Assessing yeast condition

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Before any batch of pitching yeast is used it is necessary to confirm that it is fit for the purpose. Three types of quality test may be applied. First, the yeast must be free from microbial contamination. Testing the microbiological integrity of yeast is described in Chapter 8. Second, it is usual to determine the viability of the yeast. Most breweries operate a quality reject system in which yeast is discarded if the viability falls below a pre-set value. In addition, viability measurements are used to determine the quantity of slurry required to achieve the desired viable pitching rate. Third, there is a category of quality tests, Vitality tests', which seek to probe aspects of the physiological condition of yeast. Thus, they provide information regarding the viable fraction of yeast slurries.

7.4.1 Assessing yeast viability

The classical microbiological method of assessing viability is to plate out a measured number of cells onto solid media and after a period of incubation, count the resultant colonies (see Section 8.3.2). Each colony is derived from a single live cell. Therefore, the proportion of colonies formed in relation to the total number of cells present in the original sample provides a measure of viability (Institute of Brewing Analysis Committee 1962; ASBC Analysis Committee, 1980). It is possible to accelerate the procedure by counting micro-colonies on a slide culture; however, at best several hours are needed to obtain a result (ASBC Analysis Committee, 1981; Pierce, 1970). This is too long to meet the needs of production brewing where a rapid answer is essential.

Rapid methods for viability assessment rely on the use of vital stains. In the brewing industry, the most commonly used procedure is methylene blue staining, although several others have been used. For example, crystal violet, aniline blue, Rhodamine B and Eosin Y (King et al., 1981; Evans & Cleary, 1985; Koch et al., 1986; Hutcheson et al., 1988). In the methylene blue test, viable cells remain colourless, whereas dead cells are stained blue (see Fig. 7.15). The physiological basis of the test is that viable cells take up the stain at a sufficiently slow rate for it to be

Yeast Microscope Dye
Fig. 7.15 Viability staining of yeast using methylene blue. (This figure is repeated in the colour section)

oxidised to the colourless 'leuco' form. Conversely dead cells cannot exclude the dye or perform this reaction (Chilver et al., 1978; Jones, 1987). Viability is determined by preparing a suitable dilution of slurry and counting total and stained cells using a haemocytometer and a light microscope.

It is well recognised that the methylene blue method tends to over-estimate viability when compared with plate counting techniques. This effect becomes progressively more pronounced with decrease in viability and it is recommended that the test is used only when viability is greater than 90% (Pierce, 1970; Parkkinen et al., 1976; Chilver et al., 1978; King et al., 1981). The test requires skill on the part of the operator to obtain reproducible results and problems are encountered counting flocculent yeast. Occasionally, particularly in samples containing stressed yeast, cells may stain to a slight extent only, making interpretation somewhat subjective. To overcome some of these problems it has been suggested that the decolourisation reaction could be used as the basis of a spectrophotometric procedure (Bonora & Mares, 1982). However, at high viabilities, where the methylene blue test is valid, the small change in colour makes this test impracticable. Attempts have been made to overcome some of the failings of the methylene blue staining method by use of an alkaline pH and contact with yeast for 15 minutes at 25°C (Sami et al., 1994). The rationale here was that, since entry of the dye is influenced by membrane potential, the lowering of the external H+ concentration would favour entry into very stressed cells, which normally give a false positive viability. Data was provided that supported this contention.

It has been suggested that the disparity between viability determined by methylene blue and plate counting might be overcome by a double staining technique using methylene blue and safranin O (Nishikawa & Nomura, 1974). In this test, a gradation of colour was reported to correlate with physiological condition. Thus, a cell in good condition stained blue/purple, whereas dead cells stained pink/red. In between these two extremes were slightly deteriorated cells, which stained reddish purple and grossly deteriorated cells, which stained brown.

Smart et al. (1999) compared the efficacy of viability measurements by plate count, methylene blue/safranin O double staining, citrate methylene blue, alkaline methylene blue, citrate methylene violet and alkaline methylene violet. Samples of ale, lager and cider yeast in various physiological conditions were used in the assessment. These were exponential and stationary phase 'healthy' cells, starved cells and non-viable heat-killed populations.

Predictably, the plate count approach underestimated true viable cell counts in the case of chain-forming strains; however, it was the most reliable of the techniques tested for identifying dead cells. Citrate methylene blue gave a good correlation with healthy and starved yeast but dramatically over-estimated viability using heat-killed cells. Citrate methylene blue and citrate methylene violet were of equal utility where viable populations were tested. However, only the latter was considered to be capable of unequivocal differentiation between viable and non-viable cells.

The methylene blue/safranin O double staining technique was found to give very variable results, even in replicate samples, and was not recommended. Alkaline methylene blue reportedly measures intracellular reducing power, since the high pH removes any barrier to dye penetration (Sami et al., 1994). This method failed to distinguish viable and non-viable cells in a reliable manner, particularly those in the exponential phase of growth. Conversely, alkaline methylene violet could distinguish living, stressed and dead cells. In this sense, the method was of utility in measuring 'vitality' as well as viability, as discussed in Section 7.4.2.

The authors concluded that the methylene blue staining technique produced equivocal staining reactions because of instability of the dye. Thus, they contended that commercial preparations of the dye contain two chromaphores, azure B and Bernthsen methylene violet and some lower azures deriving from oxidative demethylation of methylene blue. The latter reaction was exacerbated by prolonged storage of prepared solutions and this should be avoided.

Tetrazolium salts have been used as indicators of viability of microbial cultures by virtue of their intracellular reduction to coloured formazan deposits (Postgate, 1967). Thom et al. (1993) reported the results of a comparative study of the application of four tetrazolium salts to the measurement of viability. The yeast Candida albicans was included in the study and although results were improved by addition of glucose this organism gave the least satisfactory response. Others have reported good correlation between respiratory activity of Saccharomyces yeasts and staining with the tetrazolium salt 2-(/Modophenyl)-3-(/?-nitrophenyl)-5-phenyl tetrazolium chloride (INT) (Trevors, 1982; Trevors et al., 1983. It is unlikely that this would be of utility in the case of repressed brewing yeast cells.

The biomass meter described in Section 6.1.4.5 provides an instantaneous measure of yeast concentration. It can be shown that the meter is responsive to that fraction of the population that would be considered viable using the methylene blue test (Boulton et al., 1989). Conversely, it does not respond to the non-viable fraction of the population and it follows that it will not provide a measure of viability per se. However, this can be achieved if the meter reading is used in conjunction with an analysis of total yeast concentration. Conveniently, this removes some of the errors due to the manual aspects of methylene blue counting. Nonetheless, it is still apparently prone to the same over-estimation in the case of very-low-viability samples as the methylene blue method.

Viability may be determined by measurement of the surface electrostatic charge, or zeta potential of yeast cells (see Section 4.4.6.7). It can be shown that a significant difference exists in the magnitude of this parameter for viable (intact membrane) and dead cells (disrupted membrane) (Brown, 1997b). The same author described an apparatus that uses a laser beam arrangement linked to a powerful software package to measure both cell size and zeta potential within a yeast population and thereby calculate the viability. It was further suggested that the magnitude of zeta potential was a general measure of the yeasts' physiological condition.

Several fluorescent dyes have been used in vital staining methods. These rely for their selective staining action on several different facets of cellular metabolism. Some dyes are taken up by all cells and fluorochromes are liberated after modification by enzymes that are present only in viable cells. Other dyes rely on membrane function. Some are excluded by cells with intact membranes, or cells capable of maintaining an electrochemical transmembrane potential. Non-viable cells are not capable of excluding these dyes and these become fluorescent under appropriate conditions. Another class of dyes that also rely on membrane integrity are taken up only by viable cells. Some of the commonly used fluorescent dyes, together with a brief description of their mode of action are given in Table 7.3.

Several authors have reported that viability measurements made with fluorescent dyes provide results that correlate closely with plate counting methods. For example, McCaig (1990) reported that viability measurements made using the fluorochrome Mg-ANS (Table 7.3) were similar to slide culture results, but were significantly different to values obtained by bright field staining with methylene blue or Eosin Y. Similarly, Trevors et al. (1983) compared several viabililty methods and reported that Mg-ANS and primulin were both of good utility; however, acridine orange was less accurate.

Table 7.3 Fluorescent dyes used for viability measurement.

Dye

Mode of action

Reference

Fluorescein diacetate

Release of free fluorescein following cleavage by nonspecific intracellular esterases in viable cells

Paton and Jones (1975); Chilver et al. (1978)

Propidium iodide Ethidium homodimer

Excluded by viable cells; bind to nucleic acids in non-viable cells

Bank (1988); Donhauser et al. (1993); Hutter (1993)

Oxonol dyes

Excluded by viable cells

Dinsdale and Lloyd (1995); Lloyd et al. (1996)

Rhodamine 123

Taken up by viable cells with mitochondrial trans-membrane potential

Dinsdale and Lloyd (1995); Lloyd et al. (1996)

3,3-Dihexyloxacarbocyanine

Taken up by viable cells with plasmamembrane transmembrane potential

Lloyd et al. (1996)

Chemuchrome Y

Uptake followed by enzymic cleavage to release fluorochrome in viable cells

Raynal et al. (1994)

Mg l-anilino-8-naphthalene sulphonic acid (Mg-ANS)

Accumulates in viable cells with functional membrane and binds to proteins

King et al. (1981); McCaig (1990)

Acridine orange

Retained in viable cells

King et al. (1981); Trevors (1982); Trevors et al. (1982)

Viability measurements with fluorescent dyes can be performed in the same way as the standard methylene blue haemocyotmeter test although a fluorescent microscope is required (Chilver et al., 1978; McCaig, 1990). Much better results are obtained with automatic measuring devices, which remove the error due to the human operator. For example, Raynal et al. (1994) describe the use of fluorescein diacetate and Chem-chrome Y to measure viability in conjunction with an electronic image analyser. The most commonly used counting apparatus is the flow cytometer (Petit et al. 1993). In this device, cells in a suspension are passed singly through a narrow orifice where each is detected. In the case of fluorescent applications the cells pass through a light beam of appropriate wavelength and and fluorescence is registered by a detector. Other detectors measure total cell count by light scattering and possibly other parameters such as cell size distribution. Sophisticated instruments have the facility to separate and collect sub-populations from the stream of cells based on the response from the various detectors (Edwards et al., 1996).

Image analysers and flow cytometers provide rapid and accurate viability measurements compared to manual microscopic counting. However, there is a significant cost penalty. In particular, the more sophisticated flow cytometers cost up to £250000. Clearly, these will not find use as routine laboratory tools for assessing the quality of production yeast! Less expensive flow cytometers are also unlikely to find application purely for viability measurement; however, they offer the potential for much more delicate probing of yeast physiology, as described in Section 7.4.2.

7.4.2 Yeast vitality tests

Provided a single method for viability measurement is used and the procedure is performed in a consistent manner, it undoubtedly offers useful comparative information for the individual brewer. In particular, a test such as methylene blue staining is of great use as the basis of a simple decision to use or not use a given batch of pitching yeast. Tests such as this can also be useful, provided the viability is high (> 90%) in establishing a correction factor to ensure that viable pitching rates for all fermentations are the same. Such tests are less useful in identifying variations in pitching yeast physiology, which can produce inconsistencies in fermentation performance and beer quality.

With regard to yeast cells, there is no single definition that encompasses viability. Characteristics of viable cells would include:

(1) capability of cellular proliferation (progression through the cell cycle);

(2) capability of cellular growth (anabolic metabolism)

(3) detectable resting metabolism (oxygen uptake and carbon dioxide evolution);

(4) possession of membrane integrity (controlled selective assimilation of exogenous metabolites and excretion of by-products).

It is obvious that within a population of pitching yeast there may be cells which do not possess all these characteristics but still take an active role in fermentation. For example, they may not be capable, for whatever reason, of multiplication; however, they may still contribute to fermentation by assimilation of wort nutrients and production of beer components. It is unclear which fraction of these populations is detected by standard viability tests. It is also arguable that although the plate or slide test is considered as a standard reference method for viability determination, the test as applied to pitching yeast does not necessarily equate to subsequent growth and fermentation performance.

Simple viability tests are not capable of providing information regarding possible differences in the physiological status of the viable fraction of the yeast population. These differences could span the spectrum from extremely stressed or dying cells through to those which are in a physiological condition that would allow 'superperformance' in fermentation. For example, cells which contain high sterol con centrations would be predicted to undergo more rounds of budding during fermentation than those with basal sterol levels.

Several 'vitality' tests have been proposed which provide information of the physiological condition of the entire population of yeast cells within a slurry. This is an unfortunate choice of terminology since it has no strict scientific meaning other than being a synonym for viability. Of course, it is taken to imply a method that identifies yeast that will produce a vigorous rapid fermentation performance. It does not follow that such a fermentation would be efficient in terms of yeast growth and ethanol yield. It would be better to refer to the methods by the less concise but more accurate and useful definition of 'predictive fermentation tests'. However, vitality testing has become part of the established brewing literature and in order to avoid confusion it is used here.

The requirements of vitality tests are that they should be rapid, simple and preferably use apparatus available in a typical brewery laboratory. The results of such tests may be used to arrive at a simple decision regarding fitness to pitch. However, preferably the result would allow selection of an appropriate pitching rate and wort oxygenation regime, which provides optimum and consistent fermentation performance and beer quality. A plethora of vitality tests have been proposed that may be considered according to which aspect of yeast physiology they seek to probe.

7.4.2.1 Tests based on cellular composition. Glycogen is reported to provide carbon and energy for sterol synthesis during the early aerobic phase of fermentation (Quain, 1988). Pitching yeast that has been stored for too long or held under inappropriate conditions may have already expended much of its glycogen reserves, and therefore be unable to efficiently couple oxygen utilisation to sterol synthesis during early fermentation (Section 7.3.2). It would be predicted that this would be manifest as a prolonged lag phase in fermentation. Yeast glycogen content can be determined using the colour reaction with iodine either qualitatively by visual assessment (Fig. 7.16) or quantitatively by measurement of the brown coloration at 660 nm (Quain & Tubb, 1983). Skinner (1996) described another method, which took just over two hours to perform, based on infra-red spectroscopy.

Trehalose levels in yeast have also been proposed as a useful monitor of yeast condition. In particular the relationship between trehalose levels and yeast stress. For example, Majara et al. (1996b) observed a positive correlation between trehalose concentration and applied stress. These authors suggested that a sudden increase in trehalose concentration in non-growing cells could be indicative of cells that had been in some way stressed. Probably the carbon for such an increase would have derived from glycogen breakdown. A possible vitality test, therefore, would be to determine the ratio of glycogen to trehalose in pitching yeast. As with glycogen, yeast trehalose content can be determined in a rapid test using near infrared reflectance spectroscopy (Moonsamy et al., 1996). For individual yeast strains used in particular fermentations this ratio should fall within definable limits for yeast in 'good' condition. A significant deviation from this established value would be indicative of abnormal yeast.

A problem with this concept is the situation in which yeast has dissimilated glycogen during storage, in response to inadvertent exposure to oxygen. Such yeast would have a low glycogen content, and therefore appear stressed, but in actuality

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