Laboratory Manual
, Laboratory Manual for
Biotechnology and Laboratory Science
This manual explores the basic laboratory skills and concepts essential for a career in biotechnology and other
laboratory sciences. Written by four biotechnology instructors, all with over 25 years of teaching experience, it
incorporates instruction, exercises, and laboratory activities that the authors have been using and perfecting for years.
These exercises and activities help students understand the fundamentals of working in a biotechnology laboratory. As
students build skills through an organized and systematic presentation of materials, procedures, and tasks, they also
explore overarching themes that relate to all biotechnology workplaces.
Features
◾ Provides clear instructions and step-by-step exercises to make learning the material easier for students.
◾ Emphasizes fundamental laboratory skills that prepare students for the industry.
◾ Builds students’ skills through an organized and systematic presentation of materials, procedures, and tasks.
◾ Updates reflect recent innovations and regulatory requirements to ensure students stay up to date.
◾ Introduces skills important for careers in forensic, clinical, quality control, environmental, and other testing
laboratories.
,
,Laboratory Manual for Biotechnology
and Laboratory Science: The Basics,
Revised Edition
Authored By
Lisa A. Seidman
Faculty Emeritus, Madison Area Technical College
Mary Ellen Kraus
Faculty, Madison Area Technical College
Diana Lietzke Brandner
Former Laboratory Coordinator, Madison Area Technical College
Jeanette Mowery
Faculty Emeritus, Madison Area Technical College
,Revised edition published 2023
by CRC Press
6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742
and by CRC Press
4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN
© 2023 Taylor & Francis Group, LLC
First edition published by Pearson Education, Inc. publishing as Benjamin Cummings 2011
CRC Press is an imprint of Taylor & Francis Group, LLC
Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all
materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this pub-
lication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged
please write and let us know so we may rectify in any future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic,
mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or
retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc.
(CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC please contact
Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without
intent to infringe.
ISBN: 978-1-032-41993-0 (hbk)
ISBN: 978-1-032-41991-6 (pbk)
ISBN: 978-1-003-36074-2 (ebk)
DOI: 10.1201/9781003360742
Typeset in Warnock Pro
by Deanta Global Publishing Services, Chennai, India
,Contents
Preface .................................................................................................................................................................................................................................................................. ix
Companion Textbooks............................................................................................................................................................................................................................... xi
Acknowledgments .....................................................................................................................................................................................................................................xiii
Authors ...............................................................................................................................................................................................................................................................xv
Introduction .................................................................................................................................................................................................................................................. xvii
UNIT I Safety in the Laboratory ..............................................................................................................................................................................................1
Unit Introduction .............................................................................................................................................................................................................2
Safety Part 1: Creating a Safe Workplace ...........................................................................................................................................................3
Classroom Activity 1: Performing A Risk Assessment ................................................................................................................................4
Classroom Activity 2: Exploring Safety-Related Government Websites ........................................................................................6
Classroom Activity 3: Responding to Emergencies ....................................................................................................................................9
Safety Part 2: Working Safely with Chemicals ..............................................................................................................................................11
Classroom Activity 4: Understanding the Chemicals with Which You Work ............................................................................15
Classroom Activity 5: Personal Protection ......................................................................................................................................................21
Laboratory Exercise 1: Tracking the Spread of Chemical Contamination................................................................................... 25
Classroom Activity 6: Analyzing Safety Issues in a Laboratory Procedure................................................................................. 29
Safety Part 3: Working Safely with Biological Hazards ........................................................................................................................... 34
Laboratory Exercise 2: Production of Bioaerosols and Factors Affecting Aerosol Production ...................................... 38
Unit Discussion: Safety in the Laboratory ...................................................................................................................................................... 40
Safety Contract ...............................................................................................................................................................................................................41
UNIT II Documentation in the Laboratory..................................................................................................................................................................... 45
Unit Introduction .......................................................................................................................................................................................................... 46
Classroom Activity 7: Being an Auditor ........................................................................................................................................................... 49
Laboratory Exercise 3: Keeping a Laboratory Notebook ...................................................................................................................... 53
Classroom Activity 8: Writing and Following an SOP ..............................................................................................................................61
Unit Discussion: Documentation in the Laboratory................................................................................................................................ 65
UNIT III Metrology in the Laboratory ................................................................................................................................................................................. 67
Unit Introduction .......................................................................................................................................................................................................... 68
Laboratory Exercise 4: Recording Measurements with the Correct Number of Significant Figures ..........................74
Classroom Activity 9: Constructing a Simple Balance............................................................................................................................ 79
Laboratory Exercise 5: Weight Measurements 1: Good Weighing Practices .............................................................................81
Laboratory Exercise 6: Weight Measurements 2: Performance Verification.............................................................................. 89
Laboratory Exercise 7: Volume Measurements 1: Proper Use of Volume-Measuring Devices....................................... 94
Laboratory Exercise 8: Volume Measurements 2: Performance Verification of a Micropipette ..................................104
Laboratory Exercise 9: Measuring pH with Accuracy and Precision............................................................................................108
Unit Discussion: Metrology in the Laboratory ...........................................................................................................................................119
UNIT IV Spectrophotometry and the Measurement of Light............................................................................................................................ 121
Unit Introduction ........................................................................................................................................................................................................122
Laboratory Exercise 10: Color and the Absorbance of Light ............................................................................................................126
Laboratory Exercise 11: Concentration, Absorbance, and Transmittance ................................................................................135
Laboratory Exercise 12: Preparing a Standard Curve with Food Coloring and Using it for Quantitation .............139
Classroom Activity 10: Beer’s Law and Calculating an Absorptivity Constant ......................................................................144
v
,vi Contents
Laboratory Exercise 13: Determination of the Absorptivity Constant for ONP .....................................................................148
Unit Discussion: Spectrophotometry and the Measurement of Light....................................................................................... 151
UNIT V Biological Solutions ...................................................................................................................................................................................................153
Unit Introduction ........................................................................................................................................................................................................154
Classroom Activity 11: Getting Ready to Prepare Solutions with One Solute: Calculations .......................................... 159
Classroom Activity 12: Getting Ready to Prepare Solutions with One Solute: Ordering Chemicals ........................164
Laboratory Exercise 14: Preparing Solutions with One Solute ........................................................................................................167
Laboratory Exercise 15: Preparing Solutions to the Correct Concentration ........................................................................... 174
Laboratory Exercise 16: Working with Buffers ...........................................................................................................................................178
Laboratory Exercise 17: Preparing Breaking Buffer .................................................................................................................................185
Laboratory Exercise 18: Preparing TE Buffer ...............................................................................................................................................187
Laboratory Exercise 19: More Practice Making a Buffer....................................................................................................................... 191
Laboratory Exercise 20: Making a Quality Product in a Simulated Company........................................................................192
Unit Discussion: Biological Solutions ..............................................................................................................................................................194
UNIT VI Assays.................................................................................................................................................................................................................................195
Unit Introduction ........................................................................................................................................................................................................196
Laboratory Exercise 21: Two Qualitative Assays .......................................................................................................................................199
Laboratory Exercise 22: UV Spectrophotometric Assay of DNA: Quantitative Application ..........................................203
Laboratory Exercise 23: UV Spectrophotometric Assay of DNA and Proteins: Qualitative Applications ..............207
Laboratory Exercise 24: The Bradford Protein Assay: Learning the Assay ................................................................................ 211
Laboratory Exercise 25: The Bradford Protein Assay: Exploring Assay Verification............................................................. 217
Laboratory Exercise 26: The Beta-Galactosidase Enzyme Assay....................................................................................................221
Laboratory Exercise 27: Comparing the Specific Activity of Two Preparations of Beta-Galactosidase ..................227
Laboratory Exercise 28: Using Spectrophotometry for Quality Control: Niacin ...................................................................233
Unit Discussion: Assays............................................................................................................................................................................................238
UNIT VII Biological Separation Methods .......................................................................................................................................................................... 241
Unit Introduction ........................................................................................................................................................................................................242
Classroom Activity 13: Planning for Separating Materials Using a Centrifuge .......................................................................244
Laboratory Exercise 29: Separation of Two Substances Based on Their Differential Affinities for
Two Phases .....................................................................................................................................................................................................................258
Laboratory Exercise 30: Separation and Identification of Dyes Using Paper Chromatography .................................261
Laboratory Exercise 31: Separating Molecules by Agarose Gel Electrophoresis ..................................................................265
Laboratory Exercise 32: Using Agarose Gel Electrophoresis to Perform an Assay ..............................................................273
Laboratory Exercise 33: Optimizing Agarose Gel Electrophoresis ................................................................................................278
Laboratory Exercise 34: Quantification of DNA by Agarose Gel Electrophoresis ................................................................281
Laboratory Exercise 35: Introduction to Ion Exchange Column Chromatography ............................................................286
Unit Discussion: Biological Separation Methods .................................................................................................................................... 294
Unit VIII Growing Cells ................................................................................................................................................................................................................295
Unit Introduction ........................................................................................................................................................................................................296
Laboratory Exercise 36: Using a Compound Light Microscope .....................................................................................................299
Laboratory Exercise 37: Aseptic Technique on an Open Lab Bench.......................................................................................... 304
Laboratory Exercise 38: Working with Bacteria on an Agar Substrate: Isolating Individual Colonies ..................... 311
Laboratory Exercise 39: Gram Staining ..........................................................................................................................................................320
Laboratory Exercise 40: Preparing Phosphate-Buffered Saline.......................................................................................................324
Laboratory Exercise 41: The Aerobic Spread-Plate Method of Enumerating Colony-Forming Units ......................326
Laboratory Exercise 42: Preparing a Growth curve for E. coli ...........................................................................................................333
Laboratory Exercise 43: Aseptic Technique in a Biological Safety Cabinet .............................................................................337
Laboratory Exercise 44: Making Ham’s F-12 Medium from Dehydrated Powder ................................................................343
Laboratory Exercise 45: Examining, Photographing, and Feeding CHO Cells .......................................................................347
, Contents vii
Laboratory Exercise 46: Counting Cells Using a Hemacytometer ................................................................................................354
Laboratory Exercise 47: Subculturing CHO Cells .....................................................................................................................................359
Laboratory Exercise 48: Preparing a Growth Curve for CHO Cells ................................................................................................362
Unit Discussion: Growing Cells .......................................................................................................................................................................... 364
Appendix 1: Abbreviations and Acronyms Used in this Laboratory Manual ..................................................................................367
Appendix 2: Glossary ........................................................................................................................................................................................................................369
Appendix 3: Selected Bibliography .....................................................................................................................................................................................379
Appendix 4: Brief Metric Review.............................................................................................................................................................................................383
Appendix 5: Calculating Standard Deviation ..............................................................................................................................................................385
Appendix 6: Equipment, Supplies, and Reagents Required for Each Unit ........................................................................................389
Appendix 7: Recipes and Preparation Notes ...............................................................................................................................................................395
Index ................................................................................................................................................................................................................................................................ 419
,
, Preface
The authors of this manual are faculty in the Biotechnology critical to the success of cutting-edge research scientists
Laboratory Technician Program at Madison Area who probe the inner workings of nature. They are essen-
Technical College which prepares students to become lab- tial for scientists who develop ideas into practical prod-
oratory professionals. Since the inception of this program, ucts. The same fundamentals apply to laboratory analysts
we have been challenged to define the nature of the work who analyze samples in forensic, clinical, quality control,
for which we are preparing students, and the knowledge, and other testing laboratories. The way in which the basics
skills, and attitudes that our students need to achieve to be “play out” may vary in different workplaces, yet the funda-
successful. This manual is the distillation of our conversa- mentals remain, well … fundamental. Thus, this manual
tions, interactions with talented colleagues, and 25-plus aims to systematically build students’ basic skills, intro-
years of rewarding experiences teaching students. duce the fundamental principles underlying these basic
When we first developed our program, we began, as tasks, and explore overarching themes that relate to all
do many undergraduate laboratory teachers, with an laboratory workplaces.
experiment or two to introduce students to the scientific The goal of this manual is to help students establish a
method. Undergraduate experimentation teaches stu- coherent, integrated understanding of laboratory work. It
dents the important lesson that science is not a dreary is therefore organized in a particular way. At the first level
collection of facts but is an engaging process and a logical of organization are the basic tasks or skills that students
way of inquiring about nature. There is, however, a prob- explore, such as working safely and maintaining a labora-
lem inherent in much undergraduate research: experi- tory notebook. These tasks are the subject of individual
mentation that is performed badly is unlikely to answer exercises. At the next level, the exercises are organized
any scientific questions at all. In an attempt to mitigate into units, such as safety and documentation, which have
this problem, colleges sometimes hire professional staff unifying fundamental principles. At the highest level of
who maintain and calibrate equipment, prepare materials organization, certain themes of quality work, such as the
and solutions, and perform other essential tasks behind importance of reducing variability, are integrated into
the scenes in rooms that students never enter. The result every activity and discussion.
is that beginning students do not learn the basic skills that Perhaps the methods covered in this manual (such as
make laboratory investigation meaningful and they do not how to prepare a solution) seem less glamorous than such
appreciate the challenges of making things “work” in the tasks as editing DNA. However, we have learned from
laboratory. We found that this customary approach is not our students that developing a deep understanding of the
the most effective in preparing professional biotechnolo- fundamentals of laboratory work is not only essential, it is
gists. Instead, we now begin with the concept that pro- also rewarding.
ducing quality laboratory work requires understanding
fundamental principles and mastering fundamental tech-
niques. We developed this laboratory manual to be an
early stepping stone for students on the path to becoming
professionals.
This manual was originally developed for associate
degree college students. We knew that these individu-
als would be called on to perform basic laboratory tasks.
Over time, however, we have instructed teachers, labora-
tory professionals, and individuals with bachelor of sci-
ence degrees trying to enter the job market. Many of these
individuals never had the opportunity to learn and prac-
tice laboratory fundamentals, despite having academic
degrees and experience. We have realized that the same
fundamental skills, knowledge, and attitudes are essential
for any laboratory professional. These fundamentals are
ix
, Laboratory Manual for
Biotechnology and Laboratory Science
This manual explores the basic laboratory skills and concepts essential for a career in biotechnology and other
laboratory sciences. Written by four biotechnology instructors, all with over 25 years of teaching experience, it
incorporates instruction, exercises, and laboratory activities that the authors have been using and perfecting for years.
These exercises and activities help students understand the fundamentals of working in a biotechnology laboratory. As
students build skills through an organized and systematic presentation of materials, procedures, and tasks, they also
explore overarching themes that relate to all biotechnology workplaces.
Features
◾ Provides clear instructions and step-by-step exercises to make learning the material easier for students.
◾ Emphasizes fundamental laboratory skills that prepare students for the industry.
◾ Builds students’ skills through an organized and systematic presentation of materials, procedures, and tasks.
◾ Updates reflect recent innovations and regulatory requirements to ensure students stay up to date.
◾ Introduces skills important for careers in forensic, clinical, quality control, environmental, and other testing
laboratories.
,
,Laboratory Manual for Biotechnology
and Laboratory Science: The Basics,
Revised Edition
Authored By
Lisa A. Seidman
Faculty Emeritus, Madison Area Technical College
Mary Ellen Kraus
Faculty, Madison Area Technical College
Diana Lietzke Brandner
Former Laboratory Coordinator, Madison Area Technical College
Jeanette Mowery
Faculty Emeritus, Madison Area Technical College
,Revised edition published 2023
by CRC Press
6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487-2742
and by CRC Press
4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN
© 2023 Taylor & Francis Group, LLC
First edition published by Pearson Education, Inc. publishing as Benjamin Cummings 2011
CRC Press is an imprint of Taylor & Francis Group, LLC
Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all
materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this pub-
lication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged
please write and let us know so we may rectify in any future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic,
mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or
retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc.
(CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC please contact
Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without
intent to infringe.
ISBN: 978-1-032-41993-0 (hbk)
ISBN: 978-1-032-41991-6 (pbk)
ISBN: 978-1-003-36074-2 (ebk)
DOI: 10.1201/9781003360742
Typeset in Warnock Pro
by Deanta Global Publishing Services, Chennai, India
,Contents
Preface .................................................................................................................................................................................................................................................................. ix
Companion Textbooks............................................................................................................................................................................................................................... xi
Acknowledgments .....................................................................................................................................................................................................................................xiii
Authors ...............................................................................................................................................................................................................................................................xv
Introduction .................................................................................................................................................................................................................................................. xvii
UNIT I Safety in the Laboratory ..............................................................................................................................................................................................1
Unit Introduction .............................................................................................................................................................................................................2
Safety Part 1: Creating a Safe Workplace ...........................................................................................................................................................3
Classroom Activity 1: Performing A Risk Assessment ................................................................................................................................4
Classroom Activity 2: Exploring Safety-Related Government Websites ........................................................................................6
Classroom Activity 3: Responding to Emergencies ....................................................................................................................................9
Safety Part 2: Working Safely with Chemicals ..............................................................................................................................................11
Classroom Activity 4: Understanding the Chemicals with Which You Work ............................................................................15
Classroom Activity 5: Personal Protection ......................................................................................................................................................21
Laboratory Exercise 1: Tracking the Spread of Chemical Contamination................................................................................... 25
Classroom Activity 6: Analyzing Safety Issues in a Laboratory Procedure................................................................................. 29
Safety Part 3: Working Safely with Biological Hazards ........................................................................................................................... 34
Laboratory Exercise 2: Production of Bioaerosols and Factors Affecting Aerosol Production ...................................... 38
Unit Discussion: Safety in the Laboratory ...................................................................................................................................................... 40
Safety Contract ...............................................................................................................................................................................................................41
UNIT II Documentation in the Laboratory..................................................................................................................................................................... 45
Unit Introduction .......................................................................................................................................................................................................... 46
Classroom Activity 7: Being an Auditor ........................................................................................................................................................... 49
Laboratory Exercise 3: Keeping a Laboratory Notebook ...................................................................................................................... 53
Classroom Activity 8: Writing and Following an SOP ..............................................................................................................................61
Unit Discussion: Documentation in the Laboratory................................................................................................................................ 65
UNIT III Metrology in the Laboratory ................................................................................................................................................................................. 67
Unit Introduction .......................................................................................................................................................................................................... 68
Laboratory Exercise 4: Recording Measurements with the Correct Number of Significant Figures ..........................74
Classroom Activity 9: Constructing a Simple Balance............................................................................................................................ 79
Laboratory Exercise 5: Weight Measurements 1: Good Weighing Practices .............................................................................81
Laboratory Exercise 6: Weight Measurements 2: Performance Verification.............................................................................. 89
Laboratory Exercise 7: Volume Measurements 1: Proper Use of Volume-Measuring Devices....................................... 94
Laboratory Exercise 8: Volume Measurements 2: Performance Verification of a Micropipette ..................................104
Laboratory Exercise 9: Measuring pH with Accuracy and Precision............................................................................................108
Unit Discussion: Metrology in the Laboratory ...........................................................................................................................................119
UNIT IV Spectrophotometry and the Measurement of Light............................................................................................................................ 121
Unit Introduction ........................................................................................................................................................................................................122
Laboratory Exercise 10: Color and the Absorbance of Light ............................................................................................................126
Laboratory Exercise 11: Concentration, Absorbance, and Transmittance ................................................................................135
Laboratory Exercise 12: Preparing a Standard Curve with Food Coloring and Using it for Quantitation .............139
Classroom Activity 10: Beer’s Law and Calculating an Absorptivity Constant ......................................................................144
v
,vi Contents
Laboratory Exercise 13: Determination of the Absorptivity Constant for ONP .....................................................................148
Unit Discussion: Spectrophotometry and the Measurement of Light....................................................................................... 151
UNIT V Biological Solutions ...................................................................................................................................................................................................153
Unit Introduction ........................................................................................................................................................................................................154
Classroom Activity 11: Getting Ready to Prepare Solutions with One Solute: Calculations .......................................... 159
Classroom Activity 12: Getting Ready to Prepare Solutions with One Solute: Ordering Chemicals ........................164
Laboratory Exercise 14: Preparing Solutions with One Solute ........................................................................................................167
Laboratory Exercise 15: Preparing Solutions to the Correct Concentration ........................................................................... 174
Laboratory Exercise 16: Working with Buffers ...........................................................................................................................................178
Laboratory Exercise 17: Preparing Breaking Buffer .................................................................................................................................185
Laboratory Exercise 18: Preparing TE Buffer ...............................................................................................................................................187
Laboratory Exercise 19: More Practice Making a Buffer....................................................................................................................... 191
Laboratory Exercise 20: Making a Quality Product in a Simulated Company........................................................................192
Unit Discussion: Biological Solutions ..............................................................................................................................................................194
UNIT VI Assays.................................................................................................................................................................................................................................195
Unit Introduction ........................................................................................................................................................................................................196
Laboratory Exercise 21: Two Qualitative Assays .......................................................................................................................................199
Laboratory Exercise 22: UV Spectrophotometric Assay of DNA: Quantitative Application ..........................................203
Laboratory Exercise 23: UV Spectrophotometric Assay of DNA and Proteins: Qualitative Applications ..............207
Laboratory Exercise 24: The Bradford Protein Assay: Learning the Assay ................................................................................ 211
Laboratory Exercise 25: The Bradford Protein Assay: Exploring Assay Verification............................................................. 217
Laboratory Exercise 26: The Beta-Galactosidase Enzyme Assay....................................................................................................221
Laboratory Exercise 27: Comparing the Specific Activity of Two Preparations of Beta-Galactosidase ..................227
Laboratory Exercise 28: Using Spectrophotometry for Quality Control: Niacin ...................................................................233
Unit Discussion: Assays............................................................................................................................................................................................238
UNIT VII Biological Separation Methods .......................................................................................................................................................................... 241
Unit Introduction ........................................................................................................................................................................................................242
Classroom Activity 13: Planning for Separating Materials Using a Centrifuge .......................................................................244
Laboratory Exercise 29: Separation of Two Substances Based on Their Differential Affinities for
Two Phases .....................................................................................................................................................................................................................258
Laboratory Exercise 30: Separation and Identification of Dyes Using Paper Chromatography .................................261
Laboratory Exercise 31: Separating Molecules by Agarose Gel Electrophoresis ..................................................................265
Laboratory Exercise 32: Using Agarose Gel Electrophoresis to Perform an Assay ..............................................................273
Laboratory Exercise 33: Optimizing Agarose Gel Electrophoresis ................................................................................................278
Laboratory Exercise 34: Quantification of DNA by Agarose Gel Electrophoresis ................................................................281
Laboratory Exercise 35: Introduction to Ion Exchange Column Chromatography ............................................................286
Unit Discussion: Biological Separation Methods .................................................................................................................................... 294
Unit VIII Growing Cells ................................................................................................................................................................................................................295
Unit Introduction ........................................................................................................................................................................................................296
Laboratory Exercise 36: Using a Compound Light Microscope .....................................................................................................299
Laboratory Exercise 37: Aseptic Technique on an Open Lab Bench.......................................................................................... 304
Laboratory Exercise 38: Working with Bacteria on an Agar Substrate: Isolating Individual Colonies ..................... 311
Laboratory Exercise 39: Gram Staining ..........................................................................................................................................................320
Laboratory Exercise 40: Preparing Phosphate-Buffered Saline.......................................................................................................324
Laboratory Exercise 41: The Aerobic Spread-Plate Method of Enumerating Colony-Forming Units ......................326
Laboratory Exercise 42: Preparing a Growth curve for E. coli ...........................................................................................................333
Laboratory Exercise 43: Aseptic Technique in a Biological Safety Cabinet .............................................................................337
Laboratory Exercise 44: Making Ham’s F-12 Medium from Dehydrated Powder ................................................................343
Laboratory Exercise 45: Examining, Photographing, and Feeding CHO Cells .......................................................................347
, Contents vii
Laboratory Exercise 46: Counting Cells Using a Hemacytometer ................................................................................................354
Laboratory Exercise 47: Subculturing CHO Cells .....................................................................................................................................359
Laboratory Exercise 48: Preparing a Growth Curve for CHO Cells ................................................................................................362
Unit Discussion: Growing Cells .......................................................................................................................................................................... 364
Appendix 1: Abbreviations and Acronyms Used in this Laboratory Manual ..................................................................................367
Appendix 2: Glossary ........................................................................................................................................................................................................................369
Appendix 3: Selected Bibliography .....................................................................................................................................................................................379
Appendix 4: Brief Metric Review.............................................................................................................................................................................................383
Appendix 5: Calculating Standard Deviation ..............................................................................................................................................................385
Appendix 6: Equipment, Supplies, and Reagents Required for Each Unit ........................................................................................389
Appendix 7: Recipes and Preparation Notes ...............................................................................................................................................................395
Index ................................................................................................................................................................................................................................................................ 419
,
, Preface
The authors of this manual are faculty in the Biotechnology critical to the success of cutting-edge research scientists
Laboratory Technician Program at Madison Area who probe the inner workings of nature. They are essen-
Technical College which prepares students to become lab- tial for scientists who develop ideas into practical prod-
oratory professionals. Since the inception of this program, ucts. The same fundamentals apply to laboratory analysts
we have been challenged to define the nature of the work who analyze samples in forensic, clinical, quality control,
for which we are preparing students, and the knowledge, and other testing laboratories. The way in which the basics
skills, and attitudes that our students need to achieve to be “play out” may vary in different workplaces, yet the funda-
successful. This manual is the distillation of our conversa- mentals remain, well … fundamental. Thus, this manual
tions, interactions with talented colleagues, and 25-plus aims to systematically build students’ basic skills, intro-
years of rewarding experiences teaching students. duce the fundamental principles underlying these basic
When we first developed our program, we began, as tasks, and explore overarching themes that relate to all
do many undergraduate laboratory teachers, with an laboratory workplaces.
experiment or two to introduce students to the scientific The goal of this manual is to help students establish a
method. Undergraduate experimentation teaches stu- coherent, integrated understanding of laboratory work. It
dents the important lesson that science is not a dreary is therefore organized in a particular way. At the first level
collection of facts but is an engaging process and a logical of organization are the basic tasks or skills that students
way of inquiring about nature. There is, however, a prob- explore, such as working safely and maintaining a labora-
lem inherent in much undergraduate research: experi- tory notebook. These tasks are the subject of individual
mentation that is performed badly is unlikely to answer exercises. At the next level, the exercises are organized
any scientific questions at all. In an attempt to mitigate into units, such as safety and documentation, which have
this problem, colleges sometimes hire professional staff unifying fundamental principles. At the highest level of
who maintain and calibrate equipment, prepare materials organization, certain themes of quality work, such as the
and solutions, and perform other essential tasks behind importance of reducing variability, are integrated into
the scenes in rooms that students never enter. The result every activity and discussion.
is that beginning students do not learn the basic skills that Perhaps the methods covered in this manual (such as
make laboratory investigation meaningful and they do not how to prepare a solution) seem less glamorous than such
appreciate the challenges of making things “work” in the tasks as editing DNA. However, we have learned from
laboratory. We found that this customary approach is not our students that developing a deep understanding of the
the most effective in preparing professional biotechnolo- fundamentals of laboratory work is not only essential, it is
gists. Instead, we now begin with the concept that pro- also rewarding.
ducing quality laboratory work requires understanding
fundamental principles and mastering fundamental tech-
niques. We developed this laboratory manual to be an
early stepping stone for students on the path to becoming
professionals.
This manual was originally developed for associate
degree college students. We knew that these individu-
als would be called on to perform basic laboratory tasks.
Over time, however, we have instructed teachers, labora-
tory professionals, and individuals with bachelor of sci-
ence degrees trying to enter the job market. Many of these
individuals never had the opportunity to learn and prac-
tice laboratory fundamentals, despite having academic
degrees and experience. We have realized that the same
fundamental skills, knowledge, and attitudes are essential
for any laboratory professional. These fundamentals are
ix
