Biology Lab report ASAP 6 hours

Biol 390-Lab 4 Growth of E.coli in a bioreactor

Objective

· Grow E.coli in a bioreactor

· Follow growth using absorbance and calculate cell counts.

· Draw growth curves and calculate growth rate

Background

Microbes are grown commercially in stirred tank reactors. This enables large volumes of microbes to be grown efficiently. Many factors that affect growth can be controlled tightly, such as temperature, pH, aeration, and agitation.

Materials

· Bioreactor sterilized with 2 liters of Luria broth

· 100 ml of E.coli grown in Luria broth

· Sterile pipettes (4 x25 ml)

· Spectrophotometer

· Cuvettes

· Sample vials

· Distilled water for dilution

· Ethanol bottle

Before class preparation

· Sterilize the Bioreactor sterilize with 2 liters of Luria broth. The broth should contain anti foam (AF-204) at 0.1 ml / liter.

· Note special precautions need to be taken in preparing the fermentor for sterilization (see separate document). Briefly:

· The pH and DO probes need to be calibrated.

· The reactor needs to be sterilized with all the ports closed, except for the add port and the vent port. Caps should be placed on the DO probe, the pH probe, and the motor housing.

· Wrap all connections with aluminum foil, but only loosely around the add connection so it does not seal.

· Carefully read instructions for operation of the upright autoclave.

· Autoclave for 20 min. plus 25 min. = 45 min. The extra 25 minutes is added to compensate for heating up the 2 liters of media to the correct temperature.

· While still warm close the add line and make sure the aluminum foil is tight

· 2 days before class start a tube of E.coli and incubate in the shaker for 24hr at 37C.

· The night before, inoculate one 500 ml flask containing 100ml with 2 ml of the culture.

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Procedure

The day of the lab

· Set up the fermentor by attaching the air. DO probe, pH probe, heating jacket, and agitator.

· Allow the fermentor to come to temperature using the cooling jacket

· make sure the agitation is on.

· Add the 100 ml inoculum using the septum port using sterile pipettes. Take precautions. Alcohol the port and use a sterile pipette to transfer the inoculum

· Take a sample of medium as a blank.

· Collect samples using the sample port every 5 min.

· Read absorbance on the spectrophotometer. The absorbance of samples should be between 0.05 and 0.7. Dilute it with water if necessary to get it into range.

Data Analysis

· Record each set of data in a table

· time in mins 

· absorbance 

· Aligent calculated counts 

· Plate count calculated counts - use your curve

· pH

· Graph 

· log Aligent counts vs time 

· log calculated plate counts vs time 

· Calculate the maximum doubling time (generation time)

Answer question in the Assignments section of Canvas.