(1) On media preparation:
a) Explain the purpose of each ingredient found in the LB media.
- Bacto-trptone is used to provide essential amino acids for the growing bacteria.
- Yeast extract is used to provide plethora of organic compounds required for bacteria growth.
- Sodium chloride provides sodium ions to E. coli for transport and osmotic balance.
b) What is the purpose of ampicillin?
It is to inhibit the growth of microbial contaminants.
c) Why is ampicillin added only after autoclaving?
If it is added before autoclaving, the ampicillin would be degraded when the autoclaving is done and render it ineffective.
(2) On equipment preparation:
a) What is meant by calibration of the pH probe?
The calibration process correlates the voltage produced by the probe. The calibration of the pH probe is performed with at least two standard calibration buffer solutions that are based on the range of pH values to be measured. Such calibration buffer solutions are available commercially of pH of 4.01, 7.00 and 10.00.
The probe must be calibrated at a zero point and a span point. Typically a probe will be calibrated at a pH of 7.0 and 10.0 or 4.01. The 7.0 calibration buffer solution is the Zero point calibration and the 10.0 or 4.01 calibration buffer is used for the Span adjustment. If you plan to measure pH of acidic solutions, calibration buffer pH 4.01 should be used and if you plan to measure pH of basic solutions, calibration buffer of 10.00 is used instead.
Calibration is done here by first placing the probe into the pH 7.00 buffer and allowing the pH reading to stabilize. For older pH meters, turn the calibration knob till pH 7.00 is shown, for modern pH meters just set it to calibration mode instead.
After calibrating at pH 7.00, rinse the probe with distilled water and do the same thing with the calibration buffers of pH 4.01 and/or pH 10.00. The pH probe should be rinsed with distilled water when moving from one buffer to the next.
Reference: http://www.ph-meter.info/pH-electrode-calibration
b) Why is hydrochloric acid not suitable as a correction agent for pH?
It is not suitable as a correction agent for pH because depending on temperature and agitation, hydrochloric acid at a concentration above 10% will produce a hydrogen chloride vapor that is very corrosive when combined with water vapour in the air. This corrosive vapour can corrode the metals in the fermenter. If hydrochloric acid is used, there must proper ventilation where the gases can be easily dissipated.
c) What is meant by polarization of the pO2 probe?
The pO2 probes contain 2 electrodes. They are the anode and the cathode which are both in contact with an electrolyte solution. The anode and the cathode are separated from the solution being measured, by an oxygen permeable membrane in which oxygen can diffuse through.
As oxygen passes by the membrane, reduction of oxygen occurs at the surface cathode which is exposed at the tip of the electrode. Oxygen molecules diffuse through the semi-permeable membrane and combine with the KCl electrolyte solution. The current produced is a result of the following reduction of oxygen at the cathode.
The pO2 probe measures oxygen tension amperometrically. The pO2 electrodes produce a current at a constant polarizing voltage which is directly proportional to the partial pressure of oxygen diffusing to the reactive surface of the electrode.
Reference: http://74.125.153.132/search?q=cache:bLvBdqF4Vv4J:www.enzyme.chem.msu.ru/eduproc/prac/taskA/Oxygen%2520Analyzers.doc+polarization+electrode+oxygen&cd=8&hl=en&ct=clnk&gl=sg
d) What is a peristaltic pump?
It is a type of positive displacement pump used for pumping a variety of fluid. It is used to remove liquid from the fermenter or to add acid or base, antifoaming reagent and nutrients to the culture gradually.
(3) On seed preparation:
a) What is the purpose of arabinose?
Arabinose is used as an inducer for the production of green fluorescent protein.
b) Describe the sterile techniques used in seed preparation.
The lab bench top was disinfected with 70% ethanol before the seed preparation was done. The bunsen burner was ignited to produce a flame for sterilizing of the cap of the cryovial (containing the bacteria culture) before opening it and to provide an aseptic zone to work in. A sterile plastic inoculating loop was used to inoculate the bacteria on to the agar plate. The agar plate cover was left half open when streaking was done instead of fully open to reduce the chance of contaminants from falling into the agar plate.
c) Why do we perform step wise scale up instead of transferring directly to the fermenter?
We perform step wise scale up to allow the cells to adapt to the culture conditions and grow to a large enough number to seed the fermenter. If we transfer the cell culture from the cryovial directly into the fermenter, there would be too little cells and the cells would not be able to grow or might have a very long lag phase.
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