Yeast viability drop during storage and measurement methods To make a long story short, last year I did some experiments and viability drop tests with the methylene staining procedure and I posted the results to this list for comments some time ago, and come up with to my limited understanding some conclusions but I really would like your more authoritative opinion on this. The main reason for my confusion was that the amazingly high apparent survival rates I found in Wyeast smack packs! I did a viability drop experiment with collected primary yeast and found a quick viability drop. I definitely expected the smack pack to last longer, but the difference surprised me.
My preliminary conclusion was:
- Viability as per methylene blue staining seems to generally give slightly higher numbers as compare to platings, even accounting for the fact that one colony/count may be generated by a small floc of cells.
- The viability was higher than I though in the 19 month old pack. Though possibly 86% was and overestimate. The question is exactly how much?
As it seems, when the health drops, some cells that are technically alive, or at least enough "alive" to reduce the dye, are not healthy enough to complete a budding cycle - thus the deviation between method are expected to increase with poor health and low viability. In principle this made sense, but the question is
Q1. How big deviation in numbers can you expect? typically?
Methylene blue relies on the yeast being able to exclude the dye then if the dye does enter the cell the ability to decolorize the Methylene blue by using it as an electron acceptor (like O2 in respiration). However, there are many peculiarities with Methylene blue staining that still escape me. It is considered unreliable when the viability is less than about 85%. Counting under the microscope often seems to give higher numbers than you see with plating and this goes beyond flocculation/clumping of the cells. For flocculation to be the reason for the discrepancy between sight and plate then every cell has to clump with other cells. This is unlikely even in the worst cases.
Methylene blue is widely believed to overestimate cell viability. However, this is a simplistic view of the matter and the subject is far from being this straightforward. In reality, the accuracy of viability measurements by staining with methylene blue decreases as the number of viable cells drops below 85-90% (sometimes higher according to the strain). Unfortunately there is no accurate way of knowing the standard error of this deviation. The error is unlikely to be large (e.g. more than 20%), but enough to reduce confidence in results. For an example, see the Figure below which indicates the performance of a variety of methods. The main thing to note is the size of the error bars for methylene blue. It should be noted that these experiments were performed under laboratory conditions and factors such as the vitality of cells, ethanol, wort composition, pH and beer were not taken into consideration, which may act to skew the data (and increase error) further.
It is also worth commenting that you are correct in your supposition that the vitality of cells also influences the ability of yeast to reduce the stain to its colorless form. However, it should be noted that the principle of methylene blue staining is still a subject for debate. It has been proposed that the cell membrane may also play a lesser role by acting to prevent the stain from entering live cells, particularly under alkaline conditions (in alkaline conditions the proton differential becomes impaired).
Out of interest, many people observe a variety of shades of colors when using methylene blue. This is because the commercially available methylene blue also contains impurities in the form of azure B (light blue color), Bernthsen methylene violet and several other lower azures (which arise from the oxidative demethylation of methylene blue). The presence of these compounds becomes more apparent when studying a population of lower vitality and this can result in differences in staining intensity, leading in turn to increased subjectivity.
The first test I did was to stain a 19 month old Kolsch pack. I opened the bag, without smacking the nutrient bag, and the viability was ~86%.
Q2. Is this a normal survival rate if cells are good and O2 kept away, or is this to be considered exceptional? OR is something wrong with the staining method? The pack was stored refrigerated the whole time, around 5C or so.
I would think that 86% viability in anything at 19 months old would be a pretty good figure. You get what you get with methylene blue. The drawback may be that you have 86% viability but most of those cells are hanging on, viability and vitality (activity) are two different things, they are related in some ways, hurt the viability and you damage the vitality, however poor vitality need not mean poor viability.
Once the pack was opened I stored the remaining slurry in the fridge, however it was unavoidable to expose it to oxygen. Once the pack was opened, and I assume, exposure to oxygen. The viability drop rate quickly adopted that of the previous fridge slurry test I did.
After another 45 days, the 19 month pack originally 86%, dropped to 50% *as per the methylene blue staining method*. But after this 45 days I correlated with a plating and found the colony count / #plated cells to be 15%. Then I found by microscope inspection that the average floc size was around 1.8 cells.
So correcting for this, the 15% would actually be 27% as compare to the 50% from the staining procedure.
All of the numbers are averaged, so I am a little doubtful that all of the missing 23% are due to measurement errors. And the flocc size is already corrected for.
I would not be so sure the 23% is not measurement error. In experience it is quite easy to have this error consistently on a yeast when comparing plate counts with microscopic observation.
Q3. The question is now, if this deviation could be expected due to differences in the methods? Methylene blue staining vs. plating? Can you please comment?
Counting under the microscope often leads to overestimation of cell numbers as compared with plating. Why is less easy to understand.
From personal experience I never saw an overestimation of (total) cell numbers using microscope counts, so I presume this is just a viable cell issue. It is possible that plate count errors can arise as a consequence of poor/inaccurate dilutions. However, I believe that the difference you are seeing is probably due to methylene blue (see my answer to Q1).
(4) I'm trying to simply the some regulatory control of the main parts of metabolism and in standard bio text you read about some of the main regulatory step, but I think I also read a claim that the main regulating step for glycolysis in yeast is the uptake of sugars, or transport of sugars through the cell membrane? Is this so, in the sense that the regulatory role of the hexokinases are negligible? For example, I've read general, non-yeast specific biotexts says that the typical hexokinase enzymes for phosphorylation of hexoses have about 20 times higher affinity for glucose than for fructose.
Q4. Now my questions in this example is if yeast would ferment fructose significantly slower than glucose? If not, is this because the phosphorylation of fructose or glucose is not main limiters, and that the sugar transport through the cell wall is limiting?
This will depend on the yeast strain. In general the yeast transporters for glucose and fructose have different sensitivity to the two sugars and as such glucose my be taken up faster than fructose. I am not certain of the hexokinase kinetics but glucose has the advantage of a glucokinase which is more specific for glucose, whereas hexokinase is less loyal to glucose.
I had planned to make a comparison with a 100% glucose batch, and 100% fructose batch, do you think there would be any differences in the fermentation profile or acid production?
Would be interesting to see your results....on average I would say that on 100% glucose or 100% fructose you would see little difference. The differences may become more apparent when you mix these two sugars together. Then you would see the preference for either sugar at the level of the transporter. To enter glycolysis everything has to pass through some form of transport mechanism. Glucose has an advantage in that it can encourage the yeast to produce everything that it requires to cope with it while excluding the use of other sugars. It can vary from yeast to yeast but fructose can use the same systems as glucose so will compete with glucose at the level of the transporter, if I recall correctly glucose normally wins this battle, and in wine fermentations where both these sugars are found, along with their parental disaccharide sucrose, fructose normally is always left behind in the fermentation. For some yeast strains this slower utilisation can lead to problems in the fermentation and only addition of glucose allows the residual fructose to be consumed.
Q5) Can you elaborate on factors that affect the production of organic acids during fermentation? I have made a few sugar brew experiments some time ago and found a clear correlation between
final pH and amount of sugar used. I am assuming that this may have more than one reasons. Lacking amino acids might be one? but would ammonium nitrogen prevent excess acid production? Or does the simple and "quick" sugars cause a higher flow into the glyolysis pipelines
than the yeast can get rid of? and that this overflow in glycolysis and perhaps acetyl-CoA pools cause elevated acids being released into the beer?
>From the pH/sugar data I had the impression that there was a threshold of simple sugars where pH started to drop. How does the cell respond when pH drops? Do it increase secretion of acids into the cytosol to maintain a pH gradient? Could this be a factor, accelerating acid production in the case of pH drop due to poor buffering capacity of the growth media?
pH drops as a response to transporting some of the sugar and many of the other nutrients. Reduction of pH outside of the cells becomes one of the many stresses that the yeast has to endure in poorly buffered fermentation medium. The yeast has to maintain a higher pH internally and thus as transport of nutrients causes a reduction in extracellular pH so the cell have to expend more energy to keep internal pH high. Saccharomyces produces some organic acids, acetate is the biggest one but others can come from TCA cycle intermediates that accumulate due to the lack of TCA cycle activity itself. Nutrition of the yeast can be addressed to alleviate some of the acetate issues.