Wine Microscopy Workshop

November 8 and 11th, 2013Molly 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 techniqueIland 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

14www.pc.maricopa.edu 

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18

3 shapes of bacteria

• cocci – spherical

• bacilli – rod

• spiral - helical, comma, twisted rod, spirochete

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Methods in bacterial identification

1. Microscopic morphology2. Macroscopic morphology – colony appearance3. Physiological / biochemical characteristics4. Chemical analysis5. Serological analysis6. Genetic & molecular analysis

Haemocytometer

• Counting chamber• Yeast cell concentration• Budding yeast cells• Yeast viability

• See Iland pgs. 92-94

Neubauer Hemocytometer

Results

• Methylene blue stain:Methylene blue stain:• Clear cells-viable (they reduce the blue dye to its colorless Clear cells-viable (they reduce the blue dye to its colorless

form)form)• Blue cells-deadBlue cells-dead

• Prior to addition to must, yeast must be expanded Prior to addition to must, yeast must be expanded such that final viable cell numbers are such that final viable cell numbers are 2-5 x 10 2-5 x 10 6 6 cells/mlcells/ml

• In actively growing starters, budding cells should In actively growing starters, budding cells should comprise 60-80% of total cell numbercomprise 60-80% of total cell number

Yeast viability stain

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Salvage maneuvers

• Resuscitate: If yeast viability is still greater than 25% , still Resuscitate: If yeast viability is still greater than 25% , still producing CO2, temp is above 55 whites and 60 redsproducing CO2, temp is above 55 whites and 60 reds

• Stir up tank-resuspend yeast cellsStir up tank-resuspend yeast cells• Yeast hulls-0.5-1#/K gallonsYeast hulls-0.5-1#/K gallons• Yeast extract (sterols, fatty acids)might helpYeast extract (sterols, fatty acids)might help• Regulate temp (65-75F, no more than 80)Regulate temp (65-75F, no more than 80)

• As lose CO2, blanket with argon, CO2As lose CO2, blanket with argon, CO2• Protect wine from Acetobacter and other aerobic bacteriaProtect wine from Acetobacter and other aerobic bacteria• Check for Lactobacillus and add lysozyme if neededCheck for Lactobacillus and add lysozyme if needed

• Watch alcohol and VA levelsWatch alcohol and VA levels• As Vas get close to LL (1.2g/L reds, 1.1 g/L whites) and alcohol As Vas get close to LL (1.2g/L reds, 1.1 g/L whites) and alcohol

gets close to 15%, reinoculation gets hardergets close to 15%, reinoculation gets harder• RO or blendingRO or blending

Reinoculation

• Create a high concentration yeast starter with a Create a high concentration yeast starter with a very strong yeastvery strong yeast

• Add some juice (1-5 % volume of total stuck Add some juice (1-5 % volume of total stuck volume of wine) so start turning the juice to volume of wine) so start turning the juice to alcoholalcohol

• Acclimates membranes to alcoholAcclimates membranes to alcohol• Add incremental amounts of your stuck wine to Add incremental amounts of your stuck wine to

the newly fermenting starterthe newly fermenting starter• Monitor the starter for fermentation rate and only Monitor the starter for fermentation rate and only

adding stuck wine while the starter is fermenting adding stuck wine while the starter is fermenting stronglystrongly

Success of restarting

• Most successful when:Most successful when:• Less than 25% yeast viabilityLess than 25% yeast viability• Delle Units are under 65 ([% alc x 4.5]+%RS)Delle Units are under 65 ([% alc x 4.5]+%RS)• No Lactobacillus infectionNo 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

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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

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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

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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

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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)

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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

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Brettanomyces

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Brettanomyces intermedius

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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

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Oenococcus oenii

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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

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Pediococcus

Pediococcus and Acetobacter

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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

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Lactobacillus kunkeii

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Acetic Acid bacteria• Acetobacter aceti,

pasteurianus• Gluconobacter oxydans

• Gram negative• Obligate aerobe (needs

oxygen) • Non-motile• Gluconobacter is sensitive to

high alcohol conditions

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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

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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

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Acetobacter

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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

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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. anamola

P. membranefaciens

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

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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

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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 from pad filter

Cellulose fibers with DE

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Sediments and HazesProtein Haze

Pigment phenolic complexes

Diatoms from cellulose/DE padEnartis Vinquiry

Crystals

Calcium tartrate crystals

K-bitartrate crystals

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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.