Tissue Culture Facilities Flashcards
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.
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
3
Q
Gelrite
A
- a gelling agent
- an agar substitute
- a polusaccharide derived from Pseudomonas bacteria
- used to support the explant
4
Q
Glass Bead Sterilizer
A
- a device used to sterilize work tools
- heated beads at 250C sterilized instruments in 10 to 15 seconds
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
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
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
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
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
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
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
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
- up to 12% can be used
- glucose has been used
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!
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
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
