ASEPTIC TECHNIQUES

SUITABLE WORKING AREA

Possible flow of unfiltered air over the disinfected working area.

Air currents must be avoided because of airborne spores of contaminating microorganisms.

An interior room, similar in layout to a photographic darkroom, is an excellent choice for aseptic procedures.

Post a NO ADMITTANCE sign on the door during aseptic procedures.

An open laboratory bench can be used in a draft-free room.

Under these conditions it may be prudent to use either a face mask or a plastic biohazard shield.

A transparent plastic shield parallel to the top of an open bench to prevent spore fallout.

Sterile operations within some type of transfer chamber, bacteriological glove box, or laminar flow cabinet.

FLAMMING INSTRUMENTS

Ethanol is highly inflammable, flaming instruments in a laminar flow cabinet should be done with caution.

The air flow from the cabinet would direct a flash fire toward the worker.

A few minutes before the start of any sterile procedure, the working area should be thoroughly scrubbed with a tissue soaked with ethanol or isopropanol (70%v/v).

Ethanol is a bacteriostatic agent and disinfects a working area, but the treated area is not sterile.

LAMINAR FLOW CABINET

Air is forced through a dust filter and then passed through a high-efficiency particulate air (HEPA) filter.

Depending on the type of cabinet, the air is directed either downward or outward over the working area.

The gentle flow of sterile air is designed to prevent any spore-laden unfiltered air from entering the cabinet.

STERILIZATION AND DISINFECTION

The term “sterilization” is an absolute one that implies the total inactivation of all forms of microbial life in terms of the ability of the organisms to reproduce.

“Disinfection”, on the other hand, means the reduction of bacterial numbers to some arbitrary “acceptable” level.

GERMICIDAL LAMPS

Aseptic cabinets and transfer rooms are often equipped with one or more germicidal lamps emitting ultraviolet (UV) light.

UV is useful in eliminating airborne contaminants and for surface disinfection.

The use of UV radiation should be kept to a minimum since it is not a substitute for cleanliness.

If such a lamp is used, switch it on about 30 min prior to culture time.

Do not leave the UV lamp on for several hours or overnight because the accumulation of toxic ozone in a confined space creates a hazard.

UNCLEAN HANDS

Simply rinsing the hands with water is insufficient. It is necessary to scrub them vigorously with soap and

hot water for several minutes. Attention must be given to the fingernails and to any part

of the forearm that extends into working area. After a hot-water rinse, blot the skin partially dry with

paper towel. It is not advisable to use strong disinfectants that could

produce a skin rash. The hands may be dipped in a dilute solution of ethanol

or isopropanol, although this can cause skin dryness and must not be used indiscriminately around an open flame.

METHODS OF STERILIZATION

DRY HEAT WET HEAT MICROWAVE MICROFILTRATION CHEMICAL

DRY HEAT

This method is used only for glassware. In calculating the time required for dry-heat

sterilization, three time periods must be considered:

Approximately 1 hr (heating-up period) is allowed for the entire load to reach the sterilization temperature of 180 oC (350 oF).

A minimum of 2 hr at this temperature is required to kill all organisms.

Finally, a cooling-down period is advisable in order to prevent the glassware from cracking due to a rapid drop in temperature.

BOILING WATER BATH Another form of wet-heat sterilization is a

boiling-water bath. The bath is filled with distilled water and heated

to 100 oC. Instruments placed directly into the water. They should remain in water for 20 min. Microorganisms in the vegetative state are

destroyed, spores will be unaffected.

AUTOCLAVE

This procedure employs an autoclave operated with steam under pressure.

Standard procedure: Steam pressure of 15 Ib /in. (103.4 k Pa) and a chamber temperature of 121

oC (250 oF) [Paper products, glassware, instruments, and

liquids]. The time required for the sterilization of liquids

varies, greater the volume, the longer it will take for the contents to reach sterilization temperature.

At the end, the pressure must be permitted to return to the atmospheric level slowly because rapid decomposition will cause the liquids to boil out of the vessels.

Prolonged autoclaving must be avoided because it results in the degradation of certain components of the medium.

Steam in the autoclave chamber must penetrate the materials.

Demineralized water should be used in boilers of autoclaves.

MICROWAVE

Liquid and agar media can be sterilized using a household- type microwave oven.

The required microwave treatment is somewhat empirical since it depends on:

1. The energy produced by the magnetron.

2. Vessel type

3. Volume of medium

3. The presence of energy-sink water reservoirs. 

MICROFILTRATION

It is the process of removing contaminants in the range of 0.025-10 µm from fluids by passage through a microporous medium, such as membrane filter.

Heat sensitive chemicals The appropriate volume of the sterile liquid is

added directly to the autoclaved medium with a graduated syringe.

If an agar medium is used, this is done while the agar is in the liquid state (45 oC).

The pore diameter should measure at least 0.22 µm for the complete removal of all bacteria, yeasts, and molds.

Disposable filtration units composed of polystyrene with 0.1-or 0.2- µm cellulose acetate membranes are practical for infrequent filtrations.

Durapore membrane filter (hydrophilic) and Fluoropore (Millipore) filter (hydrophobic) are excellent choice for plant tissue culture work.

CHEMICALS

Ethanol or isopropanol (70% v/v): disinfected working area.

Ethanol (80% v/v): inflaming instruments.

Sodium hypochlorite (NaOCl) or Calcium hypochlorite (Ca[OCl]2): surface sterilization of plant material.

Calcium hypochlorite (Ca[OCl]2): surface sterilization of seeds.

ANTIBIOTICS Not widely used in plant tissue culture Antibiotics should be regarded as a form of

prophylaxis. Prophylactic use of antibiotics in medicine

depends on three conditions:

1. A single known pathogen is targeted

2. The pathogen remains sensitive to the drug

3. The period of exposure to the drug is short and limited to the period of maximum risk.

These criteria are helpful, to some extent, to plant scientists

No known antibiotic is effective against all microorganisms that might cause contamination.

Selection of an effective agent cannot be made until the contaminants are known.

HEALTH HAZARDS

Hypochlorite solutions: Inhalation can produce severe bronchial

irritation and skin contact can be harmful. Never use mouth to pipette any chemical.

Use pipette fillers. Never use hypochlorite in the presence of UV

radiation. The resulting release of chlorine gas is a

serious risk.

Ethanol

Ethanol is highly inflammable. Extremely careful about spilling of

alcohol in the vicinity of an open flame.

The test tube containing alcohol should be kept in a metal can to avoid the release of burning alcohol in case of glass breakage.

Mercuric Chloride: This chemical is an extremely dangerous

poison. Its aqueous solution is slightly volatile at room

temperature and can cause mercury poisoning to laboratory workers.

Phenols: Lysol or volatile phenols can have an adverse

effect on cultures.

Ethylene oxide: Gas sterilization should not be used in a class room

Laboratory. It is highly irritating to the eyes and mucous membranes, and

high concentrations can cause pulmonary edema.

Antibiotics: Special care must be taken in handling antibiotics.

Vancomycin and chloramphenicol are toxic to humans and some ᵝ-lactams are allergenic in humans.

Ultraviolet radiation: UV radiations poses some serious health risk. UV burns to eye are very painful. A glass barrier

between the eyes and the UV source provides complete protection.

Avoid placing the hands in the cabinet when the lamp is on, it can produce irritation to unprotected skin.

Another problem is the formation of ozone (O3) resulting from the photochemical reaction with atmospheric oxygen. It can cause severe irritation to the respiratory tract and eyes.

AVOIDING CONTAMINATION

Elimination of drafts Keep all doors and windows closed. Not to have any other person. Avoid breathing or coughing Use surgical face mask Hands, wrists, and forearms be

carefully clean.

All sterile open surfaces should be placed as far back in the hood.

Keep the hands as far as possible from the open tube or Petri dish.

Remove all unnecessary glassware, instruments, aluminium foil, and other materials that have been used.

Remove all contaminated cultures from incubators and plant growth chambers.

With a plastic squeeze bottle, add a few ml of ethanol (70% v/v) to each of the contaminated cultures.

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