Team:Newcastle/1 July 2010

Aim of this experiment

The aim of this experiment was to determine if Bacillus subtilis 168 is able to produce urease and to degrade urea.

Materials and methods

• Christensen's Urea Agar

Make up 1 liter of the agar mixture containing:

1. Gelatine peptone 1.0g
2. Potassium dihydrogen phosphate 2.0g
3. Sodium chloride 5.0g
4. Agar 20g

Top up to 1 liter and apply gentle heating to dissolve. Sterilize at 115°C for at least 20min.

Add the following to the molten base:

1. D(+)-Glucose 1.0g
2. Phenol red, 0.2% 6ml

Note: Ensure that the work was done using aseptic technique.

Transfer 10ml of the molten base to universal tube and allow the agar to set in a slanted position. Store the harden agar in the fridge.

• Pipettes
• Universal tubes
• Streaking loop

Urease test

1. Perform the experiment using aseptic technique.
2. Pick up single colony of B. subtilis 168 and streaking it onto the slanted agar tube.
3. Loosen up the cap to allow air exchange between the interior of the tube and the external environment.
4. Incubate the tubes overnight at 37°C.

• Negative control - Without B. subtilis 168

• Test 1 (Duplicate) - Inoculated with B. subtilis 168
• Test 2 (Duplicate) - Inoculated with B. subtilis 168

Expected results

Christensen's Urea Agar was formulated to detect and differentiate urolytic and urea degrading microorganisms. The addition of gelatine peptone, dextrose and reduced content of buffer supports a luxuriant growth at an early stage. Urea is the substrate and can be degraded by certain organisms, which results in ammonia building. The ammonia makes the media alkaline and therefore the indicator phenol red will change from orange color to pink-red color.

Negative control - No color change (Orange) Test 1 (Duplicate) - Pink-red color Test 2 (Duplicate) - Pink-red color

For actual results, please see 020710 lab results.

Conclusion

For experimental conclusion, please see 020710 lab results.