1. Wine Microscopy Workshop November 8 and 11 th, 2013 Molly Kelly Enology Extension Specialist

2. Overview Use of microscope in wine lab Introduction to microscopy General operating guidelines Correct use Köhler illumination

3. Overview Microscopic observation techniques Bright field Simple staining Gram stain Yeast viability stain Phase contrast Wet mount Identification of wine microorganisms Culturing

4. 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

5. 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

6. 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

7. Compound Microscope Condenser Centering Screws (x2) Condenser Knob (on left side of microscope)

8. 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)

9. Microscopy set-up Micrometer See Iland pg. 70 Troubleshooting See Iland pg. 71

10. 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

11. 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

12. Bright field microscopy Simple stain Gram stain Yeast viability

13. Simple stain Use single stain to adhere to specific cellular features to improve contrast Example: methylene blue Biology.clc.uc.edu

14. 14 www.pc.maricopa.edu

15. 15

18. 18 3 shapes of bacteria cocci – spherical bacilli – rod spiral - helical, comma, twisted rod, spirochete

19. 19 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

20. Haemocytometer Counting chamber Yeast cell concentration Budding yeast cells Yeast viability See Iland pgs. 92-94

21. Neubauer Hemocytometer

22. Results Methylene blue stain: Methylene blue stain: Clear cells-viable (they reduce the blue dye to its colorless form) Clear cells-viable (they reduce the blue dye to its colorless form) Blue cells-dead 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 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 In actively growing starters, budding cells should comprise 60-80% of total cell number

23. Yeast viability stain Braukaiser.com Enartis Vinquiry

24. Salvage maneuvers Resuscitate: If yeast viability is still greater than 25%, still producing CO2, temp is above 55 whites and 60 reds 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 Stir up tank-resuspend yeast cells Yeast hulls-0.5-1#/K gallons Yeast hulls-0.5-1#/K gallons Yeast extract (sterols, fatty acids)might help Yeast 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, CO2 As lose CO2, blanket with argon, CO2 Protect wine from Acetobacter and other aerobic bacteria Protect wine from Acetobacter and other aerobic bacteria Check for Lactobacillus and add lysozyme if needed Check for Lactobacillus and add lysozyme if needed Watch alcohol and VA levels 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 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 RO or blending

25. Reinoculation Create a high concentration yeast starter with a very strong yeast 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 Add some juice (1-5 % volume of total stuck volume of wine) so start turning the juice to alcohol Acclimates membranes to alcohol Acclimates membranes to alcohol Add incremental amounts of your stuck wine to the newly fermenting starter 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 Monitor the starter for fermentation rate and only adding stuck wine while the starter is fermenting strongly

26. Success of restarting Most successful when: Most successful when: Less than 25% yeast viability Less 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 infection No Lactobacillus infection

27. 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

28. Phase contrast Kloeckera apiculata Practical Winery.com

29. 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

30. Practical Winery.com

31. 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

33. 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

34. 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

35. 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

36. 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

37. Brettanomyces Enartis Vinquiry

38. Brettanomyces intermedius Enartis Vinquiry

39. Lactic Acid bacteria Oenococcus Pediococcus Lactobacillus Gram positive Prefer low oxygen conditions Non-motile

40. 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

41. 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

42. Oenococcus oenii Enartis Vinquiry

43. 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

44. 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

45. Pediococcus Pediococcus and Acetobacter Enartis Vinquiry

46. 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

47. 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

48. Lactobacillus kunkeii Enartis vinquiry

49. 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

50. 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

51. 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

52. Acetobacter Enartis Vinquiry

53. Acetobacter (Gram negative)

54. 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

55. 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

56. 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

57. 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

58. 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

59. 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

60. 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

61. Sediments andHazes Cellulose from pad filter Cellulose fibers with DE Enartis vinquiry

62. Sediments and Hazes Protein Haze Pigment phenolic complexes Diatoms from cellulose/DE pad Enartis Vinquiry

63. Crystals Calcium tartrate crystals K-bitartrate crystals Enartis Vinquiry

64. 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.