Author Topic: Reusable Borosilicate Screw Cap Glass Culture Tubes  (Read 10147 times)

Offline narcout

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #60 on: December 20, 2015, 07:50:10 pm »
Stir plates were already en vogue when I started homebrewing, and I have no independent knowledge of how/when/why they became popular.  Personally, I've been experimenting with skipping the stir plate and pitching at high krausen as per your advice.

However, there is a video on Youtube of Neva Parker demonstrating how to make a starter, and she is using a stir plate (she doesn't say a stir plate is necessary, but she does say that "it's a really good way to provide good gas exchange . . . .").

https://www.youtube.com/watch?v=1zUYxb-_B8A
« Last Edit: December 20, 2015, 07:51:41 pm by narcout »
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Offline klickitat jim

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #61 on: December 20, 2015, 07:51:47 pm »
Mark,

The title is Reusable... how are you cleaning the agar medium out of these tubes? Boil it out? Do you buy new caps or just clean and reuse?

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #62 on: December 20, 2015, 07:54:44 pm »
I use a plastic chopstick to break up media.  The media will rinse out of the tube after it has been broken into pieces.

Offline klickitat jim

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #63 on: December 20, 2015, 08:29:59 pm »
Great, thanks

S. cerevisiae

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #64 on: December 21, 2015, 07:44:22 am »
However, there is a video on Youtube of Neva Parker demonstrating how to make a starter, and she is using a stir plate (she doesn't say a stir plate is necessary, but she does say that "it's a really good way to provide good gas exchange . . . .").

Yes, but Neva recanted the claim in the Reddit thread.   Frankly, stir plates have become so ubiquitous in home brewing that no one except me and Stephen Deeds has dared to challenge the claim.  However, if one steps inside of a cell culture laboratory, one does not find stir plates being used for propagation, not even at White Labs.  The orbital shaker is the tool of choice for yeast cultures.  Roller tables/roller bottles are used for some lines of cells.  Brewing yeast strains do not need to be stirred because most exhibit NewFlo flocculation.  They need O2 during the lag phase, a carbon source for energy, and room to grow when propagated above the Crabtree threshold.

By the way, the forum member who posed the question regarding the value of stirring on the Reddit Neva Parker thread used the term "NewFlo phenotype."  Organisms have a genotype and phenotype.  An organism's genotype are the genetic traits that it inherited from its parents (or parent in the case of brewing yeast).  An organism's phenotype is how it expresses its genotype.    For example, it is possible for two parents with brown eyes to give birth a child with blue eyes if both parents carry a recessive gene for blue eyes.  Brown eyes are considered to be a dominant gene whereas blue eyes are considered to be a recessive gene.  The word "allele" is used to describe a variant form of a gene.

dominant allele + dominant allele = dominant phenotype
dominant allele  + recessive allele = dominant  phenotype
recessive allele + recessive allele = recessive  phenotype


The strains within the S. cerevisiae genus exhibit two different flocculation-related phenotypes.  The first phenotype is called Flo1.  It is controlled by mannose.  The second phenotype is called NewFlo.  It is controlled by mannose and glucose, and by extension, maltose (2 x glucose) and sucrose (glucose bound to fructose).   The most well-studied gene in the family is FLO11.  It is responsible for the production of a glycoprotein that resides on the cell wall that causes cell-to-cell adhesion (a.k.a. flocculation).  One can think of this glycoprotein as yeast Velcro.  Martin B. has mentioned many times that CA2+ ions promote flocculation.  That's because Ca2+ is used in the production of this compound.


Offline klickitat jim

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #65 on: December 21, 2015, 08:45:47 am »
Allele must be Latin for Doughnut, because I was always told that doughnut make my brown eyes blie.

Offline klickitat jim

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #66 on: December 21, 2015, 11:19:41 am »
Mark,

Is there any concern about breaking tubes by tightening the caps after the autoclave? IE vacuum pressure caused by sealing caps while still hot.

I made myself a step by step list.  Would you mind checking to see if I missed something? Bare in mind I'll be using pre-sterilized plastic plates.

Yeast Banking  
Prep:
Make 100ml of 5% w/v starter wort      
5g DME 100ml DI water, stirplate 2 min, microwave to boil
Make 500ml of 5% w/v and 1.5% w/v agar medium      
25g DME, 1.5g agar, 500ml DI water, stirplate 2 min, microwave to boil
Pour agar medium into 5 slants 1/2 full, cap on lose
Pour starter wort into 3 slants 1/2 full, cap on lose  
Place all 8 slants and agar vessel into autoclave 15min at 15psi
When done and cooled to 150º tighten slant caps and pour 4 plates near alcohol lamp
Lay slants at angle to cool, once cool store upside down to keep condensation away from agar

Making storage slants:
Work near alcohol lamp, flaming loop, openings and caps
Gather sample from commercial sample or old slant, streak one plate, incubate 24-48 hrs
Select a single colony and inoculate a slant, repeat for a second slant, incubate till mostly covered
Store upside down in zip lock bag in fridge up to 6 months  

Making a starter
Work near alcohol lamp, flaming loop, openings and caps
Collect a sample from best slant, streak two plates, incubate at room temp 24-48 hrs
Dispose or clean old slants
Select single colony and inoculate tube of starter wort, repeat for second tube of starter
Select single colony and inoculate new storage slant, repeat for second storage slant.
Incubate 48 hrs, store slants in zip lock bag in fridge

Step up tube starter to 100ml starters, then to 500ml starters, then decant and pitch to 1LO2HK starter  

Timing:
Slant a yeast sample if not brewing for a month or more, otherwise just step start and refrigerate the 500ml till brew day

Store slants up to 6 months before re-slanting  

Plate out a slant sample for step starters 8 days before brewday       
2 days to incubate plate       
2 days to incubate 10ml starter       
2 days to incubate 100ml starter       
2 days to incubate 500ml starter       
Morning of brew day decant and pitch to 1LO2HK Starter

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #67 on: December 21, 2015, 01:26:19 pm »
Only plates are stored upside down.  Poured and cooled plates should be sealed with Parafilm to prevent them from drying out in storage.  I allow slants  to cool to 50C/122F before tightening the caps.  Blank slants can be sealed with Parafilm before being placed in a refrigerator cap side up, but I do not bother to do so.  However, I do seal inoculated and incubated slants before placing them in ZipLoc bags in Tupperware containers in my brewing refrigerator.



S. cerevisiae

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #68 on: December 21, 2015, 01:34:51 pm »
A starter can be made directly from a slant.  There is no need to plate for singles because a slant that is inoculated from a plated colony is assumed to be a pure culture.  The process is:

1.) Inoculate 10 to 40ml of 5% w/v wort that was autoclaved in its container from an inoculated slant using aseptic technique

2.) Subculture two new slants from the slant

The only time that a culture needs to go through plating is when it passes from the non-aseptic (non-sterile) world to the aseptic (sterile) world. 

liquid culture -> plate -> slant

slant -> liquid culture

slant -> slant



Offline klickitat jim

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #69 on: December 21, 2015, 01:54:20 pm »
Outstanding!  I'm glad I asked. What I had planned wouldn't hurt anything but waste of plates and time.

This is very cool of you to do! I feel like I already know more than 3 credits at a CC would have given me. I owe you huge!

Offline purdman10

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #70 on: March 15, 2016, 12:13:59 pm »
I just got to this thread. I started a yeast bank, but need many more tubes. If they are still available, I will buy two dozen. Looks like there is another like minded soul in southern MD also!

Offline yso191

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Re: Reusable Borosilicate Screw Cap Glass Culture Tubes
« Reply #71 on: March 15, 2016, 12:15:54 pm »
I just got to this thread. I started a yeast bank, but need many more tubes. If they are still available, I will buy two dozen. Looks like there is another like minded soul in southern MD also!

Mark is on vacation from the forum for a bit.
Steve
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