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Equipment and Aseptic Technique

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used for transferring large volumes of liquid (1-50mls) ... Culture Dishes (plates) and Flasks. Flasks. Capped vessels. Prevent spills. Well-plates ... – PowerPoint PPT presentation

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Title: Equipment and Aseptic Technique


1
Equipment and Aseptic Technique
  • Helping our cells grow

2
Automatic Pipet
  • Pipettor or Pipet-Aid
  • used for transferring large volumes of liquid
    (1-50mls)
  • Important not to draw liquid up into the pipettor

3
Sterile serological pipets
  • Individually wrapped
  • Multiple sizes
  • Numbered along the sides
  • Be sure numbers face you when putting pipet into
    pipettor

4
Tubes, bottles
  • Various sizes
  • Some plastic, some glass
  • Loosen caps before starting to work in the hood

5
Micropipettor
  • For dispensing small volumes of liquid
  • 1-1000ml
  • Different pipettors dispense different ranges of
    liquid
  • Always use a disposable tip with the
    micropipettor

6
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

7
Culture Dishes (plates) and Flasks
  • Flasks
  • Capped vessels
  • Prevent spills
  • Well-plates
  • Multiple areas for experiments with multiple
    variables
  • Different sizes available
  • Dishes
  • Easy to manipulate cells
  • Easier to spill or contaminate

8
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

9
Micro-organisms
  • Bacteria
  • Most are harmless to us
  • Cell culture media will help them grow
  • Bacteria will compete for nutrients in media
  • Fungi
  • Mold, yeast
  • Float in air and are on surfaces

bacteria
yeast
mold
10
More microoganisms
  • Viruses
  • Enter into cells and destroy them in their quest
    to replicate
  • Mycoplasma
  • A bacteria that is hard to discover and hard to
    get rid of.

Viruses-transmission Electron microscopy
Mycoplasm-scanning electron microscopy
11
Sterile vs. Aseptic
  • Sterile No life at all (bacteria, fungi,
    viruses)
  • Hard to keep an area perfectly steril
  • 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

12
Controlling microorganism growth
  • The goal Prevent microbial contamination
  • Simple, common sense guidelines
  • Handwashing, using 70 ethanol to clean all items
    being used near cells
  • Additives to media
  • Antibiotics
  • Antifungals

13
Biosafety Cabinet
  • Laminar Flow hood
  • HEPA filtered air
  • Air blown down the back of the hood and then
    across the surface
  • Aseptic area
  • Must be wiped clean before and after use

14
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

15
CO2 incubator
  • maintains CO2 level (5-10), humidity and
    temperature (37o C) to simulate in vivo
    conditions.

16
Water bath
  • To warm media, TRED and PBS before placing on
    cells
  • Can harbor fungi and bacteria,
  • spray all items that were in the waterbath with
    70 ethanol before placing in the hood.

17
Vacuum pump
  • For permanent aspiration of liquids (media, PBS
    and TRED).
  • Use unplugged glass pasteur pipets, throw into
    sharps box when done.

18
Inverted Phase Microscope
  • A phase contrast microscope with objectives below
    the specimen.
  • Creates contrast in a sample without staining it.
  • Staining cells will kill them.
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