Patent Application: US-68067891-A

Abstract:
methods for the isolation and identification of a toxicant in a sample are disclosed . luminescent biological agents having sensitivity to a toxicant or an isolatable component in a sample are used to provide visually discernable zones of luminescent inhibition in the presence of a toxicant in the presence of an isolatable sample component as separated by paper or thin layer chromatography . kits for use in conjunction with the identification of a toxicant in a sample are also described , which include a luminescent biological reagent as the visualizing agent . particular examples of luminescent agents include photobacterium leoganthi , photobacterium phosphoreum , vibrio fischeri , vibrio harveyi a luminescent fungi , a luminescent fish extract , a luminescent dinoflagellate and fluorescent microorganisms , such as cypridina . potential toxicants in a liquid sample , a solid sample , or in a gaseous sample may be identified and further chemically characterized using the described methods . the isolation of potential toxicants in a sample through the processing of a sample through a separation phase matrix such as chromatography paper or tlc plate , followed by exposure to luminescent biological agent , provides for a rapid and inexpensive method for identifying pesticides , herbicides and heavy metals in a known or unknown sample .

Description:
the present invention provides methods , kits and luminescent biological ( for example , bacterial ) agents which are demonstrated to be surprisingly advantageous for the identification of specific toxicants or component substances in a sample . moreover , techniques are proposed wherein the identified component substances of a sample may subsequently be chemically characterized or additional volumes of the isolated component ingredient ( i . e ., toxicant ) be obtained employing a variety of chemical techniques in conjunction with the teachings of the present disclosure . the novel use of a luminescent biological agent together with a separation phase matrix provides a unique method for the rapid and simple identification of potentially toxic ( isolated ) substances in a sample . the inventors foresee the application of the present invention in the laboratory as well as in industry for the detection of environmental pollutants , particularly in water resources . additionally , use of the described methods in the development and identification of therapeutically valuable components in plants and organisms , such as in garlic , is also considered an important application of the described invention . where the luminescent biological agent to be used is a luminescent bacterial agent , such as the luminescent bacteria vibrio fischeri , the bacteria should constitute a suspension of bacteria at a final concentration of about 10 8 - 10 9 bacteria cells / ml in the suspension to be used , for example , where the luminescent bacterial agent is sprayed onto a chromatogram . a preferred method whereby the luminescent bacteria are prepared for use in the presently described invention is as follows . the bacteria must first be allowed to become fully &# 34 ; induced &# 34 ; in their luminescent system , i . e ., the luminescent system of the bacteria should be allowed to reach complete development prior to harvesting of the bacteria from the culture . determination of at what point a bacteria has reached full luminescent system development is well known to those of skill in the art 30 , 31 . upon full development of the luminescent system of the bacteria , the bacteria should be harvested and then placed in a centrifuge tube . the bacteria are then to be centrifuged at a speed of 10 , 000 × g for 30 minutes at room temperature . thus centrifuged , the bacteria will form a pellet of cell &# 34 ; paste &# 34 ; at the bottom of the tube . about 1 gram of this cell paste ( about 12 ml of cell &# 34 ; paste &# 34 ;= 1 gram ) of glowing bacteria is then to be diluted to a volume of 20 ml , by adding 20 ml of a diluent of choice . where the luminescent bacteria is a marine bacteria , for example , the diluent is most preferably a buffered saline solution of between 1 - 4 % nacl . as diluted to 20 ml , the cell suspension constitutes a concentration of 10 10 - 10 12 bacteria cells / 20 ml ( or 10 8 - 10 9 cells / ml ). the following examples are presented only to describe preferred embodiments and utilities of the present invention , and to satisfy best mode requirements . the examples are not meant to limit the scope of the present invention unless specifically indicated otherwise in the claims appended hereto . the following examples are provided to demonstrate various aspects of the present invention . isolation of identifiable luminescent inhibitory toxicant in garlic extract using luminescent bacteria identification of pesticides and herbicides in a sample with luminescent bacteria proposed identification of heavy metals in a sample with luminescent bacteria proposed chemical identification of a toxicant in a sample isolated with bioluminescence methods identification of toxicant in a gaseous phase sample with luminescent bacteria proposed identification of a toxicant on a solid surface sample with luminescent bacteria isolation of identifiable luminescent inhibitory toxicant in garlic extract using luminescent bacteria the present example is presented to describe a method by which components of a substance which inhibit luminescent bacteria may be isolated . the sample analyzed in the present example is a garlic extract . for this experiment , the inventors first prepared a garlic extract from garlic powder . the garlic powder was processed so as to form a liquid garlic extract . one ( 1 ) gram of garlic powder was blended with 5 ml . h 2 o . other solvents such as ethanol , chloroform , or acetone may be used to blend the sample , but h 2 o was found to be the best solvent for the garlic . a 5 ml . volume of the garlic extract was first applied (&# 34 ; spotted &# 34 ;) to tlc plates at several points equidistant from one edge of the plate . the plate was inverted in a sealed tlc solvent container with a small amount of solvent in the bottom such that spotted samples were parallel to and above the solvent interface . as the solvent ( acetonitrile : water : aqueous ammonia , 8 : 1 . 5 : 0 . 5 ) migrated up the tlc plate , the individual components in the garlic extract were sufficiently separated to detect separate zones of luminescent inhibition upon exposing the developed chromatogram to a suspension of luminescent bacteria , vibrio fischeri applied in a suspension of 0 . 5 m nacl ( see fig1 and fig2 ). the inventors applied the luminescent bacteria , vibrio fischeri to the chromatogram specifically by spraying the described suspension of bacteria ( contained in a buffered salt solution of 3 % ( 0 . 5 m ) nacl at a ph of about 7 ) onto the developed chromatogram after the solvent in which the sample was contained had evaporated . zones of luminescent inhibition were located prior to the dehydration of the bacteria on the chromatogram , i . e ., at least within 1 hour after application of the bacteria . the inhibition of bioluminescence of the bacteria caused by the presence of toxicants in isolated components of the garlic extract was then visualized . the bioluminescent inhibition effect of any toxicant in the garlic extract became apparent generally within a few minutes in the form of a clearly demarcated zone of bioluminescent inhibition ( see fig2 ). these zones of bioluminescent inhibition are areas on the chromatogram which were dimmer ( i . e ., less brightly emissive ) than the more brightly emissive surrounding areas on the chromatogram ( which did not include isolated components of the garlic extract which were capable of inhibiting the luminescence of the vibrio fischeri ). the areas wherein the chromatogram demonstrated greatest amounts and intensity of blue bioluminescence from the applied vibrio fischeri bacteria identified areas of no component substances or instead isolated components of the garlic extract which were not toxic to the bioluminescence of the bacteria , and therefore according to the described method were considered not to constitute toxicants . as stated , the inhibition of bacterial luminescence which occurs when a toxicant is detected , becomes apparent very soon , often within a few minutes , and grows more distinct with time and reaching a pronounced peak effect in the minutes before the chromatogram dries out , i . e ., the zones of decreased luminescence show more contrast relative to the surrounding luminescence with time , prior to the chromatogram drying out . when the chromatogram is dried out , of course , all the luminescence of the bacteria on the chromatogram will be extinguished with the dehydration of the bacteria . curiously , with the described methods , those positions on the chromatogram to which the toxicants have migrated ( i . e ., the &# 34 ; zones of inhibition &# 34 ;) appear to dry out faster than the remainder of the chromatogram which , for example , remains highly luminescent . alternatively , the identification of the different individual components of the garlic extract could have been accomplished using paper chromatography as the separation phase matrix for the sample or other such techniques well known to those of skill in the art . this is a photograph of a tlc plate viewed by fluorescence . the actual plate was 20 cm by about 4 . 5 cm . in the photograph the plate is seen reduced to 13 . 0 cm by 2 . 95 cm . dimensions below refer to dimensions of the photograph not of the original plate . aqueous garlic extract . preparation : 1 . 0 g of powdered garlic suspended in 5 . 0 ml of h 2 o . mixed with vortex mixer for 1 minute . centrifuged in table top centrifuge on high ( about 1 - 2000 rpm , 60 seconds ) to obtain straw yellow supernatant : the sample . five μl applied at origin of plate : pencil lines seen near 1 . 7 cm from bottom of plate . the application zone is seen as a circle ( faint ) of about a 6 mm diameter centered on the line . the solvent system used was acetonitrile : water : 25 % ammonia ( aqueous ); 80 : 8 : 12 . the sample was chromatographed in a closed chamber for approximately 20 minutes . the solvent front traveled about 4 / 5 of the distance of the plate . a faint demarkation line is seen at about 10 . 5 cm from the bottom of the plate showing the location of this solvent front . major features of the chromatogram viewed by fluorescence excitation are a pronounced dark line at about 7 . 9 cm from the bottom of the plate several chevron or v - shaped dark areas in the 5 . 8 - 7 . 7 cm from the bottom region and a faint roughly circular shaped zone centered at about 8 . 8 cm from the bottom . the chevron shaped darkenings represent chemical components in the garlic resolved by the chromatographic process . the more or less circular zone at 8 . 8 cm ( which can be more dramatically revealed by moving the photograph back and forth about 2 cm in the plane of the photograph ) is the zone or near the zone of bioluminescence inhibition seen in photograph 2 . this is a photograph of a tlc plate ( not the same one as in photograph 1 , but a plate developed in an identical fashion except for a longer time ) viewed by the emission of vibrio fischeri luminous bacteria . the actual plate was 20 cm by about 4 . 2 cm . in the photograph the plate dimensions are 12 . 9 cm by 2 . 8 cm . aqueous garlic extract : the identical sample used in chromatogram of photo 1 . same as in photo 1 except chromatogram ran longer , front reaching near the end of plate , near 12 . 5 cm in photograph . the origin was centered on the pencil line visible at about 2 . 4 to 2 . 5 cm from bottom of plate . a very dark , nearly circular zone is seen centered at about 10 . 5 cm from bottom of plate . a faint second zone is seen at about 6 . 7 cm from the bottom . several darkened regions can be seen at the edges of the plate . the dark areas which appear at the edges are artifacts , and represent places on the chromatogram sheet which were not adequately sprayed with the luminous bacterial suspension . the zone at 10 . 5 cm represents the lumotox effect i . e ., the determination of the location of the component in garlic which inhibits the luminous bacteria . routinely , for preliminary analysis of the chromatograms , the plates were irradiated with a lamp emitting uv ( 254 nm ) radiation . the tlc plate used had the f 254 backing and were therefore fluorescent everywhere that no uv absorbing samples or components existed . this preliminary detection system also revealed component substances as dark spots on a light background where heterocyclic or other uv absorbing compounds were present . however , fluorescent extinction and luminescence inhibition were often not in parallel . for example , some samples presented as very dark zones , as viewed by fluorescence ( for example , garlic ), had little or no bioluminescence inhibition , while other zones presented very faint or non - existent fluorescence extinction but had substantial ability to inhibit ( extinguish ) bioluminescence ( e . g ., garlic , lindane ®, roundup ®). particular sources of tlc plates and chromatography sheets include sargent welch ( no . s18953 - 10 - tlc plate with f 254 fluorescent material ), analtech ( uniplate taperplate silica gel g - f , no . 81013 ), and eastman - kodak ( kodak chromatogram sheets silica gel absorbent with fluorescent indicator , catalog no . 122 - 4294 ) and whatman ( absorbent plates flexible - backed aluminum polyester , catalog no . 4410 - 22 ( contains fluorescent indicator )). the albert et al . article 22 provides a description of analyzing mevinolin , a fungal metabolite employing standard laboratory techniques such as mass spectroscopy , nuclear magnetic resonance and x - ray analysis . these alternative standard laboratory techniques could be utilized to analyze eluted components from an unknown sample . upon isolation / separation of the various components in the garlic extract sample by a chromatography method , the inventors then applied the luminescent bacteria to the developed chromatogram . most preferably , the luminescent bacteria is applied to the developed chromatogram in the form of a suspension contained in a buffered salt solution ( about 0 . 3 m na + / k + phosphate buffered saline ( 3 % nacl by weight ) ph 7 . 0 ). prophetic example 2 -- proposed chemical identification of toxicants in a garlic extract the present prophetic example is provided to outline one proposed method by which the toxicant ( s ), as identified according to the method of the procedure outlined in example 1 may be further characterized to identify the chemical structure of the isolated toxicant ( s ). this method may also be used where additional amounts of the isolated substance are desired or where the purity of the isolated substance is to be determined . the particular &# 34 ; zones of luminescent inhibition &# 34 ; described above , which provide for the isolation of the component substances ( i . e ., toxicant ) in the test sample , are used as reference points to isolate each component substance from an adjacent spotted sample which was run on the same or a separate tlc plate with the same sample . unsprayed sections of the tlc plate , which correspond to zones of luminescent inhibition on the sprayed portion , may be scraped off and added to a sufficient volume of an appropriate solvent ( i . e ., distilled water , acetone , ethanol , ether , ethyl acetate - chloroform or other solvent mixtures ) such that the isolated component substance of the sample may become dissolved in the solvent . subsequent removal of the solid tlc scrapings from the liquid eluate can be accomplished by various methods known in the art such as centrifugation or filtration . if necessary , the eluates containing dissolved toxicants may then be concentrated using standard techniques . these separated , ( and in some cases , concentrated ) isolated substances of the sample may be further resolved in other tlc solvent systems ( or hplc , paper chromatography , and the like ) to verify purity or to obtain suitably pure isolated substances . these substantially pure isolated substances can then be identified using standard chemical and spectral methodologies . for example , such standard chemical and spectral methodologies include as hplc , ms , ir , nmr , and the like . alternatively , two dimensional ( 2d ) thin layer ( tlc ) can be run for higher resolution of the sample for more explicit identification of components therein . in the 2d method , a sample is spotted near one corner of the tlc sheet or plate , and run successively in two , usually perpendicular , directions , using different solvent systems or conditions . for example , the sample is chromatographed in the usual way ( described above ) on the tlc medium in the first direction using solvent system no . 1 ( e . g ., a basic non - polar system , ammonia : butanol : hexane in a 5 : 20 : 75 ratio ). the chromatogram , containing components resolved in a linear fashion in this solvent system no . 1 , is then to be removed from the chromatography chamber , dried fully to remove solvent molecules of this system no . 1 solvent , and then the thus dried chromatogram is rotated 90 ° to the orientation first used and chromatographed in the new orientation using a solvent system no . 2 ( e . g ., a polar , acetic system , such as acetic acid , acetone , ethanol in a 10 : 50 : 40 ratio ). the components resolved into a linear array by system no . 1 move in the perpendicular direction with the solvent system 2 to provide even greater resolution of individual component substances in the sample . this same basic approach can be utilized where luminescent bacteria are used to identify isolated component substances of a sample separated by paper chromatography systems , either 1d or 2d . as those in the art will appreciate , in using such systems , there are various ways to achieve separation such that toxicants can be obtained in relatively pure form . for example , another version of 2d paper chromatography may employ electrophoresis in one dimension and gravitational flow paper chromatography or isoelectric focussing in another dimension , or other two - dimension combination thereof ( i . e ., 1st d = paper chromatography , 2sn d = isoelectric focusing , etc .) the present example is provided to demonstrate the use of the claimed methods and reagents for the identification of a pesticide in a sample of known substances . in this example , the pesticides identified were diazanon ®, lindane ® and sevin ®. the luminescent bacteria used in the present example was vibrio fischeri ( atcc 7744 ). identification of these individual pesticides and herbicides was achieved essentially according to the same methods described in example 1 . a suspension of vibrio fischeri in a saline diluent was sprayed , using an aspirator bottle , on the developed chromatograms . zones of luminescent inhibition appeared surrounding those areas on the plate where the diazanon ® had migrated . similar , less dim zones of inhibition , where lindane ® had migrated ( see fig6 and fig7 ). in a similarly run tlc with sevin ®, the chromatogram also demonstrated zones of luminescent inhibition at those areas on the chromatogram where sevin ® had migrated . these are photographs of the same tlc plate taken by two different conditions : fluorescence and bioluminescence , respectively . 5 μl samples of ( 1 / 32 by diazanon ®) and ( 1 / 8 lindane ®). the diazanon ® sample was produced by serial dilution of the commercial diagram ( 25 % w / v ) 0 , 0 , diethyl - 0 -[ 2 - isopropyl - 6 - methyl -≮- pyrimidinyl ] phosphorsthionate , ortho products . the diazanon ® was diluted with ethanol by factors of 2 until a dilution of 1 / 32 commercial strength was reached . the lindane ® ( ortho products ) was diluted in ethanol from the commercial 20 % ( w / v ) gamma isomer of benzene hexachloride , until a final strength of 1 / 8 was reached . fig3 represents the results from this study using diazanon ® and lindane ® on a tlc plate viewed by 254 nm excitation . a prominent dark zone for diazanon ® is located at 8 . 8 cm from bottom of fig3 . about 3 quite faint zones for lindane ® at 8 . 2 , 9 . 2 , and 10 . 3 cm from bottom of fig3 are demonstrated . diazanon ® origin ( application spot ) at 2 . 5 cm from bottom of photo , lindane ® origin at 3 . 0 cm . fig4 viewed by bioluminescence from vibrio fischeri . dark zone for diazanon ® very close to zone for fluorescence extinction ( at about 8 . 1 cm from photobottom ). several very dark zones for lindane ® at about 8 . 0 , 8 . 8 , and 10 . 0 from photobottom . also seen is slight inhibition zone at origin of lindane ® sample . the several zones for lindane ® indicate that several isomers or different inhibition compounds are present in the lindane ® sample . the present example is provided to demonstrate the sensitivity of the claimed invention to detect relatively low concentrations of a pesticide . an exemplary pesticide for demonstrating the sensitivity of the assay used here is diazanon ®. spot tests of diazanon ® at several dilutions were performed at the following strengths : full strength ( 25 % w / v diazanon ®), 1 : 128 ; 1 : 256 ; 1 : 512 ; and 1 : 1024 . no chromatography was done . 5 μl samples of the various diazanon ® dilutions were applied to tlc plate material , sprayed with a suspension of vibrio fischeri in a saline solution ( 3 % nacl wt / vol .) and photographed . marked inhibition occurred up to and including the d / 256 dilution ( d / 252 appears by clerical mistake on sheet instead of d / 256 which was used ) of full strength ( 25 % w / v ) diazanon ®. faint inhibition is seen at dilution 1 : 512 and dilution 1 : 1024 ( see fig6 r ). the tlc plates with diazanon ® demonstrate that the methods described herein are sufficiently sensitive to identify a pesticide in a sample at concentrations in which they are likely to occur in a land or water sample obtained in the environment . the present example is presented to demonstrate the effect of varying the solvent polarity on the detection patterns , or &# 34 ; zones of inhibition &# 34 ; of vibrio fischeri in the presence of diazanon ®, a pesticide . fig7 and fig8 provide photographs of tlc plates viewed by 254 nm irradiation ( fig7 ) and by bioluminescence ( fig8 ). in each case , 5 μl of ( diazanon ®/ 8 ) was applied at origin on left and 5 μl of lindane ®/ 8 was applied at right origin . three solvent systems used . all composed of hexane : thf mixtures . in fig7 the left chromatogram was hex : thf , 70 : 30 the middle chromatogram was hex : thf , 80 : 20 the right chromatogram was hex : thf , 90 : 10 . ( middle chromatogram contains clerical labeling error of 80 thf : 20 hex , which should be 80 hex : 20thf ) fig7 shows the decrease in polarity as the proportion of thfs lowered causes the diazanon ® and faint lindane ® spots or zones to be progressively diminished in mobility ; to have smaller r f values ; to migrate shorter distances from the origin . fig8 shows only the left - hand and right - hand tlc plates seen in fig9 . dark bioluminescence zones of inhibition are seen in photo 8 for diazanon ® and lindane ® samples . identification of pesticides and herbicides in a sample with luminescent bacteria the present example is provided to demonstrate the use of the claimed methods and reagents for the identification of pesticides and herbicides in a known test sample using a luminescent biological agent . in this example , the herbicides roundup ® and weed - b - gon ® and the pesticides diazanon ® and lindane ® are identified in a test sample with the luminescent bacteria , vibrio fischeri ( atcc 7744 ). each sample was run on an individual tlc sheet . photographs of the resulting 4 individual chromatograms are presented at fig9 ( room light ) and fig1 ( bioluminescence -- chromatogram with luminescent bacteria ). two solvent systems were used . the solvent systems used to identify the herbicides ( roundup ® and weed - b - gone ®) was 100 % ethanol . a 5 ml sample of an 8 - fold dilution of these commercially available herbicides was used in the spotting of the tlc plates . the solvent system used for the pesticides diazanon ® and lindane ® was hexane : thf , 90 : 10 . the pesticides were spotted at a concentration of 1 . 8 . a 5 ml sample of an 8 - fold dilution of these commercially available pesticides was used in the spotting of the tlc plates . two solvent systems were employed as no single system has yet been found to adequately resolve all compounds ( i . e ., the two pesticides and the two herbicides ). use of 100 % ethanol causes diazanon ® and lindane ® to run at the front of the solvent system . use of 90 % hexane , 10 % thf causes roundup ® and weed - b - gone ® to stay at the origin . the tlc plates photographed are in the following order ( left to right ) ( one sample per plate ): diazanon ®, lindane ®, roundup ®, and weed - b - gone ®. in each case , the commercial strength was diluted by a factor of 8 . the diazanon ® sheet did present an entirely distinct &# 34 ; zone of inhibition &# 34 ;, but the fig1 only marginally indicates this characteristic , perhaps due to partial bacteria dehydration . the lindane ® chromatogram presented as a distinct zone of inhibition culminating in a dark spot center about 2 . 9 cm from the bottom of the plate . the roundup ® chromatogram presented a clear zone of inhibition as seen at the origin , and at least one other inhibition zone centered at 4 . 5 cm from the bottom of the plate . the weed - b - gone ® chromatogram presented as a large oval zone of luminescent inhibition ( perhaps comprised of several components ) starting at about 1 . 8 cm from the bottom of the tlc plate and stretching to beyond 6 cm from the bottom of the plate . the following example presents the results of three separately run experiments by the inventors . these data demonstrate the reliability of the described methods for consistently identifying a component substance in a sample . the following list represents a description of the particular herbicides and pesticides , and the percent dilutions used thereof , in the described 3 separately run tlc plates . 10 . 8 % w / v dimethylamino salt of 2 , 4 dichlorophenoxyacetic acid 11 . 6 % w / v dimethylamino salt of 2 -( 2 - methyl - 4 chlorophenoxy ) propionic acid the following table presents the results obtained for identifying diazanon ®, lindane ®, roundup ®, and weed - b - gon ® in three different tests conducted by the inventors . these data demonstrate that the described method provides a system which possesses the ability to detect , with varying sensitivity , a variety of herbicides or pesticides in a sample on a consistent and reliable basis , as demonstrated by the closely corresponding &# 34 ; spots &# 34 ; for each run of the same component substance between the three separately run chromatograms . table 1______________________________________ ratio of herbicide / distance spot spot spot front stan . pesticide of front 1 2 3 fluor biolu dev . ______________________________________test 1 diazanon ® 2 . 38 0 . 63 -- -- 0 . 26 0 . 26 0 . 02 lindane ® 2 . 38 . 88 1 . 38 1 . 66 0 . 37 0 . 37 0 . 02 0 . 58 0 . 57 0 . 00 0 . 70 -- roundup ® 2 . 36 0 . 00 0 . 13 1 . 38 0 . 00 0 . 00 0 . 00 0 . 06 -- -- 0 . 58 -- -- weed - b - 2 . 36 0 . 66 1 . 64 1 . 88 0 . 28 0 . 28 0 . 02 gon ® 0 . 69 0 . 69 0 . 04 0 . 80 -- -- test 2 diazanon ® 2 . 75 0 . 68 -- -- 0 . 26 0 . 25 0 . 01 lindane ® 2 . 19 0 . 83 1 . 23 1 . 55 0 . 38 0 . 35 0 . 03 0 . 56 0 . 57 0 . 00 0 . 70 -- -- roundup ® 2 . 00 0 . 00 0 . 12 1 . 22 0 . 00 0 . 00 0 . 00 0 . 06 -- -- 0 . 61 -- -- weed - b - 2 . 25 0 . 65 1 . 35 1 . 79 0 . 28 0 . 28 0 . 020 gon ® 0 . 66 0 . 66 . 01 0 . 80 -- -- test 3 diazanon ® 2 . 79 0 . 68 -- -- 0 . 24 0 . 24 0 . 00 lindane ® 2 . 09 0 . 83 1 . 23 1 . 59 0 . 39 0 . 40 0 . 01 0 . 58 0 . 60 0 . 03 0 . 73 -- -- roundup ® 2 . 13 0 . 00 0 . 21 1 . 29 0 . 00 0 . 00 0 . 00 0 . 10 -- -- 0 . 60 -- -- weed - b - 1 . 84 0 . 38 1 . 17 1 . 55 0 . 21 0 . 21 0 . 05 gon ® 0 . 64 0 . 63 0 . 02 0 . 84 -- -- ______________________________________ r . sub . f values represented in the reported values in the table ; r . sub . f = relative to the front ; a fractin of the total distance which th solvent front migrated . proposed identification of heavy metal salts in a sample with luminescent bacteria the present prophetic example is provided to present a use of the claimed methods and reagents for the identification of a heavy metal in a sample . specifically , the inventors hypothesize that the described methods would be useful in the identification of the heavy metals such as mercury , lead and cadmium using the described luminescent biological reagents , such as the bacteria , vibrio fischeri ( attc acc . no . 7744 ). in the present example , the inventors spotted the various metals on to a chromatography paper sheet , but did not run them through a chromatography separation process . upon spotting of the various metals along one side of a chromatography paper sheet , the sample spots were allowed to dry . upon drying , the spotted sheets were exposed to the luminescent bacteria vibrio fischeri . employing this method , the inventors were able to visualize the presence of the heavy metal salts of mercury , lead , and cadmium . to isolate the heavy metal spotted on the chromatography paper , the paper edge at which the sample was spotted should be exposed to a solvent system , most preferably an acidic solvent system . specific reference is made here to the rainin ® catalog 29 , wherein a standard technique ( for the separation of heavy metals ) is described using an ion chromatography metals column . resolution of pb ++ and cd ++ is demonstrated in the reference rainin ® catalog . successive equal volumes of a heavy metal could be eluted using the hplc procedure from the hplc machine and spotted in an array or in a linear fashion on a sheet of ( whatman ) chromatography paper . after the carrier solvent is evaporated or otherwise removed by drying , the sheet could be sprayed with a suspension of luminescent bacteria , such as vibrio fischeri , as described . zones of bioluminescent inhibition could be similarly visualized to identify the metal . chemical identification of a toxicant in a sample isolated with bioluminescence methods the present prophetic example is provided to outline a proposed method whereby the identified region provided on a chromatography sheet with the described luminescent agent , particularly a luminescent bacteria may be analyzed to ascertain the chemical identity of an isolated component substance of a sample . a volume of sample containing sufficient concentration of toxicants would be applied to a chromatography paper , such as whatman 1m or 3m and chromatographed using a solvent system which provides maximum separation of the sample components . various solvent systems may be utilized and tested for separation efficiency as well understood by those skilled in the art . small amounts of sample may be used to test for improved resolution in one dimensional ( 1d ) chromatography solvent systems . those solvents found most effective may then be utilized for larger scale separation on large sheets of chromatography paper for two - dimensional chromatography ( 2d ). two dimensional chromatography may be necessary to resolve sample ingredients for subsequent identification of substantially pure compounds . by determining a combination of two solvent systems which effectively resolve the component toxicants , 2d chromatography can be run in duplicate . following the chromatography , the luminescent bacteria may be sprayed onto one of two identical sample sheets . areas on the sheet which demonstrate a decreased luminescence would then be used to mark the corresponding areas of the unsprayed sheet . the corresponding areas on the unsprayed sheet are cut out and eluted with distilled water , appropriate solvents such as acetone or ethanol or a solvent mixture to provide individual , substantially pure toxicants for identification . this procedure can be repeated , and / or multiple 2d sheets may be run simultaneously , in order to accumulate sufficient quantities of various substantially pure toxicants . in this manner , appropriate amounts of toxicants in a sample may be separated and then identified using standard chemical procedures . for example , small amounts of the purified component substances may be run on high pressure liquid chromatography ( hplc ) and compared to known standards for identification 15 . as will be appreciated by those skilled in the art , additional standard techniques used for chemical identification may be employed such as spectral analysis : mass spectra , infrared spectra ( ir ), nuclear magnetic resonance ( nmr ), and the like . it will understood by those skilled in the art that multiple 2d chromatography sheets can be run simultaneously in which different sheets are sprayed with different luminescent bacteria . this would provide a more thorough analysis of toxicants which may be detectable by one luminescent bacterium , but not by another . additionally , combinations of different luminescent bacteria in one spray solution may facilitate the thorough identification of most or all of the detectable isolated component substances in a sample . in this manner , a thorough analysis and identification of toxicants in a sample may be undertaken . essentially this same approach can be taken using thin layer chromatography ( tlc ), instead of paper chromatography as described above , for the initial separation and identification of toxic substances in a sample . multiple tlc plates ( e . g ., whatman 4856 - 840 with 1 , 000 μm silica layer ) may be run simultaneously in the same solvent system utilizing 1d or 2d runs , as described above , for paper chromatography . in such a tlc approach to toxicant identification , toxicants would be identified by spaying the plates with luminescent bacteria , marking the zones of decreased luminescence , and scraping off the corresponding areas on the unsprayed portions of plates . the scrapings are then eluted with an appropriate solvent , such as distilled water , acetone or ethanol or a solvent mixture , concentrated ( if required ), and identified using hplc , ms , ir , nmr , and the like . by following either of the above procedures , the separation and identification of toxicants in a sample can be accomplished simply and rapidly . the standardization of this method to be used for the identification of toxicants in certain types of samples will be appreciated by those skilled in the art as providing simple , rapid , and inexpensive methodologies for toxicant identification . for example , certain types of samples ( i . e ., industrial effluent ) could be tested to determine the initial separation system , the solvent systems , the luminescent bacteria ( or combinations of luminescent bacteria ), the elution protocol , and any subsequent techniques for quantitation and / or identification . through the use of standard curves of easily quantitated known compounds , the percent recovery in a given separation system can be determined . in this manner , amounts of identified toxicants can be quantitated and extrapolated back to the original sample volume applied . for example , the use of radiolabeled compounds , of known specific activity , which are separated by paper or tl chromatography , eluted , and counted for radioactivity , would provide an indication of the percentage recovery of a given compound . by comparing various radiolabeled chemical compounds in a given identification system ( paper or tlc with different solvents and the like ), one could correct for recovery losses of a given identification system . when the separated toxicants are quantitated by certain chemical and spectral methods , the quantities may then be extrapolated to determine the quantities of individual toxicants present in the original sample . thus , this method , in many cases , would allow for toxicant quantification . these steps could be standardized into kits tailored for the analysis of specific types of samples ( i . e ., a kit for a certain industrial effluent or certain biological samples , such as foodstuffs , pharmaceuticals , and the like ). these kits would comprise certain solvents and luminescent bacteria which would effectively resolve specific sample types thereby greatly simplifying and reducing the cost of toxicant detection , identification , and quantitation . alternatively , an unknown sample may be processed by the above procedure for identification and quantitation . identification of toxicant in a gaseous phase sample with luminescent bacteria the present prophetic example is provided to outline a proposed method whereby an investigator may identify a toxicant present in a gaseous phase sample employing the methods with luminescent bacteria described herein . as an initial step , the gaseous sample would be collected by techniques known to those skilled in the art . for example , a gas sample might be collected by filtration through a solid filter such that toxicants deposit onto the filter or by aspiration into a liquid such that toxicants dissolve in the liquid . in the case of a solid filter , the filter could then be eluted with distilled water or a suitable solvent , concentrated , chromatographed by paper or thin layer chromatography , and identified using certain luminescent bacteria as described in example 6 . identification of a toxicant on a solid surface sample with luminescent bacteria the present prophetic example is provided to outline a proposed method whereby a toxicant on a solid surface sample may be identified with the described luminescent bacteria . as in example 7 , methods for removing a toxicant from a solid surface so that it is collected in a concentrated liquid form will vary depending on the nature of the solid surface . techniques for such removal will be apparent to those skilled in the art . using the procedures outlined in example 6 , one skilled in the art would be capable of identifying and quantifying toxicants which were eluted from or removed from the solid surface . alternatively , for direct detection of toxicants , the solid surface could be sprayed with a certain luminescent bacteria , or mixture of more than one luminescent bacteria , such as vibrio fischeri and the surface observed for zones of decreased luminescence ( i . e ., zones of luminescent inhibition ) substantially as has already been outlined in example 1 . of course , these isolated component substances of the sample ( potential toxicants ) could then be chemically analyzed according to laboratory techniques well known to those of skill in the art to identify the chemical structure of the isolated component . by way of example , such laboratory techniques for determining the chemical structure of an isolated component substance include hplc , ms , ir , nmr , and the like . the present prophetic example is provided to define those components which would comprise a proposed test kit useful for the identification of toxicants in a sample . such a kit most preferably would comprise a carrier means adapted to receive at least two container means and at least one chromatography paper sheet in close confinement therewith . the kit should also include at least one chromatography paper sheet and a first container means comprising a luminescent bacterial agent . while any luminescent bacterial agent may be used in conjunction with the described kit , that bacterial agent most preferred is the vibrio fischeri ( attc acc . no . 7744 ). most preferably , the luminescent bacterial agent should be in lyophilized form in the container means . the lyophilized bacteria would then be suspended in a diluent solution . for example , where appropriate nacl concentrations are within the lyophilized sample , deionized water may be employed as the diluent solution without any expected deleterious effects to the luminescence of the bacteria . in a second container means , the kit should further comprise a diluent for a luminescent bacterial agent . most preferably , the diluent should comprise a 0 . 5 m nacl buffered saline solution at ph 7 where the bacteria is a marine bacteria and has not been lyophilized to include nacl . the kit may optionally also include a separation solvent , such as acetonitrile , deionized water , or aqueous ammonia . in other proposed forms of the presently proposed kit , the kit may further comprise an aspirator spray bottle to facilitate the easy application of suspended luminescent bacteria to a separation phase matrix such as a tlc plate or chromatography paper , chromatogram . in addition , the kit may comprise several vials of lyophilized luminescent bacteria . in other proposed forms of the presently proposed kits , the kit may further comprise instructions for the suspension and application of the luminescent bacteria to facilitate visualization of the isolated component substances of the test sample , and also in regard to the reaction time to be allowed and at what point the luminescent bacteria - exposed separation phase matrix should be read . the following references are specifically incorporated herein by reference in pertinent part . 2 . vasseur et al . 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