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Wine Microscopy Workshop November 8 and 11th, 2013 Molly Kelly Enology Extension Specialist Overview • Use of microscope in wine lab • Introduction to microscopy • General operating guidelines • Correct use • Köhler illumination Overview • Microscopic observation techniques • Bright field • Simple staining • Gram stain • Yeast viability stain • Phase contrast • Wet mount • Identification of wine microorganisms • Culturing Microscope uses in wine lab • Testing for presence of spoilage organisms • Counting cells • Yeast viability • Check juice concentrate for contamination • Determine sterility status of bottling line • Assess bottled wine status Microscope use • Turn the microscope on • Place the prepared wet mount on the microscope stage • Swing in a low powered objective (10x); focus on the sample using the coarse focusing knob • Perform Köhler Illumination • Swing in the high dry objective (40x); focus on the sample using the fine focus knob only • Swing the objective out of the way and add a drop of immersion oil • Swing in the oil immersion objective (100x); focus on the specimen using the fine focus knob only • Adjust the light intensity with each new objective Microscope use • When finished with the sample swing the objectives out of the way • Lower the stage of the microscope all the down • Turn the light source to low and turn the power off • Use lens paper to gently absorb excess oil from the 100x objective (DO NOT RUB) • When storing place a dust cover over the microscope • If you need to move the microscope carry it carefully by the arm; avoid having to move the microscope regularly • A yearly maintenance by a microscope professional is recommended Compound Microscope Condenser Centering Screws (x2) Condenser Knob (on left side of microscope) Köhler illumination • Köhler illumination is a way to adjust the condenser to get the optimum light path for the microscope • Benefits: • Evenly illuminated image • No reflection or glare from the light source • Requires a microscope with a field diaphragm and lens and a condenser (see Iland pgs 68-69) Microscopy set-up • Micrometer • See Iland pg. 70 • Troubleshooting • See Iland pg. 71 Aseptic technique Iland pgs. 82-83 • Measures to prevent contamination • Pure cultures • Sterile media • You • Guidelines • Ethanol (80%) work surfaces before and after • Wash hands • Technique • Efficiency • Attention to sterility • Inoculating loops, forceps, open neck containers • Disposables • Autoclave waste Preparing smears for staining • • • • • • • Use dyes to make microorganisms more visible To aid in identification Sterile water and colony from plate (clean toothpick) Liquid culture Clean slides, aseptic technique Air dry, heat fix Stain • See Iland pg. 108 Bright field microscopy • Simple stain • Gram stain • Yeast viability Simple stain • Use single stain to adhere to specific cellular features to improve contrast • Example: methylene blue Biology.clc.uc.edu www.pc.maricopa.edu 14 15 3 shapes of bacteria • cocci – spherical • bacilli – rod • spiral - helical, comma, twisted rod, spirochete 18 Methods in bacterial identification 1. 2. 3. 4. 5. 6. Microscopic morphology Macroscopic morphology – colony appearance Physiological / biochemical characteristics Chemical analysis Serological analysis Genetic & molecular analysis 19 Haemocytometer • Counting chamber • Yeast cell concentration • Budding yeast cells • Yeast viability • See Iland pgs. 92-94 Neubauer Hemocytometer Results • Methylene blue stain: • Clear cells-viable (they reduce the blue dye to its colorless form) • Blue cells-dead • Prior to addition to must, yeast must be expanded such that final viable cell numbers are 2-5 x 10 6 cells/ml • In actively growing starters, budding cells should comprise 60-80% of total cell number Yeast viability stain Enartis Vinquiry Braukaiser.com Salvage maneuvers • Resuscitate: If yeast viability is still greater than 25% , still producing CO2, temp is above 55 whites and 60 reds • • • • Stir up tank-resuspend yeast cells Yeast hulls-0.5-1#/K gallons Yeast extract (sterols, fatty acids)might help Regulate temp (65-75F, no more than 80) • As lose CO2, blanket with argon, CO2 • Protect wine from Acetobacter and other aerobic bacteria • Check for Lactobacillus and add lysozyme if needed • Watch alcohol and VA levels • As Vas get close to LL (1.2g/L reds, 1.1 g/L whites) and alcohol gets close to 15%, reinoculation gets harder • RO or blending Reinoculation • Create a high concentration yeast starter with a very strong yeast • Add some juice (1-5 % volume of total stuck volume of wine) so start turning the juice to alcohol • Acclimates membranes to alcohol • Add incremental amounts of your stuck wine to the newly fermenting starter • Monitor the starter for fermentation rate and only adding stuck wine while the starter is fermenting strongly Success of restarting • Most successful when: • Less than 25% yeast viability • Delle Units are under 65 ([% alc x 4.5]+%RS) • No Lactobacillus infection Phase contrast microscopy • Used to increase contrast when viewing unstained cells • Increase contrast by making use of small differences in refraction of light passed through cells • Cells have higher refractive index and density than water • Appears dark against light background • Used for living cells, motility and viability Phase contrast Kloeckera apiculata Practical Winery.com Wet mount • Procedure: • Place one drop of water on a microscope slide. • Sterilize an inoculation loop with a flame. • Touch a colony with the cooled inoculation loop. • Mix the cells with the water. • Place a cover slip over the sample. • Place slide on the microscope stage; focus with a low powered objective, then add a drop of immersion oil and focus with 100x objective. • See Iland pgs. 66 and 109 Practical Winery.com Microorganisms • Yeast • • • • • Saccharomyces cerevisiae Brettanomyces bruxellensis Kloeckera apiculata Other See Iland pgs. 10-12 • Bacteria • Lactic acid bacteria • Acetic acid bacteria • See Iland pgs. 28-31 Saccharomyces cerevisiae • Colony color: white or cream colored, may take up the media pigment • Colony size: medium to large • Colony shape: smooth and convex • Incubation time: 48 hours • Media: WL, YM, or YEP Enartis vinquiry Saccharomyces cerevisiae • Cell shape: ovoid, globose, or elongate • Cell size: 5-10 μm • Cells occur singly or in small groups • Reproduces by multilateral budding • Vigorously ferments sugars Enartis vinquiry Brettanomyces spp. • Colony color: cream • Colony size: medium • Colony shape: smooth and convex • Incubation time: 5-7 days • Media: YM+Actidione (30 ppm to inhibit Saccharomyces yeast growth) Enartis vinquiry Brettanomyces spp. • Cell shape: spheroidal to ellipsoidal, often elongated • Cell size: 4-22 μm • Cells occur singly, pairs, short chains, or clusters • Reproduces by budding • Bud scars are visible on older cells • Spoilage yeast Enartis vinquiry Brettanomyces Enartis Vinquiry Brettanomyces intermedius Enartis Vinquiry Lactic Acid bacteria • Oenococcus • Pediococcus • Lactobacillus • Gram positive • Prefer low oxygen conditions • Non-motile Oenococcus oeni • Colony color: clear to white • Colony size: 0.1-1.0 mm diameter • Colony shape: round and slightly convex • Incubation time: 5-7 days • Media: Apple Rogosa media plus 30 ppm Actidione to inhibit yeast growth Enartis vinquiry Oenococcus oeni • Cell shape: small coccobacillus • • • • Cell size: 0.5-0.7 x 0.7-1.2 μm Cells occur in pairs or chains Alcohol tolerant Converts malic acid into lactic acid • Contributes to VA Enartis vinquiry Oenococcus oenii Enartis Vinquiry Pediococcus spp. • Colony color: clear • Colony size: 0.1-1.0 mm diameter • Colony shape: round, convex • Incubation time: 5-7 days • Media: Apple Rogosa media plus 30 ppm Actidione to inhibit yeast growth Enartis vinquiry Pediococcus spp. • • • • • Cell shape: coccus Cell size: 1.0-2.0 μm (diam) Cells occur in pairs or tetrads Good alcohol tolerance Converts malic acid into lactic acid • Contributes to VA and ropiness Enartis vinquiry Pediococcus Pediococcus and Acetobacter Enartis Vinquiry Lactobacillus spp. • Colony color: clear or white • Colony size: 0.25-1.5 mm diameter • Colony shape: round, convex • Incubation time: 5-7 days • Media: Apple Rogosa media plus 30 ppm Actidione to inhibit yeast growth Enartis vinquiry Lactobacillus spp. • Cell shape: large brick/rod shaped • Cell size: 0.5-1.2 x 1.0-10 μm • Cells occur mostly single, pairs or chains • Good alcohol tolerance • Converts malic acid into lactic acid • Forms VA • Stuck/sluggish fermentations Enartis vinquiry Lactobacillus kunkeii Enartis vinquiry Acetic Acid bacteria • Acetobacter aceti, pasteurianus • Gluconobacter oxydans • Gram negative • Obligate aerobe (needs oxygen) • Non-motile • Gluconobacter is sensitive to high alcohol conditions Enartis vinquiry Acetobacter spp. • Types: Acetobacter and Gluconobacter • Colony color: clear • Colony size: 0.1-1.0 mm diameter • Colony shape: round, convex • Incubation time: 5-7 days • Media: Apple Rogosa media plus 30 ppm Actidone to inhibit yeast growth Enartis vinquiry Acetobacter spp. • Cell shape: small rod • Cell size: 0.5-2.0 μm • Cells occur mostly single and in pairs or chains • Good alcohol tolerance • Forms VA • Contributes to mousiness Enartis vinquiry Acetobacter Enartis Vinquiry Acetobacter (Gram negative) Other yeasts:Zygosaccharomyces • Colonies: cream colored, medium to large in size • Cells: ovoid, ellipsoidal, cylindrical, size 3.5-7.0 x 5.5-14.0 μm • Reproduces by multilateral budding • Difficult to distinguish from Saccharomyces Enartis vinquiry Other yeasts:Zygosaccharomyces • Forms conjugation tubes on malt agar • Classic dumbbell shape • Highly tolerant to harsh conditions • Sugar: >70% v/v • Ethanol: >18% v/v • SO2: > 3 mg/L, molecular Z. baillii • Turbidity and possible refermentation in bottle Z. baillii Z. baillii Other yeasts:Film yeast • Pichia anamola (formerly Hansenula anamola) • Pichia membranefaciens • Pichia fermentans • Candida vini • Oxidative yeast • Colonies appear contoured or wrinkled • Cells are ovoid and elongate • Daughter cells remain attached during budding; producing a surface film P. membranefaciens P. anamola C. vini Other yeasts • Hanseniaspora uvarum (aka Kloeckera apiculata) • Indigenous yeast of grapes • Can survive but not grow at higher alcohol levels • Cells are ovoid to lemon shaped (apiculate) or irregularly elongate • Can form large amounts of VA and ethyl acetate Enartis vinquiry Malolactic Fermentation • The microscope can be used to monitor MLF • Oenococcus oeni forms long chains in rich media • Wine conditions can change the way it typically grows Enartis vinquiry Spoilage organisms: physical impact • Sluggish/Stuck Fermentations • Produced by elevated numbers of Lactobacillus • Ropiness or increased viscosity • Produced by Pediococcus • Haze or Sediment Formation • Produced by elevated numbers of yeast or wine bacteria • Re-fermentation in bottle: can lead to haze, gassiness, and pushing out the cork • Produced by Saccharomyces yeast or Zygosaccharomyces Spoilage Organisms: sensory and chemical impact • Volatile Acidity: Acetic Acid (vinegar) • Produced by AAB and yeast • Organic Acids: Lactic Acid • Produced by LAB: Lactobacillus, Pediococcus, and Oenococcus • Acetaldehyde: smells sherry-like or nutty • Produced by AAB and yeast • Ethyl Acetate: smells like nail polish remover • Produced by AAB • 4- Ethyl guaicol and 4-Ethyl phenol: Band-Aid, horse-sweat • Produced by Brettanomyces • TCA, cork taint: smells musty, like wet cardboard • Produced by mold Sediments andHazes Cellulose fibers with DE Cellulose from pad filter Enartis vinquiry Sediments and Hazes Protein Haze Pigment phenolic complexes Enartis Vinquiry Diatoms from cellulose/DE pad Crystals K-bitartrate crystals Enartis Vinquiry Calcium tartrate crystals References • Bisson, L. University of California at Davis, University Extension, 2001. • Iland, P., et al. Microbiological analysis of grapes and wine: techniques and concepts. Patrick Iland Wine Promotions Pty. Ltd. 2007. • Ritchie, G. Fundamentals of Wine Chemistry and Microbiology, Napa Valley College, 2006. • Specht, G. Overcoming Stuck and Sluggish Fermentations, Practical Winery and Vineyard, Sept/Oct 2003. • Telloian, J. Wine Microscope Seminar. Enartis Vinquiry, May 2011. • Zoecklein, B., et al. Wine Analysis and Production, Aspen Publishers, 1999.
