The Compound Microscope

(27)

Components Parts of the Microscope

(4)

Maintenance and cleaning of the microscope

(1)

Focusing of the preparation and correct positioning of the observer

(2)

Measuring lengths with the microscope

(3)

Microscopic observation techniques

(8)

Bright-field viewing

(3)

Phase-contrast viewing

(2)

Interference contrast viewing

(1)

Dark-field viewing

(1)

Viewing by oblique or anaxial illumination

(1)

Epifluorescent viewing

(1)

Preparation and set-up for microscopic observation

(14)

Wet mounts

(5)

Dry mounting

(1)

Staining procedures

(2)

Vital staining of yeast cultures with methylene blue

(2)

Gram staining

(1)

Mycelium staining

(1)

Staining solutions

(2)

Counting bacteria and yeast cells in must and wine

(36)

General aspects

(1)

Determining microbial concentration in musts and wines

(4)

Determining the total yeast cell count microscopically using counting chambers

(8)

Determining yeast and bacterial load by growth on membrane filters

(5)

Liquid growth media

(1)

Determining the concentration of yeast and bacteria by plate count on solid media

(5)

Determining microbial load by means of epifluorescence

(13)

Sterilization techniques

(14)

Sterilization in the laboratory and ``microbiological stabilization'' in the wine cellar

(1)

Sterilization techniques used in the laboratory: Establishing a sterile environment

(4)

Sterilization of glass and metal instruments

(1)

Sterilization of natural and synthetic substrates by heat treatment

(2)

Preparation and preservation of must-substrates

(3)

Elimination of microorganisms by centrifugation and preservation of natural musts by deep freezing

(1)

Chemical sterilization

(4)

The antiseptic effect of sulfur dioxide

(1)

The technological criteria for the use of SO2 in the microbiological stabilization of wine

(1)

What is molecular SO2 fraction?

100

(4)

Calculating the molecular fractions of SO2

104

(1)

The quantity of free SO2

105

(1)

The pH

105

(1)

Alcohol concentration and temperature of must and wine

105

(3)

The concept of the ``paralyzing dose'' of molecular SO2

108

(4)

Microbiological stabilization in sweet wines, contaminated with yeast cells highly resistant to SO2. The ``initial jolting dose'' concept

112

(5)

The significance of the ``resistance level'' of a microbial cell biomass toward SO2 and other antiseptics in general

117

(2)

The concept of ``selective pressure''

119

(1)

Concept of ``abiosis'' in a stabilized wine

119

(1)

The resistance of malolactic bacteria to SO2

119

(3)

Micro biological stabilization of musts by SO2 addition

122

(3)

Correlation between pH, buffer power, oxidation-reduction potential, microbiological stabilization and the acid taste of wine

100

(43)

What is the pH?

125

(2)

Microbiological importance of pH

127

(1)

The Oxidation reduction potential

128

(2)

Microbiological importance of redox potential

130

(1)

Buffer Power (Bp)

130

(2)

Microbiological importance of buffer power

132

(1)

Extant interaction between pH and BP in the determination of the acid sensation

132

(2)

Organoleptic importance of pH and BP

134

(1)

Effects on the acid sensation in the mouth by the addition of weak organic acids, strong inorganic acids, or strong bases, or by the biological degradation of malic acid and citric acid

135

(8)

Alternative techniques to the use of sulfur dioxide for microbiological stabilization

143

(14)

Microwaves

143

(1)

Irradiation with electrons

144

(2)

Excimerlaser

146

(1)

Ultrasound

146

(1)

High hydrostatic pressure

146

(2)

Enzymatic lysis of bacterial and yeast cell walls

148

(2)

Antibiotics

150

(1)

The Fatty acids caprylic (n-octanoic; C8) and capric (n-decanoic; C10)

151

(6)

Cleansing and sanitation

157

(10)

Detersives, detergents and sanitizers

157

(1)

Sanitizers

158

(1)

Sodium and calcium hypochlorite

158

(1)

Peracetic acid

158

(1)

Steam

159

(1)

Ozone

159

(1)

Sodium hydroxide

160

(1)

Sulfur dioxide

160

(1)

Ultraviolet rays

160

(1)

Sanitizing production and bottling sites, equipment and containers

161

(3)

Hygiene of grapes before and during the crush

164

(3)

Taxonomy, cytology, biology and morphology of wine-associated yeasts

167

(26)

Taxonomy

167

(1)

The physiological classification of wine years

168

(1)

The biology of yeasts

168

(1)

Cytology

168

(1)

Vegetative reproduction

169

(2)

Sexual reproduction

171

(6)

Sporulation

177

(1)

Alternative nuclear phase and alternative life cycles

178

(2)

Homothallism and heterothallism

180

(2)

The morphology of yeasts

182

(2)

The morphology during vegetative multiplication

184

(1)

The morphology during sexual reproduction

185

(1)

The morphology of spores

185

(1)

Mycelium morphology during the formation of pseudo- and true mycelia

186

(2)

Colonial morphology

188

(1)

Subdivision of the yeasts into morphological groups

188

(5)

Isolation, selection and purification of wine yeasts

193

(26)

General aspects

193

(1)

The Concept of ``selected yeast''

194

(1)

The Selection of commercial wine yeast

195

(1)

How the enologist should proceed

196

(1)

The isolation of pure monoclonal population of yeast

196

(1)

Equipment needed and media

197

(2)

Isolation techniques

199

(2)

The preservation of yeast cultures in the laboratory

201

(1)

The National Collection of Yeast and Bacteria selected for Wine (CNLBSV-ISE)

General methodology criteria for the selection of specific yeast strains for the amelioration of wines

201

(2)

Selection of isolated strains

203

(2)

Nutritive synthetic medium (NSM)

205

(1)

Preparation of the fermentation experiments

206

(1)

Determination of the alcohologenic capability, sugar consumption and the sugar/alcohol conversion coefficient

207

(2)

Determining the rate of alcohol production and the course of fermentation

209

(2)

Fermentation and growth curves

211

(1)

Determining the ability to produce acetic acid

211

(1)

Determining the aptitude to degrade or produce malic acid or other organic acids

212

(1)

Determination of the aptitude to produce characteristics aromatic and odorous substances, foam, and the modality of growth

213

(2)

Determination of the aptitude to form sulfite and sulfide (H2S)

215

(1)

The determination of the level of resistance to SO2

216

(1)

Preparations for fermentation experiments

216

(1)

Determination of the ability to grow at low temperatures

217

(1)

Determination of the ability to produce glycerol

217

(2)

Oenological characteristics of selected yeast strains and their genetic improvement

219

(22)

Introduction

219

(2)

The Contribution of yeast to the liberation of aromatic substances and their transformation

221

(1)

Terpenes

221

(1)

Thiols

221

(1)

Volatile phenols

222

(1)

Fatty Acids

222

(1)

Yeast and bacteria enzymes considered important for the expression of the varietal characteristics of wines

223

(1)

β-glucosidase, cinnamate decarboxylase

223

(1)

Periplasmic proteases and autolysis

223

(1)

Periplasmic esterases

223

(1)

Periplasmic pectolytic enzymes

224

(1)

Periplasmic lipase and phospholipase

225

(1)

Biotechnology applied to wine microorganisms

225

(7)

Physiological and genetic features of some yeast characteristics important in enology

232

(1)

Killer factor

232

(3)

Types of growth

235

(1)

Flocculent grrowth and aggregates

236

(1)

Foaming capability

236

(1)

Ability to form films (Flor)

236

(1)

Cell autolysis

237

(1)

Alcohol production and resistance to prolonged anaerobiosis

237

(1)

The importance of cultivation and storage techniques on the efficiency of selected cultures

237

(4)

Current and prospective microbiological topics in enology

241

(12)

Evidence for the existence of interactions between vine and yeast

249

(4)

The metabolism of sugars and nitrogen by yeast

253

(16)

The alcoholic fermentation

253

(5)

The Genevois Equation and secondary products

258

(5)

The Pasteur and Crabtree effects

263

(1)

Glucophilic and fructophilic yeasts

264

(1)

Fermentation in the presence of sulfur dioxide

264

(1)

Use of the nitrogen compounds and the synthesis of amino acids

265

(4)

The oxygen requirement for wine yeast

269

(14)

The importance of oxygenation, the composition of must and the age of the cells in the inoculum

269

(1)

When is it appropriate and necessary to furnish oxygen?

270

(3)

The requirement of oxygen is essentially a function of substrate

273

(3)

Advantages and risks of aeration and excessive oxygenation

276

(1)

Oxygen, sterols and fatty acids as factors in survival and resistance to anaerobiosis and alcohol content

277

(6)

Microbiological monitoring of must and wine

283

(8)

Introduction

283

(1)

Organoleptic analysis

283

(1)

Microscopic observation

284

(1)

Chemical and physical evaluation

285

(4)

Techniques to prevent and cure microbiological alterations

289

(1)

Management and control of the first and second alcohol fermentations

289

(1)

Grape crushing and preparation of must for the primary fermentation

289

(1)

Controlling the course of the primary fermentation and outcome of re-fermentation

290

(1)

The use of selected yeast strains

291

(36)

Commercial yeast cultures

291

(1)

Yeast grown on solid media

291

(1)

Yeast in liquid culture

291

(1)

Yeast prepared as a concentrated paste

292

(1)

Lyophilized yeast

292

(1)

Dry powdered yeast

293

(1)

Testing commercial dry powdered yeast cultures

294

(3)

Immobilized yeast

297

(1)

Yeast imbedded in polysaccharide gel

297

(2)

Yeast cells confined in porous tubular membranes

299

(1)

Techniques used in the preparation of starter cultures

299

(4)

Preparation of a starter must culture for the second fermentation (tirage, prise de mousse) in a base wine

303

(1)

Preparation of base wines for the second fermentation and prise de mousse in the production of sparkling wines

304

(2)

The use of sulfur dioxide during grape crushing

306

(2)

Use of fermentation activators

308

(1)

Use of ammoniacal nitrogen

309

(4)

Use of vitamin B1 (thiamin)

313

(3)

Control of the fermentation temperature

316

(1)

Controlling the degree of anaerobiosis during the alcoholic fermentation

317

(1)

Untimely lactic and acetic bacterial activity

318

(1)

Untimely activity by Shizosaccharomyces yeasts

318

(5)

Delayed and failed starts, sluggish and stuck alcoholic fermentations

323

(2)

Control of unwanted second fermentations

325

(1)

Does a correlation exist between astronomical cycles and fermentation activity of yeasts and malolactic bacteria?

325

(2)

The formation of organoleptically important metabolites

327

(12)

Acetic acid (see Chapter 19)

327

(1)

Succinic acid

327

(1)

L(+) and D(-) lactic acid

327

(1)

L(-) and D(+) malic acid

327

(1)

Pyruvic acid

328

(1)

Gluconic acid

328

(1)

Acetaldehyde

328

(1)

Glycerol

328

(1)

Octanoic acid

329

(1)

Sulfur dioxide and hydrogen sulfide

330

(2)

Other sulfur compounds

332

(1)

Benzaldehyde

332

(1)

Acetamide

333

(1)

Urea, N-carbamyl amino acids, ethyl carbamates

333

(1)

1-Octanol, 1-octyl acetate

333

(1)

Ethyl acetate

334

(1)

Glyoxal

334

(1)

C-6 alcohol and aldehydes

334

(1)

Diacetyl

334

(1)

Long chain alcohols (higher alcohols)

334

(1)

Glycosidic colloids

335

(3)

Important metabolites produced by malolactic bacteria

338

(1)

The production of acetic acid by yeasts

339

(18)

Introduction

339

(1)

Experimental observations on the existence of metabolic factors in yeast that control the formation of acetic acid

340

(3)

Experiments that emphasize the importance of clarification in the production of large amounts of acetic acid during alcoholic fermentation

343

(3)

Importance of skin contact (maceration)

346

(1)

The importance of the deposits resulting from clarification

346

(1)

The importance of yeast strain, must pH and fermentation temperature

346

(3)

The importance of ammonium nitrogen, amino acids and vitamins

349

(1)

The importance of polyphenolic compounds

349

(1)

Importance of unsaturated fatty acids and sterols

349

(1)

Hypotheses proposed to explain the nature of the factor responsible for inhibiting the production of large quantities of acetic acid by the yeast

350

(7)

Biological degradation of malic acid

357

(22)

The maloalcoholic fermentation (MAF)

357

(1)

Effects of the MAF on the harmonious balance of taste

358

(2)

Experiments involving the use of Schizosaccharomyces

360

(1)

The malolactic fermentation (MLF)

361

(2)

Beneficial and deleterious aspects of malic acid biodegradation

363

(1)

Optimal growth temperature

364

(1)

The importance of pH, SO2 and alcohol content

365

(1)

Others factors affecting MLF

366

(3)

Vinification techniques that favor MLF

369

(2)

Controlling the malolactic fermentation

371

(1)

Inoculating with bacterial cultures for MLF

372

(4)

What the winemaker can do in the cellar to prevent MLF

376

(1)

Commercial aspects of the MLF

377

(1)

Conclusive considerations on the problem of the malolactic fermentation

378

(1)

Microbiological control of concentrated musts and sulfured musts

379

(10)

Musts from partially dried grapes

379

(1)

Concentrated Rectified Must (CRM)

380

(1)

Microbiological control of CRM during production and storage

381

(1)

Acidification experiments with tartaric acid and the addition of SO2

382

(1)

Microbiological stabilization through acidification with sulfuric acid

382

(4)

Concentrated natural must

386

(3)

The microbiological control of wine during storage

389

(20)

Changes due to yeasts

389

(1)

Controlling flor spoilage

389

(1)

Control of unwanted second fermentation (or refermentations)

390

(1)

The formation of hydrogen sulfide

391

(2)

Spoilage due to bacteria

393

(1)

Acetic acid bacteria and acetic spoilage

394

(1)

The significance of volatile acidity in the diagnosis and prevention of alterations of microbial origin

395

(2)

Bacterial oxidation of carbohydrates

397

(1)

Acetic fermentation of ethyl alcohol

398

(1)

Lactic acid bacteria

399

(1)

Normal and ferrocious lactic acid fermentation

399

(4)

Mannitic fermentation or sour-sweet conversion

403

(1)

Bacterial degradation of pyruvic and citric acids

404

(1)

Tartaric acid and glycerol degradation

404

(1)

Ropy wine

405

(4)

Methodology for the microbiological stabilization of musts and wines

409

(16)

Introduction

409

(1)

Microbiological stabilization during bottling

410

(1)

Microbiological stabilization by filtration at the bottling stage

411

(5)

The preparation of wine for sterile bottling

416

(1)

The stabilization of wine and the sterilization of bottles by injection with sulfur dioxide before filling

417

(3)

The determination of the quantity of sulfur dioxide to be add to wine

420

(1)

Sterilization of bottling equipment

421

(1)

Sterilization of natural cork stoppers and the corker

422

(2)

Other general considerations

424

(1)

Microbiological stabilization of wine through thermal treatment. Pasteurization and hot bottling

425

(8)

Destructive effect of heat on cellular life

425

(1)

Factors which influence the ``lethal temperature'' value

426

(1)

The effect of medium composition

426

(1)

The effect of hydrogen ion concentration

427

(1)

The effect of carbon dioxide under pressure

427

(1)

The effect of ethanol

428

(1)

The effect of sulfur dioxide

428

(1)

The effect of the pasteurization unit

428

(3)

Hot bottling, pasteurization in the bottling line and in-bottle pasteurization

431

(2)

Microbiological stabilization through filtration

433

(20)

Introduction

433

(4)

Relationship between shape and size of microorganisms and the types of filtering materials used

437

(1)

Characteristics and sterilizing capacity of different filtration materials

438

(1)

Deep filtering materials with sieving action

439

(2)

Filtering materials prepared as stacked disks or pleated membrances (cartridges)

441

(2)

Tests used to evaluate membrances

443

(2)

Bacterial assay

445

(2)

Filtering materials acting solely as a sieve

447

(1)

Cross-flow or tangential filtration

447

(6)

Appendix Microscopic and chemical assays used for the identification of sediment. Techniques used to determine a wine's propensity to develop turbidity

453

(8)

Potassium bitartrate

453

(1)

Calcium tartrate, racemic calcium, tartrate, calcium oxalate

454

(1)

Calcium mucate

454

(1)

Ferric phosphate

455

(1)

Copper sulfide or sulfite

455

(1)

Metatartaric acid

455

(1)

Ferric and ferrous ferrocyanide

455

(1)

Colored substances

455

(1)

Tannin-iron complex

455

(1)

Proteins

455

(1)

Protein-tannin coagulum

456

(1)

Cellulose

456

(1)

Diatomaceous earth

456

(1)

Bentonite

456

(1)

Charcoal

456

(1)

Staches

456

(1)

Crystal of mannitol

456

(1)

Paraffin

456

(1)

Identification of haze

456

(2)

Assessing wine stabilization procedures

458

(1)

Procedures to verify the resistance of wine to air

458

(1)

Testing resistance to aeration with oxygenated water

458

(1)

Testing for heat stability

458

(3)

Testing for cold stability

Bibliography

461

(22)

Index

483

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Wine Microbiology: Science and Technology

9780824705909

0824705904

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