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By Aaron Golston

Many have argued that malt or hops are the soul of beer. However, without yeast, brewers would be left with a bitter, sugary, and non-alcoholic product.

To ensure flavor consistency, brew-ers use their skills and knowledge

to control the brewing process, but at times are at the mercy of the me-chanical and biological systems in the brewery. Some of these biological processes include: yeast propaga-tion, pitching, harvest, and storage which are all equally important. Recognising the importance of yeast, some brewers say that they make wort while yeast make beer and this is certainly true. There is even one brewery that has gone so far as to call their brewers ‘Unicellular Fungal Farmers’, which is a fairly accurate description of a brewer’s job. Given the important role yeast plays in alcohol and flavor production, it

behooves the brewer to establish procedures that allow for the main-tenance of their viability and vitality during propagation, fermentation, and storage. Consistency is the basis of re-producibility and what the consumer expects when he or she purchases a brand regularly. A large part of mak-ing a brand reproducible is fermenta-tion consistency. Ensuring consist-ent fermentations is a challenging task that requires rigorous focus on brewhouse and cellaring practices, which is often why automated sys-tems are installed and robust quality programmes are implemented. In ad-dition to brand consistency, consid-eration must be given to the cost of production. While extended fermen-tor residency time is not an ingredi-ent that costs money directly, it does decrease the overall efficiency and capacity of the brewery.

Yeast pitching practicesThere are four major yeast pitching practices used in commercial brew-ing: by weight, by volume, optical, and capacitance. Each method has its pros and cons from an accuracy, ease of use, automation, and cost

perspective. However, there are a few factors that are important regardless of practice employed. The user must be trained on how the system works, all components of the system must be calibrated, and the storage vessel or yeast brink must be homogeneous. Yeast brinks are typi-cally homogenised with agitators or recirculating pumps systems. Which-ever method is used to agitate the yeast, it should generate low shear to minimise damage to yeast cells. A common global practice is the use of activated dried yeast (ADY) for pitching. ADY comes from the sup-plier compressed in a sterile foil bag ready for use after a short rehydra-tion period. The aforementioned methods for direct pitching don’t apply, but any would be acceptable were the yeast to be propagated further before pitching or harvested for re-pitching. ADY is most often found in small breweries that brew infrequently, lack laboratory capabil-ities or the ability to store harvested cultures, but it has found use in full-scale and one-off production at some larger breweries. Controlling the pitch rate with ADY is done by con-trolling the mass of yeast added, and

iStock.com/TimAwe

Yeast pitching and counting A review of the importance of this microscopic fungus

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the concentration in the final beer can be verified by any of the following laboratory methods.

Laboratory methodsRapid laboratory determination of yeast slurry cell counts can be done by hemocytometer, solids measure-ment, or using automated in-lab-oratory systems. As with pitching practice, each counting method has advantages and disadvantages. Often ease-of-use and cost are significant factors surrounding the decision of which method to use; for instance, a small brewery might have a micro-scope but not a laboratory centrifuge, so hemocytometer counts might be the method of choice. At large breweries, multiple methods may be used at different levels: operators may perform solids measurements on yeast brinks, qual-ity may use an automated system in the laboratory and have a hemo-cytometer, as a backup. There is no one correct cell-counting method, as each brewery has different needs and the technique selected should ad-dress any brewery specific concerns. Haemocytometers were originally used for manually counting blood cells under a microscope but have found use outside of hematology. Commonly, the improved Neubauer haemocytometer is found in the brewing industry, as it is speci-fied in standardised yeast counting methods9. The counting chamber is divided into nine large squares and the middle square is subdivided into 25 smaller squares which are further divided into 16 still smaller squares. The large squares are 1 mm on each side, the smaller squares are 0.2 mm on each side and the smallest

squares are 0.05 mm on each side. The height of the counting cham-ber is 0.1 mm giving a precise, fixed volume of 0.1µL over the central ruled square. There are two counting chambers on a hemocytometer and each should be precisely loaded and counted for each sample measure-ment. There are many counting prac-tices for yeast cells but the most common is to count the yeast cells inside the 0.2 mm2 smaller squares and those touching the left side and top (or the right side and bottom) of the square. This helps ensure that cells are not accidentally counted twice. The ASBC method for Micro-scopic Yeast Cell Counting9 states that a minimum of 10 of the smaller squares should be counted to give an accurate and statistically valid measurement. In the method, it also states that a minimum of 75 cells should be counted in the central ruled square and that no one square should contain more than 48 cells. Good practice dictates that the cell counts generated from each side of the hemocytometer should be within 10% of each other for the measurement to be considered valid and the reported value should be the average of the counts from both sides.

Optical methodsRapid assessment of viability for brewery yeast cell cultures is gener-ally performed by staining with dyes and evaluating microscopically under brightfield or fluorescence. The most common method is methylene blue staining under brightfield for the identification of dead or non-viable cells. After staining, all yeast cells

present are counted, as well as those that have been stained blue, on a hemocytometer. The viability of the culture is reported as a percentage of live cells. There has been controversy sur-rounding the use of methylene blue for viability staining as it has been reported to overestimate the vi-ability of the culture4, especially as the viability of the culture decreases below 90%2. Regardless, it remains the most widely utilised stain in the brewing industry. Alternative dyes, such as methylene violet and trypan blue have been introduced1,5 and are used by some as they have found them to work better under their brewery conditions. Fluorescent dyes, such as: oxonol or propidium iodide (PI), have gained popularity with the use of auto-mated cell-counting technology or fluorescence microscopy. Another fluorescent dye, magnesium salt of 8-anilino-1-naphthalene-sulfonic acid (MgANS), appears in the litera-ture but is not commonly used in the brewing industry. While PI is not prevalent in the brewing literature, it

A hemocytometer (a), and view under microscope (b) containing yeast cells

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has been widely utilised in flow cytometry and other fluorescence applications. Despite PI’s limited presence in the literature, it is used successfully with some automated cell counting systems. Oxonol is negatively-charged potentiometric fluorescent stain that binds intracellular lipids and proteins. It does not enter live cells due to the transmembrane poten-tial, which is not present when a cell is dead or non-viable. Once oxonol enters the cell, it binds lipids and proteins in the cytoplasm and the excitation of the dye at specific wavelengths causes the dead cell to fluoresce red. MgANS is a membrane-imperme-able protein-staining fluorochrome that fluoresces yellow/green, when excited6. The dye crosses the yeast membrane when it has been com-promised (i.e. after death) and binds proteins in the cytoplasm. PI is a membrane-impermeable fluoro-chrome that binds to nucleic acids. It will enter the cell under the same conditions as MgANS but its mecha-nism of action is different as it binds to the cell’s DNA; when excited at specific wavelengths, the cell will fluoresce red similar to oxonol6.

Automated cell counting in-struments, such as the Countstar, Cellometer, or NucleoCounter, use image analysis software to count cells. This saves time and ensures greater consistency of measurement between operators. These systems utilise customised plastic slides or inserts for the loading of cells instead of a glass haemocytometer. The Cellometer and Countstar count yeast cells using brightfield micros-copy and both can assess viability with brightfield staining – but the Cellometer most commonly uses fluorescence staining to detect dead cells. The NuceloCounter uses pro-pidium iodide, contained within the customised insert, for both count-ing and viability. The instrument has a unique mechanism of action: to obtain a total sample cell count, the sample is mixed with a lysis buffer and then the nucleus is stained with PI as it is introduced into the insert. If a viability measurement is desired, the lysis buffer is omitted from the sample prep and the dead cells are stained with PI upon introduction into a second insert11. After taking an image of the cells or nuclei, the software performs a series of analyses assessing cell size, shape, declustering, and color/fluorescence. The results of these analyses, with the appropriate dilu-tion factor, yields the cell count and viability for the sample. It is possible that each strain of yeast measured on the system will require a sepa-rate configuration in the software, due to differences in size, shape,

and flocculation/clumping charac-teristics, but this is largely strain dependent. A potential challenge with these instruments is the configuration re-quired to reproducibly and accurately quantify budding cells. One of the advantages of automated counting systems is that the total number of counted cells is greater than with a hemocytometer, which yields more accurate results, assuming the same sample preparation. However, two potential challenges with these systems are: the elimination of user-to-user or intralab variation when manual focusing is required and the reproducible and accurate quantifi-cation of budding cells. Training must be provided to ensure consistency.

Coulter CounterAnother automated cell counting in-strument is the Coulter Counter and it works based on the Coulter prin-ciple. The Coulter principle states that when a particle is pulled through an aperture that has an electrical current applied across it, the particle will provide resistance to the flow of current which will be proportional to the size (volume) of the particle. The probe is submersed in a low-concentration electrolyte solu-tion that conducts electrical current. When a yeast cell or other particle passes through the aperture, the measured current intensity decreas-es due to the displacement of the electrolytes by the current-impeding yeast cell. These instruments are limited to solutions with low concen-trations of analytes, as they essen-

Cell-counting devices, Countstar (a), cellometer (b) and (c) showing fluorescence view, and nucleocounter (d)

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tially count cells one at a time. If two yeast cells pass through the aperture at the same time, it will be counted as one very large yeast cell. Even though cells pass through the aperture one at a time, it oc-curs very quickly and cell counts on the order of 105 are obtained. Once the measurement is complete, a particle-size histogram is generated and a cell count returned based on pre-programmed calculations. Un-like optical methods, viability is not measured so if this is desired, it will need to be performed separately12.

Concentration by solidsYeast concentration by centrifuged solids measurement was published in the MBAA Technical Quarterly in 19693. It showed an improved cor-relation between alkali-treated yeast solids measurement and fermentor full cell count. This differed from previously-used centrifuge methods, due to the treatment of the yeast with sodium hydroxide which was thought to clean the yeast cells—but now is known to remove entrapped CO2—and reduced the standard deviation of the measurement from 2.1 x 106 cells / mL to 0.3 x 106 cells / mL. The centrifugation of harvested yeast to determine solids percentage will only provide valuable process information if there is a standard chart correlating percent solids to cells/mL. This will enable the brewer to know the cell count in the sample. Unlike the universally applicable haemocytometer cell count, a unique solids chart must be made for each yeast strain used and there is no measurement of viability. It is there-fore best to use this method with a strain that has low variability in its viability or to carry out a separate viability measurement.

Pitching calculationsPitching by weight requires the con-centration of cells in the slurry, the mass of a known-volume of slurry and a gravimetric measurement device. The yeast slurry in the brink should be homogeneous prior to being sampled into a tared, known-volume container and filled to the appropriate level. Once in the laboratory, the sam-ple must be homogenised and the cell concentration determined by one of the previously mentioned meth-ods. After the cell count is known, it can be converted from cells/mL to

cells/g based on the measured mass of the known-volume sample. Since the target pitch-rate for the beer is known, the mass of yeast needed can be determined and this information can be utilised for manual pitching or with automated systems. The simplest manual system, often used in brewpubs, is a keg on a scale and the brewer will pitch to the desired mass needed. In larger brew-eries, the brinks can be on load cells and the output from the load cells is sent to the brewhouse automation to control the valves and pumps associ-ated with the yeast pitching system. Pitching by volume requires both the concentration of cells in the slurry and a volumetric measurement device. The cell concentration can be determined by any of the aforemen-tioned methods. Once the slurry cell

concentration is known, the quantity of yeast needed to pitch the wort can be determined. This can be calculated and controlled manually or entered into the automation and controlled automatically. Manual control often requires the observation of the yeast brink level indicator with a graduated scale while the pitch is occurring and stopping the pitch when it is com-plete. Automated control takes the output from the volumetric flow meter to control pumps and valves, same as with a pitching by mass system.

Near infraredPitching by near infrared (NIR) turbidity measurement requires one or two NIR sensors and a volumet-ric flow meter. NIR pitching control can be set up in one of two ways: a NIR sensor and flow meter on the yeast pitching line, or a NIR sensor pre- and post-yeast injection and a volumetric flow meter, all on the wort line.

Coulter Counter instrument (a) and method of operation (b)

Yeast slurry in 50mL CF tubes: (a) unspun, (b) spun without NaOH, and (c) spun with NaOH

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Using NIR on the yeast pitching line has an advantage over wort line measurement as the sensor is locat-ed after the yeast supply pump where the slurry will be homogeneous and flocculation/clumping characteris-tics will be less of a factor. Wort line setups utilise two NIR sensors as previously mentioned. The pre-yeast injection sensor is used to deter-mine the baseline absorbance of the matrix from trub and particulate car-ryover while the post-yeast injection sensor is measuring both the trub and yeast. In the converter, the input from the pre-injection absorbance is subtracted from the post-injection absorbance and this is used to calculate the yeast cell concentra-tion. All sensor outputs can be fed back into the automation software for automatic fermentor full cell count calculations. Correct sensor installation is a critical part of accurate pitching con-trol. Ideally, the installation of sen-sors would be on a vertical section of the pipe on the discharge side of a pump. It is possible to install on a horizontal section of pipe, in the 3 or 9 o’clock position, but the pipe must be fully packed at all times. Instal-lation on the suction side of a pump is not recommended as the forma-tion of bubbles will negatively impact the accuracy of the measurement. As with solids measurement, each strain requires a separate calibra-tion due to its unique properties. It is important to install a sample port in close proximity to the sensor for sampling and calibration. NIR sensors are inline spectro-photometers with the measurement

cell installed in the process pipe and the user interface generally mount-ed in an electrical panel. These sensors work on the same principle as a bench-top spectrophotometer, the Beer-Lambert law, A = εbc which simply states that the absorbance (A) is directly proportional to the concentration (c) of the measured analyte in solution. The molar ex-tinction coefficient (ε) is a constant value that is unique and specific for each analyte / sample and it de-fines the relationship between the absorbance (A) and concentration (c) for a fixed path length of measure-ment (b). It is important to note that ab-sorbance (A), a unitless number, has a logarithmic relationship with trans-mittance A = −logT, and therefore, an absorbance measurement of zero means 100% transmittance of NIR waves, one (A = 1) means that 90% of waves have been absorbed or 10% of the waves has been transmitted by the sample. An absorbance read-ing of two (A = 2) equals that 99% absorbed and an absorbance of three (A = 3) equals 99.9% absorbed. When measurements get above three absorbance units, the meas-urements become less reliable due to the low level of transmitted waves. Additionally, there can be complex light interactions with particles in suspension at high concentrations which can result in deviations from linearity.

Capacitance basedThe capacitance-based measure-ment of yeast cell biomass to control pitching rates requires a biomass meter and a volumetric flow meter

NIR sensor (a) and PID of install options (b)

Yeast brink on load cells

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Whirlpool Wort Heat Exchanger Yeast Brink Fermenter

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on the yeast pitching line where the output from each can be logged in the automation software. After some simple calculations with the finished wort volume, the actual viable cell count (cells/mL) in the fermentor can be given. For each strain, with which pitch-ing control is desired, a calibration must be performed. This is due to the unique characteristics of each strain, such as flocculation/clumping, size, shape, and ability to be polarised in an electric field. To calibrate the meter, a total viable cell count from the slurry of the desired strain, performed by staining and haemocytometer count or automated cell counter, must be entered while the meter is sub-merged in the same slurry. As with NIR measurement, sensor location is critical and the same installa-tion recommendations apply8. Some manufacturers have worked to sim-plify the installation and utilisation of these meters by offering skidded solutions. Capacitance-based biomass measurement is based on the di-electric (insulating) properties of the cell’s plasma membrane. The plasma membrane separates the cytoplasm from the external environment. The cytoplasm is largely water but also contains the organelles, proteins, and ions. Given that the cytoplasm and external environment can conduct electrical current while the plasma membrane cannot, each cell can be treated as a small capacitor. The low-frequency radio waves emitted from the sensor polarise the cytoplasm and external environ-ment by separating the ions and charged compounds. The magnitude of the charge separated by the cell’s plasma membrane is measured

as capacitance (units: pF/cm) and is directly proportional to the size and number of cells present in the slurry7,8. It is important to note that these meters do not measure viability, they only measure the capacitance of viable cells; non-viable (leaky membrane or dead cells) cannot be polarised and therefore, cannot be quantitated.

Final thoughtsThere is no one right way to count cells or pitch yeast. Most breweries start with a simple methylene blue cell count with a hemocytometer but as they grow, may consider utilising the more accurate automated cell counters. With regards to pitching, this is largely a question of scale as a capacitance-based or optical sensor and the associated flow meter can be quite costly and it would make little sense to install such advanced instrumentation on a small system. However, pitching by volume or weight can be very cost effective at smaller scales. It is also worthwhile to consider the level of complexity as-sociated with automated yeast pitching systems and instrumentation as they are almost never turnkey. Yeast pitching and harvest are criti-cal processes to control when trying to achieve consistent fermentations. Since yeast are the workhorse of the brewing industry, it is important to take care that it is added in correct and sufficient levels. Under-pitching can lead to sluggish, under-attenuated and/or stuck fermentations, off flavor formation – and present the opportuni-ty for spoilage organisms to take hold. Overpitching can lead to rapid fermentations, decreased ester production, high acetaldehyde levels, excessive losses, and high diacetyl levels, that may take more time for yeast reductases to reduce. With such high stakes, paying close attention to how the yeast is pitched and counted can only yield positive benefits for the brewery.

AcknowledgmentsI would like to thank the following people for their time reviewing and commenting on this article: Bill Maca of HWM Yeast Solutions, Jessica Davis of The Bruery, Dr. Chris Powell of University of Nottingham, Dr. Jan-Maarten Geertman of Heineken Supply Chain B.V., Dr. Leo Chan of Nexcelcom, Christian Hededam Berg

of Chemometec, Dr. Aditya Bhat and Dr. John Carvell of Aber Instruments, and Al Worley of optek-Danulat.

References1. Land, M. ‘Citrate-Buffered Methylene Violet Stain as an Alternative to Conven-tional Stains Used to Determine Yeast Viability.‘ Journal of the American Society of Brewing Chemists ASBC, vol. 59, no. 4, 2001, pp. 232–233.2. O’Connor-Cox, E. et al. ‘Methylene Blue Staining: Use at Your Own Risk.’ MBAA Technical Quarterly, vol. 34, no. 1, 1997, pp. 306–312.3. Palmer, F. ‘The Determination of Pitch-ing Yeast Concentration.’ MBAA Technical Quarterly, vol. 6, no. 2, 1969.4. Smart, K. A. et al. ‘Use of Methylene Violet Staining Procedures to Determine Yeast Viability and Vitality.’ Journal of the American Society of Brewing Chemists ASBC, vol. 57, no. 1, 1999, pp. 18–23.5. Szabo, S. ‘Comparison of the Efficacy of Various Yeast Viability Stains.’ Beckman Coulter.6. Van Zandycke, S.M. et al. ‘Yeast Quality and Fluorophore Technologies.’ Brewing Yeast Fermentation Performance, 2nd ed., Blackwell Science, Oxford, UK, 2003, pp. 149–161.7. Yardley, J. E. et al. ‘On-Line, Real-Time Measurements of Cellular Biomass Using Dielectric Spectroscopy.’ Biotechnology and Genetic Engineering Reviews, vol. 17, no. 1, 2000, pp. 3–36.8. ABER Compact System User Manual. ABER Instruments, 2016. 9. ASBC Methods of Analysis, online. Yeast Method 4. Microscopic Yeast Cell Counting. Approved 1958, rev. 2011. American Society of Brewing Chemists, St. Paul, MN, U.S.A.10. ‘Yeast Viability by Fluorescent Stain-ing.’ Journal of the American Society of Brewing Chemists ASBC, vol. 63, no. 4, 2005, pp. 220–224.11. ‘NuclecoCounter Y-100 Presentation.’ Chemometec, 7 Dec. 2016. PDF.12. Sponsored by Beckman Coulter Life Sciences. ‘The Coulter Principle for Par-ticle Size Analysis.’ AZoM.com. Beckman Coulter, 11 Feb. 2015. Web. 15 Nov. 2016.

ABER meter installed in a pipework

Yeast cells polarised in radio waves