Appendix A Analytical Methods and Procedures in the Small Winery Laboratory 1. Acetaldehyde Determination 2. Agar Slant Preparation 3. Alcohol Determination by Distillation 4. Alcohol Determination by Salleron-DuJardin Ebulliometer 5. Balling Determination 6. Brix Determination by Hydrometer '7. Brix Determination by Refractometer 8. Carbon Dioxide Determination by Piercing Device 9. Copper and Iron Determination by Spectrophotometry 10. Differential Stain Procedure 11. Extract Determination by Hydrometer 12. Extract Determination by Nomograph-Dessert Wines 13. Extract Determination by Nomograph-Table Wines 14. Gram Stain Procedure 15. Light Transmission (Color Intensity) by Spectrophotometry 16. Malo-Lactic Fermentation Determination by Paper Chromatography 17. Microscopy 18. Organoleptic Analysis 19. Oxygen Determination 20. pH Determination 21. Plating Procedure 22. Sulfur Dioxide-Free 23. Sulfur Dioxide-Total 24. Total Acidity Determination by Titration 25. Total Acidity Determination by Titration-pH Meter 26. Viable Microorganisms in Bottled Wines-Millipore Method 27. Viable Yeasts in Bottled Wines-Rapid Method of Detection 28. Volatile Acidity Determination by Cash Volatile Acid Apparatus Courtesy of Champagne News and Information Bureau 311 312 COMMERCIAL WINEMAKING 1. ACETALDEHYDE DETERMINATION When analyzing wines for total acetaldehyde content, a small percentage (3-4% in wines containing 20% ethanol and less than 1% in table wine containing 12% ethanol) is bound as acetal. This is not recovered in the usual procedures. The procedure given below is that of Jaulmes and Ham­ elle as tested by Guymon and Wright and is an official method of the AOAC. Modifications to consider the acetal concentration can be made. The air oxidative changes taking place during the alkaline titration step are pre­ vented by addition of a chelating agent (EDTA) to bind copper present. Copper is a catalyst for the oxidation reaction. Addition of a small amount of isopropyl alcohol also inhibits the oxidation. Procedure Potassium Metabisulfite Solution.-Dissolve 15 g ofK2S20 5 in water, add 70 ml of concentrated hydrochloric acid, and dilute to 1 liter with water. The titer of 10 ml of this solution should not be less than 24 ml of 0.1 N iodine solution. Phosphate-EDTA Solution.-Dissolve any of the following combina­ tions and 4.5 g of the dis odium salt of EDT A in water and dilute to 1 liter. 200 g of NaaP04 . 12H20, or 188 g of Na2HP04 . 12H20 + 21 g of NaOH, or 72.6 g of NaH2P04 . H20 + 42 g of NaOH, or 71.7 g ofKH2P04 + 42 g of NaOH. Sodium Borate Solution.-Dissolve 100 g of boric acid plus 170 g of sodium hydroxide in water and dilute to 1 liter. Pipet 50 ml of the wine sample containing less than 30 mg of acetaldehyde into the distilling flask and add 50 ml of a saturated borax solution to bring the pH to about 8. Distill 50 ml into a 750-ml Erlenmeyer flask containing 300 ml of water and 10 ml each of the potassium metabisulfite and the phosphate-EDTA solutions; the pH of the solution in the Erlen­ meyer flask prior to distillation should be in the range of 7.0-7.2. After the distillation add 10 ml of 3 N hydrochloric acid and 10 ml of a freshly prepared 0.2% starch solution to the Erlenmeyer flask. Mix and then immediately titrate with 0.1 N iodine solution just to a faint blue end point. Add 10 ml of the sodium borate solution and rapidly titrate the liberated bisulfite with 0.02 N iodine solution, using a 25-ml buret, to the same blue end point. Avoid direct sunlight. The pH ofthe solution should be 8.8-9.5. Acetaldehyde, mg/liter = (V) (N) (22.0) (1000) v APPENDIX A 313 where V = volume of iodine solution used for titration after the addition of the sodium borate solution, in ml N = normality of the iodine solution v = volume of wine sample, in ml Ifthe first titration with iodine, to remove the excess bisulfite, takes only a few drops, then either the bisulfite solution was not up to proper strength, or the wine contains excessive amounts of acetaldehyde; if the second titration consumes excessive amounts of iodine and yellow iodo­ form formation is noted, the buffer was improperly prepared and the solution is too alkaline. 2. AGAR SLANT PREPARATION Agar slants are glass test tubes filled about halfway with agar having been allowed to solidify while the test tube was held in a slanting position. This increases the surface area upon which microbial cultures can be grown. For the most part, agar slants are used for the storage of pure yeast and bacteria cultures in the winery laboratory. Procedure Clean test tubes thoroughly, rinsing several times with hot water. Prepare and add enough yeast dextrose agar to fill test tubes about half full or slightly less. In the upright position, stuff test tubes closed with cotton, or apply screw cap if so equipped. Sterilize in autoclave. Carefully remove sterilized test tubes from autoclave while they are still hot and lay tubes down so that tops are raised about 2 in. or so-enough that a good surface area is made, but not so as to allow the agar to touch the cotton or the screw top. Cover with a clean towel and allow to cool. After agar has sufficiently cooled and solidified, the slants may be inocu­ lated with cultures administered by a sterile loop. As the cotton or screw top is removed the tube should be "flamed", and flaming should be re­ peated after the closure is re-applied. Paraffin film may then be applied over the closure and flamed carefully to form a seal. Following the desired level of growth, the slants may be refrigerated. Figure A.l pictures agar slants in storage. 3. ALCOHOL DETERMINATION BY DISTILLATION Wine of almost any type may be accurately analyzed for alcohol content by the distillation method as long as any remaining carbon dioxide gas is removed by careful agitation or filtration. The rationale for the distillation method is to separate the alcohol from the wine into an inert liquid medium that has no dissolved solids to affect 314 COMMERCIAL WINEMAKING FIG. A.1. AGAR SLANTS the Tralle scale hydrometer. Figure 7.2 illustrates the apparatus that is needed. Apparatus and Reagents 200 ml Kohlrausch flask 1000 ml Erlenmeyer flask Berl saddles 400 ml Allihn or Graham condenser % in. ID Tygon tubing % in. ID glass tubing Rubber stopper to fit Erlenmeyer flask Rubber stopper to fit condenser Water trap Tripod and wire gauze to support Erlenmeyer flask Support rods with clamps for condenser Thermometer Cold running water for condenser Distilled or deionized water Tralle scale hydrometer (in the range of the wine to be tested) Hydrometer jar or cylinder Heat source Refrigerated storage Procedure 1. Adjust sample temperature to 68°F or to whatever temperature is indicated on the 200 ml Kohlrausch flask. APPENDIX A 315 2. Fill clean 200 ml Kohlrausch flask so that the bottom meniscus rests exactly on the fill line. Shake out any bubbles that may be adhering to the sides of the flask. 3. Carefully empty contents of Kohlrausch flask into the 1000 ml Er­ lenmeyer flask and rinse out the Kohlrausch flask three times, each with about 25-30 ml distilled or deionized water. Empty all rinsings into the same 1000 ml Erlenmeyer flask. Add several Berl saddles into the 1000 ml Erlenmeyer flask. 4. Place the same clean Kohlrausch flask under the condenser outlet. 5. Insert connecting tube from the 1000 ml Erlenmeyer flask to con­ denser. Twist rubber stoppers slightly to be sure that they seal prop­ erly and will not leak. 6. Apply cold water at a proper rate through condenser and apply heat at a proper rate under Erlenmeyer flask. 7. Distill over approximately 150-175 ml of distillate. Be sure distillate runs out of condenser at a cool temperature. If not, start over clean­ ing all equipment three times with warm water, once with distilled water, and drain. Next time, increase condenser water flow rate appropriately but do not run too fast or the condenser may break from the pressure. 8. Add distilled water to distillate in Kohlrausch flask up to about 1 cm below the fill line. Mix by placing mouth of Kohlrausch flask on base of thumb in palm of hand so that no leakage takes place, inverting 6-8 times back and forth. Slide mouth of Kohlrausch flask off palm so no liquid is lost and place in refrigerated water bath. Cool to approximately 55°F. 9. Take Kohlrausch flask from refrigerated water bath and carefully dry the outside of the container with a clean towel. Adjust tempera­ ture to about 60°F and add distilled water in Kohlrausch flask so that the bottom meniscus rests exactly on the fill line. Remix contents in the same manner as Step 8. 10. Carefully empty Kohlrausch flask into a clean, dry hydrometer jar or cylinder. 11. Insert Tralle scale hydrometer carefully holding top of hydrometer stem in a pendulum effect. 12. Spin hydrometer carefully to free the instrument from the surface tension of the sides of the hydrometer jar or cylinder. 316 COMMERCIAL WINEMAKING 13. Read instrument at bottom meniscus. When using the hydrometer, take the temperature before and after reading. Preferably get two readings, as the sample is warming up and passing 60°F. Also note that it is very important to use a clean hydrometer in order to achieve accurate results. Calibration of hydrometers is desirable. Even factory calibrations should be checked by comparing hydrometers of the same range, since variations of 0.5% may be encountered. Post reading on labora­ tory analysis log.