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Monday, August 27, 2012

Ames test - Salmonella Reversion Assay

The Ames test is a short-term assay to assess the mutagenic potential of chemical compounds. In this experiment, we use Salmonella typhimurium (His-) to be the tester, so that it is also called "Salmonella reversion assay".

These kinds of bacterium carry mutations in genes involved in histidine synthesis, so that they require histidine for growth.(Without histidine they would not live)

We chose  TA102 (a kind of Salmonella) to be tester, and handled with two negative controls and two positive controls.

The two negative controls are Water-TA102 and DMSO-TA102, and the positive controls are Mitomycin C (-S9) and 2-Anthramine (+S9)



Mitomycin C is a anti-tumor medicine and 2-Anthramine is a chemical compound that may cause cancer.

And here the S-9 fraction is a substance extracted from liver, it contains many kinds of enzymes. We use the S-9 fraction to stimulate the metabolism of a liver. Then we could know if the substance that a liver metabolized would be a mutagen.



                                                                     S-9 fractions
                                                               

After we mixed some histidine/biotin,TA102,mutagen with S-9 and without S-9 and top agar. We poured the mixture onto each MA plates separately. Then put all plates into an oven (37oC) for three days.


                                                          After three days cultivate

Finally, we have count out all the CFU(colony-forming unite) the calculate if the positive controls is two times or higher more than the negative controls, that we can assure if an agent is a mutagen.


                                                Mitomycin C is exactly a mutagen




Experimental Record



Group 3
Plate1 (CFU)
Plate2 (CFU)
Plate3 (CFU)
Average(CFU)
Water
98
105
107
103
DMSO
150
113
183
149
Mitomycin C
670
851
767
763
2-Anthramine
239
174
181
198


Analysis




Group 3
Ratio with water
Mutagen
Mitomycin C (solvent:water)
7.408
YES
2-Anthramine (solvent:DMSO)
1.329
NO



Data of Whole the Class
G1



Average
NC (-S9)
160
65
78
101
PC (-S9)
513
334
624
490.3
NC (+S9)
232
6
237
158.3
PC (+S9)
178
184
181
181





G2




NC (-S9)
131
167
192
163.3
PC (-S9)
437
302
368
369
NC (+S9)
481
327
303
370.3
PC (+S9)
362
387
423
390.7





G3




NC (-S9)
98
105
107
103.3
PC (-S9)
670
851
767
762.7
NC (+S9)
150
113
183
148.7
PC (+S9)
239
174
181
198





G4




NC (-S9)
140
114
128
127.3
PC (-S9)
463
1340
463
755.3
NC (+S9)
116
96
158
123.3
PC (+S9)
108
154
262
174.7





G5




NC (-S9)
212
210
225
215.7
PC (-S9)
800
488
495
594.3
NC (+S9)
157
159
176
164
PC (+S9)
155
153
200
169.3





G6




NC (-S9)
103
139
139
127
PC (-S9)
344
330
331
335
NC (+S9)
188
180
179
182.3
PC (+S9)
104
190
197
163.7





G7




NC (-S9)
21
17
140
59.3
PC (-S9)
420
320
108
282.7
NC (+S9)
109
151
156
138.7
PC (+S9)
96
158
176
143.3





G8




NC (-S9)
80
87
51
72.7
PC (-S9)
802
720
832
784.7
NC (+S9)
238
253
261
250.7
PC (+S9)
350
387
189
308.7


Average of all eight groups
CFU(Average)
Water
121
DMSO
192
Mitomycin C (-S9)
547
2-Anthramine (+S9)
216

 


Ratio Matrix

    Numerator
Denominator
Water
DMSO
Mitomycin C (-S9)
2-Anthramine (+S9)
Water
1.0000
1.5868
4.5207
1.7769
DMSO
0.6302
1.0000
2.8490
1.1250
Mitomycin C (-S9)
0.2212
0.3510
1.0000
0.3949
2-Anthramine (+S9)
0.5601
0.8889
2.5324
1.0000
(2.0000>VALUE>1.5000)   (>2.0000)


Test Substance
Description
Water
To compare with other three substances, it is not a mutagen.
DMSO
To compare with water, its effect is just a little stronger, but not really a mutagen.
Mitomycin C(-S9)
To compare other three compounds, it is a really strong mutagen.
2-Anthramine
To compare with water, it is almost to be a mutagen, but its effect is just like DMSO. It is hard to decide that is cause by DMSO, 2-Anthramine or the metabolites of S9 from 2-Anthramine.

  


 


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