Shake Flask Fermentation

A centrifuge is a laboratory device that is used for the separation of fluids, gas or liquid, based on density. If you open a centrifuge and you notice that a sample has broken, close the lid immediately and leave the centrifuge closed for at least 30 minutes. This allows aerosols to settle and lowers your risk of inhaling any harmful aerosols released from the broken samples. Broken tubes, even plastic ones, can be sharp so you want to avoid cutting yourself, so you could use gloves to protect your hands from getting cut.

2. Once your solutions are made and set aside, you will want to create your simulated cells using dialysis tubing. Fold one end of the dialysis tubing and wrap a rubber band (tightly) around that end to seal it.

Table 1 shows the contents of the bags and the content of the concentration it was submersed in. Bags 2-4 each contain a solution of both sucrose and water. These bags were each put into beakers containing hypertonic solution. These bags gained weight over time because the water moved from its high concentration inside the beaker to the low concentration inside the membrane of the artificial cell, the membrane being the bags that consisted of dialysis tubing. The

• Serially dilute the 4 mg/ml solution with buffer A to make working solutions of 400 µg/ml and 40 µg/ml.

Using the graduated cylinder, measure 20mLs of the stock sucrose solution and 180mL of water to create a 3% sucrose solution and place it into the 250mL beaker (beaker #2). Place bags #1‐3 (red, blue, yellow) into beaker 2 and bag #4 (green) into beaker 1. Allow the bags to sit for one hour. After allowing the bags to sit for one hour, remove them from the beakers carefully open the bags, noting that often times the tops may need to be cut as they tend to dry out. Measure the solution volumes of each dialysis bag using the empty 250 ml beaker.

When lysing the cells, add 20 - 50 μl chilled lysis buffer (RIPA buffer with protease inhibitors and PhosSTOP), and keep the samples on ice for 30 min. Centrifuge the samples at 20,000 × g for 20 min at 4℃. Transfer the supernatant to new tubes,

The correct syringe is used to place 10 cm3 of the first glucose solution into the boiling tube.

There were several steps completed to prepare for the experiment. Three dialysis tubes were filled with approximately the same volume of distilled water and then were tied shut. The initial mass (in grams) of the tubes was taken using a triple beam scale. I then filled three 500 mL beakers with 400 mL of water each and dissolved different masses of solute (table sugar) in each beaker in order to make 5%, 10%, and 20% solutions. The beakers were labeled accordingly, and then 20 g, 40 g, and 80 g (respectively) of table sugar was weighed out using a digital scale and placed into the corresponding beakers. The sugar was stirred in using a stirring rod until all of the solute was completely dissolved.

Concentration of solutionsconcentrations were kept constant by using the same digital scale (with an uncertainty of 0.01 g) to measure the amount of glucose to be added to the water

The molarity of the solutions in each dialysis bag, and the percent change in mass are both included to best show effect of increasing molarities of sucrose and osmosis. The percent change in mass exhibits the process of osmosis, because osmosis must take place for water molecules to transfer from one side of the semi-permeable (in the surrounding distilled cup) to another (within the dialysis tubing), in order to reach equilibrium.

2.Sample calculation For experiment (A): - Reagent

Now put half of the suspension from the "+P" tube onto the LB/AMP plate and the other half of the "+P" suspension on to the LB/AMP/ARA plate. Using a new sterile cotton swab, spread the suspension evenly around the surface of both plates and incubate the plates at 37°C for 24

Then 50 ml beaker with the glucose and starch mixture was also tested for glucose with a glucose strip. The dialysis tube was then clipped on one end and opened on the other end. The 15 ml of glucose and starch mixture was then poured into the dialysis tube with a pipette. The dialysis tube was then twisted and clipped on the unclipped end of the tube. The mass of the tube was then measured by a quadruple beam balance. The tube was then rinsed in the sink and blotted dry by a paper towel to clean the tube in case some of the glucose accidentally spilled out of the tube. The tube was then placed into the 250 ml beaker and left there to sit for 15 minutes. After 15 minutes, the 250 ml beaker was then tested for glucose with a glucose test strip and so was the contents inside of the tube. All observations and results were recorded in the chart below the procedure. Such observations listed were things like color change in the solutions, mass changes in the dialysis tubing, and what the glucose strips would indicate about the solution

The aim of this experiment is to follow the growth of Serratia marcescens in nutrient broth at 37oCby recording the changes in turbidity (cloudiness) by measuring the absorbance of visible light (600 nm) and also to prove that there is an increase in the cell number and not just in mass during the growth.

If a cell is placed in a ________ solution of water, there will be no net movement of water in or out of the cell.