Aseptic Techniques

1
Aseptic Techniques

• Suggested reading Razdan ch. 4 Bhojwani ch. 2
• Aseptic - conditions established to exclude
  contaminants
• Axenic containing only 1 organism

2
Aseptic Techniques

• Why are aseptic conditions needed?
• Microbes grow faster than plant cells
• Microbes can produce toxic metabolites
• Where do contaminants come from?
• Environmental sources
• The plant itself (internally and externally)

3
Aseptic Techniques

• Growth conditions of donor plants
• Most contaminated to least field, greenhouse,
  growth chamber
• Watering practices overhead watering increases
  bacterial populations in greenhouses and growth
  chambers

4
Aseptic Techniques

• Disinfestation removing contaminants from
  external surfaces of an explant
• Explant part of the plant used to initiate the
  culture
• Disinfection removing contaminants both
  externally and internally from an explant
• Sterilization destruction or removal of all
  living organisms

5
Aseptic Techniques

• Techniques for establishing aseptic conditions
• Chemical treatments
• Physical treatments

6
Aseptic Techniques

• Chemical treatments - disinfestants
• Liquid laundry bleach (NaOCl at 5-6 by vol)
• Rinse thoroughly after treatment
• Usu. Diluted 5-20 v/v in water 10 is most
  common
• Calcium hypochlorite Ca(OCl)2 a powder must
  be mixed up fresh ea. time

7
Aseptic Techniques

• Chemical treatments - disinfestants
• Ethanol (EtOH)
• 95 used for disinfesting plant tissues
• Kills by dehydration
• Usu. used at short time intervals (10 sec 1 min)

8
Aseptic Techniques

• Chemical treatments - disinfestants (contin)
• Ethanol (contin)
• 70 used to disinfest work surfaces, worker hands
• Isopropyl alcohol (rubbing alcohol) is sometimes
  recommended for hobbyists

9
Aseptic Techniques

• Chemical treatments disinfectants
• Used only when necessary or when disinfestants
  are ineffective or impractical
• antibiotics common ones are carbenicillin,
  cefotaxime, rifampicin, tetracycline,
  streptomycin

10
Aseptic Techniques

• Chemical treatments disinfectants
• Used only when necessary or when disinfestants
  are ineffective or impractical
• antibiotics common ones are carbenicillin,
  cefotaxime, rifampicin, tetracycline,
  streptomycin

11
Aseptic Techniques

• Chemical treatments disinfectants (contin)
• problems with antibiotics
• tend to be selective
• resistance acquisition
• may obscure presence of microbes
• cell/tissue growth inhibition

12
Aseptic Techniques

• Chemical treatments disinfectants (contin)
• fungicides
• some benzimidizole compounds (related to benomyl)
  shown effective and nontoxic
• carbendizim has been effective in protoplast and
  callus cultures
• virus-inhibiting compounds, such as ribavirin -
  effective against some potato viruses

13
Aseptic Techniques

• Physical treatments
• Sterilization by heat
• Oven (dry heat) suitable for tools, containers
  a 160-180 C for 3 h
• Autoclave steam heat under pressure is faster,
  more effective
• Autoclaves typically generate 15 lbs/in2 and 250
  F (1.1 kg/cm2 and 121 C)

14
Aseptic Techniques

• Physical treatments (contin)
• Sterilization by heat (contin)
• For liquids (such as water, medium), autoclave
  time depends on liquid volume
• Recommended autoclaving times (sterilization time
  only)
• 250 ml requires 15 min
• 500 ml requires 20 min
• 1000 ml requires 25 min

15
Aseptic Techniques

• Physical treatments (contin)
• Sterilization by heat (contin)
• Excessive autoclaving can break down organics a
  typical symptom is caramelized sucrose
• Microwaves (off the shelf) are useful for melting
  agar (but not gellan gum types of solidifying
  agents)
• Special pressurized containers are required for
  sterilizing in a microwave

16
Aseptic Techniques

• Physical treatments (contin)
• Flaming or heating of tools
• Flaming e.g., 95 EtOH in an alcohol burner is
  useful for sterilizing metal instruments
• Bacticinerators heats metal tools in a hot
  ceramic core
• Heated glass beads

17
Aseptic Techniques

• Physical treatments (contin)
• Physical removal of contaminants filtration of
  culture medium
• Some medium ingredients are heat labile, e.g.,
  GA, IAA, all proteins, antibiotics
• Most devices use a paper cellulose filter with
  small pore spaces (0.22 µm)
• Syringes used for small volumes, vacuum
  filtration for lg volumes

18
Aseptic Techniques

• Physical treatments (contin)
• Physical removal of contaminants filtration of
  air
• Transfer hoods usu. generate wind at 27-30 linear
  m per min (or 90-100 ft per min)
• Too slow and air drops contaminants onto your
  work surface too fast causes turbulence and
  excess filter wear
• air "corridors" must be kept free of barriers to
  be effective

19
Instructions for disinfesting fern runner tips

• Collect and wash runner tips under running water
  for a few minutes
• Make up a 10 bleach solution
• Wear disp. polyvinyl gloves
• To a 400 ml screw-cap jar, add a few drops of
  Tween 20, 40 ml of bleach, and 360 ml cold tap
  water
• Place several fern runner tips into the middle of
  a small square piece of single-layer cheesecloth

20
Instructions for disinfesting fern runner tips

• Gather up the corners of the cheesecloth and
  secure with a rubber band
• Place a stir bar and the runner tips into the
  bleach solution
• Tighten the cover of the jar so that it is sealed
  and place on a stir plate
• Stir 15 min, periodically removing the jar from
  the stir plate and turning it upside-down to coat
  all inside surfaces

21
Instructions for disinfesting fern runner tips

• After 15 min, remove the jar from the stir plate
  and, holding the jar over a sink drain, carefully
  unscrew the cover till it it almost free of the
  jar
• Tip the jar to one side while holding the cover
  in place and allow the bleach solution to slowly
  drain out of the jar
• Wipe excess bleach solution from the outside of
  the jar with a paper towel

22
Instructions for disinfesting fern runner tips

• Carry the jar to the transfer hood, spray it down
  with 70 EtOH, and set it inside the hood
• Carry a covered beaker of sterile water to the
  hood, spray it down, and place it inside the hood
• Using aseptic technique, remove the jar cover and
  pour 100 to 200 ml sterile water into the jar
• Replace the cover, swirl the contents for a
  minute or two, and take the jar back to the sink

23
Instructions for disinfesting fern runner tips

• Repeat the rinsing process so that you will have
  rinsed the runner tips at least 4 times with
  sterile water
• Plate the runner tips using aseptic technique