Transcript Wine Microscopy Workshop
Wine Microscopy Workshop November 8 and 11
th
, 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. Microscopic morphology 2. Macroscopic morphology – colony appearance 3. Physiological / biochemical characteristics 4. Chemical analysis 5. Serological analysis 6. 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
Braukaiser.com
Enartis Vinquiry
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 Turbidity and possible re fermentation in bottle
Z. baillii 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 C. vini P. anamola
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.