Bacterial & Cellular Assays
For further details and references concerning cellular studies,
consult CD Tutorial, Sections 3.6.1 to 3.6.12
Based on Sequential Injection and programmable flow, FIAlab systems have been used for biological assays in situ and in the laboratory environment. The following examples illustrate system capabilities and its value as a novel tool for study of cellular events.
Monitoring of Bacteria at Remote Locations
Colifast technology combines selective growth medium with an automated analyzer, designed and constructed by FIAlab Instruments. The growth medium for coliforms and thermotolerant coliforms includes a substrate that fluoresces after hydrolysis catalysed by galatosidase present in coliform bacteria. Different incubation temperatures of 44-44.5 C and 35-37 C, are used to distinguish between thermotolerant coliforms and total coliforms. Time lag of detecting bacteria in the monitored source is 1- 12 hrs, depending on the level of bacterial contamination.
The Colifast At-Line Monitor - CALM - is a fully automated stand-alone early warning at-line analyzer for rapid and systematic quality monitoring of E. coli, total coliforms, thermotolerant coliforms, and P. aeruginosa in waters. The applications of the CALM span from raw water, in-process water, waste water, to environmental monitoring. This instrument is widely used within municipal waterworks, sewage plants, and other areas. Depending on the method chosen, CALM will present results directly in CFU/ml, Most Probable Number (MPN), and/or Presence/Absence. The CALM is equipped with user-friendly, advanced custom software, and with remote control/warning possibilities.
No laboratory facilities and minimal skills in microbiology are required to perform the analyses. Pre-filled vials in disposable trays along with reagent bottles make field servicing fast and simple. The CALM-system consists of a sampling and analysing unit, an incubator unit with a robot-arm for distribution of the samples (up to 76 samples), and a lap-top PC with instrument software. The CALM is pre-programmed to automatically sample flowing water at any location on a schedule predetermined by the user, and subsequently automatically perform the sub-sampling required to provide the results. Analytical results are transmitted instantaneously to the operator or laboratory by analogue signalling and/or GSM data, telephone-line, or standard internet connections.
For further details contact http://www.colifast.no
Study of Live Cells
Microfluidic manipulations of cells grown adherent on beads, combined with fluorescence or absorbance measurements provide insight into kinetics of cellular processes, within a time scale that cannot be studied when using manual techniques. The following examples illustrate the scope of this novel methodology:
- functional studies for measurement of pharmaceutical antagonism (Hodder 1999)
- metabolic studies, related to oxygen and glucose consumption, and lactate extrusion (Schultz 2002)
- redox studies, such as degradation of hydrogen peroxide by live cells, as well as monitoring of intracellular hydrogen peroxide (Lahdesmaki 2007)
- automated techniques for counting of bead adherent cells. (Lahdesmaki 2009)
The first two topics were studied using fluorescence microscopy, but with the advent
of the Lab-on-Valve technique, the remaining studies were done using fiber optics, photomultiplier spectrophotometry and appropriate light sources.
The key to the success of BI based cellular assays is that beads are aspirated from the same culture tube that yields several hundred cellular samples with identical properties (viability, cell cycle, etc.), since the cells are randomly distributed within the sampled material. The result is that there is no biological variability within the series of experiments, carried out in a reasonably short period of time and sampled from the same cellular culture. Also, there is no hysteresis ( memory effect) caused by stimulants, applied in previous experiments, since a fresh cell culture can be used as needed by renewing the bead column under study. Since stimulants of known potency can be used as calibrants, by bracketing a series of other investigated stimulants, a direct comparison of potency of stimulants can be made. Besides fluorescence, the cellular events have been probed by spectrophotometry and voltammetry.
Chinese Hamster Ovary cells
grown on Cytodex Beads
Beads trapped in Flow cell
configured for fluorescence
and absorbance measurement
micro SI-LOV system set up for cellular studies
CHO Cells, transfected with type 1 muscarinic receptor and loaded with fura-2 Ca-indicator were exposed to carbachol (cch), pilocarpin (pch), or acetylcholin (ach)
NOTE: Culturing mammalian cells on beads is a well established technique, routinely used in industrial scale, for production of growth hormone and other pharmaceuticals. In laboratory scale, it is practical to work with cell culture grown on suspended beads in a 10mL culture tube
