Flow Cytometric Analysis of Marine Bacteria with Hoechst 33342

Abstract

We investigated the accuracy and precision of flow cytometric (FCM) estimates of bacterial abundances using 4',6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 (HO342, a bisbenzamide derivative) on paraformaldehyde-fixed seawater samples collected from two stations near Oahu, Hawaii. The accuracy of FCM estimates was assessed against direct counts by using epifluorescence microscopy. DAPI and H0342 differ in two aspects of their chemistry that make H0342 better suited for staining marine heterotrophic bacteria for FCM analysis. These differences are most important in studies of open-ocean ecosystems that require dual-beam FCM analysis to clearly separate heterotrophic bacterial populations from populations of photosynthetic Prochlorococcus spp. Bacterial populations were easier to distinguish from background fluorescence when stained with H0342 than when stained with DAPI, because H0342 has a higher relative fluorescence quantum yield. A substantially higher coefficient of variation of blue fluorescence, which was probably due to fluorescent complexes formed by DAPI with double-stranded RNA, was observed for DAPI-stained populations. FCM estimates averaged 2.0 and 12% higher than corresponding epifluorescence microscopy direct counts for H0342 and DAPI-stained samples, respectively. A paired-sample t test between FCM estimates and direct counts found no significant difference for H0342-stained samples but a significant difference for DAPI-stained samples. Coefficients of variation of replicate FCM abundance estimates ranged from 0.63 to 2.9%o (average, 1.5%) for natural bacterial concentrations of 6 x 105 to 15 x 105 cells ml-'.

Multiyear increases in dissolved organic matter inventories at Station ALOHA in the North Pacific Subtropical Gyre

“…Bacterial cell abundance was enumerated by flow cytometry using Hoechst 33342 as the fluorochrome (Monger and Landry, 1993). Briefly, samples were collected in 15-ml polypropylene centrifuge tubes, and then 1 ml was subsampled into Cryovials (Corning) containing 0.02 ml of 10% paraformaldehyde (final concentration 0.2%).…”

Cell‐death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques

“…Hoechst 33342 and DAPI double staining provide more reliable quantification of dying cells, not requiring sample fixation or special buffers and reagents (Monger & Landry, 1993). Necrotic cells can be discriminated from cells undergoing apoptosis, making the HO342/7-AAD method applicable to experimental systems where both modes of cell death may occur simultaneously (Bottiroli et al, 1989).…”

Current and future applications of flow cytometry in aquatic microbiology

“…A frequent approach is to label with a DNA-speci¢c £uorochrome (Hoechst 33342) at 0.5 Wg ml 31 . Hoechst staining has been shown to be more useful than DAPI because the response provides a higher signal-tonoise ratio and lower coe¤cient of variation according to the protocol of Monger and Landry [36]. Comparisons between several instruments have given similar results [37], but the combination of UV excitation and DNA-speci¢c £uorochromes remains problematic primarily due to: UV source problems especially with mercury lamps, low quantum e¤ciency and background responses especially when phototrophic pigments are present.…”