Laboratory Methods in Microbiology is a laboratory manual based on the experience of the authors over several years in devising and organizing practical classes in microbiology to meet the requirements of students following courses in microbiology at the West of Scotland Agricultural College. The primary object of the manual is to provide a laboratory handbook for use by students following food science, dairying, agriculture and allied courses to degree and diploma level, in addition to being of value to students reading microbiology or general bacteriology. It is hoped that laboratory workers in the food manufacturing and dairying industries will find the book useful in the microbiological aspects of quality control and production development. The book is organized into two parts. Part I is concerned with basic methods in microbiology and would normally form the basis of a first year course. Abbreviated recipes and formulations for a number of typical media and reagents are included where appropriate, so that the principles involved are more readily apparent. Part II consists of an extension of these basic methods into microbiology as applied in the food manufacturing, dairying and allied industries. In this part, the methods in current use are given in addition to, or in place of, the "e;"e;classical"e;"e; or conventional techniques.
Front Cover 1
Laboratory Methods in Microbiology 4
Copyright Page 5
Table of Contents 8
PREFACE 6
PART I: BASIC METHODS 14
CHAPTER 1. GENERAL NOTES ON LABORATORY PROCEDURE 16
CHAPTER 2. LABORATORY REPORTS 17
CHAPTER 3. PROCEDURE FOR THE USE OF A MICROSCOPE WITH AN OIL-IMMERSION OBJECTIVE 18
CHAPTER 4. EXAMINATION OF CULTURES FOR MOTILITY BY "HANGING DROP" PREPARATIONS 19
CHAPTER 5. STAINING METHODS 20
A. Preparation of Smears for Staining 20
B. Simple Stains 20
C. Gram's Staining Method 21
D. Ziehl-Neelsen Method for Staining Acid-fast Bacteria 22
E. Staining of Bacterial Flagella 23
F. The Demonstration of Bacterial Capsules 25
G. Negative Stain 26
H. The Staining of Bacterial Spores 26
CHAPTER 6. CULTIVATION OF MICRO-ORGANISMS 27
A. Types of Culture 27
B. Incubation of Cultures 28
C. Method of Inoculation: Aseptic Technique 28
D. Maintenance of Pure Cultures in the Laboratory 28
CHAPTER 7. THE DESCRIPTION OF THE MORPHOLOGICAL AND CULTURAL CHARACTERISTICS OF AN ORGANISM 30
A. Morphological Characters 30
B. Cultural Characters 30
CHAPTER 8. PLATE CULTURES 32
A. Preparation of Plates for Streaking 32
B. Streak Plates: The Separation of Mixed Cultures 32
CHAPTER 9. DETERMINATION OF THE NUMBER OF VIABLE ORGANISMS IN A SAMPLE 34
A. Colony Count Methods 34
B. Membrane Filtration 38
C. Dilution Tube Count 40
D. Dye Reduction Methods 42
CHAPTER 10. DETERMINATION OF THE TOTAL NUMBER OF ORGANISMS IN A SAMPLE 43
A. The Breed's Smear Method for Direct Microscopic Counts 43
B. Direct Microscopic Counts by Membrane Filtration 45
C. Brown's Opacity Tubes 45
CHAPTER 11. STATISTICAL METHODS FOR THE SELECTION AND EXAMINATION OF MICROBIAL COLONIES 47
CHAPTER 12. COMPOSITION OF CULTURE MEDIA 49
A. Introduction 49
B. Dehydrated Media 49
C. The Determination of the pH of Culture Media 50
D. Examples of Non-selective Culture Media 51
E. Separation of Mixed Cultures—Enrichment Procedures, Elective and Selective Media 53
F. Examples of Selective Culture Media 55
CHAPTER 13. METHODS OF ANAEROBIC CULTURE 59
A. Robertson's Cooked Meat Medium 59
B. Shake Cultures 59
C. Semi-solid Media 60
D. Vaseline, Paraffin Wax or Agar Seals 60
E. The Anaerobic Jar 61
CHAPTER 14. CULTIVATION IN A CARBON DIOXIDE-ENRICHED ATMOSPHERE 63
CHAPTER 15. BIOCHEMICAL TESTS FOR BACTERIA 64
A. Reactions Involving Protein, Amino Acids and other Nitrogen Compounds, Including Tests for Proteolytic Activity 64
B. Reactions Involving Carbohydrates and other Carbon Compounds, Including Tests for Saccharolytic Activity 71
C. Reactions Involving Fats and Related Substances, Including Tests for Lipolytic Activity 76
D. Miscellaneous Tests 78
CHAPTER 16. CLEANING OF GLASSWARE AND APPARATUS 82
A. Treatment of New Glassware 82
B. Treatment of Used Glassware and other Apparatus 82
C. Disposable Apparatus 82
CHAPTER 17. STERILIZATION 83
A. Sterilization by Dry Heat 83
B. Sterilization by Moist Heat 83
C. Sterilization by Filtration 84
D. Chemical Disinfectants 86
E. Preparation of Glassware and Materials for Sterilization 86
F. To Test the Sterility of Laboratory Equipment 86
CHAPTER 18. LABORATORY EVALUATION OF DISINFECTANTS 88
A. The Rideal-Walker Test 88
B. The Suspension Test 88
C. The Capacity Test 89
CHAPTER 19. SEROLOGICAL METHODS 90
A. Introduction 90
B. The Agglutination Reaction 90
C. The Precipitin Test 94
CHAPTER 20. MOULDS AND YEASTS 96
A. General Conditions for the Growth of Moulds and Yeasts 96
B. Media for the Growth of Moulds and Yeasts 96
C. Examination of Moulds 96
D. Examination of Yeasts 97
E. Note on the Identification of Moulds and Yeasts 97
CHAPTER 21. THE ISOLATION OF BACTERIOPHAGE 99
PART II: TECHNIQUES IN APPLIED MICROBIOLOGY 102
CHAPTER 22. THE BACTERIOLOGICAL EXAMINATION OF WATER 104
A. Introduction 104
B. Sampling Procedure 105
C. Plate Counts 105
D. Presumptive Coliform Counts and the Eijkman Test 107
E. Enumeration of Faecal Streptococci 108
F. Detection of Clostridium welchii 108
G. Membrane Filtration Methods 109
CHAPTER 23. THE MICROBIOLOGICAL EXAMINATION OF SOIL 111
A. Introduction 111
B. General Viable Counts of Bacteria by the Pour Plate Method 111
C. Examination for Moulds and Yeasts 112
D. Buried Slide Technique (Rossi-Riccardo-Cholodny Technique) 112
E. Isolation of Nitrogen-fixing Bacteria 113
F. Demonstration of the Presence of Nitrifying Bacteria 114
G. Demonstration of the Presence of Denitrifying Bacteria 115
H. Demonstration of Cellulose-decomposing Bacteria 115
CHAPTER 24. THE BACTERIOLOGICAL EXAMINATION OF SILAGE 116
A. The Preparation of Experimental Silage 116
B. The Quantitative Bacteriological Examination of Grass or Silage 116
C. Qualitative Bacteriological Examination of Grass or Silage 117
D. Identification of Isolates 118
CHAPTER 25. THE MICROBIOLOGICAL EXAMINATION OF FOODS 119
A. Introduction 119
B. Sampling Methods 120
C. Preparation of Dilutions 123
D. The Isolation of Pathogenic and Indicator Organisms 125
E. Meat and Meat Products 132
F. Fish, Shellfish and other Seafoods 135
G. Eggs 137
H. Fruit Juices and Squashes 139
I. Sugar Syrups 140
J. Fresh Fruit and Vegetables 140
K. Salted, Pickled and Fermented Vegetables 140
L. Bread, Cakes etc. 141
M. Frozen Foods 142
N. Canned Foods 143
O. Note on the Rationale of the Microbiological Analysis of Foods 145
CHAPTER 26. THE EXAMINATION OF MILK 147
A. Determination of Total Numbers 147
B. Determination of General Viable Numbers 148
C. Dye Reduction Methods 150
D. Presumptive Coliform Test 154
E. The Laboratory Pasteurization Test 156
F. Enumeration and Isolation of Thermophiles in Milk 157
G. Enumeration and Isolation of Aerobic Spore-formers in Milk 158
H. Enumeration and Isolation of Psychrophiles 160
I. Investigation of Faults and Taints in Milk and Isolation of Causative Organisms 161
J. Examination of Mastitis Milk 164
K. The Detection of Antibiotics in Milk 167
CHAPTER 27. THE EXAMINATION OF DAIRY UTENSILS AND EQUIPMENT 171
A. Examination of Washed Milk Cans 172
B. Examination of Washed Milk Bottles – Rinse Method 173
C. Swab Method for the Examination of Milk Plant and Equipment 174
D. Examination of Farm Dairy Equipment by the Rinse Method 175
CHAPTER 28. THE EXAMINATION OF FERMENTED MILKS AND ISOLATION OF CONSTITUENT FLORA 177
A. Microscopic Examination 177
B. Isolation of Constituent Flora 177
CHAPTER 29. THE EXAMINATION OF DAIRY STARTER CULTURES 179
A. Biochemical Tests 179
B. Microscopic Examination 180
C. Cultural Examination 180
D. Demonstration of the Effect of Phage on Starter Bacteria 182
E. Demonstration of the Effect of Penicillin on Starter Bacteria and Test Organisms 183
CHAPTER 30. THE MICROBIOLOGICAL EXAMINATION OF CHEESE 185
A. Sampling 185
B. Microscopic Examination 186
C. Cultural Examination 186
D. Isolation of Propionibacteria from Swiss Cheese 188
E. Isolation of Moulds from Mould Ripened Cheese 189
F. Isolation of Brevibacterium linens from Bacterial-ripened Soft Cheese 190
CHAPTER 31. THE MICROBIOLOGICAL EXAMINATION OF BUTTER 192
A. Sampling 192
B. Cultural Examination 193
C. The Isolation and Further Study of Lipolytic Micro-Organisms 195
CHAPTER 32. THE MICROBIOLOGICAL EXAMINATION OF MILK POWDER 196
A. Sampling 196
B. Microscopic Examination 196
C. Cultural Examination 197
D. Results and Suggested Standards 198
CHAPTER 33. THE MICROBIOLOGICAL EXAMINATION OF CANNED CONCENTRATED MILK 199
A. Evaporated Milk (Unsweetened Condensed Milk) 199
B. Sweetened Condensed Milk 200
CHAPTER 34. THE MICROBIOLOGICAL EXAMINATION OF ICE-CREAM 202
A. Collection of Samples 202
B. Treatment of Samples 203
C. Cultural Examination 203
D. Methylene Blue Dye Reduction Test 204
E. Results and Recommended Standards 205
CHAPTER 35. THE MICROBIOLOGICAL EXAMINATION OF CREAM 206
A. Sampling 206
B. Methylene Blue Dye Reduction Test 206
C. Crossley's Bromcresol Purple Milk Test 207
D. Cultural Examination 208
E. Results and Recommended Standards 208
APPENDIX 1: SCHEMES FOR THE IDENTIFICATION OF MICRO-ORGANISMS 210
1. DIAGNOSTIC TABLES FOR GRAM-NEGATIVE BACTERIA WHICH CAN GROW ON NUTRIENT AGAR OR PLATE COUNT AGAR 212
2. SIMPLE KEY FOR THE IDENTIFICATION OF GRAM-POSITIVE BACTERIA 224
3. IDENTIFICATION OF YEASTS AND MOULDS 242
APPENDIX 2: RECIPES FOR STAINS, REAGENTS AND MEDIA 268
STAINS 270
REAGENTS 273
MEDIA 277
APPENDIX 3: PROBABILITY TABLES FOR THE ESTIMATION OF BACTERIAL NUMBERS BY THE DILUTION TUBE TECHNIQUE 330
TABLE 1 332
TABLE 2 333
TABLE 3 334
Determination of MPN's from Series of more than Three Dilutions 336
SELECTED BIBLIOGRAPHY 338
REFERENCES 341
AUTHOR INDEX 350
SUBJECT INDEX 356
STAINING METHODS
Publisher Summary
This chapter discusses various staining methods. In Gram’s staining method, bacteria are first stained with crystal violet and are then treated with iodine solution. The bacterial smears are next treated with alcohol or acetone, which entirely removes the violet stain in the case of Gram-negative bacteria. Dilute carbol fuchsin or safranin may be used as a counterstain. This method divides bacteria into two classes: (1) Gram-positive and (2) Gram-negative. The Ziehl–Neelsen method consists firstly of staining the organisms with a hot, concentrated dye. Once stained, the cells resist decolorization with acid; they are, thus, acid-fast. This method is used for the detection of Mycobacterium tuberculosis in body tissues and fluids. Leifson’s flagella stain may be used for bacterial capsules, the capsules staining red and the bacteria blue when methylene blue is used as a counterstain. Negative Stain is a very simple and effective method for demonstrating the external shape of bacteria in a smear preparation. The bacteria are surrounded by a thin film of black dye and appear as white objects upon a grey background.
A Preparation of Smears for Staining
Smears of bacteria from cultures on solid media are made upon clean glass slides as follows.
1. The slide may be divided up into sections, one section for each smear, using a glass-writing pencil.
2. With a wire loop, place a small drop of water on each section of the slide.
3. Sterilize the wire by holding it vertically in the Bunsen flame until it is heated to redness along its entire length.
4. Allow the loop to cool.
5. Holding the loop like a pen, pick off a little of the bacterial growth from the colony to be examined.
6. Transfer to a water drop on the slide and, using the flat of the loop, emulsify the growth, finally smearing it evenly over the area of the slide allocated to it. Aim at obtaining thin, evenly spread smears which are almost too thin to be seen when dry. The individual bacteria will then be well spaced for examination under the microscope. If the suspension does not spread evenly over the slide, but collects in small droplets, the slide is greasy and should be discarded.
7. Sterilize the loop.
8. Prepare the other smears in the same manner.
9. Leave the slide to dry in the air, then heat-fix by passing the slide twice through the Bunsen flame. This causes the bacteria to adhere firmly to the slide.
Smears of bacteria from cultures in liquid media are made in a similar manner except that dilution with water is not required.
B Simple Stains
Place the heat-fixed smear on a staining rack over the sink and flood with any of the staining solutions given below. After the time indicated, rinse the slide gently in water and blot dry with blotting paper or filter paper. Examine as described on page 5.
(a) Crystal violet (gentian violet), a 0·5 per cent solution in water. Stain for one minute.
(b) Loeffler’s methylene blue
| Potassium hydroxide solution, 1 per cent | 1 ml |
| Methylene blue, saturated solution in alcohol | 30 ml |
| Water | 100 ml |
Stain for at least 5 minutes as this stain is weaker in action than crystal violet.
(c) Carbol fuchsin (dilute). This stain is made by diluting one part of the strong Ziehl-Neelsen’s carbol fuchsin (see page 10) in 15 parts of water. Stain for 30 sec. only.
C Gram’s Staining Method
This is a differential double-staining method which forms the basis of most examinations and the preliminary identification of bacteria. In this method, bacteria are first stained with crystal violet and are then treated with iodine solution. The bacterial smears are next treated with alcohol or acetone, which entirely removes the violet stain in the case of Gram-negative bacteria. Dilute carbol fuchsin or safranin may be used as a counterstain. This method divides bacteria into two classes:
(a) Gram-positive. These do not decolorize with alcohol, thus appearing purple. Examples of Gram-positive bacteria are Staphylococcus, Bacillus;
(b) Gram-negative. The cell membranes of these bacteria have a different physico-chemical structure and alcohol treatment removes the crystal violet so that the bacteria are only stained by the subsequent carbol fuchsin. Gram-negative bacteria appear pink. Examples of Gram-negative bacteria are Pseudomonas, Escherichia.
Note that the test should be carried out on young cultures (18–24 hours) or on cultures of various ages including young cultures, since some bacteria change in their Gram reaction as the cultures age.
There are in existence many modifications of Gram’s staining method (see for example Silverton and Anderson, 1961). The reagents and times employed depend upon the nature of the specimens most commonly being examined in a particular laboratory, but user preference also plays a large part in the choice of the modification. The reagents used in the method described below are chosen for having a greater latitude than many others toward deviations from the recommended staining times, and they are thus particularly suitable for class and routine use. Nevertheless, every effort should be made to adhere to the times recommended.
Procedure
1. Prepare a heat-fixed smear from an 18–24 hour culture in the usual way.
2. Stain with crystal violet solution for 1–2 min.
3. Rinse off with Gram’s iodine solution and allow the iodine to act for 1 min.
4. Pour off the iodine, blot dry and wash the slide with 95 per cent alcohol until no more violet stain runs from the slide (only 5–15 sec in the case of well-prepared thin smears).
5. Rinse under the tap and stain with dilute carbol fuchsin solution for 10 sec.
6. Wash the slide well and blot dry.
Reagents
This may be more conveniently prepared by diluting one part of Ziehl-Neelsen’s carbol fuchsin in 15 parts of water (see page 10).
D Ziehl-Neelsen Method for Staining Acid-fast Bacteria
Members of the genus Mycobacterium, as typified by the tubercle bacillus M. tuberculosis, stain very poorly, if at all, with normal techniques because of the high proportion of lipid material in their cells.
The Ziehl-Neelsen method consists firstly of staining the organisms with a hot, concentrated dye. Once stained, the cells resist decolorization with acid; they are thus “acid-fast”. Decolorization is effected with suitably strong acid and the smear is then counterstained with methylene blue solution. Acid-fast bacteria stain red, other bacteria and the background stain blue. This method is used for the detection of M. tuberculosis in body tissues and fluids (e.g. lungs, liver, sputum, urine).
Procedure
1. Cover the slide with strong Ziehl-Neelsen’s carbol fuchsin and heat the underside of the slide with a lighted alcohol-soaked swab. Remove the heat when the slide steams. Keep the slide hot and replenish the stain if necessary, taking care not to allow the smear to become dry. Heat for 5 min, not allowing the staining solution to boil.
2. Wash well.
3. Decolorize with 20 per cent sulphuric acid. The excess stain is removed as a brownish solution, and the smear will become brown. Rinse in water, when the film will appear pink once more. Apply more acid and repeat the rinsing several times until the film appears faintly pink upon washing.
4. Wash well.
5. Counterstain with Loeffler’s methylene blue for 5 min.
6. Wash well and carefully remove the stain deposits from the back of the slide with filter paper. Blot dry and...
| Erscheint lt. Verlag | 28.6.2014 |
|---|---|
| Sprache | englisch |
| Themenwelt | Medizin / Pharmazie ► Medizinische Fachgebiete |
| Studium ► Querschnittsbereiche ► Infektiologie / Immunologie | |
| Naturwissenschaften ► Biologie ► Evolution | |
| ISBN-10 | 1-4832-7434-9 / 1483274349 |
| ISBN-13 | 978-1-4832-7434-8 / 9781483274348 |
| Haben Sie eine Frage zum Produkt? |
PDF (Adobe DRM)Größe: 22,1 MB
Kopierschutz: Adobe-DRM
Adobe-DRM ist ein Kopierschutz, der das eBook vor Mißbrauch schützen soll. Dabei wird das eBook bereits beim Download auf Ihre persönliche Adobe-ID autorisiert. Lesen können Sie das eBook dann nur auf den Geräten, welche ebenfalls auf Ihre Adobe-ID registriert sind.
Details zum Adobe-DRM
Dateiformat: PDF (Portable Document Format)
Mit einem festen Seitenlayout eignet sich die PDF besonders für Fachbücher mit Spalten, Tabellen und Abbildungen. Eine PDF kann auf fast allen Geräten angezeigt werden, ist aber für kleine Displays (Smartphone, eReader) nur eingeschränkt geeignet.
Systemvoraussetzungen:
PC/Mac: Mit einem PC oder Mac können Sie dieses eBook lesen. Sie benötigen eine
eReader: Dieses eBook kann mit (fast) allen eBook-Readern gelesen werden. Mit dem amazon-Kindle ist es aber nicht kompatibel.
Smartphone/Tablet: Egal ob Apple oder Android, dieses eBook können Sie lesen. Sie benötigen eine
Geräteliste und zusätzliche Hinweise
Buying eBooks from abroad
For tax law reasons we can sell eBooks just within Germany and Switzerland. Regrettably we cannot fulfill eBook-orders from other countries.
EPUB (Adobe DRM)Größe: 3,2 MB
Kopierschutz: Adobe-DRM
Adobe-DRM ist ein Kopierschutz, der das eBook vor Mißbrauch schützen soll. Dabei wird das eBook bereits beim Download auf Ihre persönliche Adobe-ID autorisiert. Lesen können Sie das eBook dann nur auf den Geräten, welche ebenfalls auf Ihre Adobe-ID registriert sind.
Details zum Adobe-DRM
Dateiformat: EPUB (Electronic Publication)
EPUB ist ein offener Standard für eBooks und eignet sich besonders zur Darstellung von Belletristik und Sachbüchern. Der Fließtext wird dynamisch an die Display- und Schriftgröße angepasst. Auch für mobile Lesegeräte ist EPUB daher gut geeignet.
Systemvoraussetzungen:
PC/Mac: Mit einem PC oder Mac können Sie dieses eBook lesen. Sie benötigen eine
eReader: Dieses eBook kann mit (fast) allen eBook-Readern gelesen werden. Mit dem amazon-Kindle ist es aber nicht kompatibel.
Smartphone/Tablet: Egal ob Apple oder Android, dieses eBook können Sie lesen. Sie benötigen eine
Geräteliste und zusätzliche Hinweise
Buying eBooks from abroad
For tax law reasons we can sell eBooks just within Germany and Switzerland. Regrettably we cannot fulfill eBook-orders from other countries.
aus dem Bereich
