Synscoby Co Culture

Day 0 (Wednesday PM) November 30th

1. Inoculate K. rhaeticus from glycerol stock in 5mL YPD + 1% cellulase
2. Place in shaking incubator at 30˚, 250rpm for 3 days
3. Streak S. cerevisae on YPD plates so fresh when inoculating on day 3.

To avoid contamination the YPD + Cellulase 1% were added into a centrifuge tube in the hood, then added to smaller to tubes with snap off caps.

Cellulase                                                    YPD

Outside of the hood we added the K.Rhaeticus with tooth picks into 30 ml tubes.

There were a total of 6 tubes so we can start co coulture of (3x BY4741p + 3 x Venus Ura)

Streaking new fresh yeast from the October batch into Agar plates 

Isolating yeast at this point is as simple as taking a very small amount of your culture and rubbing (streaking) it on to an agar plate. Because of the stable, non-liquid agar medium, once streaked, single colonies of microbes are essentially stranded by themselves.

After a few days or weeks (depending on incubation temperature and microbe population), they’ll multiply and grow large enough to be seen with the naked eye. At that point, it’s a matter of selecting the colonies you like and growing them up to larger amounts.

It’s impossible to stress enough how important cleanliness and sanitation are at this point in the process. If you truly want a pure strain, you need to ensure you’re not contaminating what you’re trying to isolate with other cultures.

Day 2 (Friday PM) December 2nd

1. Inoculate S. cerevisiae from plate in 5mL YPD
2. Place in shaking incubator at 30˚, 250rpm for 1 day

Day 3 (Saturday) December 3rd

1. Centrifuge K. rhaeticus at 3,220 g  (5119 rpm) for 10 minutes. 
2. Resuspend K. rhaeticus cells in YPS to OD600=2.5. This helps to remove cellulase.
3. Dilute S. cerevisiae in YPS to OD600 = 0.01
4. Combine K. rhaeticus and S. cerevisiae in 50mm petri dish, inoculating K. rhaeticus 1/50 and S. cerevisiae 1/100 in fresh YPS-optiprep. Final volume should be 15mL.
5. Incubate at 30˚ for 3 days under static conditions. Do not move co-cultures while growing or BC layers will come apart.

*****

YPS

• 5g yeast extract

• 10g peptone

• 10g sucrose

HS Recipe x 500 ml

• 2.5g yeast extract

• 2.5g peptone

• 1.35g sodium phosphate

• 0.75g citric acid

• 10g sucrose

• 500ml distilled water

**Snap off cap works best for K. Rhaeticus growth 

K.Rhaeticus

Growing critters ins Hestrin Schramm HS {brown media)  (look recipe)

K. Rhaeticus (bacteria) :  HS (hestrin schramm)  30c

HS Recipe x 500 ml

HS 

• 2.5g yeast extract
• 2.5g peptone
• 1.35g sodium phosphate
• 0.75g citric acid
• 10g sucrose
• 500ml distilled water

---------------

Pour from the stock to a separate tube is done in the sterile hood 

Streaking from Plates. with the microbes from (Part1) : 

Taking a loopful of microbe culture (a colony) from the plates and putting them in Media

 

October 25 LEFT                                       November 2nd RIGHT pellicle is visible - grew in 30C shaker

   

November 2nd

We run a test to see which tube top worked better a snap off cap and a screw top

The Snap off seems to be better as it lets more air come in

November 4th - moving forward we will only use Snap off tops

Nov7 - Testing to see if the K. Rhaeticus we took from the frozen stock will grow on HS media

We just took a bit from the frozen stock that we put in the -80C

Growing the pellicle in motion or still. Both will be growing at 30C, One in the shaker and one in the incubator

Nov 14 , The one on the shaker grew, the one in the incubator did not grew.