​​​​​Microbiology​ Methods


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  • Yeast

    Yeast 1. Sampling

    Since yeast slurries, suspensions, and compressed cakes can easily become stratified, sampling must be done in a manner that ensures homogeneity. This method presents the basic procedures for obtaining representative samples and preparing them for laboratory analysis.

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    Yeast 2. Physical Examination

    The homogeneous yeast sample may be subjected to a variety of tests and examinations to determine its quality and suitability for brewing. The first sensory steps will be to record its color, character (flocculent vs. nonflocculent), odor, and taste. This will be followed by microscopic examination, as described in this method, to determine cell morphology and the presence of undesirable microorganisms, e.g., bacteria.

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    Yeast 3. Yeast Stains

    Numerous yeast staining procedures have been investigated, either to supplement information acquired by trial under brewing conditions or to reduce the time required for such evaluation. This method describes dead yeast cell stain, yeast spore stain, the use of triphenyltetrazolium chloride for identification of respiratory-deficient cells, and determination of yeast viability by fluorescent staining.

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    Yeast 4. Microscopic Yeast Cell Counting

    This method determines yeast cell concentration by using a hemocytometer and a microscope. The procedure relies on the enumeration of cells within a specific number of microscopic fields to determine the concentration of yeast in a population.

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    Yeast 5. Yeast Solids

    This method determines yeast solids as total by dry weight (for liquid and pressed yeast samples) and as percent by spin-down (for slurries).

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    Yeast 6. Yeast Viability by Slide Culture

    Viability of culture yeast is of utmost importance. This method measures viability by a slide-culture technique employing a medium containing malt extract, yeast extract, glucose, and peptone supplemented with maltose and zinc.

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    Yeast 7. Yeast Sporulation

    Yeast sporulation is useful for identification of some species of wild yeast, because lager yeast generally sporulates poorly, if at all. Sporulation can also be used to help characterize yeast and to produce spores for genetic studies. This method determines percentage of sporulated cells and typical number of spores per ascus in a sample.

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    Yeast 8. Killer Yeast Identification

    The capacity to be killed by a killer yeast is an easily scored and well-defined phenotype. An agar plate seeded with a sensitive strain and then inoculated with a killer strain produces a clear zone of inhibition within the seeded sensitive lawn. This technique, a simple screening assay to determine whether a yeast secretes killer toxin, is described in this method.

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    Yeast 9. Morphology of Giant Yeast Colonies (International Method)

    This method produces giant yeast colonies on wort gelatin medium. Comparative analysis of the morphology of such colonies can be used as a means of differentiating brewing yeasts.

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    Yeast 10. Differentiation of Ale and Lager Yeast

    This method differentiates ale and lager yeasts by measuring growth of colonies on X-(-gal medium, by determining growth at 37°C, and by determining growth on melibiose.

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    Yeast 11. Flocculation

    This method measures flocculation of a yeast sample by the Helm assay, in which sedimentation of a yeast sample is observed in a calcium sulfate solution buffered at pH 4.5 to determine sedimentation volume and flocculation type, and by the absorbance method, in which flocculation is measured by absorbance after a settling reaction in a calcium sulfate solution buffered at pH 4.5.

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    Yeast 12. Vitality By Fluorescence

    This method is included as a Provisional Method and should provide a guideline for the use of the Yeast Activity Monitor instrument for vitality analysis at the discretion of the user. The method was not recommended for inclusion in Methods of Analysis due to the criteria outlined in the method.

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    Yeast 13. Differentiation of Brewing Yeast Strains by PCR Fingerprinting

    Brewing yeast strains can be differentiated on the basis of their DNA structure and composition. While there are many DNA fingerprinting methods available, polymerase chair reaction offers a quick and simple process for the differentiation of strains. This method describes the process in which oligonucleotide primers that bind to complementary regions within yeast delta sequences are used to produce a DNA fingerprint.

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    Yeast 14. Miniature Fermentation Assay

    Small-scale wort fermentation (15-mL volumes) can be used to assess malt for premature yeast flocculation behavior and to compare the fermentability of yeast strains. This method may also be applied to malt treatments (or problem versus control malts) by performing the method simultaneously on all treatments. The method also includes a system for assessing (or comparing) malt performance quantitatively.

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    Yeast 15. Differentiation of Ale and Lager Yeast Strains by Rapid X-α-GAL Analysis

    The melibiose analog 5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside (X-α-gal) has previously been used to differentiate lager strains (Saccharomyces pastorianus) from ale strains (S. cerevisiae). Differentiation of these two types of yeast is based on the capacity of lager strains to cleave the X-α-gal molecule through the action of melibiase. This results in the release of indol, which oxidatively polymerizes to give an insoluble blue-green dye. After incubation with X-α-gal solution, a lager yeast suspension produces a blue-green coloration while the color of an ale yeast suspension remains unchanged (cream or white). This method is a variation of a previous method with modifications to the format and reagent and yields results in ≥ 30 min

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    Yeast 16. Phenolic Yeast Detection By Decarboxylation Of Ferulic Acid

    This method is intended for the detection of yeast able to decarboxylate ferulic acid, resulting in the formation of 4-vinyl guaiacol (4-VG or 2-methoxy-4-vinylphenol). Detection is based on a sensorial aromatic assessment to determine whether a phenolic clove-like or smokey attribute is present. The method is limited to the sensorial skills of the assessor and the amount of 4-VG produced by a yeast strain.

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    Yeast 17. FastOrangeTM Yeast Detection

    This method is intended for enrichment of yeast strains utilizing FastOrangeTM​ yeast agar media and application of the agar media to analyze samples. The media is designed to inhibit bacteria and allow for yeast growth. Growth typically occurs in conjunction with a color change from violet to yellow. Some bacteria may be capable of growth in the media. Microscopic examination should be used in conjunction with growth and color change for all assessments to determine whether growth is yeast or bacteria.

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  • Microbiological Control

    Microbiological Control 1. Aseptic Sampling

    This method analyzes microbial populations in brewery air (reported as number of colonies/volume of air examined) and provides a general swabbing procedure to be utilized to determine the hygiene of brewery surfaces using adenosine triphosphate (ATP) bioluminescence detection instrumentation.

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    Microbiological Control 2. Detection of Microorganisms

    This method provides incubation and plating procedures for the detection, enumeration, and partial characterization of viable microorganisms in liquid samples and a membrane filtration technique that is recommended for examination of beer, rinse water, or similar samples that contain relatively few bacteria, yeast, or molds.

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    Microbiological Control 3. Differential Staining

    Identification of bacteria may be facilitated by differential staining. This method identifies brewery contaminants by using the Gram stain procedure.

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    Microbiological Control 4. General Culture Media

    This method provides instructions for preparing two media (universal beer agar and brewers’ tomato juice medium) that can be used for general microbiological control work in the brewery. Most culture yeast, nonculture yeast, and Gram-negative rods commonly found in breweries grow well on these media under standard conditions. Lactic acid organisms commonly found in breweries, such as Lactobacilli and Pediococci, grow well on these media under anaerobic conditions. When supplemented with cycloheximide, these media differentially suppress the growth of brewing yeast. A safer alternative to cycloheximide is


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    Microbiological Control 5. Differential Culture Media

    Differential media have provided additional means for identifying microbiological contaminants in the brewery. This has been achieved by differential antibiotic action or by medium composition that suppresses the growth of one type of microorganism or enhances the growth of another. This method provides instructions for preparing cycloheximide medium, Lee’s multi-differential agar, Raka-Ray lactic acid bacteria medium, lysine medium, Lin’s wild yeast differential medium, Barney-Miller brewery medium, De Man Rogosa Sharpe medium, MYGP + copper medium, CLEN medium for wild yeast, selective medium for Megasphaera and Pectinatus, nystatin in selective media, Hsu’s Lactobacillus and Pediococcus medium, and FastOrangeTM Brett agar media for Dekkera/Brettanomyces.

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    Microbiological Control 6. Water and Rinse Water Hygiene Using ATP

    Bioluminescence provides a rapid, real-time assessment of microbial load (bacteria and yeast) in brewery water, including rinse water samples obtained after sterilization or cleaning of equipment. This test utilizes the reaction of adenosine triphosphate (ATP) with luciferin/luciferase to generate quantifiable bioluminescence that is detected using a luminometer. The data obtained may be used to provide an immediate assessment of hygiene or to complement traditional routine microbiology tests.

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    Common Brewery-Related Microorganisms

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