BIOE 202 Section AB1

1
BIOE 202Section AB1

• Lab 1 Cell Culture and Aseptic Technique
• January 14, 2008

2
Lab 1 Overview

• Introductions
• Laboratory and Safety Rules
• Lab Tour and General Information
• Course Grading Procedure
• Laboratory Equipment Practice Exercises
• Intro to Aseptic Technique and Culture Media
• Video In Vitro Insights
• Solution Transfer and Media Prep Exercises
• Microscope Lesson
• Clean Up!

3
Introductions

• Joanne Manaster, Course Coordinator
• Nell Keith, Teaching Assistant
• Stephen Barman, TA Assistant

4
Introductions Nell Keith (TA)

• From Tulsa, OK 9 hours SW of UIUC
• BS Chemical Engineering
• University of Oklahoma, May 2005
• Hobbies Running, Volunteering, Skiing
• Getting married August 9th in Little Rock, AR
• 3rd Year Bioengineering PhD Student at UIUC
• - work under Dr. Rohit Bhargava
• - studying breast cancer using infrared imaging

5
Introductions Nell Keith (TA)
F.N. Keith and R. Bhargava, Technology in Cancer
Research and Treatment, 2008
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Introductions Nell Keith (TA)

• Contact Information
• fkeith2_at_uiuc.edu
• Beckman Institute 4159 244-6271
• Office Hours
• Thursdays 4-5pm
• (Im also available during return days or by
  appointment.)

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Rules of the Laboratory

• Follow general lab safety guidelines from the
  Division of Research Safety at UIUC and the
  specific lab guidelines for the Bioengineering
  Cell Culture Room
• Hang coats on coat hooks behind door and keep
  backpacks out of the way
• No eating or drinking during lab exercises or in
  the cell culture facility at any time
• You may bring a snack for the lecture at the
  beginning of the lab period, but you must finish
  it and dispose of all trash before starting any
  exercises or experiments
• Bring safety glasses or goggles to lab when
  required
• Always wear closed toed shoes on lab and return
  days
• Always tie back long hair and remove all jewelry
• Always clean up after yourself

8
Safety Procedures

• Highlights from the UIUC Division of Research
  Safety and Bioengineering Cell Culture Room
  guidelines
• - Wash hands before leaving the lab
• - Label and properly store all containers
• - Dispose of supplies appropriately
• - sharps container pasteur pipets, glass
  microscope
• slides, razor blades, needles, scalpels
• - trash can kimwipes, plastic serological
  pipets, micropipettor tips, used snap cap tubes
• - Use a bleach solution to disinfect anything
  that
• contained live cells prior to disposal in trash
  can
• - Dispose of chemical wastes in designated waste
• containers
• Take online safety quiz link found on course
  website

9
Lab Tour

• Cell Culture Room
• - 4 cell culture hoods
• - Microscopes
• - Water bath used to warm media and culture
  materials
• Safety Items
• - First aid kit
• - Fire extinguishers
• - Shower and eye wash station
• Bathrooms
• - Mens room on right at hall entrance
• - Womens room at same location on 2nd floor
• Vending machines in DCL basement

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Grading Procedure
More course information is available at the
course website http//www.bioen.uiuc.edu/courses/b
ioe202/index.html
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Other Notes

• Course schedule
• Lab Monday 1-5pm
• Return Day Thursday 2-4pm
• Lecture Thursday 1-2pm 243 MEB
• Will work in groups of two for this semester
• TAs will monitor that each member of the group
  takes turns in the hood so both will learn all
  techniques
• Group number (on your box) determines order of
  hood use each week-changes each week (Week 1,
  groups 1, 2, 3 and 4 start. Week 2, groups 2,
  3,4 and 5 start, etc.)

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Introduction to Cell Culture

• Part 1 Lab Equipment

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Laboratory Equipment Overview

• Pipet Aid

• Sterile Serological Pipets

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Laboratory Equipment Overview

• Conical and round bottom tubes

• Glass bottles

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Laboratory Equipment Overview

• Micropipettor

• Culture Dishes and Flasks

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Now for some practice.

• Three Pipetting Practice Exercises

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Pipetting Practice Exercise 1 (lab manual p. 4)

• Complete table in lab manual (p.4)
• Column B answers are displayed here
• When you are finished, raise your hand and
  Stephen or I will check your work

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Pipetting Practice Exercise 2 (lab manual p.
4-5)

• Label eppendorf tubes
• - Tube Rack Row 1 6 0.5 mL tubes (A-F)
• - Tube Rack Row 2 6 0.5 mL tubes (A-F)
• - Tube Rack Row 3 6 1.5 mL tubes (A-F)
• Use designated colored fluids to pipet volumes
  from your table in exercise 1
• Dispose of tips in designated waste container
• Stephen or I will initial results when you finish

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Pipetting Practice Exercise 3 (lab manual p. 5)

• Assigned volumes are as follows
• Pipet assigned volume onto mass balance and take
  4 readings for each volume
• Dont forget to zero the scale between each
  reading
• Change parafilm between each liquid color
• Find average mass, standard deviation, and error
• Error average mass theoretical mass
• Calculate theoretical value using water density
  of 1 g/mL
• Complete this exercise by the 4th lab period

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Introduction to Cell Culture

• Part 2 Aseptic Technique

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Invisible life forms are everywhere!

• Include bacteria, fungi, and viruses
• Present on skin, fall off of hair, clothing, in
  saliva, tears, breath.
• Present on surfaces and in water
• Especially warm water

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Sterile vs. Aseptic

• Sterile Devoid of all life forms (bacteria,
  fungi, viruses)
• Difficult to achieve once people start
  manipulating the cultures
• Aseptic Reduced numbers of life forms
• Most equipment and reagents provided to you comes
  sterile
• gamma irradiation
• autoclaving
• High temperature and high pressure steam
• filtration

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Controlling microorganism proliferation

• The goal Prevent microbial contamination
• Simple, common sense guidelines
• Handwashing, using 70 ethanol to clean all items
  being used near cells
• Additives to media (philosophies vary from lab to
  lab)
• Antibiotics
• Antifungals

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Biosafety Cabinet

• Laminar Flow hood
• HEPA filtered air
• Air blown down the back of the hood and then
  across the surface
• UV light to kill bacteria
• also mutates human DNA, so it cannot be on when
  using the hood
• Aseptic area
• Must be wiped clean before and after use

http//ohs.uvic.ca/biosafety/biosafetycabinets.htm
l
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Aseptic Technique

• work in culture hood
• roll back long sleeves, no rings, watches,
  bracelets, etc
• hair pulled back, caps off
• wash hands with antibacterial soap, spray down
  with 70 ethanol
• wash all surfaces with 70 EtOH
• dont cough, talk, sneeze, sing into hood, no
  eating or gum chewing
• lids on bottles when not in use
• sterile tips change if they touch anything other
  than media.
• keep tips IN hood
• back surface of hood is cleanest

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Video

• In Vitro Insights

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Aseptic Solution Transfer Practice

• Procedure
• 1) Add 4 mL yellow solution to red solution and
  mix
• 2) Add 2 mL green solution to 10 cm dish and
  spread
• 3) Tip dish to pipet green fluid back into snap
  cap tube
• 4) Add 4 mL red solution to 6 cm dish, 8 mL to
  10 cm dish, 6 mL to flask
• 5) Transfer 200 µL from each plate into separate
  1.5 mL eppendorf tubes
• 6) Add 200 µL blue solution to each tube and mix
  by gently tapping side of closed tube

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Aseptic Solution Transfer Practice

• Important Tips
• - Loosen all caps before beginning aseptic
  transfer
• - Keep fluids covered as much as possible
• - Avoid pulling solutions up into cotton plug at
  top of serological pipets (see Stephen or I if
  this happens)
• - Change pipets between each fluid transfer
• - Use smallest volume pipet possible to improve
  volume accuracy
• - Pipet up and down slowly and keep pipet tip
  below solution surface when mixing to prevent
  bubble formation
• - Avoid touching sterile eppendorf tubes not in
  use

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Introduction to Cell Culture

• Part 3 Media

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Media Components

• Dulbeccos Modified Eagle Media (DMEM)
• Media for growing cells in vitro. Contains
  glucose, glutamine, sodium pyruvate, and
  essential salts
• Contains a pH indicator (phenol red) which gives
  the media its pink/red color at pH 7.2.
• Acidic -yellow or orange (cell growth, bacterial
  growth)
• Basic -purple (no cell growth, not enough CO2)

Acidic
Basic
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Media components, continued

• Penicillin/Streptomycin (Pen/Strep)
• added to prevent bacterial contamination
• Sodium Pyruvate
• A by product of glucose utilization in the Krebs
  cycle
• Helps create more ATP (energy) for the cells
• Sodium Bicarbonate
• Added to some media to help cells grow at a
  higher CO2 concentration
• Our basic DMEM already contains this so we will
  not supplement

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Serum

• Cell nutrient source
• Fetal Bovine Serum (FBS)
• Or Fetal Calf Serum (FCS)
• Newborn Calf Serum (NCS)
• FBS is more nutrient rich than NCS
• NCS content is more consistant and is used more
  commonly

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Aseptic Mixing of Media

• Procedure
• 1) Wash hands and spray them down with 70
  ethanol
• 2) Wipe down hood surface with 70 ethanol and
  large kimwipe
• 3) Spray and wipe bottles and tubes from water
  bath
• 4) Loosen all tube caps before beginning fluid
  transfers
• 5) Add 10 mL NCS to media (labeled DMEM-)
• 6) Add 1 mL pen/strep (p/s) to media
• 7) Add 1 mL sodium pyruvate (NaP) to media
• 8) Label media with initials, date, and DMEM,
  10 NCS, and place it in refrigerator
• 9) Remove all empty bottles and trash from hood
  and wipe it down with ethanol for the next user

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Media Preparation and Inverted Phase Microscope
Lesson

• I will demo media mixing in biosafety hood, then
  you will mix your own media
• While Stephen is helping students working in the
  biosafety hood I will demo the use of the
  inverted phase microscope to view live cells
• After completion of the media prep exercise and
  microscope lesson you can leave
• Dont forget to clean up your lab bench and cell
  culture hood
• See you at lab lecture on Thursday 1-2pm