Transcript Wine Microscopy Workshop

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
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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
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3 shapes of bacteria
• cocci – spherical
• bacilli – rod
• spiral - helical, comma, twisted rod, spirochete
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Methods in bacterial identification
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2.
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5.
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Microscopic morphology
Macroscopic morphology – colony appearance
Physiological / biochemical characteristics
Chemical analysis
Serological analysis
Genetic & molecular analysis
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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
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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
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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
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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.
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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.