Tissue Culture Techniques
Media Preparation
1. Organize your work before you start. This will help eliminate wasted time.
Determine the quantity of each medium and the amount of plant growth regulator
needed for each experiment are determined before mixing medium. Make
slightly more medium than is required. A media check off list is helpful in making
medium to keep track of ingredients.
2. Remove media stock solutions and plant growth regulator stock solutions to be
used from the refrigerator. Make up fresh stock solutions if adequate quantities
are not available or if solutions have precipitated.
3. Plant growth regulators stock solutions are prepared biweekly and stored at 4º C
in the dark. Cytokinins (thidiazuron and BA) are dissolved in approximately KOH
prior the addition of deionized water. Gibberellins and auxins (2,4-D, IBA and
NAA) are dissolved in 100% ethanol prior the addition of water.
4. Volumetric flasks are filled ½ to ¾ full with deionized water. Proper aliquots of
stock solution and plant growth regulators are added and each item is marked on
the check off list as used. Sucrose, 30 g 1-1, is added and stirred until dissolved.
Flasks are brought to volume with deionized water and emptied into a larger
beaker or Erlenmeyer flask.
5. The pH meter is calibrated with 7.0 and 4.0 pH buffers prior to adjusting medium
pH. The medium pH is adjusted to 5.8 with 1 N KOH or HC1.
6. Sigma agar, 1 g 1-1, is added to the medium unless otherwise noted. The
medium is placed on high heat and stirred until the agar dissolves. The medium
appears translucent when the agar is in solution. CAUTION: there is only a
slight difference between the temperature at which agar goes into solution and
boils over. DO NOT LEAVE MEDIUM UNATTENDED. Do not heat in
volumetric flasks. If the medium boils over, wait until the hot plates have cooled
and clean off the hot plates with soapy water if necessary.
7. Proper aliquots are poured into culture vessels; test tubes contain 15 ml medium,
glass baby food jars contain 25 ml and GA7s contain 50 ml. Culture vessels are
capped and placed in the autoclave for 20 minutes at 121º C and 1 kg cm -2.
8. Autoclaved medium is stored on the clean medium racks in the laboratory. Avoid
storing medium for more than 2 weeks, it is better to make fresh medium for
successful, repeatable experiments.
9. Put away equipment, throw away trash, rinse glassware and leave the lab
CLEANER than it was when you started.
Special preparation for medium containing antibiotics and bialaphos
Medium containing filtered sterilized ingredients (antibiotics and bialaphos) is prepared
differently since autoclaving may destroy antibiotic or balaphos activity.
1. Antibiotics and bialaphos are dissolved in water or phosphate buffer (pH 7.0) as
indicated. Stock solutions are filtered sterilized through a 22 µm filter disk
attached to a syringe into sterile glass baby food jars under sterile conditions in a
laminar flow hood. Filtered sterilized stock solutions are stored in sterile baby
food jars at 0º C in the dark.
2. Medium preparation is the same as above except after the addition of agar, the
medium is directly placed into Erlenmeyer flasks and autoclaved for 20 minutes.
Do not fill Erlenmeyer flasks more than 2/3 to 3/4 full with medium.
3. Medium is removed from the autoclave. When the temperature reaches
approximately 60º C, filtered sterilized ingredients are added to the medium
under sterile conditions in a laminar flow hood. After mixing, the medium is
poured into sterile culture vessels.
4. Medium is stored at 4ºC in the dark for no longer than 2 weeks.
Laminar Flow Hood Instructions
1. Organize your work before you start. This will help eliminate wasted time.
Prepare media ahead, have plenty of sterile distilled water (if needed) and have
all instruments, plant material(s) and/or cultures to be used assembled.
2. Hands and arms are thoroughly washed with an antibacterial soap prior to
working in the hood and are occasionally sprayed with 70% ethanol while
working in the hood. Note, alcohol will dry skin, therefore you may want to use
hand cream when finished.
3. Ethanol (70%) is used to disinfest the laminar flow hood, culture containers,
instruments and anything that is placed into the hood. WARNING: ethanol is
VERY FLAMMABLE, so be careful around flame or Bacti-cinerators. Fire
extinguishers are located near the hoods. Know the fire extinguisher location
and how to use the fire extinguisher before beginning use in the laminar flow
hood. If the fire extinguisher is used, clean up the mess afterwards.
4. Turn on laminar flow hood (if not already on) and spray all interior surfaces with
ethanol. Place Bacti-cinearators and other electronic equipment (if used) into the
hood. Avoid spraying electronic instruments with ethanol. Thoroughly spray
instruments (i.e. forceps, scalpels), containers or anything else that will go into
the hood with ethanol. Place sprayed items into the hood.
5. Forceps, scalpels and scissors are sterilized in Bacti-cinerators or a Steri-matic
sterilizer for approximately 20 to 30 seconds. Bacti-cinerators should be glowing
red inside before using. Also, make sure alcohol has evaporated before placing
instruments into Bacti-incinerators. Instruments are placed on a disinfected
stainless steel test tube rack for cooling. Sterilization of instruments is performed
after each use to help ensure that cross contamination from other cultures would
be prevented.
6. Plant material is dissected on a glass plate. The glass plate is sprayed with 70%
ethanol and wiped with a clean Kimwipe prior to dissection. Do not wipe with
Kimwipes alone, Kimwipes are not sterile and must be wetted with alcohol.
7. Equipment and culture vessels are placed in a manner as to assure a clear wide
path between the working area and the filter surface. All tissue culture
procedures, (i.e. explant initiation and transfers) are performed as close to the
back of the hood as possible. Working near the hood edge is avoided. The
closer you work to the outside hood edge, the greater the chance that you may
contaminate your cultures.
8. Avoid talking, singing or coughing into the hood. Avoid touching items (and
yourself, such as scratching your nose) that have not been sprayed with alcohol.
If you are sick (cold or flu) avoid working in the hood if possible. This often lead
to contaminated cultures.
9. To avoid contamination, do not rest instruments on the hood surface or contact
any other surfaces once instruments are sterile. Work as quickly and efficiently
as possible.
10. Once finished, clean up the hood and surrounding areas. Put away equipment,
throw away trash, wash glassware and leave the lab CLEANER than it was when
you started. If you poured media in the hood, clean up spilled media from the
hood’s surface.
Murashige and Skoog Stock Solution Preparation
These stock solutions are grouped together based on their compatibility. Nutrients in
each stock solution are weighed, dissolved with deionized water and brought to one liter
final volume.
Organics
Dispense 10 ml stock per liter medium
Final
concentration
mg 1-1
Myo-inositol
Glycine
Nicoltinic acid
Pyridoxine HC1
Thiamine HC1
100.0
2.0
0.5
0.5
0.1
10.0 grams
0.2
0.05
0.05
0.01
Phosphates
Dispense 10 ml stock per liter medium
KH2PO4
H3B03
KI
Na2Mo04 ●2H2O
CoC12 ● 6H2O
17.0
0.62
0.083
0.025
0.0025
170.0
6.2
0.83
0.25
0.025
Nitrates
Dispense 20 ml stock per liter medium
KNO3
NH4NO3
95.0
82.5
1900.0
1650.0
Calcium
Dispense 20 ml stock per liter medium
CaC12
22.0
440.0
Sulphates
Dispense 20 ml stock per liter medium
MgSO4 ● 7H2O
MnSO4 ● 7H2O
ZnSO4 ● 7H2O
CuSO4 ● 5H2O
18.5
1.115
0.43
0.00125
370.0
16.9
8.6
0.025
Iron
Dispense 20 ml stock per liter medium
Na2-EDTA
FeSO4
1.865
1.39
37.3
27.8