a cassette ( fig1 ) was constructed consisting of a polycarbonate ( makroion , amari plastics , u . k .) or acetal frame ( 1 ) which was 2 mm thick and had outer measurements of 130 mm × 145 mm and a window ( 2 ) within the frame that measured 100 mm × 100 mm , sealed within a sleeve ( 3 ) of polyvinyl chloride ( pvc ) packaging film ( tsb 11 , borden , u . k .). this film was 17 μm in thickness , and was manufactured with an integral 10 % stretch along its longitudinal and transverse axes . a cassette was made by enveloping the frame within a sleeve of film that was heat sealed on three sides to form an envelope that measured 1 . 1 times the length and 1 . 1 times the width of the frame . the frame was inserted into the envelope and a strip of domestic baking parchment inserted between the sheets of pvc in order to avoid adhesion of the two inner faces of the film . the envelope was sterilised by autoclaving at 121 ° c . for 20 mins , the cassette was removed from between the supporting stainless steel plates , the baking parchment removed aseptically , and the remaining side of the envelope around the cassette sealed . the pvc film sheet on either side of the sterile cassette was made taut in a stream of hot air . a filling port ( 4 ) was provided by way of an aperture in the frame coextensive with the space between the pvc films enclosing the window ; thus allowing filling tube access . this port in turn was accessed by making an incision in the film adjacent it which was sealed with tape before scanning . filling of cassette . the cassette was filled with microbiological culture medium that was composed of trypticase soy broth ( tsb , baltimore biological laboratory ) with added yeast extract and giucose ( tsbyg ) and gelatin ( approx . 225 bloom from bovine skin , sigma ). the tsbyg was prepared at twice the final concentration and was sterilised by filtration . the gelatin was was prepared as a 20 % ( w / v ) aqueous solution , adjusted to ph 7 . 0 by the addition of naoh , 5 mol / l . and sterilised by autoclaving at 121 ° c . for 20 mins . for use in a cassette the gelatin was melted by incubation at 55 ° c . for 20 mins , and mixed in equal volume with double strength tsbyg . in some cases the composition of the culture medium within the cassette was modified by the addition of nacl to the tsbyg or by adjustment of the ph by the addition of sterile hcl ( 1 mol / l ). bacteria were added to give a suspension that contained approximately 10 3 viable bacteria ml - 1 . at this concentration the bacteria , and hence the colonies derived from them , were on average 1 mm apart so that seen in 2 dimensions in the cassette they appear on average to be 0 . 6 mm apart with a 2 mm thick gel . addition of medium to the cassette was carried out by making an incision in the pvc adjacent an inoculation port outside the window area and pumping the medium in through a tube with a needle or nozzle in its distal end under influence of a syringe and a three way tap . the cassette was cooled in order to solidify the gelatin / culture medium mixture within and then mounted in the laser gel scanner ( lgs ) system in a room strictly controlled at 20 ° c . a cassette as described in example 2 was filled layer by layer with liquid gel medium of increasing nacl content ; each layer being allowed to set before addition of the next . analysis of nacl content at given areas of the window was carried out after each experiment to determine the gradient lifetime for a given gradient . using this technique ph and chemical gradients can be successfully established for several hours . the growth of salmonella typhimurium in this cassette at 20 ° c . was studied using the laser scanner and the results of this study are set out in fig5 . such results provided from a panel of microbes may be used as standard ` calibrations ` against which growth of unknown microorganisms may be compared in the second aspect of the invention . a gradient former was constructed from stainless steel ( see fig3 ) having front and back plates which engage a standard gel filled cassette having incisions across the width of one pvc windows at it top and bottom . the solidified contents of the cassette were brought into contact with former reservoirs containing liquid medium having composition of study component equal to the extremes of the range being studied such that a gradient forms from top to bottom . the medium in the reservoirs was maintained at respective constant study levels by pumping medium between them and larger reservoirs containing the desired composition ; the reservoir medium being sterile as opposed to the inoculated cassette medium . leakage was prevented using rubber gaskets / seals which interface the periphery of the incisions in a liquid tight manner . in fig3 there is shown a cross section through the arrangement of front and back plates of the gradient former prior to their association with a gel cassette of the invention and appropriate gaskets . the gel cassette frame ( 16 ) and gel body therein ( 17 ) are held between the front ( 18 ) and back ( 19 ) plates of the former such that elongate gaskets ( 20 , 21 , 22 , 23 ) are provided to engage the pvc around slits made in the pvc film , within the cassette windows adjacent their top and bottom , and the former plates . the gaskets ( 21 , 23 ) between the cassette and the back plate are such that they are aligned with inlets ( 24 , 25 ) from small reservoirs therein which are in turn connected to larger reservoirs of medium of desired chemical or physical property . plate guides ( 26 , 27 , 28 ) are provided on the back plate for engaging holes in the front plate such that windows in each plate which correspond in size approximately to the window in the cassette are aligned with each other such as to allow laser light to pass generally in the direction from the laser source side ( 29 ) to the detector side ( 30 ). it will be realised that in use the cassette and the plates will be at about 45 ° and thus the line ( 29 ) ( 30 ) passes at an angle through the combination . one of the plates has mounting holes for attaching the complete assembly to the x / y stage . fig4 shows a front elevation of the back plate wherein window ( 31 ) is provided between inlets ( 32 , 33 ) from reservoirs which are in turn connected by inlets from larger reservoirs ( 34 , 35 ) and flush outlets ( 36 , 37 ). holes ( 38 ) for mounting the plate onto the x - y stage are provided on an extension on one side of the reservoir / window area , while the guides ( 26 , 27 , 28 ) are provided about the window and bottom reservoir . the light processing apparatus ( see fig2 ) consisted of a vertical mount plate ( 5 ) in which the cassette was held vertically on a motorised x - y stage ( micro - control ): x - stage ( 6 ) and y - stage ( 7 ). the cassette was illuminated by a 30 mw he - ne laser ( 8 ) ( spectra physics ) at a wavelength of 632 . 8 nm . the laser beam was collimated by a 200 μm pinhole ( 9 ) mounted as close to the cassette ( 2 cm ) as possible . this provided a well defined diameter and a sharp beam profile , and illuminated each pixel as evenly as possible . measuring total light scattered through 90 ° from each pixel did not maximise sensitivity . contributions to the total scattered light intensity from any point on the cassette when illuminated by a 0 . 2 mm laser beam were from many sources , including the 2 cassette windows and the gel itself . light scattering contribution made by a few bacteria of combined diameter of approximately 10 μm is very small but if the path of the laser through the cassette is interrogated a small section at a time , for example 200 μm , then the contribution from the bacteria is much greater . in the order to facilitate this the laser beam was passed through the cassette at an angle of 45 ° giving a path length in the gel of 2 . 8 mm . light scattered through 90 ° from the laser beam was collected and focused by a × 10 microscope objective ( 10 ) placed 2 cm from the cassette . the scattering angle was set at 90 ° because this was the only way of maintaining a focused image of the laser beam throughout the thickness of the cassette and to keep the distance between the scattering centre and detector as similar as possible along the length of the beam in the gel , thus providing a similar solid angle for light collection at all points along the beam . the cassette was mounted such that light was reflected away from the detection system so that only scattered light was detected . the lens focused the image of the laser beam going through the cassette onto a 400 μm pinhole ( 12 ). between the lens and the pinhole was a scanning mirror ( 11 ) which could move the focused image of the laser beam across the pinhole . directly behind the pinhole was a narrow band pass filter ( 13 ) to remove any stray background light . light passing through the filter was detected by a photo - multiplier tube ( 14 ) ( hamamatsu ) ( pmt ). the signal from the pmt was fed to a fixed gain preamplifier and thence to a variable filter ( kemo ltd .) and 12 bit analogue to digital converter ( adc ). the filter was a variable 24 db butterworth type set to low pass with a cutoff at 2 khz . the filter had a variable gain amplifier which could be set to 1 , 3 or 10 times gain , allowing an effective increase in the dynamic range available through the adc by a factor of 10 . the dynamic range in intensity available to the controlling computer was 4095 , in any given run . however , over a series of runs such as would be made when following colony size as a function of time this could be improved upon . in the early stages of growth the scattered intensity was low and high gain was required and the filter gain was set to 10 . in the later stages of growth the scattered intensity was high and low gain was needed so the filter gain was set to 1 . this approach gave an effective dynamic range of 40950 . the x - y stage was driven by a cc1 . 2 2 - axis stepper motor controller ( micro - control ) and the scanning mirror controlled through a 12 bit digital to analogue converter ( dac ). the three peripheral devices , the adc , dac and the cc1 . 2 were driven from a vme based microcomputer ( microsys ). control software was written in both fortran and 68000 assembler code . the computer accessed the peripherals via an on board 68230 parallel interface . a beamstop ( 15 ) was provided for capturing unscattered light . for the purposes of measurement the scan window ( area of the cassette to be interrogated ) was broken into pixels the size of which were set by the laser beam diameter passing through the cassette ( i . e . 200 μm ). the cassette was scanned in horizontal raster fashion by driving the x - y stage 0 . 28 mm in the x direction or 0 . 2 mm in the y direction from one pixel to the next . at each pixel position the scanning mirror moved the laser beam image across the detector pinhole and back again . this scan was broken down into 100 separate readings but for reasons of phase lag only 60 were used . each reading took approximately 100 μs so the effective mirror drive frequency was 100 hz which gave a phase lag between the mirror drive signal and the mirror position of about 1 ms or 10 readings . the stages have a resolution of 10 μm per step and a maximum start frequency of 400 hz . because the mirror scan was slower than one half step the stage could only be run at the maximum start speed . moving the 28 steps from one pixel to the next thus took 70 ms plus the mirror scan of 10 ms giving a pixel scan frequency of about 12 hz or 2 μl / s in a 2 mm thick cassette . at each pixel position the minimum intensity value plus the threshold value were subtracted from all 60 data values and resultant negative values were set to zero . any peaks in the remaining data were then stored so that the final data was in the form of an x - position , a y - position and an intensity value . the upper limit on the number of peaks detected is 25000 due to memory limitations . the growth of bacterial colonies was monitored by regular measurement of the light scattered through 90 ° from the colonies . the scattered intensity was then related to colony radius and colony viable count from previous calibration measurements . measurements of scattered intensity were also made as a function of position within the cassette . it was assumed that the scattered intensity was proportional to the square of the colony radius and that the number of viable bacteria in a colony was proportional to the cube of the radius . thus in theory we have a dynamic range of about 200 for radius and about 8 × 10 6 for numbers of viable bacteria . intensity data was treated simplisticly by integration over all pixels in the area of the cassette window that was scanned and conversion to intensity per unit volume ( ml ) as in equation 1 . equations 2 and 3 were then applied to give colony radius and numbers of viable bacteria at the time of each measurement . where r is the colony radius and vc is the number of viable bacteria per ml . i is the measured intensity per ml , k n and k r , are proportionality constants , n col : is the number of colonies per ml , i o is the integrated background scattering from everything except the bacteria . the exempliifed lgs was not designed to detect single bacteria and hence provided no useful data in the lag phase . the latter part of the exponential and the stationary phases could be monitored , and if the initial inoculation concentration in the cassette was known then the lag phase could be deduce . in order to provide any of these parameters the lgs intensity data must be converted into growth curves by the application of equation 3 . the value of k n from this equation must be deduced empirically as must the value of k r in equation 2 . cassettes were constructed that contained culture medium adjusted to ph 7 . 0 , and with either 0 . 5 or 3 . 5 % ( w / v ) nacl and inoculated with s . typhimurium at a concentration of approximately 10 3 ml - 1 . one cassette was scanned in the lgs constantly throughout incubation . other cassettes , prepared and incubated in parallel , were used for the determination of the numbers of viable bacteria , using conventional plating techniques , or for the measurement of colony diameter using a light microscope fitted with normarski optics . lgs data from cassettes that contained 0 . 5 or 3 . 5 % ( w / v ) nacl show the typical form for a bacterial growth curve with an initial lag phase followed by an exponential growth phase and finally a stationary phase . in order to extract the values for k n at the two different nacl concentrations these curves were fitted to the corresponding plate count data using standard nonlinear least squares techniques . the following conditions were applied when fitting for k n : firstly i o the background had to be included as a fit parameter , and secondly as the times for the lgs and the viable count data did not coincide the lgs results had to be interpolated to the times for the viable counts . the interpolated lgs data were then fitted to the data via equation 3 . the result of equation 3 at the original lgs times were calculated using the values of k n and i o obtained from the fit . the only variable parameters in the fit were k n and i o , the values for which were 4 . 9 × 10 - 3 and 1 . 41 × 10 5 respectively for the 0 . 5 % nacl and 4 . 94 × 10 - 3 and 1 . 0 × 10 5 for the 3 . 5 % nacl ; both curves being in agreement . a similar approach was taken to the radius data , and the interpolated lgs data was fitted using equation 2 to the measured colony radii . in the fits of equation 2 the values of i o used were those obtained from the fits of the viable count data and k r was the only floating parameter , n col was obtained from a viable count of a cassette at the time of inoculation . for the 0 . 5 % nacl lgs cassette the initial inoculum was 800 ml - 1 and the value obtained for k r was 13 . 0 . the initial inoculum for the 3 . 5 % nacl cassette was a little lower at 300 ml - 1 and the fitted k r was 5 . 8 . the growth parameters obtained from all four curves show good agreement between the measured size and that calculated from the lgs data . the data obtained demonstrated the usefulness of the lgs system for the continuous automated determination of the rate of growth of bacteria within a colony . the mean size of stationary phase colonies in medium containing 3 . 5 % nacl was larger than in medium containing 0 . 5 % nacl . the numbers of viable bacteria in colonies growing in the higher salt concentration was lower , however , than in the low salt . this may have been due to death of cells within the colony , or differences in the size or packing of cells , but demonstrated the necessity of assigning different values to the proportionality constants in equations 2 and 3 .