Evaluation of the LIVE/DEAD Bac Light Kit for Detection of Extremophilic Archaea and Visualization of Microorganisms in Environmental Hypersaline Samples

Abstract

Extremophilic archaea were stained with the LIVE/DEAD BacLight kit under conditions of high ionic strength and over a pH range of 2.0 to 9.3. The reliability of the kit was tested with haloarchaea following permeabilization of the cells. Microorganisms in hypersaline environmental samples were detectable with the kit, which suggests its potential application to future extraterrestrial halites.Numerous archaea (archaebacteria) thrive in hostile conditions such as salt brines, hot springs, and acidic or alkaline environments (20). Their membrane lipids differ from those of (eu)bacteria and other organisms because they contain ether linkages instead of ester linkages, are composed of regularly branched phytanyl and biphytanyl chains instead of fatty acyl chains, and possess glycerol ethers, which are sn-2,3 substituted rather than sn-1,2 substituted (13). These properties are thought to contribute to the greater chemical stability of archaeal lipids (13,22) and the generally low permeability of archaeal membranes (5,14). The LIVE/DEAD BacLight bacterial viability kit (henceforth referred to as the LIVE/DEAD kit) from Molecular Probes is widely used for the enumeration of bacteria (2,8,12) and provides an indication of the fraction of active cells. The kit consists of two nucleic acid stains: SYTO 9, which penetrates most membranes freely, and propidium iodide, which is highly charged and normally does not permeate cells but does penetrate damaged membranes. Simultaneous application of both dyes therefore results in green fluorescence of viable cells with an intact membrane, whereas dead cells, because of a compromised membrane, show intense red fluorescence (10). Archaea have not been treated with the LIVE/DEAD kit, except for one psychrophilic isolate (15); in view of the low permeability of their membranes and their existence in habitats that often border on the physicochemical limits of life, it was of interest to determine if the LIVE/DEAD kit would detect extremophilic archaea and provide reliable information about their viability.Archaeal strains were purchased from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany), except Halobacterium sp. strain NRC-1 ATCC 700922, which was obtained from LGC, London, United Kingdom. Haloarchaea were grown at 37°C in M2 medium (27), except Halococcus and haloalkaliphiles, which were grown in M2S medium (25) or Tindall's medium (26), respectively, and Halobacterium sp. strain NRC-1, which was grown in ATCC medium no. 2185 (http://www.lgcpromochem .com/atcc/). Acidianus brierleyi DSM 1651T (21) and Sulfolobus acidocaldarius DSM 639 T (31) were grown at 65 to 70°C in DSM medium no. 150 (http://www.dsmz.de/media/med150 .htm) and ATCC medium no. 1723 (http://www.atcc.org /SearchCatalogs/Search.cfm), respectively. The dyes of LIVE /DEAD BacLight kit L-7012 (Molecular Probes, Inc., Eugene, Oreg.) were freshly diluted with water and used as previously described (3, 10). Staining with 4Ј,6Ј-diamidino-2-phenylindole (DAPI) was done as described b...

Phosphorus Deprivation Responses and Phosphonate Utilization in a Thermophilic Synechococcus sp. from Microbial Mats

“…Cell viability was monitored with the Live/Dead BacLight kit L-7012 (Molecular Probes, Inc., Eugene, OR), as previously described (24).…”

“…For analysis of transcript levels from putative Pho regulon genes over the time course of growth and under different P conditions (ϩP, ϪP, ϩPϩMePhn, and ϪPϩMePhn), cells were harvested at 24-h intervals throughout the logarithmic phase of growth (24,48,72, and 96 h). For experiments in which P i was added to cultures following 72 h of growth in ϩP or ϪP medium, the P i was filter sterilized and added to a concentration of 0.77 mM (if the concentration in the culture after the initial growth period was not considered).…”

Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties

“…Bacteria present in the biofilms were stained with the LIVE/DEAD BacLight ™ kit (Invitrogen), which contains two dyes Syto-9 and propidium iodide and facilitates determination of the ratio of live (stained with Syto-9) to dead (stained with propidium iodide) cells (Leuko et al 2004). For this purpose, 24-h bacterial cultures in 79CA were equilibrated to OD 600 =0.6 and subsequently diluted 1000-fold in the same medium.…”

Nanoarchaeum equitansandIgnicoccus hospitalis: New Insights into a Unique, Intimate Association of Two Archaea

“…In contrast, propidium iodide (reducing the green SYTO 9 fluorescence to red) only penetrates damaged cell membranes. Thus, under UV light (excitation, 360 to 370 nm), cells with an intact membrane (living cells) exhibit a green color whereas cells with a damaged membrane (dead cells) stain red (19,30).…”