New and Amended Methods The second edition (2005) of the EBC Analytical Microbiologica has been updated with 19 new methods: Method Selection Guide 2.2.5.7 Anaerobic Sample Handling 2.3.9.4 Oxidase test 2.3.9.5 Phenolic Off Flavour (POF) 2. 4 Molecular Techniques 2.4.1 Polymerase Chain Reaction (PCR) Techniques 2.4.1.1 Detection and Identification of Microorganisms from Turbid Liquid Samples or Cultured Colonies by Real-Time PCR 2.4.1.2 Detection and Identification of Microorganisms from Filterable Samples by Real-Time PCR 3.2.1.1 Methylene Blue / Violet Stain 3.4 Yeast Strain Storage, Supply and Propagation 3.4.1 Yeast Storage at Ultra-Low Temperatures 3.4.2 Yeast Sub-Culturing for Short Term Storage 3.4.3 Yeast Strain Transport 3.4.4 Yeast Propagation (Laboratory Stages) 3.5 Yeast Characterisation 3.5.1 Detection and Enumeration of Respiratory Deficient Mutants 3.5.2 Fermentation in EBC Tall Tubes 4.4.4 Chromogenic / Fluorogenic Method for Escherichia coli and Coliform Bacteria 4.4.5 Clostridium perfringens (including Spores) in Water 4.6 Detection of Contaminants in Other Brewing Materials 4.6.1 Detection of Contaminants in Additives 4.6.2 General Aerobic Count in Dilute Sugars 4.6.3 General Aerobic Count in Process Gases 4.7 Plant Hygiene 4.7.1 General Aerobic Count in Acid and Caustic Recovery Systems ANNEX II Archived Methods IV
In addition, amendments have been made to 13 existing methods: 2.2.1.4 On-Line Sampling by Membrane Filtration 2.2.4.1 Sampling of Contaminants from Compressed Gases 2.2.4.2 Sampling of Contaminants from Atmospheric Air 2.2.5.1 Sampling of Washed Bottles, New Bottles and Cans 2.2.5.2 Sampling from Filling Machine 2.2.5.3 Rinse Water from Kegs and Casks 4.2.2 General Aerobic Count on Samples of Yeast or Fermenting Beer 4.2.5.2 Heat Differentiation 4.3.1.4 Enrichment 4.3.2.1 General Aerobic Count on Beer 4.3.2.2 General Anaerobic Count on Beer 4.3.3.3 Selective Enrichment of Pectinatus and Megasphaera ANNEX I Hygiene Legislation V
Method Selection Guide SECTION 1 Microbiology 1.1 Principles of Microbiology 1.2 Brewery Microbiology 1.2.1 Classification of Microorganisms 1.2.2 The Microflora of Barley, Malt and Beer 1.2.2.1 Barley 1.2.2.2 Malthouse 1.2.2.3 Hops 1.2.2.4 Water 1.2.2.5 Brewhouse 1.2.2.6 Fermentation 1.2.2.7 Maturation and Filtration 1.2.2.8 Flash Pasteurisation 1.2.2.9 Beer Spoilage Microorganisms 1.2.2.10 Tunnel Pasteurisation 1.3 Controlling Brewery Hygiene 1.4 Yeast 1.4.1 General Description of Yeasts 1.4.2 Brewers' Yeast: Saccharomyces pastorianus and Saccaromyces cerevisiae 1.4.3 Wild Yeasts VIII
1.5 Bacteria 1.5.1 General Description of Bacteria 1.5.2 Gram-positive Spoilage Bacteria 1.5.3 Gram-negative Spoilage Bacteria 1.5.3.1 Anaerobic Rods 1.5.3.2 Anaerobic Cocci 1.5.3.3 Acetic acid Bacteria 1.5.3.4 Enterobacteriaceae 1.6 Moulds 1.7 Literature SECTION 2 Microbiological Techniques 2.1 Sterilisation Methods 2.1.1 Dry Heat Sterilisation 2.1.2 Autoclaving 2.1.3 Intermittent Sterilisation (Tyndallisation) 2.1.4 Pasteurisation 2.1.5 Sterile Filtration 2.2 Sampling Methods 2.2.0 General Rules for Hygienic Sampling 2.2.1 Sampling of Wort and Beer from Tanks and Pipes 2.2.1.1 Sampling by Diaphragm Samplers 2.2.1.2 Sampling by Plug Type Sample Cocks 2.2.1.3 Sampling by Valves IX
2.2.1.4 On-Line Sampling by Membrane Filtration 2.2.1.5 Counterpressure Sampling 2.2.1.6 Sampling for Yeast Cell Counts 2.2.2 Sampling of Thick Yeast 2.2.2.1 Sampling of Thick Yeast from Open Vessels 2.2.2.2 Sampling of Thick Yeast from Closed Vessels 2.2.2.3 Sampling of Compressed Yeast 2.2.3 Sampling of Water 2.2.3.1 Sampling of Water from Taps 2.2.3.2 Sampling of Water from Wells 2.2.3.3 Sampling of Open Water Resevoirs 2.2.3.4 Neutalisation of Chlorinated Water 2.2.4 Sampling of Air and Gases 2.2.4.1 Sampling of Contaminants from Compressed Gases 2.2.4.2 Sampling of Contaminants from Atmospheric Air 2.2.5 Sampling for Cleaning Control 2.2.5.1 Sampling of Washed Bottles, New Bottles and Cans 2.2.5.2 Sampling from Filling Machine 2.2.5.3 Sampling from Crowner 2.2.5.4 Rinse Water from Kegs and Casks 2.2.5.5 Rinse Water from Tanks and Pipes, etc. 2.2.5.6 Swab Tests 2.2.5.7 Anaerobic Sample Handling 2.3 Laboratory Techniques 2.3.1 Dilution of Samples 2.3.2 Membrane Filtration 2.3.2.1 Laboratory Membrane Filtration Technique X
2.3.2.2 Filter Membrane Growth Technique 2.3.3 Plating Techniques 2.3.3.1 Pour Plate Technique 2.3.3.2 Surface Spread Plating Technique 2.3.3.3 Isolation of Single Colonies 2.3.4 Incubation 2.3.4.1 General Rules for Incuabtion 2.3.4.2 Aerobic Incubation 2.3.4.3 Anaerobic, Catalysed Carbon Dioxide Incuabtion 2.3.4.4 Anaerobic, Carbon Dioxide Purge Incubation 2.3.5 Microscopy 2.3.5.1 Bright Field Microscopy 2.3.5.2 Phase Contrast Microscopy 2.3.5.3 Fluorescence Microscopy 2.3.5.4 Stereo Microscopy 2.3.8 ATP Bioluminescence Methods 2.3.8.1 ATP Analysis of Swab Samples 2.3.8.2 ATP Analysis of Rinse Water 2.3.8.3 ATP Analysis of Final Product 2.3.9 Characterisation of Contaminants 2.3.9.1 Gram Staining for Differentiation of Bacteria 2.3.9.2 KOH Method for Gram Differentiation 2.3.9.3 Catalase Test 2.3.9.4 Oxidase Test 2.3.9.5 Phenolic Off Flavour (POF) 2.4 Molecular Techniques 2.4.1 Polymerase Chain Reaction (PCR) Techniques 2.4.1.1 Detection and Identification of Microorganisms from Turbid Liquid Samples or Cultured Colonies by Real-Time PCR XI
2.4.1.2 Detection and Identification of Microorganisms from Filterable Samples by Real-Time PCR SECTION 3 Yeast Analysis 3.1 Yeast Mass Determination 3.1.1 Yeast Cell Count 3.1.1.1 Haemocytometry 3.1.1.2 Electronic Counter 3.1.1.3 Photometric Determination 3.1.2 Yeast Mass 3.1.2.1 Centrifugation 3.1.2.2 Dry Weight 3.2 Yeast Viability 3.2.1 Staining Methods 3.2.1.1 Methylene Blue / Violet Stain 3.2.1.2 Fluorescence Stain 3.2.2 Growth Techniques 3.2.2.1 Slide Culture Techniques 3.3 Yeast Morphology 3.3.1 Microscopic Analysis 3.3.1.1 Cell Morphology 3.3.2 Cell Colony Morphology 3.3.2.1 Yeast Giant Colonies 3.3.2.2 Morphology on WLN Agar 3.4 Yeast Strain Storage, Supply and Propagation 3.4.1 Yeast Storage at Ultra-Low Temperatures XII
3.4.2 Yeast Sub Culturing for Short Term Storage 3.4.3 Yeast Strain Transport 3.4.4 Yeast Propagation (Laboratory Stages) 3.5 Yeast Characterisation 3.5.1 Detection and Enumeration of Respiratory Deficient Mutants 3.5.2 Fermentation in EBC Tall Tubes SECTION 4 Detection of Contaminants 4.1 Detection of Contaminants in Wort 4.1.1 Wort Forcing Test 4.1.2 General Aerobic Count 4.1.3 Specific Detection of Bacteria in Wort 4.1.3.1 Enterobacteriaceae 4.1.3.2 Lactic Acid Bacteria 4.2 Detection of Contaminants in Yeast and Fermenting Beer 4.2.1 Enrichment 4.2.2 General Aerobic Count on Samples of Yeast or Fermenting Beer 4.2.3 General Anaerobic Count on Samples of Yeast or Fermenting Beer 4.2.4 Specific Detection of Bacteria in Yeast 4.2.4.1 Enterobacteriaceae 4.2.4.2 Lactobacillus and Pediococcus 4.2.4.3 Acetic Acid Bacteria 4.2.5 Saccharomyces Wild Yeasts 4.2.5.1 Cu-differentiation 4.2.5.2 Heat Differentiation 4.2.6 Non-Saccharomyces Yeasts XIII
4.2.7 Dekkera (formerly Brettanomyces) 4.3 Detection of Contaminants in Beer 4.3.1 Shelf Life Test 4.3.1.1 Shelf life of Beer in Bottles 4.3.1.2 Shelf Life of Beer in Cans and Kegs 4.3.1.3 Shelf life of Kegged Beer Transferred to Bottles 4.3.1.4 Enrichment 4.3.2 Non-Specific Detection of Microorganisms in Beer 4.3.2.1 General Aerobic Count on Beer 4.3.2.2 General Anaerobic Count on Beer 4.3.3 Specific Detection of Bacteria in Beer 4.3.3.1 Lactic Acid Bacteria 4.3.3.2 Pectinatus and Megasphaera 4.3.3.3 Selective Enrichment of Pectinatus and Megasphaera 4.4 Detection of Contaminants in Water 4.4.1 General Aerobic Count in Water 4.4.2 Escherichia Coli and Coliform Bacteria 4.4.3 Enterococci 4.4.4 Chromogenic/Fluorogenic Method for Escherichia coli and coliform Bacteria 4.4.5 Clostridium perfringens (Including Spores) in Water 4.5 Detection of Contaminants in Barley and Malt 4.5.1 Fusarium 4.5.2 Storage Fungi 4.5.3 General Method Cultivation on Wet Filter Paper XIV
4.6 Detection of Contaminants in Other Brewing Materials 4.6.1 Detection of Contaminants in Additives 4.6.2 General Aerobic Count in Dilute Sugars 4.6.3 General Aerobic Count in Process Gases 4.7 Plant Hygiene 4.7.1 General Aerobic Count in Acid and Caustic Recovery Systems SECTION 5 Media and Reagents 5.1. Media and Reagents for Microbiological Analyses 5.1.1 Inhibitors in Brewery Microbiology 5.1.2 Commercial Media 5.1.2.1 Preparation of Growth Media 5.1.2.2 Dehydrated Media 5.1.3 Media Recipes 5.1.3.1 Carr s Medium 5.1.3.2 Czapek-Dox Iprodione Dicloran Agar (CZID) 5.1.3.3 Dekkera Medium 5.1.3.4 Malt Salt Agar (MSA) 5.1.3.5 YM (or YMPG) + CuSO 4 5.1.3.6 PYF Medium 5.1.3.7 SMMP Medium 5.1.3.8 Supplemented YM (MYGP) Medium 5.1.3.9 Wort Agar 5.1.3.10 Yeast Extract Broth 5.1.3.11 Phosphate Buffered Agar (PBA) 5.1.3.12 m-cp Agar 5.1.4 Reagents in Brewery Microbiology 5.1.4.1 1-8 ANS 5.1.4.2 Physiological Saline 5.1.4.3 Gram Stain reagents 5.1.4.4 Methylene Blue XV
ANNEX I Hygiene Legislation 1. Council Directive 93/43/EEC on the Hygiene of Foodstuffs 2. Council Directive 98/83/EC on the Quality of Water 3. Existing ISO/EN Methods for Water Analysis 4. Countries outside EU and EEA ANNEX II Archived Methods XVI