Yeast Starters: Stirred vs Not

If you have heard anything about stirred yeast starters you've probably heard that stir plates produce more yeast than starters that are not agitated.  This seems to be a misconception that has been unfortunately widely propagated. 

I'm sure Jamil Zainasheff  had the best intentions when creating his Mr. Malty Starter Calculator, however it seems that brewers have taken the results out of context and drawn conclusions from the calculator that I doubt Jamil intend.  The data behind the vastly popular Mr. Malty equation are from growth that had no aeration and no agitation.   (Details of this experiment can be found in the widely acclaimed book: Yeast by Dr. Chris White and Jamil Zainasheff.  [1] If you haven't read this book I highly recommend you get a copy.)  There is an anecdotal reference to increased yield from agitation in the book Yeast, however without more detail on how the yield was compared we can only guess as to what the author observed.  It is also common in scientific papers to use "specific growth rate" (a measure of the speed of propagation) interchangeable with yield.  [2]  Without further explanation I can only assume this is what Dr. Chris White may be refereeing to.

The lack of aeration make Chris White's experiments questionable when used to predict growth on a stir plate.  Oxygen is critical for sterol production in yeast.  Without aeration an the onset of fermentation the yeast membranes will lack the pliability they need to bud new cells.  Given proper aeration at inoculation yeast can double four times, growing to sixteen times the initial population. [3]  Without aeration, as was done by Chris White for the experiments that Jamil used for his calculator, the data show that cell budding was extremely limited. This limitation follows what may be expected given the volume of dissolved oxygen.

Stir plates produce yeast faster, but do not produce more yeast. 

In scientific papers on yeast propagation you will generally see that the time frame used is 48 hours.  [4][5]  Propagation occurs much more rapidly with a stir plate so at the 48 hour mark there is often substantially more yeast produced with some form of agitation.  However, given enough time, without agitation a similar amount of yeast is produced. [6]

Yeast can metabolize sugar much more efficiently in the presence of oxygen aerobically than without anaerobically.   [7]  Using a stir plate oxygen is constantly introduced.  The common conclusion from these two facts is that with a stir plate considerably more sugar will be metabolized aerobically than without.  This is, however, not the case due to the Crab Tree Effect.[8]  When the sugar concentration is typically above 0.5% w/w (0.5°P or 1.002 SG)[9] yeast will metabolize sugar using anaerobic fermentation.  The balance between aerobic respiration and anaerobic fermentation is similar independent of agitation.

[1] Yeast

[2] J. Novak, G. Basarova, J. A. Teixeira and A. A. Vicente,

Monitoring of Brewing Yeast Propagation Under Aerobic and Anaerobic Conditions Employing Flow Cytometry. J. Inst. Brew. 113(3), 249–255, 2007

[3] Greg Doss, Yeast Propagation A Practical Approach

[4] L. Camacho-Ruiz, N. Pérez-Guerra, R. Pérez Roses, FACTORS AFFECTING THE GROWTH OF SACCHAROMYCES CEREVISIAE IN BATCH CULTURE AND IN SOLID SATE FERMENTATION

[5] C. R. Murray, T. Barich and D. Taylor, The Effect of Yeast Storage Conditions on Subsequent Fermentations,

[6] Woodland Brew, Yeast Growth

[7] Yeast Propagation with Aerobic Respiration

[8] Crab Tree Effect

[9] R. H. DE DEKEN, The Crabtree Effect: A Regulatory System in Yeast

The 15 Minute Brew Process

The final article in the series is up on the Homebrew Talk main page.  Check it out here:
http://www.homebrewtalk.com/15-minute-brew-process.html

Automated cell counting of yeast using ImageJ

Cell count automation isn’t as easy as I expected, and I’ve learned quite a bit in the processes getting it to work well.  Hopefully what I’ve figured out will help you to be automatically counting yeast cells and viability in less time than it took me!

Sample Preparation

It takes more than fiddling with the algorithm settings in ImageJ to get good results. It all starts with sample preparation.  The cells should not be clumped up and should be sparse across the field of view.  If you aren’t staining for viability 5% acetic acid (white vinegar) can be used to unclump the cells.  Unfortunately most viability stains, such as Methylene Blue and Trypan Blue are very sensitive to pH and will not be absorbed by the cells at a low pH.  Alternatively malt extract can be used.  In the presence of fermentable sugars yeast will naturally separate in order to create more surface area to metabolize the sugar.  For a good image of the yeast the cells should also be sparse enough to make counting easy.  50-200 cells in the field of view is a good target.  This corresponds to a cell density of about 10 to 50 Billion cells per liter which will appear slightly turbid in a test tube.

If the foreground stands out well from the background ImageJ can better differentiate the cells.  Contrast can be enhanced by staining.  Some common stains are as follows:

• Methylene Blue – Stains dead cells blue.  A one to one ratio of cell suspension and a 0.1% solution is commonly used.
• Iodine – Stains cellular glycogen brown.  The intensity of the color is proportional to the glycogen content.  0.2% solution is commonly used. [1] 

Imaging

It’s possible to take a picture through a microscope by holding a camera at just the right spot over the eyepiece, but it’s very hard to take a good image this way.  The simplest way to get a good picture is by purchasing a USB microscope camera.  An alternative is to use a camera adapter for your cell phone.  Most cell phones have pretty nice cameras.  The iPhone camera is certainly better than most web cameras, and even better than most USB microscope cameras.  If you have it, why not use it?  To solve this problem I used a 3D printer to make one.  It attaches to the eyepiece and uses rubber bands to attach to the phone.  

Brightfield imaging works well if the microscope is setup well.  Darkfield provides a more crisp image, but color is difficult to distinguish.  A dark field accessory is available for many microscopes.  For mine I printed a disk that can be placed in the light path normally used for color filters. 

Some tips for getting a good image:

1. Use a fixed focal length on your camera of about 10”
2. Zoom in on the camera as far as possible then adjust focus on the microscope
3. Adjust focus on the camera if needed.
4. Zoom out on the camera so that most of the field is in view.
5. Turn the brightness setting on the camera down (I use -1) to compensate for the black corners of the image.
6. Use a low iso and long exposure time for the best image quality.
7. Adjust the iris aperture to accentuate the cell membrane, but not so much as to entirely loose the cell color.  (Mine is normally almost closed)
8. Adjust the focus so that the centers of the cells are bright, but not so bright that the color is washed out.
9. Take a picture of an empty field to be used for the background removal.

Algorithm

It can take hours and hours to really hone a process, and can be quite frustrating if you are working with poor images.  I’ve played with this for way more hours that I should have, and come up with the following procedure:

1. Make a background image by blurring a copy of the specimen image.
2. Subtract the background with the image
3. Connect the shapes with maximum, minimum and blur functions.
4. Make a mask by using the Auto Threshold function.
5. Find the particles and add them to the ROI manager
6. Make the measurements.

Place these files in your ImageJ macro directory to use them.  The "Batch Yeast" macro processes all of the images in a folder with the "One Yeast" Macro.  The results are saved in a coma separated log file as well as annotated images.  Cells detected as being alive are circled in white, dead cells are circled in blue, and trub is boxed in red.

 

One Yeast 

Batch Yeast

The FIJI build of ImageJ can be downloaded here:
http://fiji.sc/Downloads

[1] V.E. Chester, Heritable Glycogen-storage Deficiency in Yeast and its Induction by Ultra-violet Light. 1967 J. gen Microbiol (1968), 51, 49-56

Starter Calculator

How time effects cell growth is a factor that I have felt is missing in the existing calculators, which led me to create this starer calculator.   Another personal gripe I have with the commonly used calculators is their unrealistic implied accuracy.  For honesty I have included one sigma error bars on the cell calculations.  I've collected data from dozens of experiments and factored it all down into this Excel calculator.

This calculator also lets you select which strain of yeast you are using.  Right now it is limited to US-05 and S-04, but I plan to expand this as resources allow.

You will need to enable macros to use this calculator.

This is the Beta Release, so give it a try and let me know what you think!

Download the Starter Calculator