Experiments in microbiology, plant pathology and biotechnology
- 4
- New Delhi New Age International Pvt. Ltd. 2012
- xxiii, 607p.
SECTION 1 Scope and Historical Development of Microbiology • Introduction • Scope of Microbiology • History of Microbiology—Milestones of Progress • The Discovery Era • Transition Period • Golden Age of Microbiology (1857-1911) • Fermentation • The Germ Theory of Disease • Immunization • Chemotherapy and Wonder Drugs • Microbiology in the 20th Century: Era of Molecular Biology • Nobel Prize Awards for Research Involving Microbiology (1901-2016) • Taxonomy and Nomenclature of Living Organisms • Classification of Bacteria
SECTION 2 Laboratory Rules, Tools, Equipment and Other Requirements in Microbiology and Biotechnology Laboratories • Laboratory Rules • Equipment Requirements in Microbiology and Biotechnology • Bunsen Burner • Laminar Flow Hood • Inoculation Chamber/Sterile Chamber • Autoclave • Pressure Cooker • Oven • Microbiological Incubator • BOD Incubator • Fermenter (Bioreactor) • Constant Temperature Water Bath • Shaking-water Bath • Hot Plate Stirrer • Quebec Colony Counter • Refrigerator (Fridge) • Deep-Fridge (Deep Freezer) • Centrifuge • Spectrophotometer • pH Meter • Photomicrographic Camera • Agar Slants • Agar Deep Tubes • Agar Plates • Pipette • Inoculating Loops and Inoculating Needles • Throat Swab • Ocular Micrometer • Stage Micrometer
SECTION 3 Microscopy • Introduction Experiment 1 The components, use and care of the bright-field compound microscope Experiment 2 Use and care of the dissecting microscope
SECTION 4 Micrometry Introduction Experiment 3 Calibration of an ocular micrometer for different objectives (low-power, high-power and oil-immersion) of a microscope Experiment 4 Measurement of microorganisms by the use of an-ocular micrometer
SECTION 5 Counting of Cells/Spores of Microorganisms • Introduction Experiment 5 Measurement of cells concentration of bacteria or fungal conidia by the use of a counting chamber. Experiment 6 Enumeration (counting) of bacteria by plate count or serial dilution agar plate technique Experiment 7 Counting of bacterial population by the use of spectrophotometer
SECTION 6 Microscopic Examination of Living Microorganisms Introduction Experiment 8 Temporary wet mount (TWM) technique for observation of living microorganisms Experiment 9 Hanging drop technique for demonstrating motility of bacteria Experiment 10 Microscopic examination of free-living protozoa of a pond Experiment 11 Microscopic examination of parasitic protozoa Experiment 12 Demonstration of bacterial lysis by bacteriophages Experiment 13 Slide culture technique for studying morphology of molds Experiment 14 Cover slip culture technique for preparing permanent fungus mounts Experiment 15 Scotch tape preparation for studying morphology of fungi
SECTION 7 Preparation of Bacterial and Blood Smear Introduction Experiment 16 Preparation of a bacterial smear Experiment 17 Preparation of a blood smear and differential blood count
SECTION 8 Staining Methods Introduction Experiment 18 Simple staining of bacteria Experiment 19 Negative staining of bacteria Experiment 20 Gram staining of bacteria Experiment 21 Acid-fast staining of bacteria Experiment 22 Bacterial cell wall staining Experiment 23 Cytoplasmic membrane staining Experiment 24 Bacterial spore (endospore) staining Experiment 25 Capsule staining Expt. 25.1 Capsule staining by negative staining technique Expt. 25.1I Capsule staining by Anthony staining method Experiment 26 Flagella staining Experiment 27 Staining of nuclear material of bacteria Experiment 28 Viability staining technique for bacteria Experiment 29 Lactophenol cotton blue mounting of fungi. Experiment 30 Nuclear staining in filamentous fungi Experiment 31 Staining of actinomycetes Experiment 32 Staining of spirochetes by Fontana’s method Experiment 33 Staining of mycoplasmas (PPLO) Experiment 34 Staining of vesicular arbuscular mycorrhizae (VAM)
SECTION 9 Control of Microorganisms Introduction Experiment 35 Physical agents of control: Moist heat Experiment 36 Physical agents of control: Dry heat (hot air) Experiment 37 Physical methods of control: Ultraviolet radiations Experiment 38 Mechanical removal methods: Sterilization by filtration Experiment 39 Chemical agents of control: Evaluation of antiseptics by filter paper disk method Experiment 40 Chemical agents 136 of control: Evaluation of alcohol effectiveness as a skin antiseptic Experiment 41 Chemical agents 139 of control: Evaluation of disinfectants (phenol coefficient)
SECTION 10 Preparation of Culture Media • Introduction Experiment 42 Preparation of basic liquid media (broth) for the routine cultivation of bacteria Experiment 43 Preparation of basic solid media, agar slants and agar deep tubes for the routine cultivation of microorganisms Experiment 44 Demonstration of selective and differential media
SECTION 11 Cultivation Techniques for Isolation and Enumeration of Microorganisms • Introduction Experiment 45 Isolation and enumeration of microorganisms from soil by the serial dilution-agar plating method (or viable plate count method) Experiment 46 Isolation of fungi by Warcup method Experiment 47 Isolation of vesicular-arbuscular mycorrhizal (VAM) spores from soil Experiment 48 Isolation of Azotobacter from soil Experiment 49 Isolation of algae from soil Experiment 50 Isolation of protozoa from soil Experiment 51 Isolation of microorganisms from air Experiment 52 Effect of dusting and sweeping on the indoors microbial population of a laboratory Experiment 53 Isolation of microorganisms from rhizosphere Experiment 54 Isolation of microorganisms from phyllosphere (phylloplane) Expt. 54.1 Study of phylloplane microflora by serial dilution plate method Expt. 54.11 Study of phylloplane microflora by leaf impression method Experiment 55 Isolation of rhizobia from root nodules Experiment 56 Isolation of cyanobacteria (blue green algae) from paddy fields Experiment 57 Isolation of aquatic fungi Expt. 57.1 Isolation of aquatic fungi by baiting in the laboratory Expt. 57.II Isolation of aquatic fungi by baiting in the field Experiment 58 Isolation of slime molds Experiment 59 Isolation of mycoplasmas
SECTION 12 Methods of Obtaining Pure Cultures of Microorganisms • Introduction Experiment 60 Streak-plate method Experiment 61 Pour-plate method Experiment 62 Spread-plate technique Experiment 63 Subculturing (or picking off) technique Experiment 64 Broth cultures Experiment 65 Cultivation of anaerobes Expt. 65.1 Shake-culture method for culturing anaerobic microorganisms Expt. 65.11 Pyrogallic acid-sodium hydroxide method for culturing anaerobic microorganisms
SECTION 13 Maintenance of Pure Cultures • Introduction Experiment 66 Paraffin method Experiment 67 Freeze-drying (lyophilization) method Experiment 68 Maintenance (storage) of mold cultures on distilled water Experiment 69 Control of mites in fungal cultures by fumigation
SECTION 14 Measurement of Growth and Enumeration of Microorganisms and Factors Influencing Growth • Introduction Experiment 70 Measurement of fungal growth by linear determination Experiment 71 Measurement of fungal growth by mycelial weight determination Experiment 72.1 Determination of population growth by turbidometry (spectrophotometric method) Experiment 72.1] Determination of population growth curve in bacteria by viable plate count Experiment 73 Determination of algal growth Experiment 74 Effect of temperature Expt. 74.1 Effect of incubation temperature on microbial growth Expt. 74.11 Determination of thermal death point (TDP) Expt. 74.01 Determination of thermal death time (TDT) Experiment 75 Effect of pH Experiment 76 Effect of relative humidity Experiment 77 Effect of molecular oxygen (air) Experiment 78 Effect of Osmotic pressure Experiment 79 Effect of salt concentration Experiment 80 Effect of metals Experiment 81 Effect of dyes Experiment 82 Effect of different wavelengths of light Experiment 83 Effect of light on growth and sporulation of fungi Experiment 84 Lethal effects of ultraviolet radiations (UV rays)
SECTION 15 Biochemical Activities of Microorganisms • Introduction Experiment 85 Amylase production test (or demonstration of starch hydrolysis) Experiment 86 Cellulase production test (degradation of cellulose) Experiment 87 Production of pectolytic enzymes (degradation of pectin) Experiment 88 Hydrolysis of gelatin, a protein (production of gelatinase) Experiment 89 Casein hydrolysis Experiment 90 Urease test Experiment 91 Hydrogen sulfide production test Experiment 92 Carbohydrate catabolism by microorganisms (oxidation and fermentation of glucose) Experiment 93 Fermentation of carbohydrates Experiment 94 Microbial reactions in litmus milk Experiment 95 IMViC tests Expt. 95.1 Indole production test Expt. 95.11 Methyl-Red and Voges-Proskauer tests Expt. 95.11 Citrate utilization test Experiment 96 Catalase test
SECTION 16 Identification of an Unknown Microorganism (Bacterium, Mold, Protozoan) • Introduction Experiment 97 Identification of an unknown bacterial culture Experiment 98 Identification of an unknown fungus: Mold Experiment 99 Identification of an unknown protozoan
SECTION 17 Medical Microbiology and Immunology • Introduction Collection and handling of specimens for microbiological examination Experiment 100 Normal bacterial flora of the skin Expt. 100.1 Demonstration of bacterial flora of skin Expt. 100.11 Testing of antibacterial activity of skin Experiment 101 Pathogenic fungal flora of skin Expt. 101.1 Direct examination of dermatophytes 236 Expt. 101.11 Isolation and identification of dermatophytes Experiment 102 Microorganisms of wounds Experiment 103 Normal flora of oral cavity Expt. 103.1 Study of normal flora of mouth Expt. 103.11 Study of normal flora of the teeth crevices Experiment 104 Determination of dental caries susceptibility Experiment 105 Microorganisms of upper respiratory tract-I (throat) Experiment 106 Microorganisms of upper respiratory tract-I] (nasopharynx) Experiment 107 Microorganisms of respiratory tract-III (examination of sputum) Experiment 108 Examination of sputum for tuberculosis Experiment 109 Microorganisms of gastrointestinal tract Expt. 109.1 Primary isolation of enteric pathogens—Salmonella and Shigella Expt. 109.11 Preliminary identification of enteric pathogens, triple Sugar iron agar medium Experiment 110 Microorganisms of urinary tract (urine) Expt. 110.1 Estimation of urine bacteria by calibrated loop direct streak method Expt. 110.1 Estimation of urine bacteria by pour-plate (dilution) method Expt. 110.11] Examination of bacteria by urine dip-slide Experiment 111 Microorganisms of genital tract (normal and pathogenic) Expt. 111.1 Examination of direct smears for gonococci Expt. 111.11 Isolation and identification of gonococcus from vaginal and anorectal specimens Experiment 112 VDRL and RPR serological tests for syphilis Expt. 112.1 VDRL test for syphilis Expt. 112.1. RPR card test for syphilis Experiment 113 Estimation of hemoglobin (Hb) by Sahli’s hemoglobinometer Experiment 114 Blood group determination: Slide agglutination Experiment 115 Rh factor determination Experiment 116 Slide-agglutination test for serotyping pathogens Experiment 117 Tube agglutination test: The Widal test
SECTION 18 Water Microbiology • Introduction Experiment 118 Determination of total dissolved solids of water Experiment 119 Determination of dissolved oxygen (DO) of water Experiment 120 Determination of biochemical oxygen demand (BOD) of water Experiment 121 Determination of chemical oxygen demand (COD) of water Experiment 122 Determination of total bacterial population by standard plate count technique Experiment 123 Sanitary analysis of water by multiple-tube fermentation test (or multiple-tube test) Expt. 123.1 Presumptive coliforms test Expt. 123.II Confirmed coliforms test Expt. 123.111 Completed coliforms test Experiment 124 Detection of coliforms to determine water purity using membrane filter method
SECTION 19 Food, Dairy, Industrial and Agricultural Microbiology • Introduction Experiment 125 Detection of arsenic by microbiological methods Experiment 126 Bioassay of nicotinic acid Experiment 127 Detection of number of bacteria in milk by SPC Experiment 128 Detection of number of bacteria in milk by breed count Experiment 129 Determination of quality of raw milk by methylene blue reduction test Experiment 130 Microbiological examination of food Experiment 131 Production of alcohol from grape juice Experiment 132 Role of yeasts in bread-making Experiment 133 Isolation of antibiotic-producing microorganisms from soil Experiment 134 Antibiotic production and testing of its antimicrobial activity Expt. 134.1 Production of penicillin in the laboratory Expt. 134.II Antibiotic sensitivity test by Kirby-Bauer method Experiment 135 Testing of nodulation ability by rhizobia Experiment 136 Seed inoculation with rhizobia Experiment 137 Production of wine from grapes Experiment 138 Isolation of lipolytic microorganisms from butter Experiment 139 Lipase production test (or demonstration of lipid or fat hydrolysis by microbes) Experiment 140 Production of sauerkraut by lactic acid bacteria
SECTION 20 Bacterial Genetics • Introduction Experiment 141 Isolation of antibiotic-resistant bacterial population by gradient—plate method Experiment 142 Isolation of streptomycin-resistant mutants by replica plating technique Experiment 143 Demonstration of genetic recombination in bacteria by conjugation Experiment 144 Isolation of plasmids from bacteria Experiment 145 UV induced auxotrophic mutants production and isolation of mutants by replica plating technique Experiment 146 The Ames test: for detecting potential carcinogens Expt. 146.1 The Ames test by plate incorporation method Expt. 146.11 The Ames test by spot method Experiment 147 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of proteins
SECTION 21 Plant Pathological Methods • Introduction Experiment 148 Isolation of fungal pathogens from plant materials. Expt. 148.1 Isolation of fungal pathogens from leaves Expt. 148.11 Isolation of fungal pathogens from stems, fruits and other aerial plant parts Expt. 148.11 Isolation of fungal plant pathogens from soil Experiment 149 Isolation of bacterial plant pathogens Experiment 150 Isolation (purification) of plant viruses Experiment 151 Proving plant pathogenicity and Koch’s postulates Expt. 151.1 Demonstration of Koch’s postulates for a bacterial plant pathogen Expt. 151.11 Demonstration of Koch’s postulates for a fungal plant pathogen Experiment 152 Cellulases from plant pathogens Expt. 152.I Extraction of cellulase from a plant pathogen (in vitro) Expt. 152.II Extraction of cellulase enzyme from diseased plants (in vivo) Expt. 152.III Measurement of CMCase or β (1-4) glucanases (or Cx) by viscometric method Expt. 152.IV Measurement of endoglucanase or carboxymethyl cellulase by reducing sugars determination Experiment 153 Pectinases or pectolytic enzymes from plant pathogens Expt. 153.1 Extraction of pectolytic enzymes from diseased plants (in vivo) Expt. 153.11 Extraction of pectolytic enzymes from a plant pathogen (in vitro) Expt. 153.111 Assaying of polygalacturonase Expt. 153.1V Assaying of pectin transeliminases (PTE, PATE) Experiment 154 Screening for antagonism by dual culture method
SECTION 22 Plant Tissue Culture • Introduction • Equipment and other requirements in a tissue culture laboratory • Steps generally followed in tissue culture technique. Experiment 155 Sterilization of plant materials Experiment 156 Preparation of tissue culture media Experiment 157 Plant cell culture techniques Expt. 157.1 Isolation of single cells from intact plant organs Expt. 157.11 Single cell culture by Bergmann’s cell plating technique Experiment 158 Plant regeneration from callus or plant tissue Experiment 159 Isolation and culture of plant protoplasts Experiment 160 Protoplast fusion (somatic cell hybridization) Experiment 161 Production of pathogen-free plants through meristem culture
SECTION 23 Mushroom Production Technology • Introduction • What are mushrooms? • Food value of edible mushrooms • Cultivated edible mushrooms. • Poisonous mushrooms • Diseases of mushrooms • Cultivation of mushrooms Experiment 162 Spawn production for white 488 button mushroom (Agaricus bisporus) Experiment 163 Commercial cultivation 489 of white button mushroom Experiment 164 Cultivation of paddy 493 straw mushroom (Volvariella volvacea) Experiment 165 Cultivation of Oyster mushroom (Pleurotus spp.)
APPENDICES Appendix A-I. 506-576 Classification of Prokaryotes According to Bergey's Manual of Systematic Bacteriology, Second edition, 2001-2012 (5 Volumes) Appendix A-II, Classification of 506 Prokaryotes: According to Bergey’s Manual of Systematic Bacteriology, First Edition, 1984—19g9 52] Appendix B. World’s major microbial ‘culture collections Appendix C. Units of Measurement and conversion factors Appendix D. Atomic numbers and weights of the elements Appendix E. Preparation of biochemical test reagents Appendix F, Preparation of indicators Appendix G Preparation of stains Appendix H. Preparation of buffers Appendix I. Culture Media Appendix J. Identification common bacteria—cultural and biochemical characteristics Appendix K, Some milestones in biotechnology and recombinant DNA technology