A flow cytometry method for safe detection of bacterial viability.

Flow cytometry is a relevant tool to meet the requirements of academic and industrial research projects aimed at estimating the features of a bacterial population (e.g., quantity, viability, activity). One of the remaining challenges is now the safe assessment of bacterial viability while minimizing the risks inherent to existing protocols. In our core facility at the Paris-Saclay University, we have addressed this issue with two objectives: measuring bacterial viability in biological samples and preventing bacterial contamination and chemical exposure of the staff and cytometers used on the platform. Here, we report the development of a protocol achieving these two objectives, including a viability labeling step before bacteria fixation, which removes the risk of biological exposure, and the decrease of the use of reagents such as propidium iodide (PI), which are dangerous for health (CMR: carcinogenic, mutagenic, and reprotoxic). For this purpose, we looked for a non-CMR viability dye that can irreversibly label dead bacteria before fixation procedures and maintain intense fluorescence after further staining. We decided to test on the bacteria, eFluor Fixable Viability dyes, which are usually used on eukaryotic cells. Since the bacteria had size and granularity characteristics very similar to those associated with flow cytometry background signals, a step of bacterial DNA labeling with SYTO or DRAQ5 was necessarily added to differentiate them from the background. Three marker combinations (viability-DNA) were tested on LSR Fortessa and validated on pure bacterial populations (Gram+ , Gram- ) and polybacterial cultures. Any of the three methods can be used and adapted to the needs of each project and allow users to adapt the combination according to the configuration of their cytometer. Having been tested on six bacterial populations, validated on two cytometers, and repeated at least two times in each evaluated condition, we consider this method reliable in the context of these conditions. The reliability of the results obtained in flow cytometry was successfully validated by applying this protocol to confocal microscopy, permeabilization, and also to follow cultures over time. This flow cytometry protocol for measuring bacterial viability under safer conditions also opens the prospect of its use for further bacterial characterization.

Phospholipid FA from Indian Ocean tunicates Eudistoma bituminis and Cystodytes violatinctus.

Two tunicates (Eudistoma bituminis and Cystodytes violatinctus, family Polycitoridae) were investigated for the FA content of their phospholipids. GC-MS analysis of their methyl esters and N-acyl pyrrolidides revealed 40 FA in E. bituminis, and 26 in C. violatinctus. In both cases, the most abundant FA were the saturated ones (C10 to C18). Cystodytes violatinctus contained considerable oleic acid (20%). Both E. bituminis and C. violatinctus contained phytanic acid and delta10-unsaturated FA, which had not previously been found in such organisms. The two tropical tunicates contained only trace amounts of PUFA, which are usually predominant in this phylum.

Cycloshermilamine D, a new pyridoacridine from the marine tunicate cystodytes violatinctus.

A new alkaloid, cycloshermilamine D, was isolated from the marine tunicate Cystodytes violatinctus, and its structure (1), which is closely related to shermilamine D, was established mainly on the basis of NMR spectroscopic data.

Polycitone B and prepolycitrin A: two novel alkaloids from the marine ascidian Polycitor africanus.

Two novel compounds, polycitone B (4) and prepolycitrin A (3), were isolated from the marine ascidian Polycitor africanus. The structures of these new dibromotyrosine metabolites were established mainly on the basis of NMR spectroscopic data. Isolation of compound 3 supports the earlier suggestion that it is the bioprecursor of polycitrin A (2).

Hydroquinone antioxidants from the Indian ocean tunicate Aplidium savignyi.

Three hydroquinone compounds have been isolated from the Indian Ocean tunicate Aplidium savignyi. Two are already known: geranylhydroquinone and the 2-(3-hydroxy-3,7-dimethyloct-6-enyl)-1,4-benzenediol as a new stereoisomer 2-(2Z)-(3-hydroxy-3, 7-dimethyloct-2,6-dienyl)-1,4-benzenediol. The structures were determined using spectroscopic data ((1)H NMR, (13)C NMR, MS, UV, and IR). These compounds are potential natural antioxidants.

New 9,11-secosterols from gorgonia Subergorgia suberosa of the Indian Ocean.

Three new 9,11-secosterols, 3 beta,6 alpha,11-trihydroxy-9,11-seco-5 alpha-cholest-7-ene-9-one (1), 24S- (2a), and 24R-methyl-3 beta,6 alpha, 11-trihydroxy-9,11-seco-5 alpha-cholest-7,22E-diene-9-one (2b), were isolated from the Indian Ocean gorgonia, Subergorgia suberosa. Their structures were established by spectroscopic data.

Bebryazulene, a new guaiane metabolite from the indian ocean gorgonian coral, bebryce grandicalyx

Bebryazulene (1), a new sesquiterpene having the guaiane skeleton, was isolated from the methanol-chloroform 1:2 extract of the Indian Ocean gorgonian coral Bebryce grandicalyx (Kuekenthal) collected in the lagoon of Mayotte. Structure assignment was based on interpretation of spectroscopic data. This new metabolite was very labile and reacted with 4-phenyl-3H-1,2,4-triazoline-3,5-dione to yield an unexpected adduct.

Several new isoprenoids from the soft coral sinularia erecta

Eight compounds were isolated from the Indo-Pacific soft coral Sinularia erecta (Tixier-Durivault, 1945) collected from the lagoon of Mayotte, Comoros Islands, northwest of Madagascar. Four of the compounds, namely the sesquiterpene germacrene D, the diterpenoids nephthenol and decaryiol, and norcembrene, are known. The other four are the new secondary metabolites germacrene E (1), epi-norcembrene (3), and two bis-pukalide diterpenes, mayotolides A and B (4 and 5). The structure of each of the compounds was determined by means of 1D and 2D NMR and MS spectroscopy in addition to some chemical transformations.

Sodwanones K, L, and M; new triterpenes from the marine sponge Axinella weltneri.

Three new triterpenes, sodwanones M, K, and L (4, 5, 6), together with three known sodwanones (A, B, and D) have been isolated from the sponge Axinella weltneri from the Comoros Islands, in the Indian Ocean. The three new structures were determined by interpretation of NMR spectra and, in the case of sodwanone K, also by oxidation to the known sodwanone D. Sodwanone M, the major isolated compound, was found to be cytotoxic to P-388 murine leukemia cells at a concentration of 1 microgram/mL.

Novel 3-beta-methoxysteroids from the Senegalese sponge Microscleroderma spirophora.

The Senegalese sponge Microscleroderma spirophora has been found to produce exclusively very unusual 3 beta-methoxysteroids in place of the common 3 beta-hydroxysteroids. Six different methoxysteroids (three of which are novel compounds) have been isolated and identified by spectroscopic means (MS, IR, 1H and 13C NMR), and their taxonomic significance discussed. A new method for the determination of the configuration at C-24 in saturated 24-ethyl side chains is also proposed.