This study presents an automated, image-based cytometry method for determination of total counts and viability of yeast cells by using a newly synthesized DNA fluorescent dye PO-TEDM-1 and a new Easycounter YC instrument. The synthesized polycationic asymmetric monomethine cyanine dye PO-TEDM-1 penetrates only into dead cells. The new fluorescent dye has high nucleic acid sensitivity and rapid interaction kinetics. The optimal concentration of the fluorescent dye for staining dead cells was 1 mg mL À1. The Easycounter YC system was used to determine the total cell count and viability of Saccharomyces carlsbergensis. The actual viability measured using the proposed method significantly correlated with the theoretical viability (R 2 of 0.9988). The optimal linear interval was from 1 Â 10 5 to 1 Â 10 7 cells mL À1. The coefficient of variation with Easycounter YC in the optimal range was 2.5-4%, whereas that of the manual hemocytometry method, in the same range was higher, 15-23%. We tested the procedure in a study of the total cell count and viability of yeast cells from the propagators in beer production as a function of the dilution. The proposed method can be used in assays involving simple cell counting and quality assurance in sample bioprocessing.

Ochratoxins are possible human carcinogens. The aim of this study is to develop a rapid and sensitive competitive immunofluorescent analysis for determination of ochratoxin A (OTA) on the base of immobilized polyclonal antibody against ochratoxin and immobilized F(ab′) 2 fragment on magnetic nanoparticles (MNPs). F(ab′) 2 fragment of anti-OTA antibody was obtained by pepsin hydrolysis of polyclonal antibody against OTA. The competitive fluorescent conjugate OTA-OVA-FITC (OTA coupled to ovalbumin (OVA) and then conjugated to fluorescein isothiocyanate (FITC)) was prepared and purified by size-exclusion chromatography. Competitive immunoassay was performed by using obtained immobilized antibody or F(ab′) 2 fragment on magnetic nanoparticles and the conjugate OTA-OVA-FITC. The analytical characteristics of the analysis with immobilized polyclonal antibody and F(ab′) 2 fragment were compared. The linear measuring range of OTA in milk, obtained with immobilized whole antibody, was from 0.1 to 2.5 ng/mL OTA and with immobilized F(ab′) 2 fragment from 0.1 to 7.5 ng/mL OTA. The detection limit of immunoassay with immobilized whole antibody was 0.1 ng/mL OTA and with immobilized F(ab′) 2 fragment was 0.08 ng/mL OTA. Milk samples were spiked with OTA at different levels. The recovery rates when using immobilized F(ab′) 2 fragment were between 99.4 and 118.0%, and the relative standard deviations (RSDs) were 6.5-7.7%. The results indicate that this fluorescence immunoassay with MNPs was accurate and has good reproducibility.

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This Research Communication describes the relation between somatic cells and microbial content in milk from Jersey cattle. Milk samples were classified in groups: healthy, dirty and mastitic (from Staphylococcus spp., Escherichia coli, Coliforms). The somatic cells in each of those groups were analysed by two methods – flow cytometric and automatic fluorescent cell counting. Those methods were compared. Total somatic cell count (SCC), neutrophil count, and lymphocytes with cluster of differentiation 4 (CD4+cells) were determined. There was a positive relationship between microbes and somatic cells. It was noticed that the neutrophil count was generally increased together with SCC, whilst the CD4+ cell count was higher in healthy milk samples (about 8%) compared to mastitic ones (about 3%). Lower number of CD4+ cells (from 1 to 4%) was determined in samples positive for Staphylococcus spp. but with lower SCC (from 2.7 to 4.0 × 105 cells/ml). Also, the number of CD4+ cells in Staphylococcus spp.-positive samples increased (to 4.8%) together with higher SCC, something that was not observed in the other mastitic samples. Knowledge of those relations could be useful for veterinary medical tests in the initial phase of inflammation.

Background: Antibiotic residues are a problem of increasing importance and have direct consequences for human and animal health. The frequent use of antibiotics in veterinary practice causes their excretion in milk in dairy cattle. This way, they can easily enter the human body through the consumption of milk and dairy products. Objectives: This induces the need for accurate and sensitive methods to monitor antibiotic levels in milk. The aim of this study was to develop a rapid and sensitive magnetic nanoparticle-based fluorescence immunoassay for the simultaneous detection of chloramphenicol and penicillin G in milk. Methods: Magnetic nanoparticles were synthesized and functionalized with (3-aminopropyl)triethoxysilane. Chloramphenicol-Ovalbumin and Chloramphenicol-Ovalbumin-Fluorescein-5-isothiocyanate conjugates were prepared. Penicillin G – ATTO 633 fluorescent conjugate was synthesized. Antibodies against chloramphenicol and penicillin G were immobilized onto the magnetic nanoparticles. The competitive fluorescent immunoassay was developed. The optimal concentration of the antibody-magnetic nanoparticles and the fluorescent conjugates for the assay was determined. The calibration curves for the antibiotics in buffer and milk were plotted. Fluorescent immunoassay for the simultaneous determination of chloramphenicol and penicillin G in milk was developed. Results: The limit of detection by the simultaneous immunoassay of chloramphenicol and penicillin G in milk was 0.85 ng/mL and 1.6 ng/mL, respectively. The recovery of different concentrations of chloramphenicol and penicillin G in milk samples varied from 98% to 106%. Conclusions: A rapid and sensitive magnetic nanoparticle-based immunofluorescent assay for the simultaneous determination of chloramphenicol and penicillin G in milk was developed. The magnetic nanoparticles ensured rapid and easy procedure.