How to grow Chlamy quickly in TAP_41877614

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title: "TheNessLab : How to grow Chlamy quickly in TAP"

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TheNessLab : How to grow Chlamy quickly in TAP

Created by Wen Chen, last modified on Oct 09, 2019

AKA Alice's not so secret recipe

Follow this protocol to grow Chlamy quickly to target growth phase in almost any plate/flask size. Both methods propagate Chlamy culture every time it reaches mid-late exponential phase to a less dense culture that is in early exponential phase and, therefore, maintain the culture at the maximum growth rate until it reaches exponential phase in the target plate/flask size.

Incubator conditions:

• Infors incubator: 100 rpm, 25C

Method A

Day	Plate type	Action
0	96-well	Inoculate any (>5 days old) single colony grown in 1.5% agar in TAP to 250 uL TAP in a 96-well plate.
2	48-well	Transfer 40 uL culture from 96-well plate to 460 uL TAP in a 48-well plate.
4	48-well	Tranfer 100 uL culture from 96-well plate to 400 uL TAP in a 48-well plate.
6	-	48 well plate culture is in exponential phase

Method B

Day	Plate type	Action
0	96-well	Inoculate any (>5 days old) single colony grown in 1.5% agar in TAP to 250 uL TAP in a 96-well plate.
2	48-well	Transfer 40 uL culture from 96-well plate to 460 uL TAP in a 48-well plate.
4	6-well	Tranfer 320 uL culture from 48 well plate to 7.68 mL TAP in a 6-well plate.
6	Flask	Transfer 5 mL TAP to 500 mL TAP in 2L flask. Alternatively, 6-well plate culture is in exponential phase and ready for use.
9	-	Flask culture is in exponential phase.

1. To calculate the Day # that a culture needs to reach stationary phase, add 2 days to the day it requires to reach exponential phase.  (Exceptions: Some GB strains can be slower growing so you will need to test these strains out.)

2. Propagate the cultures at approximately the same hour +/- 2 hours on each day of culture transfer to acheive the best results.

3. The Flask culture on Day 9 (Method B) is ~1/10 of the cell density in the 48 well plate culture on Day 6 (Method A) even though both cultures are in exponential phase. 

4. If you want to grow 48-well plate culture (volume ≥500 uL) in the Infors incubator for more than 2 days, the recommended humidity level is 90% because of evaporation issues. Try surrounding the plate with uncovered plates filled with ddH2O to resolve this problem.  

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