8/29 Testing the Effect of the Shape of Growth Vessel on BC Growth
Updated 9/14/2019 06:51 PM
Introduction
Materials
- Rectangular vessels
- small pyrex box (250mL, "Rect 2")
- large pyrex box (400mL, "Rect 1")
- Round vessels
- conical test tube
- small circular pyrex bottle (500mL)
- large circular pyrex bottle (250mL)
- 1.2 um filter (for media)
- 1 syringe
- 50mL pipette + tips
- K. rhaeticus stock
- Yamanaka + Glucose Media
Procedure
Procedure
- Seed each vessel with K. rhaeticus stock and Yamanaka media at 1:10 ratio, respectively. Remove the air filter on the end of the tubing and replace it with the media filter. Using the syringe, pump in the appropriate amount of Yamanaka + Glucose Media. Removing the media filter, use the micropipette to pipette in K. rhaeticus cells. Do not pump in K. rhaeticus cells through filters. Place the air filter back onto the tubing to act as an air vent when media and cells are in the vessels.
Rect 1 : Media + Culture: 400mL of space
Rect 2: Media + Culture: 250mL of space
Cylin 1: Media + Culture: 100mL of space
Cylin 2: Media + Culture: 50mL of space
Above is the given amount of space that that should be filled by media and culture for each container. This is half of the overall volume of each container, so that the media level doesn't touch the tubing that will be used to sterilely put in media and culture, and cause irregular BC growth.
- Incubate at 30C for 10-14 days.
- Follow harvesting and drying protocol to collect BC and mass. Record masses in the below table.
Results
Table1
A A | B B | C C | D D | E E | F F | G G | H H | I I | |
1 1 | Container | amount of media (mL) | amount of K.rhaeticus (mL) | Total Volume | Observations | Wax Paper Mass | Wax Paper + BC Mass | BC Mass | BC Mass/Total Volume (column D) |
2 2 | 500 mL bottle (cylin 1) | 91 | 10 | 101 | |||||
3 3 | 250 mL bottle (cylin 2) | 43 | 5 | 48 | |||||
4 4 | Large flat pyrex (rect 1) | 421 | 47 | 468 | |||||
5 5 | Small, taller pyrex (rect 2) | 270 | 30 | 300 |
- Compare the BC mass to Total Volume Ratios with each other, to see if the square container design will work just as well as the circular container.
Observations (9/14)
- 500mL bottle had a clump of BC that appears to be growing, under the white spot of that clump were a lot of wispies which I think might be contamination...
- 250mL bottle had a little more BC in it, and had a more obvious pellicle start to form, because there appeared to be a whole thin sheet forming over the top
- large flat pyrex had a lot of BC growin in it. The coloration was appropriate (darkers, phlemmy color) but it was still splotchy
- Small taller pyrex had less bc clumps
The purpose of this experiment is to test how different shapes of growth vessels affect the growth of BC in our desired shape. We will be testing two general categories of vessels, rectangular and circular, and within those, surface area. Specifically, we will be observing the rectangular vessels to see how BC growth is affected by the corners. (large and small below refer to surface areas) Because the containers vary greatly in volume, we just made sure that they had the same surface area to overall volume ratio. We settled on a 1:2 ratio. The overall volume includes both seed stock used, and media volume. We used a 1:10 Ratio of seed stock volume to overall media volume. Because the volumes differ, we expect the higher volume samples to produce larger quantities of BC. In order to be able to compare the results between the containers, we need to compare the ratio of BC mass to the total volume of media+seed stock. This way we can see which container shape produces proportionally more BC. Area to research related to this: can we try out placing the sparge stones in different areas around the vessels? Because this is a one time test we will record the data in the protocol and not turn this into a notebook, just because it's easier to have it all in the same place.