Monitoring motility, spreading, and mortality of adherent insect cells using an impedance sensor.

An emerging sensor technology referred to as electric cell-substrate impedance sensing (ECIS) has been extended for monitoring the behavior of insect cells including attachment, motility, and mortality. In ECIS, adherent cells were cultured on an array of eight small gold electrodes deposited on the bottom of tissue culture wells and immersed in a culture medium. Upon the attachment and spreading of cells on the gold electrode, the impedance increased because the cells acted as insulating particles to restrict the current flow. Experimental data revealed that insect cells interacted differently with various proteins used to precoat the gold electrode with concanavalin A as the best promoter to accelerate the rate of cell attachment. After the cells were fully spread, the measured impedance continued to fluctuate to reflect the constant motion and metabolic activity of the cells. As the cell behavior was sensitive to external chemicals, the applicability of ECIS for inhibition assays was demonstrated with HgCl2, trinitrotoluene, trinitrobenzene (TNB), and 2-amino-4,6-dinitrotoluene as model systems. Unlike conventional assays, the quantitative data obtained in this study are taken in real time and in a continuous fashion to depict cell motility and mortality.

Digoxigenin-labeled probe for rapid identification of nisinogenic Lactococcus lactis strains.

From the nisZ gene sequence, a non-radioactive digoxigenin-labeled DNA probe, was tested for detection of nisin-producing strains using polymerase chain reaction amplification. The digoxigenin-labeled DNA probe clearly discriminated between nisin-producing and non-producing strains with a high degree of sensitivity and specificity. By agarose gel electrophoresis, 1.4 ng of nisin DNA was detected using the digoxigenin-labeled DNA probe compared with 11 ng using direct polymerase chain reaction amplification. A colony hybridization method using digoxigenin-labeled DNA to selectively detect nisinogenic bacteria showed that the nis-probe was specific and did not react with any other non-bacteriocinogenic and non-nisinogenic strains.

Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence.

A highly specific antisera was produced in New Zealand white rabbits against nisin Z, a 3400 Da bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719. A dot immunoblot assay was then developed to detect nisin Z in milk and whey. As few as 1.5 10(-1) international units per ml (IU ml-1), corresponding to 0.003 microgram ml-1 of pure nisin Z, were detected in carbonate-bicarbonate buffer within 6 h using chemiluminescence. When milk and whey samples were tested, approximately 0.155 microgram ml-1 (7.9 IU ml-1) of nisin Z was detected. The detection limit obtained was lower than that of traditional methods including microtitration and agar diffusion.

Note: genetic and biochemical characterization on nisin Z produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719.

The bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719 was purified and characterized. Two peaks exhibiting antimicrobial activity were obtained after purification. Primary structure of the peptide of major peak 2 was identical to that of nisin Z when determined by Edman degradation and confirmed by DNA sequence analysis. The molecular mass as determined by mass spectrometry was 3346.39 +/- 0.40 Da for peak 1 and 3330.39 +/- 0.27 Da for peak 2, which suggests that peak 1 may correspond to an oxidized form of nisin Z. The two purified peaks exhibiting antimicrobial activity appear to correspond with oxidized and native forms of nisin Z.

Regulation of nisin biosynthesis by continuous cultures and by resting cells of Lactococcus lactis subsp. lactis.

Nisin production by Lactococcus lactis subsp. lactis has been investigated using lactose as carbon source. Whether or not continuous cultures were lactose-limited, maximum nisin titre was observed at an intermediate mu value with a sharp peak of activity between 0.2 and 0.3/h. The maximum specific growth rate obtained in the medium used was 0.6/h and the maximum titre of nisin at mu = 0.25/h (160 AU/ml) was about nine-fold higher as compared with activity obtained at a dilution rate of 0.05/h or 0.4/h. With a constant dilution rate of 0.25/h and varying initial lactose concentrations from 3 to 40 g/l, there is an increase in nisin biosynthesis with increasing lactose concentration correlated with higher rates of sugar consumption. A Ymax value of 0.2 g bacterial dry weight and a maintenance coefficient of 124 mg lactose/g bacterial dry weight/h were determined. Lactose consumption increased from 1 to 3.28 g of lactose/g (dry wt) of cell mass/h and the nisin titre from 12.5 to 164.2 AU/ml. At higher values, nisin production declined. This implies that biosynthesis of nisin is regulated by a system of repression and derepression. Addition of lanthionine and beta-methyllanthionine precursors to the medium decreased the nisin titre when either threonine, threonine-cysteine, or cysteine-serine-threonine was added at the optimal dilution rate of 0.25/h; however, simultaneous addition of serine and cysteine elicited a slight increase in nisin activity. Studies with resting cells confirm that the biosynthesis of nisin is tightly regulated, since the production rate can be 5.6-fold higher than in cells grown in continuous culture. In addition, cell-adhered nisin appears to play a role in the production of the enzyme: low levels of cell-adhered nisin elicited high production rates, whereas high levels were not associated with nisin biosynthesis. In addition to pH, magnesium sulphate and lactose concentrations, nitrogen sources were also able to interfere in cell-adherence nisin.