Monday, December 31, 2012

Isolating a Single Yeast Strain by Plating

Plating is a wonderfully easy way to separate the good from the bad.   After only two days you should be able to see distinct round colonies of yeast.  These are what you will want to remove with a pipette to start a larger culture.

One of my recent batches of beer was used to propagate the S-04 strain. When it came time to harvest the slurry I grabbed what I thought was the sterilized jar I had prepared, but at the end of the day I realized I had grabbed jar I intended.  The jar I had picked up from the counter was in line for the dish washer and had only been rinsed out after being used as a jam jar.  I saved the slurry for a week, just in case things might work out for me, but when I did the cell count I found that the viability was very low, and there were significant signs of cocci bacteria.  Later, over the course of about a week I drank some of the beers and save the dredges in a container in the fridge.  Unfortunately, dredges, without the protection of the beer, spoil quickly.  When I looked at the dredges under a microscope half of the cells were the characteristic finger shaped mold cells.  This was not going to work for bottle conditioning directly.  You would think at this point I would give up.  S-04 is only about $4 at my local home brew store, but I didn't.  In the spirit of experimentation and experience I pressed on.  I diluted and plated the yeast so that I can separate good yeast colonies from the mold colonies.  So here is how I did it:

Making The Plates

Isolating a single yeast cell isn't as hard as it might sound, and is very possible even without a microscope.  Your first job will be to make gelatin plates.  You will only need one for each strain.

Gelatin Plate Recipe:

2 tablespoons of water
3/4 teaspoon of DME
1/2 teaspoon of Gelatin or Agar.

Combine all ingredients in a shallow dish with a top and microwave on high for 30 seconds with the top slightly ajar.  Slosh or shake the container until everything is dissolved.  Microwave on high for another 30 seconds.  This should bring the contents to a boil.  I gave a plate an extra 30 seconds recently, "just to be safe" and it boiled over.   Close the lid and place it in the refrigerator.  If the lid is air tight you may need to let it breath a few times as the liquid cools.  In a few hours your plate will have jelled and you will be ready to move on.  Plate will keep in the refrigerator for quite a while, but the sooner you use the plate, the lower the risk of contamination.  It's easy enough to make the plate a day ahead.


The first time I tried plating I thought the best way to do it would be to dilute the cells so that I would have individual colonies across the entire plate.  Dilution, however, is over kill for this application.  If you were trying to assess the types and amounts of bacteria in solution then dilution would be useful.

The easiest way to isolate single yeast strains is by streaking.  The idea is that you start with a concentrated amount and as you streak it across the plate the cell concentration will diminish.  The longer the streak the further the cells will be apart.  It's best to avoid digging into the media on the plate.  This will bury the colonies beneath the surface.  A glass stir rod, or a pipette might be easier to use than a culture loop for the streaking processes.

1) Pipette one drop of your slurry or bottle dregs onto the upper right corner of the plate.  (One drop is all you will need to cover the plate.  The water will not be absorbed by the media so the less you have the better)

2) Use a sterile dry pipette (or stir rod) to draw a "C" shaped line across the top of the plate and down the left hand side and across the bottom.  It is important that the tool is dry.  If it is wet you will end up with pools of mixed cells instead of distinct cultures.  See the image at the bottom of this post.
4) Starting from the bottom working your way up draw lines from the left hand line across to the right working your way up the plate. These should be about a centimeter or half inch apart.

Cover the plate and put it out of the way somewhere, preferably slightly warm such as on top of the refrigerator.  Try not to be curious at this point.  It is very important that the plate is not moved for 48 hours.  The yeast cells need to implant on the media.  If they are disturbed they will continue to float around in the shallow puddle and not form individual colonies. 

In 4 to 10 days you should see distinct circular colonies that are yeast.  Suck these up with a pipette to grow the culture.  Look out for large fuzzy growths hazy cloud shapes.  These are mold and bacteria.

Saturday, December 29, 2012

Fermentability of Crystal Malt

For the most part you are safe assuming that crystal malt will ferment the same as your base malt, especially considering that the crystal will be a relatively small portion of your grain bill.  Additions of lactose and sucrose will effect final gravity much more,  but when it really comes down to fine tuning your recipe calculations you might want to consider how fermentable crystal malt is.  It could change your final gravity by a couple of points.

A few years back some fairly extensive tests were run by Nilo over the course of several months.  The results of these tests are a look into how fermentable crystal malt is when mashed with a base malt. 

The Average attenuation from Nilo's data:
80 % for 2-row
77% for 50% C10, 50% 2-row
70% for 50% C40, 50% 2-row
67% for 50% C120, 50% 2-row

If we assume that the 2-row ferments the same regardless of being mashed with the crystal the 100% crystal fermentability numbers can be derived.

74% for C10
60% for C40
54% for C120

If you really want to fine tune your recipes you can use the difference in fermentability to adjust your final gravity calculation.  These would be:
-6% for C10
-20% for C40
-26% for C120

For example, say you were making a dark beer with 9 lbs of 2-row, and 1 lb of crystal.   Based on your mash temperature and type of yeast you can determine your base malt fermentability as described on the "Final Gravity in Recipe Formulation" post.

Let's say that came out to 75%
OG would be  (note that mash efficiency has been rolled into the pppg numbers)
9 lbs of 20row * 24 pppg  = 216 GU
1 lb of C120 *  23 pppg  =  23 GU
216+23 / 5 = 48 GP or a specific gravity of 1.048

FG would be as follows
216 GU * (1-75%) = 54 GU
23 GU * (1-75%-26%)  = 4 GU
54+4 / 5 gallons = 11.6 GP or a specific gravity of 1.012

If the fermentability of the crystal was not considered then the contribution would be 6 GU instead of 4.  Because this change is very small, the FG of the five gallon batch would still be 1.012
So, as you can see, most of the time the change in attenuation caused by crystal malt is insignificant.
Here is Nilo's thread: (post number 108 has the data)

Thursday, December 27, 2012

Yeast Health When Kept At Ambient Temperatures

 WLP004 (blue) is read on the right axis.  WLP566 (red) is read on the left axis

Yeast maintains it's viability very well outside of the refrigerator.  Even after three days outside of the refrigerator viability will not change more than 3%.  The results from a viability test of yeast even shipped across the country should be fairly accurate.

However, there is much more to yeast health than viability.

When considering yeast health the first thing that comes to mind is viability: the percentage of live cells of the total cell population.  However, there are other factors when considering yeast stored at in the food danger zone.  In this range of temperatures from 40 degrees F to 140 degrees F bacteria thrive. 

Yeast that has been packaged for storage in a refrigerator is typically not stored with any food for the yeast because it is intended to stay dormant.  When the yeast warms up to room temperature it will become active, and during this time the cell population will increase slightly, however it quickly runs out of energy reserves.  The bacteria, on the other hand, will feed on the dead yeast cells, and continue to propagate even after the yeast have stopped growing.

During a test of two strains of yeast, WLP004 and WLP566 It was noted that within the first 12 hours both strains grew in population.  Initial inspection of the yeast when it was removed for the refrigerator showed no signs of bacteriological activity, but after only 12 hours pairs of bacteria were observed indicating cell growth. 

When the yeast is pitched into an environment that it thrives in, such as a typical wort, they can out preform the bacteria and the defects produced by the bacteria may go unnoticed.  If the bacteria are given a head start, such as a day or two at ambient conditions, they will dominate the flavor profile.

In conclusion, I wouldn't pitch a wort with yeast that has been left out for an extended period of time, but the viability count and cell count should be very accurate even if the sample has been warm for several days.

Tuesday, December 25, 2012

Refrigeration Effects on Yeast Viability

Some subjects have a plethora of information available allowing the home brewer to evaluate different pieces of data and come to their own conclusion.  However, other subjects have one piece information that misinformation is inferred from.  The effects of refrigeration on viability is one of the latter.   Mr. Malty's slurry viability slider is often misrepresented in this manner.  For a new slurry this slider shows the viability as 94% and for every day it drops 1.6%, and bottoms out at 10% after 53 days.  While Jamil likely had good intentions when designing this into the calculator, it is commonly misused.  Perhaps it was an attempt to show that a slurry cannot be kept in the refrigerator indefinitely.  There are a number of other consideration when deciding to repitch a slurry such as contamination and overall health of the yeast. 

The fact is that it is documented in very reliable brewing literature that yeast stored in a broth can be kept for six months, and yeast stored on an agar slant can be kept for over a year.   (1)(2)

There are much more important considerations when it comes to the viability of a starter than the amount of time it has been refrigerated.  Fruit(3) has a drastic impact on viability as does alcohol.(4)

The linear decay has actually been propagated over to Yeast Calc, another trusted source.  Rather than simply propagate information, I did the tests for myself.  Over the course of a month data was collected on seven different slurries from two different strains used to make a variety of beers.

Yeast Strain
Yeast Layer
Viability loss per day

While the initial viability can vary greatly, the viability over time dose not change a measurable amount over the course of one month.

(1) The Practical Brewer,Yeast Strains and handling techniques, Sources and Maintenance of Pure Yeast Cultures. p276
(2) Kirsop (1991)
The italic text above is a paraphrase of the texts referenced as (1) and (2)


Sunday, December 23, 2012

Yeast Cell Growth Observations

Recently I plotted the viable suspended cell density and specific gravity of a normal fermentation.  It was one gallon 1.036 wort pitched with 7 billion cells per liter.  The wort was 1 gallon composed of all sucrose except for 1/2 tsp of yeast nutrient.  The yeast pitched was 25 billion cells of EC-1118 Champagne Yeast. 
  • The red curve is the specific gravity that can be read with the left hand axis. 
  • The blue is the concentration of viable cells measured in billions of cells per liter.  It can be read on the right hand axis.

Yeast Growth as a function of available sugar.

Note the hyper attenuation that resulted from this combination.  Not only is champagne yeast known for it's ability to fully ferment a wort, but when provided with yeast nutrients and a wort of simple sugars, it has no problem converting all of the sugar to alcohol.  Because alcohol is lighter than water the resulting solution is lower than 1.000 specific gravity.

Notice that the highest rate of cell production directly correlates to highest concentration of sugar .  This is seen as the second through fifth data point. As the available sugar decreases the cells fall out of suspension faster than they are being produced.  (data points 6 though 8) When fermentation is nearly complete the cells maintain a concentration of about 3 billion cells per liter.

The peak cell density suspended in the wort was 35 billion cells per liter, or about five time the initial cell count.

Viable cells vs non-viable cells.

See graph below
  • The number of dead cells per liter is shown in red and can be read on the left hand axis in billions of cells per liter. 
  • The number of viable cells per liter is shown in blue and can also be read on the left hand axis in billions of cells per liter. 
  • The Viability is shown in green and can be read on the right hand axis.

The yeast that was pitched was approximately 5% viable, as can be observed on the fist point of the viability graph.  After approximately half a day the yeast began dividing as can be seen by the increase in both viability and viable cell density.

The number of non-viable cells in suspension tracks that of the viable population.  I suspect that this is caused by simple mechanical stimulation.  As the living yeast cells produce CO2 a small bubble forms on the cell causing it to float.  On it's way to the surface it bumps into dead yeast cells and carries them upward. 

The peak viability is reached at the same time the food source has been consumed.  As the fermentation completes the viability percent also increases but the cell count in suspension is about a tenth of the peak value.  So although the non-viable cells do flocculate faster after fermentation has completed there are very few viable cells on top of the layer of dead cells.

Friday, December 21, 2012

How Fruit Effects Yeast Viability

There are a number of factors that can reduce the viability of a yeast cake that you may have heard.  These include, high gravity, highly hoped, and dark color, but one I have not heard of is the effect of fruit on viability.  In the past few months I have made several fruit beers, and three of them I collected viability data on the yeast slurries taken from these brews.

I have been surprised to find that yeast slurries from beer made with fruit have a drastically lower viability!

On one of these fruit beers I took a cell sample during fermentation just before adding the fruit and the viability was in the high nineties as you would probably expect during active fermentation.  When the beer was bottled it was down to 1% viability.

The viability of the yeast that was taken from beers that did not have fruit has always been substantial higher. It looks like from this point forward, in order to save the yeast, I will be racking off of the yeast cake before adding fruit. 

The EC-1118 Champagne yeast that I recently harvested off of my Strawberry Champagne contained 19 billion suspended viable cells per litter during fermentation, but after adding the strawberries, the thick slurry only contained 6 billion viable cells per liter.  There were three times as many viable cells suspend in the wine during fermentation than there were in the thick slurry after fermentation completed.  This indicates that there was massive cell death after adding the strawberries.

Although this evidence seems fairly conclusive, I am hesitant to say this is always the case.  But independent of what the causes is, there are significantly less viable yeast cells per liter of slurry.  If a microscope was not used to asses viability before pitching, and a standard cell density was assumed, the wort would end up drastically under pitched, even if using a starter. 

The fruit in these beers has pasteurized and has the cellulose removed with a strainer before being adding to the secondary fermentor.  You can see exactly how it was done in the blog post called "Fruit The Easy Way." In the juice that is transferred to the fermentor there are likely still a plethora of fruit cells.  It is possible that this large cellular mass is being counted as dead yeast cells and thus the viability show as being low. 

Perhaps there is some merit to a secondary fermentation vessel.

Raspberry Cream
Strawberry Champagne
Sasion Terr
Raspberry Wheat

Wednesday, December 19, 2012

Sporulation in Brewers Yeast

After spending uncounted hours looking through a microscope at yeast I can identify yeast from some other microorganisms, I've seen enough protein globs to distinguish them from fibers and hop partials, and I know the tricks to search for bacteria hiding amongst the yeast.  Most of the time, what I see is what I expect. However...

The other day I found something unexpected.

Generally speaking, yeast can reproduce both sexually (by sporulation) and asexually (by budding).  Brewers yeast, primarily reproduces by budding.  (1) However it seems that the cells at mark (B) may be another form of reproduction.  The cells at mark (A) is budding.  A new cell can be seen forming on the upper left side.  Also notice one birth scar in the middle and a second scar on the lower right of the cell.  The cells at mark (B) seem to be conjoined cells, although this coud be a budding cell that has not separated.  These do not appear to be flocculation, as flocculation look similar to grapes in a bunch.  Floculating cells are very close to each other and may have slightly flat sides because of the proximity.  An example of this are the dark cells above mark (B) and to the right of mark (A) The cells at mark (B) the membranes seem to be pulled toward each other instead of being pressed back toward the cell.  Although with the resolution limits of a bright field microscope this could be an optical illusion.

In Sporulation, two haploid cells, each having one copy of each chromosome, combine to form a diploid cell.  This can occur when the yeast is under stress.  The resulting is four yeast spores that each have different DNA from the two parents.  The yeast cells that result from sporulation are therefor genetically different than the parents rather that the direct copies that are formed during the much more common budding reproduction. 

It's really quite a fantastic world that yeast live in, and since getting this microscope my understanding of it has increases several fold.

This is likely simply budding that is further along in development.  See the comment below.

(1) The Practical Brewer, Yeast- Strains and Handling Techniques. Life Cycle of Brewers Yeasts p265

Monday, December 17, 2012

Final Gravity as an Indication of Sweetness

After fermentation has completed a nearly all of the simple sugars such as sucrose and glucose, have been converted to alcohol.  Most of the maltose has also been converted.  What is left is mostly long chain sugars such as dextrin sugars and maltotriose.  (1)  Dextrins are relatively taste less, but Maltoriose can be broken down by enzymes in your mouth to form glucose (2)(3)

Because some of the sugars remaining in beer cannot be tasted, drinking beer with and noting the level of sweetness and final gravity is likely the best way to gauge how sweet your beer may turn out.  However these comparisons may give you a new way to think about residual sweetness.

How much sugar do you put in your tea or coffee?

The alcohol and hop level of a beer balance out the residual sugar the same way bitterness in tea or coffee can balance the sugar that you might add.  Coffee is a closer comparison for highly hopped beer as they are both quite bitter without a little sugar, and tea might be a good comparison for a beer with low alcohol and hops. 

Although, I like my coffee without any sugar, but prefer a Belgian Quad to a IIPA.  This might give you a new way to think about final gravity, but it's certainly not a perfect comparison.

Final Gravity
 sugar in 1 cup of water
1 tsp
2 tsp
3 tsp
4 tsp

Saturday, December 15, 2012

Final Gravity in Recipe Formulation

The attenuation of beer styles listed in the BJCP style guide range from 63% for a  9E Strong Scotch Ale up to 97% for the 1A Light American Lager! 

Having control over your attenuation is necessary for these styles and actually fairly simple. 

There are three main contributing factors to attenuation during fermentation.  These are the yeast's attenuation range, mash temperature, and fermenability of added sugars.

Attenuation of a given yeast is essentially it's ability to ferment more complex sugars.  Today's brewing yeast provides a range from about 65% for the English Ale Yeasts to 80% for Belgian Sasion strains.  This alone give the brewer almost enough room to over the whole spectrum.  However, these numbers can be a little misleading.  If you took almost any brewing yeast and made a wort of nothing but corn sugar and sucrose the result would be complete attenuation.  Therefore, this attenuation factor should be only applied to the gravity provided from the malt.  In my experience crystal malt adds very little to the final gravity of the beer.  Especially when it is added to the mash as the enzymes will convert some of the complex sugars that would have otherwise be left unconverted in the event that they were steeped.

The mash temperature has one of the largest impacts on fermenability of the wort.  Higher temperatures will break down the beta enzymes and also speeds the rate at which the alpha enzymes work.  Higher temperatures leave more complex sugar in the wort which are less fermentable.  Mash times longer than 60 minutes will also lead to more attenuation of the sugar during fermentation.  The results of fairly extensive experiments, as well as my own experience, indicate that each degree above 151 will yield 1% less attenuation.
Fermentability of the sugar, in some ways, trumps mash temperature and attenuation.  Sucrose will ferment out completely dry regardless of mash temperature or yeast strain.  Conversely lactose will is just about completely unfermentable.

From these three factors the final gravity can be calculated.  Let's use a simplified recipe for a poorly designed milk stout as an example. 

This is not a recipe you would want to brew.

5 gallon batch.
7 lbs 2-row.
1 lb sucrose.
1 lb lactose.
WLP004 (72% attenuation)
154 mash temperature
75% mash efficiency

The OG contribution of the 2-row is:
7lbs * 37pppg * 75% / 5 gallons = 38.85 gravity points

The OG contribution from the sucrose is:
1lbs * 46pppg / 5 gallons = 9.2 gravity points

The OG contribution from the lactose is:
1lb * 43pppg / 5 gallons = 8.6

The original gravity is therefor:
38.85 + 9.2 + 8.6 = 56.65 gravity points, or a specific gravity of 1.057

The final gravity of the two row is effected by both the yeast and the mash temperature.  The temperature is 2 degrees about 152, so that means the malt will be 4% less fermentable.

72% - 4% = 68% attenuation of the malt contribution.

The FG contribution of the two row is:
38.85 * (1 - 68%) = 12.4

The FG contribution of the sucrose is zero because it will ferment out dry

The FG contribution of the lactose is 8.6 because it will not ferment at all.

The final gravity is therefor:
12.4 + 0 + 8.6 = 21 gravity points, or 1.021 specific gravity.

The apparent attenuation is much lower than what is specified for the yeast alone.
1 - (21/57) = 63%

Even with the sucrose in the recipe to dry it out, the final gravity may be high from some people.  To fix this a higher attenuating yeast could be selected, the mash temperature could be lowered, or the lactose could be reduced.  Any of these would work, but the most appropriate solution for the style would be to lower the mash temperature.

This is a very nice grain chart that I refer to often:


Thursday, December 13, 2012

How Many Cells Are Produced By A Starter?

The above table is a summary what is expected.  It is not derived from the results of this experiment.

There are a few calculators that you can use to figure this out fairly simply base on the number of cells that you are starting with, and the volume of wort that is fermenting. 

But how accurate are these calculators?

Tell me you are interested by commenting on this post, or send me an email :
When I added a microscope to my brewing equipment I was surprised to learn how far off these type of estimates can be. 

There are some factors that remain the same from one setup to the other, and some that change.  Most people propagate cells at ambient air conditions, so in these experiments temperature will not be used as a variable.  Also, not everyone has access to a stir plate, so no stir plate will be used in these experiments.

One of the things that people often seem to change without thinking about the consequences is the specific gravity of the wort.  Because most starter calculators seem to be based on data collected with a 1.036 specific gravity wort, a range around this gravity will be used.

The next factor is the ratio of cells pitched to wort volume, also known as the inoculation rate.  The yeast, being very small, have no idea how big the starter is.  It could be 10ml or 5 gallons, and they would function the same.  What will effect them is how crowded the vessel is.  This is essentially the inoculation rate.  If there are 1 billion cells in a 10 ml wort, or 10 billion cells in 100 ml of wort the growth will be the same.  In both cases the the cell population grows by about 50%.

Another factor that is not considered by the calculators is the strain of yeast.  I'll be testing some common strains and some not so common ones so that you can have a better idea of how your yeast may preform.
A total of 50 tests will be run.

A 5x5 matrix of specific gravity vs inoculation rate.  The Gravities will be 1.010, 1.025, 1.036, 1.045, and 1.100.  (2, 6, 9, 12, and 24 degrees Plato) The inoculation rates will be 5, 55, 65, 75 and 200 million cells per ml.  I expect that the 1.036 gravity will correlate closely with Mr. Malty.  Dr. Chiss White at White Labs did some expements for the Mr. Malty calculator.  This data was taken and also used for Yeast Calc.  These calculators both show that an inoculation rate of 65 million cells per mililiter should exibit the most growth per volume of wort.  These test will all use Safale S-04 as it is a very widely used and commonly available yeast.

The same inoculation rates and the 1.036 gravity will be used with five additional yeast for the remaining 25 tests.  The yeast will be WLP004 Irish Ale, WLP862 Cry Havoc, WLP566 Belgian Saison, EC-1118, and for variety, Fleischmann's Bread Yeast.

I would be happy to add your yeast to the list.  Just send me a message and we can work out the details.

Tuesday, December 11, 2012

When More Grain Doesn't Add More Sugar

It's common knowledge that if you want more fermentables, then you should add more grain.  It makes simple sense, but every brewing system has its limitation.  Regardless of the system you use there is a point when adding more grain does not add more fermentables.  In fact, eventually it will result in less fermentables. 

You read that right, more grain can equal less fermentables.

The limit comes when the water absorption of the grain approaches that of the water that can be added to the mash vessel.  Grain absorbs about 0.8 quarts of water per pound.  So as your mash out thickness approaches this more and more of the converted sugars are trapped with the water absorbed by the grain.

For example we can compare 9 lbs of grain to using 7 pounds.  It would be easy to assume that using 9 pounds of grain would produce more sugar in the final wort, but this in not always the case.

Say your mash tun holds 4 gallons like my small batch system.  If you add 9 pounds of grains they will displace 0.72 gallons.  Leaving a 0.25 gallon head space for stirring, this limits the water you can add to 3.03 gallons.  Grain absorption will be 1.8 gallons, leaving you with first runnings of 1.23 gallons.  This is 40% of the water that was added meaning that only 40% of the extracted sugars are in these runnings.  40% * 34 GU/lb * 9 lbs = 122.4 gravity units of sugar

If only 7 pounds of grain were used, they would displace 0.56 gallons.  Leaving the same space for stirring as previously the limit to the water added is a little more at 3.19 gallons.  Grain absorption is a little less at 1.4 gallons.  This leaves first runnings a fair amount higher at  1.79 gallons.  The runnings are 56% of the total water.  56% * 34 * 7 = 133.28 gravity units which is about 11 points higher! 

These examples are for a mash without a sparge.  Each sparge that is added will increases effeciciancy.  If you typicaly do one sparge then the higest final gravity will be achived with 2 pounds of grain per gallon of mash volume.

Number of sparges
Maximum pounds per gallon
No sparge
1.75 lb/gallon = 2.3 qt/lb
1 sparge
2 lb/gallon = 2.0 qt/lb
2 sparges
2.25lb/gallon = 1.8 qt/lb
3 or more sparges
2.5lb/gallon = 1.6 qt/lb

Sunday, December 9, 2012

Hemocytometers Side By Side

The price of a hemocytometer range from $20 to over $200.  The Bright-Line (image on left) sells for about $260 on Amazon right now, but luckily I was able to borrow one to evaluate.  For my own use I purchased a $33 hemocytometer made my Qiujing. (image on right) 

The difference under a microscope is night and day!

The cell phone pictures here don't do it justice, but I was absolutely amazed how much the quality of the slide has to do with the crispness of the image!  Subtle detail that apear to be birth scars on viable yeast cells were visible, and texture inside the nucleus of the dying cells could be seen with the Brite-Line.  The Qiujing will work fine for counting viability, but the processes is much slower.  It's hard to find a focal depth where the cells are clear and the lines can be distinguished.  Also, smaller objects, such as bacteria, are harder to resolve.
But for for homebrew, I can't justify spending more on a slide than I spent on the microscope.  The squares aren't exactly square, but the ruling is close enough not to add much error to the cell count.  There are other sources of error that can cause more deviation in measurements. 

With some practice, and learning how to best adjust the microscope the Qiujing is more than adequate.  If you are are a microbiologist, or a lab technician you may be disappointed with the quality.  However, for the money, I don't think you can beat it.  It has been of great value for me!

For more recent pictures of yeast on the Qiujing Hemocytometer see the lab reports:

Friday, December 7, 2012

Yeast Washing Exposed

Yeast washing is a means of cleaning yeast to separate the viable yeast cells from unwanted partials.
To wash yeast, The yeast is harvested from a fermentation vessel and combined with water.  It is allowed to settle until the water has separated to the top and the yeast to the bottom.  The water and the top of the yeast is then poured into several jars.  The yeast and debris that has settled to the bottom of the first container is then discarded.

Normally I don't wash my yeast, I just pour the cake into three or four quart size mason jars, but I have been curious as to what benefit may be achieved from yeast washing.  My understanding is that the intent is to separates the hops and dead yeast cells from the live yeast cells.  In the last few days I have been running experiments on this technique to find out what it does, and if it is worth the extra time. 

Yeast washing is beneficial, but not for the reasons that I had anticipated.

After the yeast has settled into the container it divides into roughly three sections.  Common brewing wisdom indicates that the top portion is mostly water, the light colored middle section contains viable yeast, and the darker bottom contains dead yeast, hops, and other debris.  However, it seems that this in not the case.

The viability throughout the container is roughly the same.

In four test cases the viability was not statistically different in these layers.  Tests were run with three slurries with 10%, 50% and 90% viability.  In all tests the viability of the yeast in each section did not vary more than one standard deviation.

What was interesting was that the bacteriological content was much higher in the top portion of the yeast containers than in the lower parts.  There was about 100 times more bacteria per live yeast cell in the top "liquid" section.

Another strange finding was that the concentration of non-yeast debris followed the cell density.  While the cell concentration at the bottom of the container was twice what it was in the middle, the viability was the same, and the concentration of non-yeast material per yeast cell was virtual identical.  The hops and other partials did not separate from the yeast.  So when you are throwing out the junk, it takes just about as much viable yeast with it as it takes debris.

The above experiments were done in test tubes.  To make sure this was repeatable, I tried yeast washing with a full sized batch.  The results were the same.  The viability of the part that would have been thrown away was the same as that of the part that would have been kept.  The viable cell density was also very similar. 

For further information on washing a a full sized batch see the new blog post: Yeast Washing Revisited.

The tests were conducted with WLP566, WLP004, EC-1118 and S-04.
When washing yeast, discard the liquid when washing yeast to remove bacteria.  Keep the thick slurry and add clean water on top of it for storage and to wash out additional bacteria.

I would be happy to talk through the numbers with anyone interested.

Wednesday, December 5, 2012

ABV from Priming Sugar

One thing that I have neglected, although it seems readaliy apparent, is that adding dry sugar or malt extract will increase the level of alcohol of the beer.  It would be easy to assume that adding a few ounces of sugar will not effect the alcohol level much considering pounds of grain were used in making the initial wort. 
However a few ounces of priming sugar could be considered significant. 
My fear was that dissolving the sugar in additional water would water down the beer.  My wife, however, prefers to drink not more than 4% ABV.  With this in mind, the target for my last brew was 3.5% ABV and about 3 volumes of CO2.  The OG and FG were right on the money (a light 1.030 and dry 1.003 respectively) leaving the ABV at 3.6%.  Tasty Brew indicates that 3.3oz of sugar should be added for this 3 gallon batch.  3.3 oz of sugar in 3 gallons of beer has a specific gravity of 1.003.  That's 10% of the initial 1.030.  The resulting ABV by adding this sugar is 4.0%!

So either assume that the priming sugar will increase the ABV by a 0.5%, or add water when priming.

To maintain the ABV of the beer the sugar should be in a solution equal to that of the original gravity of the wort.  Knowing that white sugar adds 46 gravity points per pound per gallon, and a little algebra the equation can be worked out.

(oz of sugar) / (original gravity in gravity points) * 46 = cups of water

For a starting gravity of 1.030 add 1 and 1/2 cups of water for every ounce of sugar.
For a starting gravity of 1.040 add 1 and 1/8 cups of water for every ounce of sugar.
For a starting gravity of 1.050 add 1 cup of water for every ounce of sugar.
For a starting gravity of 1.060 add 3/4 of a cup of water for every ounce of sugar.
(3.3) / (30) * 46 = 5.06
For the 1.030 original gravity batch that I made the 3.3oz of sugar should be dissolved in five cups of water.

If you want to skip the math, just dissolve the priming sugar in about a quart of water for a five gallon batch.

Monday, December 3, 2012

An Alternative To Starters

Sure, making a starter is cheaper than buying multiple vials of yeast, but the accuracy, scheduling, and waste of fermentables bothers me.  However, it may seem like a necessity to better approximate the pitch rate required if you don't have a microscope.  I don't think any one will disagree when I say that yeast is a major cost contributor to the beer. 

So what's a better way to get the ensure the correct cell count for your beer?

For a 5 gallon batch of a standard ale with an initial gravity of 1.060, 200 billion cells are required.  There are several ways this can be achieved.

1) buy two yeast vials (2 x $8 = $16)
2) buy one yeast vial and use a 2 liter starter ($8 + ~$2.00 = $10)
3) buy one yeast vial and pitch into less wort. ($8)

With the third method listed the cost of yeast is half of what it could have been!  Starters themselves are not as accurate as they may seem.  Sure Yeast Calc and Mr. Malty give you three digits of precision, but I would be surprised if they are within 25% of the cell count predicted with a wide variety of yeast strains.  (I'll be doing a post on that soon)

When pitching yeast, the important thing is to have the correct number of cells per volume of wort.  Instead of increasing the number of cells needed for the wort, the amount of wort can be reduced to the appropriate volume for the number of cells you have.  For the 5 gallon 1.060 case here, one vial of yeast has half the cells we need so these should be pitched into 2.5 gallons of the wort.  The following day, once fermentation has picked up, the remainder of the wort can be added.  Viable cell count can double within a few hours of the start of cell division.  (blog post showing that coming up as well)

Viability of the yeast you pitch may be a concern.  Sure, when the yeast left White Labs for your local home brew store it had very close to 100 billion cells, but what is the condition when you picked it up for your brew?  Test I have conducted indicate that loss of viability is very slow with refrigerated yeast.  In fact, I have seen no noticeable drop in viability over the course of a month of the slurries in my refrigerator.  I would imagine that professionally packaged yeast would only preform better.  (Blog post detailing this on this coming December 25th)

One concern you may have with this method is infection.  Because a large percentage of the wort remains for 24 hours without being inoculated the chance that something else may get a head start is a possibility.  However, if you make sure to sanitize everything, there should be no issue with holding the wort for a day before pitching.

On brew day sanitize two fermentors.  Chill the wort and pour half into each one.  Pitch the yeast into your primary fermentation vessel, and seal both containers.  The following day, just like a starter, your yeast will have grown significantly.  Pour the wort from the second container into the primary fermentation vessel.  As a bonus, pour as vigorously as you like.  Aeration is beneficial at this stage of fermentation.

An alternative to a second pail would be to keep the wort in your boil kettle and seal it up.  My boil kettle is a pressure caner, so this is an easy task.  If you are using a traditional kettle you may need to get creative.  An alternative would be to pour the hot wort into a large expandable water container as done in the no-chill method.

It's that easy.  No starter make or clean.  No delicate scheduling to ensure you have enough cells on brew day.  There is only one more thing to sanitize which should take an insignificant amount of time when done in parallel to sanitizing you primary fermentation vessel.

Apparently this is similar to a German technique known as "Drauflassen"

For discussion on this topic see this thread started on the AHA forum:

Saturday, December 1, 2012

Fermentation temperature control

A swamp cooler, or water bath, is one of the simplest and most effective ways to regulate fermentation temperature.  Because of the excellent thermal conductivity properties of water the temperature of the fermentation tracks that of the water temperature within one degree.  Fermentation temperature has a major impact on the flavor of the beer.  Fermenting toward the high end of the yeasts range (65-75 for Ales) will add more characteristic phenols.  Fermenting toward the lower end (60-65) will leave a more clean taste and can leave more residual sugar.  Lagers are typically fermented even cooler at 45 to 55 degrees, and yet other yeasts are designed to work best at temperatures near 80 degrees.

Control of the fermentation temperature is therefore a wonderful tool to have on your homebrewing belt.

In a previous blog post using ice to ferment at low temperatures.  In this post a method for fermenting at higher temperatures will be described in detail.

If you keep your swamp cooler in the basement the temperature of the water bath will likely be in the low sixties or below when no heat is added.  If a way to add a temperature controlled amount of heat is introduced, fermentation at temperatures above the ambient air temperature are possible without any interaction during the fermentation processes.  The easiest, and best way that I have discovered to do this is by using an aquarium heater. 

When selecting a fish tank heater for your water bath there are several factors that you will want to evaluate.  The heater you select needs to be effective for heating the volume of water in your swamp cooler.  A 50W heater is good for 10-15 gallon fish tank, This is a reasonable size for keeping a 5 gallon batches of beer about 10 degrees above air temperature, but if you want a larger temperature difference you might consider a 100W heater.  Next is temperature.  Make sure to select an aquarium heater that has a thermostatic control, not just a heat adjustment knob.  During the initial phases of fermentation the yeast will produce a fair amount of heat, and toward the end it will not be producing much heat at all.  In order to maintain a constant temperature it is therefore important that the heater can regulate the amount of heat it adds to the tank based on the temperature of the water.  Temperature range is also an important consideration.  The fermentation temperature of yeast is close to that of a typical fish tank, but check that the heater has setting for the range you will be using. 

After doing a lot of research and evaluating different products the best one I have found is the Fluval M Submersible Heater.  The temperature is settable from 66 to 86 degrees which is perfect for ales.  It also stretches into higher temperatures that many Sasion and Belgium yeasts prefer.  The best part is that it is less than $20 at Amazon as I write this.
Today I popped open a bottle of a "Sasion Terri" that I made with the Fluval M 50W submersible Heater, and it't came out perfect!  Without this heater I would have to wait until May of next year to try this!

Thursday, November 29, 2012

Counting Yeast Cells to Assess Viability for Brewing

Pitch rates, as you have likely heard, are very important to creating the desired flavor profile in a beer.  Pitching too much yeast will result in a bland lifeless flavor, while under pitching the yeast will result in hot alcohols and possible a stuck fermentation.  Generally if you are within a factor of two of the target pitch rate the resulting beer will match your target.
How to count yeast cells with a hemocytometer.
What you need:

A microscope with 400x magnification
A hemocytometer
2 test tubes or small vials
A graduated 2ml volumetric pipette
A 250ml (1 cup) or larger container with a tight lid.
Methylene Blue (which can be purchased at a pet store)

Step one, making a stock 0.01% Methylene Blue solution.
Don't worry, you only have to do this once.  Staining is used to distinguish the live cells from the dead cells.  Methylene Blue is attracted to acids such as the deoxyribonucleic acid (DNA) in the yeast cell. Yeast cell have the ability to reject Methylene blue that has entered through the cell membrane.  The result is that the dead yeast cells will stain blue, while the live yeast cells will stay clear.  Because Methylene blue is toxic to yeast the concentration should be kept lower than 0.1%, and once the cells have been stained the should be counted within half an hour.  When working with methylene blue keep in mind that it is a very dark dye that stains very well.  If you spill even a drop you may have a blue dot on your counter forever.  Methylene Blue sold to treat aquariums is typically 2.303%. 

To dilute this to the 0.01% concentration that is recommended by White Labs (1)(2)
1) add 1ml of the 2.303% Methylene blue to the 250 ml container. 
2) Fill the container with 230ml of water (230 grams, 7.77floz, 47 tsp, or 1 cup less 1 teaspoon)

Only 0.5ml are needed per measurement, so this container of stock solution that you have prepared will likely be enough for several years of testing.

Step two, preparing the sample.
With the hemocytometer you will only be looking at a tiny fraction of the yeast that you are evaluating, so it is important the the slurry is completely homogeneous before taking a sample.  Every time a sample is drawn with a pipette it should be pulled up and dispensed three times.  A pipette will hold 5% of the previous sample on its walls.  Pipetting three times ensures that the sample is not diluted by the previous washing or sample.

1) Shake the container of yeast until the everything is completely mixed up.  (If the slurry is not too thick a stir plate works great for this)
2) Shake the container some more.
3) using a pipette, remove 1ml of yeast from the slurry container.  If the slurry has large particles it may block the pipette.  If this happens you can use a drinking straw to remove the sample.
4) add the 1ml sample to one of the test tubes.  The accuracy of this volume measurement will have a direct impact on the accuracy of your data.
5) add 19ml of water to the yeast to dilute the sample 20:1
6) mix the sample by shaking vigorously, or by drawing in and out of the pipette at least 10 times.  Pulling and pushing the yeast through the small orifice of the pipette will break apart most floccs.
7) pipette 0.5ml of yeast from the sample test tube to the second test tube.
8) sterilize your pipette.
9) pipette 0.5ml of 0.01% methylene blue into the second test tube. Note that this dilutes an additional 2:1.

Step three, loading the hemocytometer.
1) place the hemocytometer on a paper towel and put the cover slip on.
2) mix the stained yeast with the pipette by pulling in and out at least three times.
3) remove a small amount with the pipette (you will only need about 0.1ml, or a fraction of a drop)
4) bring the pipette up to the hemocytometer without letting the tip touch.
5) dispense a hanging drop and touch the drop to the hemocytometer sample loading point.  The counting chamber should be filled to the point where it begins to spill into the center overflow area.  If some of the sample falls into the side spill areas that is okay, but these side moats should not be filled.  If the center area has no sample in it the count will be low.  If the side moats are filled then the count will appear high.
Step four, counting the cells.
If you haven't use a microscope before you might want to spend some time fiddling with the controls and doing some reading as to how to adjust everything properly.  I was surprised by the number of adjustments on a modern microscope, and how crisp the image can be when you have everything adjusted correctly.  There is a lot more to focus than just the focus knob!  When you look at the cells you should note the amount of clumping.  Clusters, or clumps, of cells indicate that the sample was not adequately mixed, and is thus an indication that your count may not be representative of the entire slurry.  When focusing on the cells, tighten the diaphragm all the way.  If the focus is too high you will see halos  around the cells.  If the focus is too low the cells will look blurry.  Once the cells are in focus open up the diaphragm until the blue cells are clearly blue and the clear cells should still have defined membranes.  If the diaphragm is opened too much, the cells will appear washed out and you may miss some in your count.  Focus is important.  It the focus is too high, the halo can make a dead cell appear alive.

1) Locate the upper left counting chamber.  (This is called box 1)
2) Count and record the viable cells (clear) and the dead cells (blue).
3) Repeat this process with the upper right, middle, lower left, and lower right chambers
4) If you want to have an idea of your accuracy you can preform statistical analysis on these five data points.

Step five, calculating.
1) viability is the percentage of living cells.
v = total number of Viable cells counted adding all five boxes
d = total number of Dead cells counted adding all five boxes
viability = v / (d+v) 
example: for v = 432 and d = 10
viability = 432 / (432+10) = 98%

2) viable cell density is the number of living cells per volume
df = dilution factor = 20
sf = stain factor = 2
vol = volume of boxes counted = 4nl x 5 boxes = 20nl
cd = cell density in billions of cells per liter
cd = v * df * sf / vol = v * 20 * 2 / 20
cd = v * 2 
example 432 * 2 = 864 billion cells per liter

3) pitching volume is the amount of slurry needed to achieve your pitch rate
pv = pitching volume in ml
c = billions of cells to pitch
pv = c / cd * 1000 
example: c = 200 billion cells needed to pitch
cd = 864 billion cells per liter
pv = 200 / 864 * 1000 = 231 ml (or roughly 231 grams) 

There is a plethora of information available, but these are some of the best suited for this task that I have found:

A wonderful tutorial with pictures of what you should see while counting:

(1) Step by step procedure from White Labs:

Some more nice photos of what to expect and some bacteria that you don't want to see:

(2) Currently I am using 0.03% MB as it stains more strains consistently.
Methylene blue (Left) Test Tubes (Right) Left to Right are Alcohol, slurry sample, 20:1 dilution, stained sample, and water.

Loading the Hemocytometer (Left) Cell counting (Right)Live cell circled in green.  Dead cells circled in red. Blue arrow on the top points to trub.  Blue arrow on left points to a fiber from cleaning the slide.