Tissue Culture Facilities Flashcards

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1
Q

Agar

A
  • a gelling agent
  • an algal extract that solidifies after heating
  • an extract from red algae
  • most commonly used
  • it is used as a support for explants in tissue culture.
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2
Q

Autoclave

A
  • used to sterilize media, containers, and tools
  • needs to be capable of reaching 120C
    • to keep water from boiling, has to have a pressure aspect also
  • a household pressure cooker can be used by amateurs or small specialty growers
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3
Q

Gelrite

A
  • a gelling agent
  • an agar substitute
  • a polusaccharide derived from Pseudomonas bacteria
  • used to support the explant
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4
Q

Glass Bead Sterilizer

A
  • a device used to sterilize work tools
  • heated beads at 250C sterilized instruments in 10 to 15 seconds
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5
Q

Growing Area

A
  • the area where the cultures grow between subcultures
  • should be a separate area from Preparation Area and Transfer Area
    • often have to walk through separate room before can go in
    • kept very clean!
  • should have temperature and light control
    • light for 16 hours but irradiance is kept down
    • temp kept between 70-85F
    • humidity control is not important b/c each container is sealed
      • but not air tight so no high air flow in room b/c can force spores under the container caps
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6
Q

Hepa Filter

A
  • the filter through which air is pushed in a laminar flow hood
  • stands for High Efficiency Particulate Air (HEPA) filter
  • it removes microbial spores
  • must turn on hod at least 30 minutes before use
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7
Q

Laminar Airflow Hood

A
  • a cabinet that filters air through a HEPA filter to eliminate fungal and bacterial spores from tissue culture work spaces
  • open-sided hood where filtered air is passed from the rear of the hood outward on a positive pressure gradient
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8
Q

Media

A
  • a basic tissue culture medium contains inorganic salts, organic compounds, and a gelling agent
  • may be purchased as pre-mixed formulations or made from scratch
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9
Q

Media Components - Hormones

A
  • auxin and cytokinins are 2 most important hormones used to control organ and tissue development
    • usually stable and added prior to autoclaving
  • gibberellins sometimes used to promote shoot elongation
    • should be filter sterilized and not autoclaved
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10
Q

Media Components - Miscellaneous Materials

A
  • vitamines
    • may or may not be used
  • antioxidents - to prevent excessive browning
    • may or maynot be added
    • citric acid, ascorbic acid, activated charcoal, PVP
  • materials of unknown composition
    • may add organic nitrogen or hormones
    • like: coconut milk, casein hydrolysates, malt, yeast extracts
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11
Q

Media Components - Salts

A
  • provide the macroelements (nitrogen, phosphorus, potassium, calcium, and magnesium) and microelements (boron, cobalt, copper, manganese, iodine, iron, and zinc)
  • these salts can be made up in stock solutions by group and stored in a refridgerator
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12
Q

Media Components - Sugar

A
  • carbohydrates as an energy source
  • added at time of making he medium
  • usually use sucrose at 2-4%
    • up to 12% can be used
      • but sugar is osmotically active so can get low water potential
  • glucose has been used
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13
Q

Preparation Area

A
  • a lab where media is prepared and sterilized, glassware is cleaned and supplies are stored
  • looks like kitchen or wet lab
  • a separate area from the Transfer or Growing Area
  • needs equipment to heat the media, weigh out chemicals, measure pH, and dispense media
  • kept very clean!
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14
Q

Stock Solution

A
  • the concentrated solutions that are used to store inorganic and organic ingredients for tissue culture media preparation
  • usually 100 times the concentration found in the final medium
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15
Q

Transfer Area

A
  • where explants are initially extablished and all subcultures and transfers occur
  • a separate room from the Preparation or Growing Area
  • needs to be steril and free of microbial contamination
  • has a laminar flow hood with an HEPA filter and glass bead sterilizers
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