Ames Test

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Back to Micro 5

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NOTE: I still need to work on this lab to be more clear about it

Per Student Pair

* 1mL Salmonella typhimurium TA1535 (Az =M strain) **\*** in VB salts **OR** 1mL Salmonella typhimurium TA1538 (Nitro=F strain)\* in VB salts
	+ half of the class will have M and half will have F
* test substance 1mL in orange mt (no mutagenic properties nor histidine or protein)
* Positive control (sodium azide—1mL in blue tubes with a +M on top)
* Positive control (nitro-phenyl… -- 1mL in pink tubes with a +F on top)
* Negative control (Sterile media water, 1mL in yellow tube with – on top)
* 3 minimal medium (mm) plates

* 3 complete medium (cm) plates
* forceps and spreader in beaker with EtOH
* 6 sterile filter disks
* P-200 micropipetor, yellow tips, and used tip beaker

### **Methods**

**\*\*Grow Samonella in a BHI tube**: 100uL in 7mL of broth.  Disperse to 6 centrifuge tubes, centrifuge, pull off broth.  Suspend pellet in 1mL of1x VB salts.

**1x VB Salts**

9.8mL Sterile
water and 0.2mL VB Salts = 10mL of 1x VB Salts

\*\*Has
been growing in BHI broth direct from cryofreezer.  Work ahead…..

### Sodium azide

1mL in 12 tubes to cover 2 labs.

**4-Nitro-o-phenyleenediamine**

 Put in beaker, add
drops of HCL (1M) to start dissolving then add H2O

1mL in 12 tubes to
cover 2 labs.

### **Complete Medium (CM)**

Materials

* 0.75g beef extract
* 1.25g peptone
* 1.25g NaCl
* 3.75g Agar
* 250mL media water

Methods

1. mix well and boil until completely dissolved.
2. Autoclave (15lbs, 121oC) for 15 minutes.
3. Remove from autoclave cool and pour into plates, 20mL/plate.

### **Minimal Medium (MM) (250mL bottles)**

Materials

* 5.0g Dextrose
* 5.0mL 50x Vogel-Bonner salts \*see recipe below\*
* 0.00016g Histidine
* 0.00025g Biotin (see below)
* 3.75g Agar
* 245mL media water

Methods

1. Prior to preparation of the medium:
	1. Add 0.0016g histidine to 10.0mL media water and filter sterilize.
	2. Add 0.0025g biotin to 10.0mL media water and filter sterilize.
	3. Prepare the 50x Vogel-Bonner salts solution by adding the ingredients to just enough water to dissolve them while heating and stirring.  After the ingredients are dissolved, add enough water to bring the total volume up to exactly 1 liter.  Make in a big beaker then divide equally into 4-250mL glass grad cylinders and bring water up to the 250mL mark for 1L total.

1. Suspend, mix, and boil the agar in 184.0mL of media water until completely dissolved.
2. Suspend and mix the dextrose in the remaining 61.0mL media water until completely dissolved.
3. Sterilize the 3 and 4 as separate items. (121oC for 15min)
4. Remove from autoclave and allow to cool to 80oC
5. Aseptically add 0.25mL/250mL histidine solution, 0.25mL/250ml biotin solution, and 5mL/250ml 50x Vogel-Bonner salts to the dextrose solution.  Mix Well. [250mL end result of agar solution]
6. Add the dextrose solution to the agar solution, mix well and aseptically pour into sterile petri dishes (20mL/plate).

### **50x Vogel-Bonner medium (per 500mL)  Beth find a 250mL volumetric flask!!**

#### **Materials**

* 167.5mL warm media water (45oC)
* 2.5g MgSO4•7H20 magnesium sulfate
* 25.0g Citric acid•H2O
* 125.0g K2HPO4 potasium phosphate dibasic
* 43.75g NaNH4HPO4•4H2O sodium ammonium phosphate

#### **Methods**

1. Add salts in the order indicated to warm water in a 2 liter flask placed on a magnetic stirring hot plate.  Each salt **must** dissolve completely before adding the next.
2. Adjust the volume to 500mL.
3. Transfer to glass media bottle.
4. Autoclave, loosely capped, for 20 minutes at 121oC.
5. When solutions have cooled, tighten the caps

### **1x VB medium**

* 50x(mL) = 1x(10mL)
* 50x in 9.8mL sterile H2O