A Users Guide Kit Components and Reagents The Muta-ChromoPlate kit - - PowerPoint PPT Presentation

A Users Guide

Kit Components and Reagents

• The Muta-ChromoPlate kit is a 96 well version of the

traditional bacterial reverse mutation Ames Test.

• Used for the detection of mutagenic activity.
• The test uses amino-acid requiring strains of Salmonella

typhimurium and Escherichia coli to detect point mutations.

• It detects mutations which revert mutations present in the

test strains and restore the functional capability of the bacteria allowing it to synthesize the essential amino acid.

Point Mutations involve:

• Substitutions of one or a few DNA
• Additions or Deletions of one or a few DNA

Base Pairs.

Reverse Mutations in Various Bacterial Strains Base-Pair Substitutions Positive Controls

TA100 Sodium Azide TA1535 Sodium Azide WP2 E. coli Strains 4NQO TA102 (Site A-T ) Mitomycin C

Frame-Shift Mutation

TA98 2-Nitrofluorene TA97a 9-Aminoacridine

As outlined in the OECD GUIDELINE FOR TESTING OF CHEMICALS

Developed to test mutagenic materials in water (or DMSO) soluble extracts of sediment, air, chemicals, food components, cosmetics, waste waters, potable waters and any other material that can be solubilized or placed into micro suspension in water such that the material being tested can be taken up by the test strain. The Muta-ChromoPlateT kit is generally more sensitive than the Ames pour-plate assay, because it allows testing of higher concentrations of sample (up to 75% v/v).

Basic Kit

• 12 Sterile 50ml tubes
• 10 Sterile Reagent Boats
• 12 Sterile 96 Well Plates
• 1 Sterile .22µl Filter
• Ziploc and Auto-Clave Bags
• 1 Bacterial Strain (TA100)

– Other individual strains available upon request

• 1 Positive Control
• Reagents for 12 plates

Two Strain Kit

• 25 Sterile 50ml tubes
• 20 Sterile Reagent Boats
• 24 Sterile 96 Well Plates
• 2 Sterile .22ul Filters
• Ziploc and Auto-Clave Bags
• 2 Bacterial Strain (TA100 and TA98)

– Other individual strains available upon request

• 2 Positive Controls
• Reagents for 24 plates

S-9 Activation Mix Components

• S9A

MgCl2 + KCL

• S9B

Glucose-6-Phosphate

• S9C

NADP

• S9D

Phosphate Buffer

• S9E

Sterile Distilled Water

• S9F

Rat Liver Extract

• 2AA

Positive Control(s)

S9 Activation Enzymes

• The assay procedure is simple and requires minimal

training.

• Example Applications
• Testing of industrial effluents for presence of possible mutagenic

compounds

• Screening of municipal discharges for possible routine presence or spills of

mutagenic compounds

• Screening of surface and/or groundwater for mutagenic residues
• Screening of potable water supplies for the presence of chemicals with

mutagenic potential

• Screening of water soluble air pollutants for mutagenic agents
• Evaluation of pure or complex raw mixtures for potential mutagenicity
• A convenient and easy to use teaching tool for university and college

laboratories

• Currently EBPI offer five different types of Bacterial Strains to

meet the OECD’s Guideline for Testing of Chemicals.

• TA100
• TA1535
• TA98
• TA97a
• TA102
• WP2

• The Muta-ChromoPlateTM provides a clear colour endpoint.
• Reagents, cultures and other consumable components are supplied

ready-to-use in a non-specialized laboratory.

Sterility Check Background Strong Mutagen Positive Control

(Very strong mutagen)

A possible example of the Muta-ChromoPlateTM kit on day 5 of the assay is shown above which includes all controls essential for the assays.

No need for cultures Quick and easy overnight growth of the bacteria (No need for time consuming dilutions which may lead to contamination).

Filter Sterilization of the sample for assays

Filter sterilization of the samples is recommended to be preformed prior to starting the assays. This can be done with either the 0.22µm filter unit supplied with the kit, or with a 0.22µm syringe filter (not included unless requested)

Overnight Growth of the Bacteria

1. Remove the vial of Growth Media from the fridge and remove the vial of bacteria from the freezer

2. Using aseptic techniques open G (Growth Media) and the vial that contains the bacteria. Transfer the contents from vial G to the vial that contains the bacteria.

3. Place the lyophilized stopper back on the vial that now contains the bacteria and growth media and give the vial a quick shake to ensure that the bacteria and growth media are well mixed. Incubate overnight at 37ºC for 16 to 18 hours

4. Visually examine the bacteria grown overnight for turbidity

Obtain reagents A through E and dispense as outlined in the protocol into the sterile 50ml tube included in the kit.

Basic Kit (5051)

21.62ml (A) + 4.75ml (B) + 2.38ml (C)+1.19ml (D) + 0.06ml (E) for a total

• f 30.00ml

Bacterial Strain Kit (B5051)

43.24ml (A) + 9.50ml (B) + 4.76ml (C) +2.38ml (D) + 0.12ml (E) for a total of 60.00ml

NOTE: The Bacterial Strain Kit comes with a 100ml Reaction Mixture Bottle

Label the sterile 50ml tubes as well as the sterile 96 well plates

Add 2.5ml of Reaction Mixture to each tube Add sterile water (included) and (or) sample material to be tested.

Add 100µl of the Positive Control (included) to the correct tube. Add 5µl of the bacteria to each tube, except for the blank tube.

Vortex the tube for 15 seconds to ensure that the contents are mixed well Pour contents of tube into the sterile reagents boats and dispense 200µl into each well of the sterile 96 well plate

Concentrated Water Sample Un-Concentrated Water Sample Example Dilutions Example Dilutions 50ul – 100ul of sample is added. 5.5ml-17.5ml of sample is added. 17.40-17.45ml of sterile distilled water is added (included within the kit) 0.00-12.5ml of sterile distilled water is added

Once the reagents are mixed and the bacteria and samples for analysis are added the suspension is then dispensed into the 96 well plate. The user will notice that all the wells in the plates will be a purple colour as seen below.

Place the plates into the Ziploc bags and incubate for 5 days at 37⁰C. Remove the plates and score

• Each well of the 96 well plate is considered a colony.
• If the colony reverts back to the natural state, a

mutation has occurred.

• If a reverse mutation has occurred, the bacteria in the

colony have the ability to synthesize histidine and will continue to grow turning the colour in the well from purple to yellow.

• The Muta-ChromoPlateTM kit (as with the traditional

‘Ames Test’) compares the natural background rate of reverse mutation to a rate of reverse mutation within a sample assay.

Understanding the results

• For example: There are two plates shown below after the 5 day

incubation period. (LEFT) is the “Back Ground” plate and (RIGHT) is the “Sample treatment” assay plate.

10 Wells have mutated (TA100 Strain) 86 Wells have mutated (TA100 Strain) Using Table 2 from the protocol under the “No. Wells Positive in Background Plate” column, locate the number 10 and you will find the statistical significance data for the 96-Well test.

THE NUMBER OF POSITIVE WELLS SCORED IN A 96-WELL MICROPLATE LEADING TO CLEAR SIGNIFICANCE IN THE FLUCTUATION TEST

• No. Wells
• No. Wells

Positive in No. Wells Positive Positive in

• No. Wells Positive

Background in Treatment Plate Background in Treatment Plate Plate 0.05 0.01 0.001 Plate 0.05 0.01 0.001 3 6 10 36 48 53 58 1 5 8 12 37 49 54 59 2 7 10 14 38 50 55 60 3 9 12 16 39 51 56 61 4 10 14 19 40 52 57 62 5 12 15 20 41 53 58 63 6 13 17 21 42 54 59 64 7 15 18 23 43 55 60 65 8 16 20 25 44 56 61 66 9 17 21 26 45 57 62 67 10 19 23 27 46 58 63 68 11 20 24 29 47 59 64 69 12 21 25 30 48 60 63 70 13 22 27 32 49 61 66 70 14 24 28 33 50 62 67 71 15 25 29 34 51 63 67 72 16 26 30 36 52 64 68 73 17 27 32 37 53 65 69 74 18 28 33 38 54 66 70 75 19 30 34 39 55 67 71 76

20 31 35 40 56 68 72 77 21 32 36 42 57 68 72 77 22 33 38 43 58 69 74 78 23 34 39 44 59 70 75 79 24 35 40 45 60 71 75 80 25 36 41 46 61 72 76 81 26 37 42 47 62 73 77 71 27 39 43 49 63 74 78 82 28 40 44 50 64 75 79 83 29 41 45 51 65 76 80 84 30 42 47 52 66 77 80 84 31 43 48 53 67 78 81 85 32 44 49 54 68 78 82 86 33 45 50 55 69 79 83 87 34 46 51 56 70 80 84 87 35 47 52 57 71 81 84 88 72 82 85 89 84 91 94 95 73 83 86 89 85 92 94 96 74 83 87 90 86 93 94 96 75 84 87 90 87 93 95

• 76

85 88 91 88 94 95

• 77

86 89 92 89 94 96

• 78

87 89 92 90 95 96

• 79

87 90 93 91 96

• 80

88 91 93 92 96

• 81

89 91 94 93 96

• 82

90 92 94 83 90 93 95

• No. Wells Positive in Treatment Plate

0.05 0.01 0.001 19 23 27

The background rate of reverse mutation is compared to the treatment plate of mutation. Since the treatment rate of reverse mutation is greater than 27 (86 in our case), it will be noted that there is a less than a 0.001 chance that 10 and 86 are the same

• result. Thus suggests that our treatment plate contains a strong

mutagenic material producing a very significant difference in reverse mutation rate from that observed in the control.

This suggests that our treatment plate contains a strong mutagenic material producing a very significant difference in reverse mutation rate from that observed in the control.

Bio-informatics Toolkit

EBPI provides a spreadsheet with each kit that allows for simple data entry and analysis Shown here is the data entry form which allows you to enter all information about your tested samples

Bio-informatics Toolkit

Once you have entered sample information, the Bioinformatics Toolkit will analyze your results, colour-coding each plate result according to the significance of the response, as well as generating graphs for each bacterial strain for your reference

• The Muta-ChromoPlateTM kit is available with
• r without the S9 Activation Enzymes.
• The S9 Activation Enzymes are from the male

Sprague-Dawley rat liver – Aroclor 1254 induced.