Sterile methods.

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Contaminants
When growing orchids in-vitro it's important to keep the cultures axenic. Instruments and working spaces must be sterilized so that no contaminants can spread to the cultures. Single spores can destroy a culture easily. An orchid medium containing sugar (2-3%), vitamins, and micro- and macro-nutrients; and which is also humid and kept in warm temperatures, makes the perfect growing place for a large amount of microbes. Spores and bacteria are naturally occurring in air and can easily be spread around. There are many methods to keep things sterile. The absolutely best way is the one that works.

Bench

Working straight on a bench or sterilised table is somewhat risky. It's extremely difficult to keep instruments, containers, media, and plant explants sterile. Some simple rules are however a must if one is to succeed...

Close all doors, windows, and fans and vents. Tie up your hair, if possible; use a hair net. Use a clean working robe. Wash your hands carefully. If possible, use a sterilant solution (kitchen gloves are recommended). Sterilize the bench (70% ethanol, isopropanol). Only use sterile instruments. Keep calm and work systematically, close to the bech. Don't talk, avoid laughing, and whatever you do, don't sneeze. Burn all instruments off and keep re-sterilizing all instruments again and again during the lab.

A gas- or alcohol burner is used to flame off the instruments (candles are OK if nothing else is available). The burning flame will also create a steady pillar of hot air, keeping microbes away (if you work close to the flame, that is).

Laminar flow-hood

A laminar flow-hood (LAF) is used by serious orchid growers. It's basically a bench with walls and a cover (opening in the front). A contineous flow of sterile air is directed towards the worker, keeping contaminants away. The air is filter sterilized using a HEPA-filter which stops all aerosol-particles with a diameter of ca. 0.3m. It's still important to know how to work correct and to keep the hands steady (don't wave around). This kind of equipment gives a lot of freedom. Everything inside the LAF is sterilized before the work begins. Chemical and/or UVsterilisation is used. All objects and instruments that are taken into the sterile area has to be sterilized before entering. Working in the bench, one must recognise that placing hands and objects behind cultures etc. increases the chance of contamination, as particles that still are on the objects or on the hands might be carried away by the air-stream and land in the culture.

Aquarium - Sterile box

A smaller aquarium or terrarium (ca. 60dm) can be turned over on the side, be sterilized on the inside and be used as a sterile working area. A box can be built with a minimum of material (a cardboard-box and some plastic- bags or film, or a simple transparent plastic-box). Long plastic gloves can be fastened on the sides of the box. Thus one can access the sterile area without bringing new contaminants inside. Another method is to hang a wet rag over the opening as a curtain, stopping the movement of air (suitable solution to wet the rag with: 1:10 bleach:water; that is: 1% sodium hypochlorite - NaOCl). All instruments must be sterilized before they're placed into the box . One should try to place all the necessary equipment; containers and instruments, inside the box before the work begins. After that, one can sterilize everything again, just to make sure that nothing unwanted is there (or rather to minimize the occurrence of contaminants). 1% sodium

hypochlorite or alcohol (ethanol or isopropanol) can be sprayed to sterilize the objects and the surface. To sterilize with a gas, a beaker with ca. 5% sodium hypochlorite can be placed inside the box. A few drops of concentrated hydrochloric acid (HCl) are added to the beaker with the hypochlorite and a gas of mainly Cl2 is produced. It's important to seal the box before this is done. The gas is not very nice to breathe. When opening the box, after the work has been done, consider doing it in some kind of fume-hood or by an open window or so. See to it that nothing can be spoiled by being exposed to such high contaminant environments.

Steam pillar
One way of getting a sterile atmosphere to work in is simply to place a container of water on a heating element (the stove perhaps ?) and then bring the water to a boil. A metal net or a similar object is placed on the container to support as an area to place jars, instruments etc. on. Boiling water produces a steady flow of hot water vapour (120C). This pillar of gaseous water is pretty much sterile. Don't move around too quickly while working. Another tip is to use some kinds of gloves, since the vapour not only is burning hot but also will condense to liquid water and, well, mess things up for you.

Chemical sterilization
Hypochlorite oxidises and destroys organic materials. Sodium hypochlorite is easily found, is efficient and rather cheap. When sterilizing explants, a 1% solution of sodium hypochlorite is used (that's 1 part bleach (5%) and 4 parts of water). The time it take to sterilize is usually 15 minutes. A surfactant is often added to make the contact between explant and solution more easy (liquid detergent e.g. for kitchen use, may be used). Calcium hypochlorite (Ca(ClO)2) can also be used. In fact, there may be advantages in using it since no other things have been added to it (it's sold as powder for pool cleaning). About 35g/L mass/volume solution of distilled water is a good concentration; sterilization time ca. 5 minutes. Hydrogen peroxide (H2O2) is a good disinfectant (concentration 2-3% for ca. 10 minutes). A great advantage, using hydrogen peroxide, is that it eventually breaks down to oxygen and water. Before introducing explants that have been sterilized, a rinse with sterile water may be advantageous. Sterilize the water in a pressure cooker, in the microwave or in an oven (150C ugn, 2-3h). Using hydrogen peroxide, you can skip the rinsing. Table* on approx. sterilizing times for plant-explants (*source: Sigma Aldrich). Chemical Concentration (%) Time (min) Calcium hypochlorite, Ca(ClO)2 9-10 5-30 Sodium hypochlorite, NaOCl 0.5-5 5-30

Hydrogen peroxide Ethanol Isopropylalcohol

3-12 70-95 70-95

5-15 quickly quickly

Autoclave

Efficient killing of microbes and spores of microbes can be accomplished by autoclaving instruments, jars, containers, and nutrient media. An old pressure cooker can be used. It's important to let the autoclave reach a pressure high enough. The inside of it should be over-saturated with water vapour (the temperature reaches 121C). Let the hot air escape before 'hanging the knob on the vent'. When the autoclave has reached its full pressure and been let to whistle a bit, turn the heat off and let the autoclave cool. When the pressure difference is close to none (pressure difference between the atmosphere on the outside and the interior of the autoclave), the autoclave can be opened and evacuated. Media to be autoclaved is prepared and distributed to clean vessels. The containers are plugged with lids, cotton stoppers (covered with aluminum foil), or whatever autoclavable there is to use. (n.b. Don't fill the containers to more than of the total volume.)

Oven heat
An oven is useful when nothing else is available for sterilizing. Metal instruments (scissors, forceps etc.) are excellent to sterilize by baking in an oven. Clean instruments can be wrapped into aluminum foil. 'Let them sweat' in 150C during 2-3 hours. Media can naturally be oven sterilized as well. However, one should be cautious about it, since a lot of water can evaporate.

Microwave oven
Some growers use a microwave oven for sterilizing (mainly media and other solutions). Metal doesn't sterilize in microwaves (well, it does, but you can destroy the microwave)

Radiation

UV-light kills microorganisms and is used to sterilize working areas. It can take time to sterilize with UV and it's not very convenient, since UV seriously may harm all organisms. Many chemicals and materials are affected by exposure of UV-light. Sunlight has in some cases been used to fight back contaminants in newly inoculated cultures. The temperature will probably rise quickly if a contained culture would be placed in sun light. The inocula can be hurt. Also remember never to place your cultures in sun light.

Sterilizing seed

Orchid seeds are extremely small (just like dust). Dried seeds are contaminated. The seed capsule bursts as it's drying. The seeds must be sterilized before sown onto medium. Chemical sterilants are usually used. There are however a few different methods of sterilizing the seeds:

Drowning
An easy way of sterilizing seeds is simply to pour the dust-like particles into a small beaker and then add a sterilant. The seeds are rinsed with sterile water before sown onto a sterile nutrient medium.

The package method

More sensitive (expensive and rare) orchid seeds are usually placed on a piece of paper or filterpaper. The paper is folded into a package and sealed. A sterilizing fluid is prepared and to sterilize the seeds, the packet is simply placed into the solution. The package is rinsed in sterile water, opened in a sterile environment and the seeds are placed onto a medium.

Syringe
Seeds are placed into a small syringe. A liquid sterilant is sucked up and the syringe is agitated. The solution is evacuated from the syringe (without loosing too many seeds). Sterile water is sucked up into the syringe to rinse the seeds. To sow, the syringe is either opened in a suitable environment, or one can let the seeds follow with the water out down directly onto the medium.

Pre-treatment with a sugar solution

Some growers think it's best to pre-treat seeds with a sugar solution. Some microbes might be resting or in a spore state and are thus less affected by chemical sterilants. If the microbes were to wake up, one could kill them more easily. Incubating seeds in a solution containing sugar (during 12-24h) on surely do wake the microbes from their sleep. A suitable sugar concentration is a half-saturated solution. A saturated solution is prepared and decanted. The solution is measured for its volume and an equal volume of sterile water is added to it. The solution with seeds is incubated at room temperatures (12-24h). After the pretreatment, the seeds are rinsed and treated with a sterilant.

Green capsule
A seed capsule doesn't need to be mature to be used. A dry capsule is contaminated, a green one is still (hopefully) sterile on the inside. The only thing one has to do is to sterilise it properly on the outside. The capsule can be opened in a sterile area and the pre-mature seeds can be sown. The capsule is washed with a washing agent (detergent). It's then sterilized in a solution of sodium hypochlorite (1-5%) (yes, 'pure' bleach can now be used) during ca. 15-30 minutes. The capsule is dipped into alcohol and flamed. The time for a capsule to mature vary between orchids (may be from 3 to 10 months (some takes ca. 20 months to fully mature)).

Practical tips
Aluminum foil is a valuable material when working with sterile techniques.

It can be autoclaved, oven sterilized, and sterilized with chemicals. A sterilized packet of aluminum foil, containing instruments, can be opened in sterile conditions. The instruments need no further sterilization before the work can be started. A foil of aluminum can be folded and sterilized to be used as a sterile working area, to be folded up when needed. Aluminum foil can be used to cover openings of smaller containers, be wrapped around stoppers, and be used to keep instruments etc. together when autoclaved or sterilized in any other way. Aluminium foil is not expensive, is very easy to get hold of and can be recycled.

Sterile surface
As mentioned aluminum foil can be used as a sterile working area. Another way of having a sterile surface is to completely soak a rag or filter paper in a liquid sterilant.

Teaball
A teaball can be used to sterilize explants. Sometimes it's hard to really soak explants sufficiently but the tea ball will surely do the job. Just place the plant parts into the ball and soak the thing in a suitable solution during the time it takes to sterilze the explant (e.g. 15 minutes).

Spray bottle
A spray bottle will definitely help sterilizing larger areas. A bottle is simply filled with a suitable solution. A 10% bleach can be used to spray out on surfaces. (A smaller amount of acetic acid may be added to neutralize the pH a bit . (CH3COOH) (replace some of the water with acetic acid (conc. 12%): 1 part bleach, 1 part 12% acetic acid and 8 parts water + some surfactants, a detergent). Of course, one must always be careful when dealing with chemicals, especially when they're sprayed like this. Bleach does not only bleach things, it's toxic and can seriously damage all organisms. One shouldn't bread too much of those fumes either. Alcohol (ethanol or isopropanol) can also be sprayed. lower concentrations leave more water than higher concentrations. Higher concentrations evaporates more readily. and costs more to spray. Alcohol is very efficient, but can seriously harm the worker. Higher concentrations of alcohol ignites easily. It's however important not to use methanol, as it's poisonous.