Sterilization techniques in Plant Tissue Culture

Physical Sterilization

• By Using Heat

Dry heat

• Flaming In this method heating instruments over the fire until they become hot in red. Instruments that are
used such as point of forceps, Spatulas, inoculating loops, and Wires.
• Incineration It is a process that involves the combustion of organic substances contained in waste materials.
Items such as contaminated cloth, animal carcasses, and pathological material. PVC and polythene can be
dealt with.
• Hot air oven Hot air ovens are the electrical devices used for sterilization. The oven uses dry heat to sterilize
items. Generally, they can be set at minimum to maximum temperature from 50˚C to 300˚C. The thermostat
is present to control the temperature. This is the most widely used method of sterilization by dry heat.
Moist Heat

Categorized into 3 groups:

Temperature below 100° C : Pasteurization of milk: This method is done by holding period at 63˚C for 30
minutes or 72˚C for 15-20 minutes followed by cooling. This method is not suitable for killing all spores.
Temperature around 100 ° C : Steam at atmospheric pressure is used to sterilize culture media. This is an
inexpensive method. The principle of this method is first exposure kills vegetative bacteria and next exposure
will kill vegetative bacteria that mature from the spore. It is intermittent sterilization by holding at a
temperature of 100˚C for 20 minutes on three consecutive days.
Temperature above 100 ° C: An autoclave or steam sterilizer is an instrument that uses steam to sterilize
culture media, equipment and other objects. This implies that all microscopic organisms, infections, parasites,
and spores are inactivated. In any case, prions may not be annihilated via autoclaving at the regular 134˚C for 3
minutes or 121˚C for 15 minutes. It is suitable for the Items such as dressings, instruments, laboratory ware,
media, and medical products.
Filtration

• Filtration assists with eliminating microorganisms from heat-labile fluids such as sera and
solutions of antibiotics.
Types of Filters
.
•Asbestos filters are disposable and single usage. They tend to alkalinize filtered fluids. Their usage
is less because of their carcinogenic property.
•Sintered glass filters These filters have low absorptive properties. They are brittle and costly.
•Membrane filters These filters are made of cellulose esters or other polymers. They are usually used
for water purification and analysis, sterility testing, and preparation of solutions.
HEPA is a type of pleated mechanical air filter. It is an acronym
for "high efficiency particulate air [filter]" (as officially defined by
the U.S. Dept. of Energy). This type of air filter can theoretically
remove at least 99.97% of dust, pollen, mold, bacteria, and any
airborne particles with a size of 0.3 microns (µm)
Radiation
There are 2 types of radiation: Ionizing radiation & non-ionizing radiation

•Non-ionizing radiation In the non-ionizing method infrared is used for rapid mass
sterilization of prepacked items such as syringes, falcon tubes and other plastic
consumables. UV is used for disinfecting enclosed areas such as halls, operation
theatres, and labs.

•Ionizing radiation In ionizing radiation, gamma rays and x-rays are used for
sterilizing plastics, syringes, swabs, catheters, animal feeds, cardboard, oils, and
metal foils.

Chemical Sterilization

•Chemical agents: The action of chemical agents include protein coagulation and disruption of cell membrane
resulting in exposure, damage, and loss of contents.
•Alcohols: Commonly used are Ethyl alcohol, and Isopropyl alcohol and must be utilized at concentrations of 60-
95%. Isopropyl alcohol is used in the sanitization of the clinical thermometer. Methyl alcohol is viable against
contagious spores, treating cabinets, and incubators. It is toxic and inflammable.
•Aldehyde Formaldehyde: It contains bactericidal and sporicidal and it has a great effect on viruses.
•Phenols: The deadly impacts include- it can cause cell membrane damage, releasing cell contents, and causing
lysis.
 E.g. Seed sterilization

Sterilization •contaminated by various microorganisms like fungi and bacteria.

•Sodium hypochlorite solution

NaOCl 0.02% Triton X100

dynamic balance
NaOCl + H2O NaOH + HOCl Na+ + OH- + H+ + OCl-
Hypochlorite ions
NaOH : Saponification reaction
Fatty acid/ lipids + NaOH Soap FA salt + glycerol

NaOH : Neutralization reaction

neutralizes amino acids forming water and salt

Hypochlorous acid / HOCl :Chloramination reaction

•HOCl in contact with organic tissue releases chlorine
• Chlorine combines with protein NH2 groups: Chloramines
•Irreversible oxidation of SH group (sulphydryl group)
•inhibiting bacterial enzymes activity
•interfere in cell metabolism
 Sodium hypochlorite is a strong base (pH>11).

•Inhibition of essential enzymatic sites, such as those in the

membrane

0.02% Triton X100 (0.02% v/v)

Detergent to permeabilize the membranes
Gases

Types of gases used for sterilization are:

•Ethylene oxide works because of its alkylating of the amino, carboxyl, hydroxyl, and sulfhydryl groups in
protein molecules and furthermore on DNA and RNA. It very well may be utilized on instruments such as
heart/lung machines, respirators, dental equipment, books, and clothing.

•Formaldehyde gas This is generally utilized for fumigation of operational theatres and other rooms in
clinics. Formaldehyde is formed by the addition of 150g of KMnO4 to 280ml of formalin for each
1000cu.ft of room volume, in the wake of shutting the windows and different outlets. After fumigation,
the doors ought to be sealed and left unopened for 48 hours.
 Plant Tissue culture

• Involves use of small pieces of plant tissue called explant which are cultured in nutrient medium under appropriate
growth condition
• Explant can rapidly produce callus, somatic embryos, suspension cells, new shoots and new roots (hormonal
balance)
• Needs various combination of nutrients, minerals, plant growth substance, vitamins and sugar (as carbon source)

Suitable for biological contamination (rapid growth of bacteria and fungi)

• Depletion of nutrients
• Production of toxins

Requirements:
Sterilization of the culture medium and the culture container
Surface sterilization of the seeds or plant tissue to be cultured
Sterilization of the instruments used to handle or manipulate the plant tissue
Sterilization of room and/or cabinet and filtered-air environment like laminar flow cabinet

Killing or removing all form of microbial life (including endospores) in a material or an object is defined as the process
of sterilization.
 Aseptic Sterilization techniques

• Media and apparatus are rendered sterile by autoclaving at 15 Psi,(121 deg C ) for 15 min
• Sterile plastic ware
• Filter sterilization is employed for heat-labile substances
• Aseptic transfer by using standard bacteriological techniques using a laminar flow hood.
• Plant tissues collected from fields needs surface sterilization with ethanol and/or clorex
and added surfactant. ( conc. of the surface sterilant and exposure time are determined
empirically depending on the type of explant used.
 Sterilizing Culture Rooms and Transfer Hoods
• Tissue culture lab is compartmentalized into work elements in separately specific areas
such as, media preparation, glassware washing, sterilization, microscopy, and aseptic
transfers.

• Large transfer rooms are sterilized by exposure to UV light (time may vary according to
size or room)
• Transfer rooms can be sterilized by washing them 1-2 times a month with commercial
brands of antifungal liquids.
• Small transfer rooms and hoods can be sterilized with UV light.
• Laminar flow hood : wiping all surface with 95% ethyl alcohol and UV treatment for 15-
20mins.
• Culture room should be initially cleaned with detergent and then carefully wiped down
with a 2% sodium hypochlorite solution or 95% ethanol
• Formaldehyde gas is an excellent disinfectant for large culture area and rooms
• Formalin (commonly used) inactivates viruses and bacteria by inactivating proteins by
forming covalent cross-links with several functional groups.
Fumigation- A disinfection process to decontaminate Tissue culture laboratory
most common method used to decontaminate the tissue culture rooms by using the fumes of various toxic chemicals

• commonly used chemicals in fumigation are formaldehyde solution (Formalin) and potassium permanganate (KMnO4)

“basic principle is that the addition of excess formaldehyde solution to potassium permanganate
(KMnO4) generates fumes of formaldehyde (HCHO and methanol present in formaldehyde solution will
oxidized by potassium permanganate (KMnO4) and leads to the generation of formaldehyde fumes.”
Sterilization of Nutrient Media

1. Steam Sterilization
• Nutrient media are autoclaved at 15 psi and 121 deg C
• Small vol (100ml or less) require 15-20min- large quantity (2-4 lt) may require 30-40min

• The pressure should not exceed 20 psi as high pressure may decompose carbohydrate and other thermo-
labile components of a medium.

Autoclaving can cause the following changes in the medium

❑ Lowering of the pH of the medium by 0.3-0.5 units
❑ Caramelize sugars which may have toxic effect
❑ Volatile substances may be destroyed by the use of autoclave.
❑ Precipitate salts

.
 2. Filter Sterilization
• Proteins, vitamins, amino acids, plant extracts, hormones and carbohydrates are thermo-labile and
decompose during autoclaving
• Millipore/ Nalgene/ Sartorious/ Seitz filters are used with porosity no large than 0.2 microns
• Most filters are of cellulose acetate or cellulose nitrate and are available pre-sterilized, plastic disposable
units.
• During filter sterilization, all the particles, microorganisms and virus that are bigger than the pore
diameter of the filter are removed.

Nutrient media with thermo-liable components

1. A solution of the heat-stable components is sterilized by autoclaving and the cooled to 50-60 deg C under sterile
condition
2. The thermo-liable components are filter-sterilized
3. The sterilized solutions are then combined under aseptic conditions to give the complete media.
 Glass bead sterilizer (steripot) and infra-red sterilizer

• A high watt element heats up the glass beads contained in a

brass crucible at the centre of the box.
• The temperature of the beads is raised to 250°C in 15-20
min. Sterilization of instruments is effected by pushing them
into the beads for 5-7 s.
• A regulator maintains the temperature at 250 °C by a 15 s
cut off

• The infrared sterilizer has a cavity where temperature rises to almost 700 °C.
• Here sterilization is effected by a 2-5 s exposure at this temperature.
• Being well insulated, these sterilizers do not spill out large quantities of heat.

These instruments are also safe compared to a Bunsen burner which could cause heat burns and may also be a fire
hazard.
 Sterilization of Plant Materials

1. Actively growing healthy part of the plants and seeds are surface sterilized

Contaminants • Bacteria
• Fungi
• Yeast

✓ Once explant have been obtained they are washed in mild soapy detergent before treatment with a sterilization
solution
✓ After washing, the explant is rinsed under running tap water for 10-30 min and then submerged into the
disinfectant under sterile condition.
✓ After allotted time, the explant is washed at least three times in sterile distilled water
Flow chart for sterilization of explant for tissue culture
Few recent advances sterilization techniques

Plasma sterilization (low-temperature hydrogen peroxide gas plasma sterilization)

• Plasma: ionized gas- application of electric field to a gas results in ionization into electrons and ions

• utilizes a low-temperature, low-pressure environment to create a unique plasma state that eradicates
microorganisms through a combination of physical and chemical processes.
• sterilizes and inactivates micro-organisms with Hydrogen Peroxide, (H2O2), vapor acting as the sterilizing
agent. It sterilizes medical devices by diffusing H2O2 into the chamber and then “exciting” the
H2O2 molecules into a plasma state.

Benefits:
• Gentle on Sensitive Equipment
• Rapid Turnaround Time
• Reduced Environmental Impact
• Effective Microbial Eradication
 Pulsed light sterilization
• use of xenon lamps for microbiological decontamination
• The first patent for pulsed light technology was granted in 1984 (Hiramoto)

The Claranor pulse is produced in 2 steps:

– A 20 kV pulse lasting a few nanoseconds makes the xenon-filled lamp conductive while creating an electric arc in the
lamp
– The capacitor charged at 3000 V discharges in this arc during a 300-microsecond pulse, which ionizes the gas in the
lamp and generates a plasma emitting a very high-intensity white light (20,000 times greater than sunlight on the
earth’s surface)
The pulse covers the full white light spectrum and has a particularly high UV
output (UV light is well-known for its anti-microbial properties).
 Hydroclave

And unlike traditional autoclave technology which has to cool down and to heat up again, the Hydroclave
temperature is retained at a minimum of 121°C during 40-minute cycles, thanks to the unique steam jacket.

Stage One – Loading Stage Two – Sterilizing

The Hydroclave can process:

• Powerful rotators mix the waste and breaks it into small
Bagged waste, in ordinary bags. pieces.
Sharps containers. • Steam fills the double wall (jacket) of the vessel and heats
Liquid containers. the vessel interior.
Cardboard containers. • The liquid in the waste turns to steam.
Metal objects. • After 20 minutes the waste and liquids are sterile.
Pathological waste.
Stage Three – Dehydration

•The vent is opened, the vessel de-pressurizes via a condenser, and sterile liquid drained into sanitary sewer.
•Steam heat and mixing continue until all the liquids are evaporated and the waste is dry.
•Guaranteed sterility of all waste particles!
•No pre-shredding of infectious waste necessary!
•No special waste bags or chemicals required!

Stage Four – Unloading

•The unloading door is opened.

•The mixer now rotates in the opposite direction, so angled blades on the mixer can push the waste out the unloading door.
•The dry sterile waste can be fine-shredded further or dropped in a waste disposal bin.
The waste is now ready for safe disposal!