Briefly, 50 ,u of each dilution of bacteria-seeded CSF was filtered through a polycarbonate membrane filter (pore size, 0.2 ,um; Nuclepore Corp., Pleasanton, Calif.).…”

“…Detection of bacteria in clinical samples by filtration represents an alternative approach to direct specimen antigen testing. A sensitive filter Gram stain procedure was developed for the detection of bacteria in blood (18), and a direct epifluorescent filter technique has been applied to the enumeration of bacteria in food (14,15,17). In this study, a filter-fluorescent-antibody (FFA) staining procedure was developed for rapid detection of bacteria in CSF.…”