Rapid identification of bacterial viability, culturable and non-culturable stages by using flow cytometry

The protocol details methods developed to optimize staining procedures and instrument settings for the rapid identification and unbiased enumeration of viable but non-culturable (VBNC) and viable-culturable (VC) cells as well as the membrane integrity of bacteria by flow cytometry (FCM). To detect viability, various Gram-negative bacteria were stained with numerous fluorescent membrane permeable (SYTO 9, SYTO 13, SYTO 17, SYTO 40) and impermeable (propidium iodide, PI) probes, and then quantified using the FCM. FCM data are then integrated with specific plate count results to calculate VC cells in different growth states. The cells were also exposed to heat (72° C) to monitor cellular membrane integrity. At late log phase (at 18 h incubation) of bacterial culture, the culturable, non-culturable and cells with damaged membranes varied from 40-98%, 1 to 64% and 0.7% to 4.5%, respectively. In this study, the cells with damaged cell membranes were considered dead (non-viable). This robust method preserves cell viability and takes a little more than an hour allowing the simultaneous quantification of phenotypes or cellular functions that is compatible with downstream cell-sorting and RNA-based assays.

Evaluation of an Alternative Staining Method Using SYTO 13 to Determine Reticulated Platelets

Reticulated platelets reflect the rate of platelet turnover and represent the youngest circulating platelets in peripheral blood. Reticulated platelets contain residual ribonucleic acid (RNA) from megakaryocytes which is lost in a time-dependent manner and can be transcribed into proteins even in the absence of a nucleus. An increased proportion of reticulated platelets is associated with higher platelet reactivity, cardiovascular events and mortality. At present, a fully automated assay system (SYSMEX haematology analyser) is available for analysis. This method, however, is not suitable for extended laboratory investigations like subsequent cell sorting. Flow cytometry analysis after staining with thiazole orange (TO) is frequently used in such settings despite several limitations. Here, we describe a new assay for determination of reticulated platelets by flow cytometry using the nucleic acid staining dye SYTO 13 and compare it with SYSMEX and TO staining as current standards. A significant correlation between immature platelet fraction (IPF) determined by SYSMEX XE-2100 analyser and results obtained with the SYTO 13-based assay was observed (r = 0.668, p < 0.001) which was stable during a reasonable time period. In contrast, the correlation between TO staining and IPF was weaker (r = 0.478, p = 0.029) and lost after 90 minutes of staining. SYTO 13 staining of platelets enabled sorting of RNAlow and RNArich platelets which was confirmed by RNA quantification of sorted platelets. Except for fixation of platelets, sorting of these platelet sub-populations was stable under various experimental settings. In summary, determination of reticulated platelets with the new SYTO 13 assay offers distinct technical advantages enabling further laboratory processing.

Nonenzymatic Cryogenic Isolation of Therapeutic Cells: Novel Approach for Enzyme-Free Isolation of Pancreatic Islets Using In Situ Cryopreservation of Islets and Concurrent Selective Freeze Destruction of Acinar Tissue

Cell-based therapies, which all involve processes for procurement and reimplantation of living cells, currently rely upon expensive, inconsistent, and even toxic enzyme digestion processes. A prime example is the preparation of isolated pancreatic islets for the treatment of type 1 diabetes by transplantation. To avoid the inherent pitfalls of these enzymatic methods, we have conceptualized an alternative approach based on the hypothesis that cryobiological techniques can be used for differential freeze destruction of the pancreas (Px) to release islets that are selectively cryopreserved in situ. Pancreata were procured from juvenile pigs using approved procedures. The concept of cryoisolation is based on differential processing of the pancreas in five stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water to distend the acinar; 3) freezing the entire Px solid to lt; −160°C for storage in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen Px into small fragments; 5) thawing the frozen fragments, filtering, and washing to remove the CPA. Finally, the filtered effluent (cryoisolate) was stained with dithizone for identification of intact islets and with Syto 13/PI for fluorescence viability testing and glucose-stimulated insulin release assessment. As predicted, the cryoisolate contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact and viable (>90%) embedded islets. Islets were typically larger (range 50–500 μm diameter) than their counterparts isolated from juvenile pigs using conventional enzyme digestion techniques. Functionally, the islets from replicate cryoisolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7. An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze destruction of acinar tissue is feasible and proposed as a new and novel method that avoids the problems associated with conventional collagenase digestion methods.

Effect of nutrient and stress factors on polysaccharides synthesis in Proteus mirabilis biofilm.

The extracellular matrix in biofilm consists of water, proteins, polysaccharides, nucleic acids and phospholipids. Synthesis of these components is influenced by many factors, e.g. environment conditions or carbon source. The aim of the study was to analyse polysaccharides levels in Proteus mirabilis biofilms after exposure to stress and nutritional conditions. Biofilms of 22 P. mirabilis strains were cultivated for 24, 48, 72 hours, 1 and 2 weeks in tryptone soya broth or in modified media containing an additional amount of nutrients (glucose, albumin) or stress factors (cefotaxime, pH 4, nutrient depletion). Proteins and total polysaccharides levels were studied by Lowry and the phenol-sulphuric acid methods, respectively. Glycoproteins levels were calculated by ELLA with the use of selected lectins (WGA and HPA). For CLSM analysis dual fluorescent staining was applied with SYTO 13 and WGA-TRITC. In optimal conditions the levels of polysaccharides were from 0 to 442 μg/mg of protein and differed depending on the strains and cultivation time. The agents used in this study had a significant impact on the polysaccharides synthesis in the P. mirabilis biofilm. Among all studied components (depending on tested methods), glucose and cefotaxime stimulated the greatest production of polysaccharides by P. mirabilis strains (more than a twofold increase). For most tested strains the highest amounts of sugars were detected after one week of incubation. CLSM analysis confirmed the overproduction of N-acetyloglucosamine in biofilms after cultivation in nutrient and stress conditions, with the level 111-1134%, which varied depending on the P. mirabilis strain and the test factor.

Detection of BCR-ABL Mutation by High Resolution Melting Analysis Is Affected by the Sequence Content, Position and Type of Nucleotide Change and DNA Dye Together with Salt Composition of PCR Mixes.

Abstract 1565 Poster Board I-588 Mutations in the ABL kinase domain of the BCR-ABL gene are one of the important mechanisms of resistance to tyrosin kinase inhibitor imatinib, now used as a standard first-line treatment of newly diagnosed CML patients. Various methods with different sensitivity are used to detect BCR-ABL mutations including High-resolution melting analysis (HRM) which allows to screen DNA samples for single-base changes, insertions/deletions, or other unknown mutations based on their dissociation behavior as they transit (melt) from double stranded (dsDNA) to single stranded DNA (ssDNA) form with increasing temperature. To validate previously reported HRM analysis (Poláková KM et al., 2008, Leuk Res 32, 1236–1243) as a routine screening test for detection of mutations in ABL kinase domain we used LCGreen dye combined with Fast Start Taq polymerase and a PCR mix from Roche Applied Science. Four primer pairs divide the ABL kinase domain into four amplicons (HRM1–HRM4); HRM1 (221bp) covers amino acids P216-E275, HRM2 (225bp) amino acids F283-M343, HRM3 (239bp) amino acids A350-K415 and HRM4 (241bp) amino acids L429-T495. Sixteen different mutations at different ratios (previously identified and quantified by direct sequencing) each in at least four samples were used in duplicates for HRM analysis. HRM results were concordant with those obtained by direct sequencing for 9/16 mutations. For 3/16 mutations we were not able to detect in samples with 100% mutant template content. This issue could be improved by addition of exogenous wild-type DNA after PCR to form heteroduplexes. One mutation in HRM1 and three mutations in HRM3 regions were not detected reliably and presumably PCR reaction conditions need to be changed. Our results proved that HRM analysis is more sensitive than direct sequencing since mutations were detected by HRM earlier in 6 of 9 tested cases. As a next step we assessed the HRM curves of templates amplified with 4 different PCR mixes combined with several DNA dyes. We tested 6 dyes (SYBR Green, SYTO-9, SYTO-13, EvaGreen, LCGreen, ResoLight) and 4 PCR mixes differing in their salt content (24 combinations in total) to evaluate their use in HRM analysis with respect to mutation detection sensitivity and reliability. For this test, HRM1 was applied to detect E255K mutation in samples containing 100%, 50%, 15%, 5% and 0% of the mutant BCR-ABL. HRM analysis was performed immediately after PCR, and normalized melting curves and difference graphs were calculated. Mutant templates representing 15%, 50% and 100% of mutation were distinguished using all combinations of dyes and mixes. Enhanced variations of HRM scoring values in individual experiments of 100% mutant templates, reflected by higher SD values, were noticed in some PCR mix-DNA dye combinations. HRM analysis of samples containing 5% of mutated templates showed that various HRM dyes differed significantly in their ability to detect mutants. LCGreen, SYTO-9 and SYTO-13 in all mixes significantly detected 5% mutant templates (P<0.05 or P<0.01). HRM performed with SYBR Green did not reliably detect 5% mutant templates in none of mixes tested whereas other dyes differed in their capabilities to detect this mixture depending on PCR mix used. In conclusion, our data indicate that the sequence content of HRM1-4, type of mutation, composition of PCR mix as well as DNA-binding dye affect HRM performance indicating careful choice of DNA with PCR mix and primer design for successful HRM assay reliably detecting mutations in BCR-ABL kinase domain. We showed that HRM is a helpful tool for sequencing data confirmation and earlier detection since observed by sequencing. Supported by MZOUHKT2005. Disclosures No relevant conflicts of interest to declare.