Environmental Research Applications:

In virtually every country around the world there is concern about the increasing number of new chemicals that are entering our environment. Many of these chemicals have been tested for acute toxicity prior to their being approved for use in society. Many of these chemicals however undergo changes as they move through societal use and through the natural environment when they are:

Recycled in Water Systems

Incinerated

Decomposed in Landfills

And are modified by microorganisms in surface waters, ground waters, soils or sediments

In a number of universities and research institutes, scientists are screening for the presence of acutely toxic, genotoxic or mutagenic materials so that they can be isolated, identified and environmental and public health risks evaluated. EBPI test kits are being used in over 30 countries in these applications.

Toxi-ChromoTest Kit - Testing for Toxicity

This test kit is simple and easy to run in a standard laboratory or simple field laboratory. It can be used to test for the presence of toxic materials in water and a special kit has been developed for the direct testing of soils, sludge's or sediments for the presence of toxic materials. The kit measures the ability of a rough mutant of E.coli to activate the LacZ gene and excrete beta galactosidase into solution where its presence is measured by its reaction with a chromogen. The test thus has a simple colourimetric endpoint. If toxicity is detected further sample fractionation ,using standard TIE (Toxicity Identification Evaluation) protocols, can be undertaken to isolate and identify the responsible material(s). Good laboratory practice should be followed as with all testing using bacteria.

SOS ChromoTest Kit - Testing for Genotoxicity

This test has been designed so that the bacteria is grown the evening before the morning the test is to be carried out and the test can easily be completed within a 3 hr time period the following day. The test relies on the use of a genetically engineered E. coli bacterium in which the promoter for the SOS gene repair complex has been linked to the LacZ gene. The theory is based upon the fact that in all cells when DNA is damaged by a “genotoxic agent” the cell tries to repair the damage through the induction of one or more DNA repair systems such as the SOS system. In this test when the DNA of the test bacteria is damaged the bacteria “tries to activate” the SOS gene repair complex but because the promoter that turns on the SOS system has been linked to the LacZ gene, the Lac Z gene is activated instead and the enzyme beta galactosidase is produced and excreted into the growth well. A chromogen is introduced to the growth well and if the cell has suffered DNA damage and it is “trying to repair it” using the SOS repair system a blue color will be produced. The test kit is supplied with a positive and negative control to ensure that acute toxicity has not occurred during the test and that it is functioning properly. The strength of the response of “unknowns” can be directly compared with the response of the “known” genotoxic positive control. The appearance of genotoxicity following metabolism can also be determined through the activation of genotoxicity following exposure to the S-9 liver enzyme. The laboratory requires only a micro pipette, a 37 0 C incubator, bench space, a spectrophotometer to measure the bacteria density in the test suspension and an autoclave or chlorine bath to kill the bacteria following the testing. Good laboratory practice should be followed as with all testing using bacteria.

Muta-ChromoPlate Kit - Kit Test for Mutagenicity Based on the Ames Tests

This test kit has been designed to determine if material being tested results in a significant increase in mutation rate above background rates. The test will be set up on the first day and the results read after the bacteria have been allowed to grow and mutate for a 5 day period. The test is based upon the AMES “reverse mutation” assay where a mutation in the wild strain of Salmonella has resulted in the loss of the bacteria’s ability to metabolize histidine. As the bacteria “reverse mutate” back they gain the ability to metabolize the histidine present in the growth media and this results in a color change in the wells in the microtiter plate in which the bacteria are growing. A simple chart is provided by which a statistically significant difference between the natural background rate of mutation and the rate of mutation when exposed to different chemicals can be determined. The TA-98 and the TA-100 strains are the most commonly used but other strains are available. The test can also be used with S-9 activation if the formation of mutagenic materials through the metabolism of less mutagenic materials is suspected. The test kit comes with a positive control compound.

Pharmaceutical Applications:

The development of new drug compounds involves the screening of a large number of both natural products and synthetic products or derivatives. These compounds are screened not only for efficacy but also for potential side effects. Some of the earliest screens involve rapid screening tests using bacteria looking for evidence of “toxicity,” “genotoxicity” and “mutagenicity.” To serve this need, EBPI has developed a number of sensitive screening technologies that have been incorporated into easy, to-use test kit systems. Pharmaceutical companies in the USA, Canada, Europe and Asia are currently using the tests listed below.

Toxi-ChromoTest Kit

This test can be completed in a three hour time period in a standard laboratory and requires only a micro pipette and a 37˚C incubator. All other materials needed are supplied with the kit. The test measures the ability of a rough mutant of the E. coli test organism to successfully synthesize and excrete the enzyme beta galactosidase into the liquid medium it is growing within. If the material being screened interferes with the induction of the lacZ gene and its subsequent expression, the test will show a positive result. The test has both positive and negative control endpoints. The results expressed as a blue color in the growth medium can be read qualitatively by eye or quantitatively using a micro-plate reader at 615nm. The test is supplied with both a positive and negative control to ensure that the kit is functioning properly.

SOS-ChromoTest Kit

This test can be completed in a two hour time period following an initial grow-up of the test bacteria overnight or within either four hours the day of the test. In order to run the test the user must have access to a micropipette, an incubator operating at 37˚C and a meter to measure the optical density or turbidity of the bacterial suspension to be used in the test. All other required materials are supplied with the kit. The test is based on a strain of E. coli in which the SOS gene promoter has been linked to the LacZ gene. As a result, when there is DNA damage to the cell, which would normally result in the SOS gene repair complex being activated to repair it, the Lac Z gene is activated and beta galactosidase is excreted into the surrounding growth medium. The enzyme then reacts with a chromogenic substrate which results in a blue color being expressed. The extent to which the test material is causing the cell to try and repair DNA damage can then be read qualitatively by eye or quantitatively using a micro-plate reader at 615nm. The test is supplied with both positive genotoxic and negative genotoxic controls to ensure that the kit is functioning properly.

The SOS ChromoTest kit can be use with or without S-9 activation.

Muta-ChromoPlate Kit

This test requires a five day period to complete. The test is set up on day one and the results read on day five. The user must have access to a micropipette and an incubator running at 37˚C. The test itself is a microplate format of the reverse mutation 'AMES TEST.' The Salmonella strains have a mutation that does not allow them to metabolize histidine. As the bacteria under the test conditions “reverse mutate” back to the wild strain and regain their ability to metabolize the histidine present in the growth medium, the medium will turn from a purple to a yellow color. The test is set up with both a background plate to evaluate the natural rate of reverse mutation and a positive control plate where exposure to a known mutagen occurs. A control chart is also supplied so that the user can easily determine whether or not there is a statistically significant difference in the mutation rate between the background rate and the rate of mutation when exposure to the material of interest occurs.

The Muta-ChromoPlate kit can be use with or without S-9 activation.

Educational Organizations

Colleges and Universities around the world are continually exploring ways of both training their students in the theory of new bio-molecular technologies and techniques and providing hands on training in the use of the tools of modern biotechnology. To this end, EBPI has taken its screening test kits used by the research communities around the world and adapted them for post secondary classroom use. We have worked with a number of post secondary educational institutions across North America and customized the kits for use in classes of varying sizes and operate on under a range of budgetary constraints. The following are a few example of how EBPI’s kits have been used in this application.

Toxi-ChromoTest Kit

This test kit has been used to illustrate how a number of different environmental compounds can, at lower concentrations affect, gene expression and at higher concentrations cause acute toxicity. The test is supplied with both a standard reference toxicant known to the students and one or more unknown single compounds or mixtures of compounds with “unknown” effects. Students, after receiving some theory in the nature of genes and their induction, can measure the induction of the LacZ gene directly in a laboratory period lasting under three hours. Techniques involving the use of micropipetors, microplates and incubation can be taught along with the calculation of LC­­50­ ­or LC20 concentrations from response curves the students plot from the data they obtain for both the reference and test materials.  The laboratory teaching method requires only a micro pipette and a 37˚C incubator, plus bench space and an autoclave or chlorine bath to kill the bacteria following the testing. Good laboratory practice should be followed as with all testing using bacteria.

SOS-ChromoTest Kit

This test has been designed so that the lab instructor can begin to grow the SOS bacteria the evening before the lab and the students can complete the laboratory exercise easily within a three hour time period the following day. The laboratory provides an introduction to the use of a genetically engineered E. coli bacterium in which the promoter for the SOS gene repair complex has been linked to the LacZ gene. The theory is based upon the fact that in all cells when DNA is damaged by a “genotoxic agent” the cell tries to repair the damage through the induction of one or more DNA repair systems, such as the SOS system. In this test, when the DNA of the test bacteria is damaged, the bacteria “tries to activate” the SOS gene repair complex, but because the promoter that turns on the SOS system has been linked to the LacZ gene, the Lac Z gene is activated instead and the enzyme beta galactosidase is produced and excreted into the growth well. A chromogen is introduced to the growth well and if the cell has suffered DNA damage and it is "trying to repair it" using the SOS repair system a blue color will be produced.  The test kit is supplied with a positive and negative control to ensure that acute toxicity has not occurred during the test and that it is functioning properly. The strength of the response of “unknowns” can be directly compared with the response of the “known” genotoxic positive control.

Concepts associated with the appearance of genotoxicity following metabolism can also be taught through the illustration of the activation of genotoxicity following exposure to the S-9 liver enzyme.

The laboratory requires only a micro pipette, a 37˚C incubator, bench space, a spectrophotometer to measure the bacteria density in the test suspension and an autoclave or chlorine bath to kill the bacteria following the testing. Good laboratory practice should be followed as with all testing using bacteria.

Muta-ChromoPlate Kit

This test kit has been designed to illustrate the fact that mutations are continually occurring in cells (background rate) and that when exposure to known mutagens occurs the rate of mutation can increase. The test will be set up by the students and the instructor on the first day of the Laboratory and the results read by the students after the bacteria have been allowed to grow and mutate for a five day period. The test is based on the AMES “reverse mutation” assay, where a mutation in the wild strain of Salmonella has resulted in the loss of the bacteria’s ability to metabolize histidine. As the bacteria “reverse mutate” back they gain the ability to metabolize the histidine present in the growth media and this results in a colour change in the wells in the microtiter plate in which the bacteria are growing. A simple chart is provided by which a statistically significant difference between the natural background rate of mutation and the rate of mutation when exposed to different chemicals can be determined. The TA-100 or TA98 strain basic test kit is recommended for laboratory use. It can also be used with S-9 activation if the instructor wishes to illustrate the formation of mutagenic materials through the metabolism of less mutagenic materials. The test kit comes with a positive control compound.

The laboratory requires only a micro pipette, a 37˚C incubator, bench space, and an autoclave or chlorine bath to kill the bacteria following the testing. Good laboratory practice should be followed as with all testing using bacteria.

Please contact EBPI for university protocols and laboratory designs.