Propidium iodide (PI) and YO-PRO-1 (YPI) dyes are routinely used to determine sperm viability in many livestock species. It is commonly accepted that these dyes penetrate only sperm cells with damaged plasma membranes. Recently, however, the mechanism of dye uptake unrelated to damaged plasma membranes, but instead related to pannexin channels in dog and stallion sperm cells was demonstrated. This pilot study aimed to evaluate the role of pannexins in the uptake of PI and YPI dyes on Wallachian frozen-thawed ram spermatozoa by flow cytometry using probenecid, a specific inhibitor of pannexin channels. Additionally, the expression of pannexins in Wallachian sperm was evaluated directly (by qRT-PCR). The results demonstrate the active role of pannexin channels in the uptake of PI and YPI dyes on frozen-thawed Wallachian ram sperm. In conclusion, when using the PI or YPI exclusion assay to determine Wallachian frozen-thawed ram sperm viability, the danger of overestimating the number of spermatozoa with the damaged plasma membrane must be considered. The observed breed-specific, and more importantly, individual differences in gene expression as well as in dye uptake indicate the need for further studies.

• Klíčová slova
• PANX1, PANX2, flow cytometry, frozen-thawed spermatozoa, qRT-PCR,
• MeSH
• barvicí látky MeSH
• benzoxazoly MeSH
• chinolinové sloučeniny * MeSH
• jodidy * MeSH
• koně MeSH
• kryoprezervace metody   veterinární   MeSH
• pilotní projekty MeSH
• propidium MeSH
• psi MeSH
• sperma MeSH
• spermie MeSH
• uchování spermatu * veterinární   metody   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• barvicí látky MeSH
• benzoxazoly MeSH
• chinolinové sloučeniny * MeSH
• jodidy * MeSH
• propidium MeSH
• YO-PRO 1 MeSH Prohlížeč

• MeSH
• barvení a značení MeSH
• buněčné jádro * MeSH
• fluorescenční barviva MeSH
• fluorescenční protilátková technika MeSH
• indoly * MeSH
• lidé MeSH
• propidium MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DAPI MeSH Prohlížeč
• fluorescenční barviva MeSH
• indoly * MeSH
• propidium MeSH

Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 µM bis(benzonitrile)dichloropalladium(II) or 30 µM palladium(II)acetate at 5 °C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.

• MeSH
• azidy farmakologie   MeSH
• biotest MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• mikrobiální viabilita MeSH
• Mycobacterium avium subsp. paratuberculosis * genetika   MeSH
• palladium farmakologie   MeSH
• paratuberkulóza * mikrobiologie   MeSH
• propidium farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azidy MeSH
• palladium MeSH
• propidium MeSH

Extracellular pH has been assumed to play little if any role in how bacteria respond to antibiotics and antibiotic resistance development. Here, we show that the intracellular pH of Escherichia coli equilibrates to the environmental pH following treatment with the DNA damaging antibiotic nalidixic acid. We demonstrate that this allows the environmental pH to influence the transcription of various DNA damage response genes and physiological processes such as filamentation. Using purified RecA and a known pH-sensitive mutant variant RecA K250R we show how pH can affect the biochemical activity of a protein central to control of the bacterial DNA damage response system. Finally, two different mutagenesis assays indicate that environmental pH affects antibiotic resistance development. Specifically, at environmental pH's greater than six we find that mutagenesis plays a significant role in producing antibiotic resistant mutants. At pH's less than or equal to 6 the genome appears more stable but extensive filamentation is observed, a phenomenon that has previously been linked to increased survival in the presence of macrophages.

• MeSH
• antibakteriální látky farmakologie   MeSH
• Escherichia coli účinky léků   genetika   účinky záření   MeSH
• koncentrace vodíkových iontů MeSH
• kyselina nalidixová farmakologie   MeSH
• mikrobiální viabilita účinky léků   účinky záření   MeSH
• nestabilita genomu účinky léků   genetika   účinky záření   MeSH
• poškození DNA účinky léků   genetika   účinky záření   MeSH
• propidium farmakologie   MeSH
• průtoková cytometrie MeSH
• retardační test MeSH
• rifampin farmakologie   MeSH
• ultrafialové záření MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• kyselina nalidixová MeSH
• propidium MeSH
• rifampin MeSH

The human bed bug Cimex lectularius is one of the most prevalent human ectoparasites in temperate climate zones. The cytogenetic features of this resilient pest include holokinetic chromosomes, special chromosome behavior in meiosis, and numerical variation of chromosomes, where the diploid number ranges from 26 + X1 X2 Y to 26 + X1-20 Y. It is desirable to assess the nuclear DNA content of various cytotypes for a further detailed study of the C. lectularius genome. Detailed knowledge of the DNA content of this parasite could also clarify the origin of additional chromosomes. The average nuclear genome size C. lectularius with 2n = 26 + X1 X2 Y is 2C = 1.94 pg for males and 1.95 pg for females. There is a significant correlation between genome size and the number of chromosomes, but in some specimens with additional chromosomes, nuclear genome size decreases or remains average. Several species used as the internal reference standard were tested for further investigations of genome size in C. lectularius, and the plant Solanum pseudocaspicum turned out to be the most suitable. © 2019 International Society for Advancement of Cytometry.

• Klíčová slova
• Cimex lectularius, chromosome number variability, cytogenetics, flow cytometry, genome size, holokinetic chromosomes, internal standard,
• MeSH
• barvení a značení MeSH
• buněčné jádro chemie   genetika   MeSH
• cytogenetika MeSH
• délka genomu * MeSH
• indoly MeSH
• meióza genetika   MeSH
• metafáze genetika   MeSH
• mitóza genetika   MeSH
• pohlavní chromozomy * MeSH
• propidium MeSH
• průtoková cytometrie MeSH
• štěnice genetika   MeSH
• zastoupení bazí MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DAPI MeSH Prohlížeč
• indoly MeSH
• propidium MeSH

This study aimed to evaluate and document the excystation process of Cryptosporidium muris oocysts in various incubation media, and to monitor the behaviour of excysting and freshly excysted sporozoites. A test of oocyst viability, using fluorescent double staining with fluorescein diacetate and propidium iodide, was performed prior to each experimental assay. Light microscope observations confirmed that relatively often only three sporozoites were released; the fourth one either left the oocyst later together with a residual body or remained trapped within the oocyst wall. These results suggest that successful oocyst excystation is not limited by the viability of all four sporozoites. Darkening of oocysts to opaque and their specific movement (the so-called "oocyst dancing") preceded the final excystation and liberation of sporozoites, while the dormant oocysts appeared refractive. The process of excystation in C. muris is not gradual as generally described in cryptosporidia but very rapid in an eruptive manner. Experiments were performed using oocysts stored at 4 °C for various time periods, as well as oocysts freshly shed from host rodents (Mastomys coucha) of different ages. The most suitable medium supporting high excystation rate (76 %) and prolonged motility of sporozoites was RPMI 1640, enriched with 5 % bovine serum albumin (BSA). Our results emphasize that to reliably evaluate the success of in vitro excystation of cryptosporidia, not only the number of released sporozoites in a set time period should be taken into consideration but also their subsequent activity (motility), as it is expected to be essential for the invasion of host cells.

• Klíčová slova
• Cryptosporidium muris, Excystation rate, Motility, Oocyst, Sporozoite, Viability test,
• MeSH
• Cryptosporidium účinky léků   fyziologie   MeSH
• krysa rodu Rattus MeSH
• kultivační média farmakologie   MeSH
• mikrobiální viabilita účinky léků   MeSH
• oocysty fyziologie   MeSH
• propidium MeSH
• sporozoiti účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kultivační média MeSH
• propidium MeSH

Electrical discharge plasmas can efficiently inactivate various microorganisms. Inactivation mechanisms caused by plasma, however, are not fully understood because of the complexity of both the plasma and biological systems. We investigated plasma-induced inactivation of Escherichia coli in water and mechanisms by which plasma affects bacterial cell membrane integrity. Atmospheric pressure argon plasma jet generated at ambient air in direct contact with bacterial suspension was used as a plasma source. We determined significantly lower counts of E. coli after treatment by plasma when they were assayed using a conventional cultivation technique than using a fluorescence-based LIVE/DEAD staining method, which indicated that bacteria may have entered the viable-but-nonculturable state (VBNC). We did not achieve resuscitation of these non-culturable cells, however, we detected their metabolic activity through the analysis of cellular mRNA, which suggests that cells may have been rather in the active-but-nonculturable state (ABNC). We hypothesize that peroxidation of cell membrane lipids by the reactive species produced by plasma was an important pathway of bacterial inactivation. Amount of malondialdehyde and membrane permeability of E. coli to propidium iodide increased with increasing bacterial inactivation by plasma. Membrane damage was also demonstrated by detection of free DNA in plasma-treated water.

• Klíčová slova
• Electrical discharge, Escherichia coli, LIVE/DEAD assay, Lipid peroxidation, Viability, culturability,
• MeSH
• atmosférický tlak MeSH
• bakteriologické techniky * přístrojové vybavení   metody   MeSH
• buněčná stěna metabolismus   MeSH
• design vybavení MeSH
• dezinfekce metody   MeSH
• Escherichia coli cytologie   účinky léků   fyziologie   MeSH
• permeabilita buněčné membrány MeSH
• peroxidace lipidů MeSH
• plazmové plyny * MeSH
• propidium farmakologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• plazmové plyny * MeSH
• propidium MeSH
• reaktivní formy kyslíku MeSH

Physiological changes in populations of Clostridium beijerinckii and Clostridium tetanomorphum were monitored by fluorescence staining and flow cytometry. To estimate the number of metabolically active cells in exponential growth, a combination of the dyes propidium iodide and carboxy fluorescein diacetate appeared to be a good choice for both species. During stationary phase, these stains did not reflect physiological changes sufficiently and therefore additional labeling with bis-(1,3-dibutylbarbituric acid) trimethineoxonol was applied. Results of fluorescence staining in solventogenic batch fermentations were compared with substrate-use data, the concentration of key metabolites and growth curves. We demonstrate that measurements by all methods were mutually compatible.

• Klíčová slova
• Clostridium beijerinckii, Clostridium tetanomorphum, Flow cytometry, Fluorescence staining,
• MeSH
• barbituráty MeSH
• barvení a značení metody   MeSH
• bioreaktory MeSH
• Clostridium fyziologie   ultrastruktura   MeSH
• fermentace * MeSH
• fluoresceiny MeSH
• fluorescenční barviva MeSH
• isoxazoly MeSH
• propidium MeSH
• průtoková cytometrie MeSH
• techniky vsádkové kultivace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• barbituráty MeSH
• bis(1,3-dibutylbarbiturate)trimethine oxonol MeSH Prohlížeč
• carboxyfluoresceindiacetate MeSH Prohlížeč
• fluoresceiny MeSH
• fluorescenční barviva MeSH
• isoxazoly MeSH
• propidium MeSH

Mycobacterium avium subsp. paratuberculosis (MAP) has a high degree of resistance to chemical and physical procedures frequently used for the elimination of other bacteria. Recently, a method for the determination of viability by exposure of MAP to propidium monoazide (PMA) and subsequent real time quantitative PCR (qPCR) was established and found to be comparable with culture. The aim of this study was to apply the PMA qPCR method to determine the impact of increasing concentration or time and repeated cycles of the application of selected disinfectants on MAP viability. Different MAP isolates responded to the same type of stress in different ways. The laboratory strain CAPM 6381 had the highest tolerance, while the 8819 low-passage field isolate was the most sensitive. Ultraviolet exposure caused only a partial reduction in MAP viability; all MAP isolates were relatively resistant to chlorine. Only the application of peracetic acid led to the total elimination of MAP. Repeated application of the treatments resulted in more significant decreases in MAP viability compared to single increases in the concentration or time of exposure to the disinfectant.

• Klíčová slova
• Disinfection, Mycobacterium avium subsp. paratuberculosis, Propidium monoazide quantitative PCR, Viability,
• MeSH
• azidy * MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• chlor farmakologie   MeSH
• dezinficiencia farmakologie   MeSH
• kvantitativní polymerázová řetězová reakce veterinární   MeSH
• kyselina peroctová farmakologie   MeSH
• mikrobiální viabilita účinky léků   MeSH
• Mycobacterium avium subsp. paratuberculosis účinky léků   MeSH
• propidium analogy a deriváty   MeSH
• ultrafialové záření MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azidy * MeSH
• bakteriální proteiny MeSH
• chlor MeSH
• dezinficiencia MeSH
• kyselina peroctová MeSH
• propidium monoazide MeSH Prohlížeč
• propidium MeSH

This paper concerns the formation of biofilm in bacteria of the genus Arcobacter. A multiplex polymerase chain reaction (PCR) method was introduced and optimized for detecting biofilm while using the intercalating dyes ethidium monoazide (EMA) and propidium monoazide (PMA), first for analysis of strains of the genus Arcobacter from a collection, and then applied to samples of prepared biofilms. The results of the study indicate considerable variability among species of bacteria within the genus Arcobacter. The EMA-PMA PCR method can distinguish viable cells from dead cells and is therefore suitable for determining the viability of cells.

• MeSH
• azidy chemie   MeSH
• biofilmy * MeSH
• Campylobacter genetika   izolace a purifikace   fyziologie   MeSH
• interkalátory chemie   MeSH
• mikrobiální viabilita * MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• propidium analogy a deriváty   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 8-azidoethidium MeSH Prohlížeč
• azidy MeSH
• interkalátory MeSH
• propidium monoazide MeSH Prohlížeč
• propidium MeSH