Welcome to Bioengineering 202MCB 493JLM

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Welcome to Bioengineering 202/MCB 493JLM
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What will you do this week?

• Learn about course mechanics
• Learn about handheld equipment and supplies
• Use pipettors and micropipettors
• Transfer fluids from tubes and bottles to flasks
  and plates
• Learn about aseptic technique
• Keep your cells free of competing organisms
• Learn about components of media
• What do cells need to grow well?

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Course Coordinator

• Joanne Manaster, Laboratory Teaching Specialist
• Part time academic professional
• Split between Bioengineering and School of
  MCB/Department of CDB
• Biologist with a passion for histology and cell
  culture
• Not so great with engineeringYet!
• Contact information
• joannema_at_life.uiuc.edu
• 244-2489
• 3103 DCL

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Importance of This Course

• First laboratory course in Bioengineering major
• Fundamentals of cell culture
• Understand the concepts central to Tissue
  Engineering
• Hot topic!
• Learn to write in the scientific style

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The Course

• Lecture
• Saves TAs some time/effort
• Saves students from running over time in lab
• Lab
• Learn to handle cells proficiently
• Learn to work cooperatively
• Learn to document and write about lab work
• Return Day
• Your cells are counting on you!

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Topics

• How cells grow
• Maintaining them in culture
• How cells differentiate
• And what we can do to help them along
• How to analyze differentiated cells
• Understanding stem cells, cell lines and primary
  cultures
• Equipment used in the above procedures

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Books
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Course Website

• http//www.bioen.uiuc.edu/courses/202/
• Announcements
• TA contact information
• Syllabus
• Links to gradebook, preflight quizzes and online
  safety quiz
• Print out powerpoint presentations

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Each week in lab

• Laboratory Notebook
• Duplicate pages
• Copy lab protocol into there before coming to
  class
• Hair Ties
• Keep valuable jewelry at home
• Wear closed toe shoes
• No long flowing sleeves

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Grading

• Online safety quiz
• http//www.drs.uiuc.edu/gls/training/index.aspx?tb
  IDtr
• 5 points
• Pre-flight quizzes
• http//online.physics.uiuc.edu/courses/bioe202/fal
  l08/
• Open at ____ Thursday, Close at 1100am Monday
• 5 points/week, lowest dropped

• Participation
• 15 points/week, lowest dropped
• Lab write-ups
• TAs will explain in more detail
• 3 write ups, first two can be re-graded
• 5 points deducted for each day late
• Final exam
• No date set yet

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Introduction to Cell Culture

• Part 1 Equipment and supplies

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Automatic Pipet

• Pipettor or Pipet-Aid
• use for transferring large volumes of liquid
  (1-50mls)
• Important not to draw liquid up into the pipettor

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Sterile serological pipets

• Individually wrapped
• Multiple sizes
• Numbered along the sides
• Be sure numbers face you when putting pipet into
  pipettor

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Tubes, bottles

• Conical bottom tubes
• Blue or orange capped
• 15ml or 50ml
• Round bottom tubes
• Polypropylene
• 4ml and 14ml
• Snap cap with two stops
• Pyrex bottles
• Autoclavable
• Storage of media

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Micropipettor

• For dispensing small volumes of liquid
• 1-1000ml
• Different pipettors dispense different ranges of
  liquid
• Always use a disposable tip with the
  micropipettor

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What is a microliter?

• In the metric system, used by all scientists
• 1 gram is the weight of a cube of water that
  measures 1cm3this volume is 1milliliter
• 1 liter (l) 1000 milliliters (ml)
• 1 milliliter (ml)one-thousandth of a
  liter 0.001 l 1 x 10-3 l
• 1milliliter 1000 microliters (ml)
• 1 microliter (m l)one-millionth of a liter
  (l) 0.000001 l 1 x 10-6 l
• 1 microliter (ml) one-thousandth of a milliliter
  (ml) 0.001ml 1 x 10-3 ml

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Culture Dishes (plates) and Flasks

• Flasks
• Capped vessels
• Prevent spills
• Harder to manipulate cells in vessel
• Students most often forget to loosen cap to allow
  air into vessel
• Well-plates
• Multiple areas for experiments with multiple
  variables
• 6, 12, 24, 48, 96 well plates
• Dishes
• Easy to manipulate cells
• Large surface area could create evaporation
  issues
• Easier to spill or contaminate

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Introduction to Cell Culture

• Part 2 Aseptic Technique

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Invisible life forms are everywhere!

• 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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Micro-organisms

• Bacteria
• Most are benign, but opportunistic
• Media used for cell culture will maintain
  bacteria
• Bacteria will compete for nutrients in media
• Fungi
• Mold, yeast
• Float in air and are on surfaces
• Grow prolifically

bacteria
yeast
mold
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More microoganisms

• Viruses
• Enter into cells and destroy them in their quest
  to replicate
• May come from natural products used to grow cells
• Mycoplasma
• A bacteria that is cryptic and persistent
• Usually wont destroy host

Viruses-transmission Electron microscopy
Mycoplasm-scanning electron microscopy
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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 serological pipets IN hood while working
  with themdont swing them out when you turn
  around to ask your partner questions.
• back surface of hood is cleanest

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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

• Fetal Bovine Serum (FBS)
• Or Fetal Calf Serum (FCS)
• Newborn Calf Serum (NCS)
• Bovine Calf Serum (BCS)
• added to media as a nutrient source for growing
  cells, variable contents
• Lipids, proteins
• FBS might be more nutrient rich than the other
  two, but otherwise, these terms used
  interchangeably in literature

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Other notes

• 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.)