Micro 3004 LabManual_Fa2023

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School

Brooklyn College, CUNY *

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Course

3004

Subject

Biology

Date

Jan 9, 2024

Type

pdf

Pages

128

Uploaded by ProfTeam17414

Microbiology 3004 Lab Manual Fall 2023 V3

i Table of Contents Laboratory Schedule --------------------------------------------------------------------------------------------------------- 2 Photographic Atlas Reading ------------------------------------------------------------------------------------------------ 4 Lab Safety Instructions ------------------------------------------------------------------------------------------------------ 5 Laboratory Practice ----------------------------------------------------------------------------------------------------------- 6 Common Laboratory Equipment ------------------------------------------------------------------------------------------ 7 Scientific Method of Inquiry------------------------------------------------------------------------------------------------ 8 The Spread of Contagious Diseases ------------------------------------------------------------------------------------- 10 Glo-Germ Activity -------------------------------------------------------------------------------------------------------- 10 Questions ------------------------------------------------------------------------------------------------------------------------------ 11 Use of the Microscope ------------------------------------------------------------------------------------------------------ 13 Parts of the Microscope and Magnification ----------------------------------------------------------------------- 15 Questions: ----------------------------------------------------------------------------------------------------------------------------- 15 Culture Techniques ---------------------------------------------------------------------------------------------------------- 16 Liquid Culture Inoculation ---------------------------------------------------------------------------------------------- 18 Questions ------------------------------------------------------------------------------------------------------------------------------ 19 Streak Plate & Slant ------------------------------------------------------------------------------------------------------ 19 Questions ------------------------------------------------------------------------------------------------------------------------------ 20 Spread Plate --------------------------------------------------------------------------------------------------------------- 21 Questions ------------------------------------------------------------------------------------------------------------------------------ 22 Prokaryotic survey ----------------------------------------------------------------------------------------------------------- 23 Colony Morphology ------------------------------------------------------------------------------------------------------ 24 Cell Arrangements ------------------------------------------------------------------------------------------------------- 25 Prokaryotic Survey Table ----------------------------------------------------------------------------------------------- 26 Survey of Prokaryotes --------------------------------------------------------------------------------------------------- 27 Cytological Techniques ----------------------------------------------------------------------------------------------------- 28 The Gram-Positive Bacteria -------------------------------------------------------------------------------------------- 30 The Gram-Negative Bacteria ------------------------------------------------------------------------------------------ 31 Common Staining Techniques ----------------------------------------------------------------------------------------- 32 Gram Staining ------------------------------------------------------------------------------------------------------------- 33 Questions ------------------------------------------------------------------------------------------------------------------------------ 34 Micro Pipettor Usage ------------------------------------------------------------------------------------------------------- 35 Enumerating Microorganisms -------------------------------------------------------------------------------------------- 37 Viable Count --------------------------------------------------------------------------------------------------------------- 38 Hemocytometer Direct Count ----------------------------------------------------------------------------------------- 39 Questions ------------------------------------------------------------------------------------------------------------------------------ 42 Wet Mount Technique ----------------------------------------------------------------------------------------------------- 43 Urban Microbiome ---------------------------------------------------------------------------------------------------------- 44 Experiment ----------------------------------------------------------------------------------------------------------------- 46 Questions ------------------------------------------------------------------------------------------------------------------------------ 46 Part 1: Experiment Setup ----------------------------------------------------------------------------------------------- 46 Part 2: Bacteria Initial Viability --------------------------------------------------------------------------------------- 47 Part 3: DNA Extraction -------------------------------------------------------------------------------------------------- 47 Part 4: DNA quantification by NanoDrop --------------------------------------------------------------------------- 49 Part 5: Sample Sequencing --------------------------------------------------------------------------------------------- 49 Part 6: PCR amplification ----------------------------------------------------------------------------------------------- 47 Part 7: Gel electrophoresis --------------------------------------------------------------------------------------------- 48 Part 8 DNA sequence analysis --------------------------------------------------------------------------------------- 49 Cytological Techniques: Special Cell Stains ---------------------------------------------------------------------------- 53

ii Acid-fast Stain for Mycobacteria ------------------------------------------------------------------------------------- 53 Endospore Stain----------------------------------------------------------------------------------------------------------- 54 Questions ------------------------------------------------------------------------------------------------------------------------------ 54 Cytological Techniques: Bacteria Motility ----------------------------------------------------------------------------- 55 Flagella stain -------------------------------------------------------------------------------------------------------------- 55 Motility Stab and Oxygen Requirement ----------------------------------------------------------------------------- 56 Questions ------------------------------------------------------------------------------------------------------------------------------ 56 Eukaryotic Survey ------------------------------------------------------------------------------------------------------------ 57 Question ------------------------------------------------------------------------------------------------------------------------------- 57 Fungi ------------------------------------------------------------------------------------------------------------------------- 58 Zygomycota --------------------------------------------------------------------------------------------------------------------------- 58 Ascomycota --------------------------------------------------------------------------------------------------------------------------- 59 Basidiomycota ------------------------------------------------------------------------------------------------------------------------ 59 Chytridiomycota --------------------------------------------------------------------------------------------------------------------- 60 Deuteromycotes (abstract group not used for taxonomic identities)-------------------------------------------------- 60 Protozoa -------------------------------------------------------------------------------------------------------------------- 62 Sarcodina ------------------------------------------------------------------------------------------------------------------------------ 62 Actinopodia --------------------------------------------------------------------------------------------------------------------------- 63 Ciliophora ------------------------------------------------------------------------------------------------------------------------------ 63 Mastigophora ------------------------------------------------------------------------------------------------------------------------ 63 Apicomplexa -------------------------------------------------------------------------------------------------------------------------- 64 Euglena --------------------------------------------------------------------------------------------------------------------------------- 64 Streptomyces and Antibiosis ---------------------------------------------------------------------------------------------- 65 Questions ------------------------------------------------------------------------------------------------------------------------------ 66 Antibiotic Sensitivity Testing ---------------------------------------------------------------------------------------------- 67 Questions ------------------------------------------------------------------------------------------------------------------------------ 68 Zones of Clearing for Various Antibiotics --------------------------------------------------------------------------- 69 Bacterial Growth Curve----------------------------------------------------------------------------------------------------- 70 Questions ------------------------------------------------------------------------------------------------------------------------------ 73 Optimum Growth Temperature ------------------------------------------------------------------------------------------ 74 Questions ------------------------------------------------------------------------------------------------------------------------------ 75 Fermentative Patterns of Gram-Negative Bacteria ------------------------------------------------------------------ 76 Questions ------------------------------------------------------------------------------------------------------------------------------ 78 Further Information: Biochemical Tests----------------------------------------------------------------------------- 79 Nitrogen-Fixing Bacteria --------------------------------------------------------------------------------------------------- 89 Questions ------------------------------------------------------------------------------------------------------------------------------ 91 Enzyme Induction in E. coli ------------------------------------------------------------------------------------------------ 92 Questions ------------------------------------------------------------------------------------------------------------------------------ 95 Human Microbiome --------------------------------------------------------------------------------------------------------- 96 Questions ------------------------------------------------------------------------------------------------------------------------------ 98 Microbes and Food Science ----------------------------------------------------------------------------------------------- 99 Lactic Acid Bacteria ------------------------------------------------------------------------------------------------------ 99 Questions ---------------------------------------------------------------------------------------------------------------------------- 101 Prevalence of Antibiotic-Resistance in the Environment -------------------------------------------------------- 102 Photosynthetic Bacteria and Algae ------------------------------------------------------------------------------------ 107 Questions ---------------------------------------------------------------------------------------------------------------------------- 108 Further Information ---------------------------------------------------------------------------------------------------- 109 Cyanobacteria ---------------------------------------------------------------------------------------------------------------------- 109 Algae ---------------------------------------------------------------------------------------------------------------------------------- 111 Bacteriophage: T3 --------------------------------------------------------------------------------------------------------- 112 Questions ---------------------------------------------------------------------------------------------------------------------------- 114 Water Quality Analysis --------------------------------------------------------------------------------------------------- 116 Questions ---------------------------------------------------------------------------------------------------------------------------- 119

iii APPENDICES ----------------------------------------------------------------------------------------------------------------- 120 List of Reagents --------------------------------------------------------------------------------------------------------- 121 Biochemical Table ------------------------------------------------------------------------------------------------------ 123 Glossary of Important Terms ---------------------------------------------------------------------------------------- 124

2 Laboratory Schedule Lab Session Exercises for that Session Week 1 Lab Safety The Spread of Contagious Diseases: How To See Germs Spread (online video: https://www.youtube.com/watch?v=I5-dI74zxPg How a Virus Spreads So Easily | MythBusters online video: https://www.youtube.com/watch?v=UbQ9Kl9CqUU ) Routine Techniques Used in Microbiology: Use of the Microscope Cytological Techniques: Gram Staining Streak for Colony Isolation (aseptic culture techniques) Culture inoculation (aseptic culture techniques) Discuss Scientific Method of Inquiry (online) Week 2 Continued Lesson: Gram Staining (practice) Streak for Colony Isolation (practice) New Lessons: Enumerating Microorganisms and serial dilutions – Part A and B Spread Plating Urban Microbiome – Step 1 Week 3 Continued Lessons: Enumerating Microorganisms – Part C Practice cell enumeration New Lessons: Micropipettor Usage Cytological Techniques: Special Cell Stains – Endospore Stain Week 4 QUIZ #1 Continued Lessons: Urban Microbiome – Step 2 and 3 New Lessons: Cytological Techniques: Special Cell Stains – Acid Fast Stain Survey of Eukaryotic Microbes (online) Week 5 Continued Lessons: Observe special stains New Lessons: Cytological Techniques: Bacteria Motility – Flagella stain and Motility stab Streptomyces and Antibiosis – Part A Week 6 Continued Lessons: Streptomyces and Antibiosis – Part B Urban Microbiome – Step 4, 5, and 6 New Lessons: Bacterial Growth Curve Week 7 Midterm Practical Exam Continued Lessons: Streptomyces and Antibiosis – Part C Analysis of bacterial growth curve

3 Week 8 Continued Lesson Urban Microbiome – Step 7 New Lessons: Temperature and Growth of Microbes – Part A Fermentative Patterns of Gram (-) Bacteria – Part A (online?) Week 9 Continued Lessons: Temperature and Growth of Microbes – Part B Fermentative Patterns of Gram (-) Bacteria – Part B (online?) New Lesson: Enzyme Induction in E. coli Isolation of Nitrogen-Fixing Bacteria – Part A Week 10 QUIZ #2 Continued Lessons: Isolation of Nitrogen-Fixing Bacteria – Part B New Lessons: Food Microbiology: Lactic Acid Bacteria – Part A Human Microbiome – Part A Week 11 Continued Lessons: Food Microbiology: Lactic Acid Bacteria – Part B Human Microbiome – Part B Urban Microbiome – Analysis New Lesson: Bacteriophage: T3 – Part A Photosynthetic Bacteria and Algae Week 12 Continued Lessons: Food Microbiology: Lactic Acid Bacteria – Part C Urban Microbiome – Analysis New Lessons: Bacteriophage: T3 – Part B Sanitary Water Analysis – Part A Week 13 Continued Lessons: Urban Microbiome – Analysis Sanitary Water Analysis – Part B Practice, if available: Enumerating Microorganisms and serial dilutions Cytological Techniques: Gram Staining Week 14 Final Practical Exam Clean-up

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