This material may be copied for personal use, or for use by societies or research groups, providing that it is accompanied by a notice acknowledging authorship. © Cedric Maunder
This guide is based on myown experiences here in the UK, and the methods and materials described may not suit other people. Over a period of about two years I have had good results with a variety of orchid species and hybrids, and have suffered very little contamination of flasks. These notes are provided not because I consider myself an expert, far from it but because this comparatively cheap and simple set-up has worked well for me and might be helpful to anyone contemplating having a go at flasking. I am very grateful for help and advice received from other people - notably George Black of Brize Norton, England and Robert M. Hamilton of Berkeley, California, USA
A veryimportant factor is to be very aware that bacteria, moulds and other potential "nasties" are everywhere - in huge numbers. Awareness of this fact, together with recognition of the existence of gravity and air currents, will help to ensure that the said nasties do not gain access to your flasks.
I should say at this stage that I do not possess a laminar-flow cabinet. The initial purchases needed for starting out in flasking are sufficiently expensive without the fairly large outlay that such a cabinet requires. Having seen the results obtained by George Black - a noted orchid hybridiser and flasker - using only a very simple glove box, I decided to try that method first. After a couple of years of this I see no reason to change to anything more complex and expensive unless I become sufficiently fool-hardy to consider commercial ventures!
This is a very simple device. Various people use cardboard boxes, glass carboys etc.! I thought that wood or plastic (acrylic) would probably be the best choices for me. (A simple wooden box can have a piece of glass for a lid and does not necessarily need a frame or hinges for the glass top. George Black has raised many, many seedlings from such a set-up.)
I finally settled for using acrylic sheet; most pieces of the box can be made from off-cuts which are very cheap, though I used thicker sheets for the base and lid - these being at full price. The only drawback of this material is the adhesive it requires. This should be used out of doors or in a very well ventilated place.
The material is transparent if one does not spill too much of the adhesive on the visually critical parts - lid and front panel.
The one I made has the following dimensions (not very critical, I wanted it to fit on the end of the kitchen table when in use and on the top of the freezer for storage)
Width - 24 inches; 810mm.
Depth - 18 inches; 457mm.
Height at rear - 12 inches; 305mm. --- 14 inches; 356mm. Might be better.
Height at front - 8 inches; 203mm. --- 10 inches; 263mm. Might be better.
The lid is made of two pieces, a rear fixed part and a hinged part at the front. I also used a fair number of small off-cuts to strengthen the corners and rear part of the lid.
Diameter of arm holes :- 4.25 inches; 108 mm. (I used a saucer as a pattern!)
I used pieces of shower curtain material cut to shape and size to make sleeves. A hem is made at the inside end with elastic inserted and tightened to give a comfortable fit for the wrist, and cuts made at the other end to make radial flaps with which the sleeve can be glued to the outside rim of the box.
If one wears rubber gloves when using the box, the sleeves and gloves keep one's skin fairly free of Domestos. (See below )
This is done using Domestos or similar bleach diluted 1:4 with water. A 500ml spray bottle contains this solution so that It can be sprayed across all parts of the box to make a mist which will remove all organisms. (If the spraying is done correctly, all interior surfaces will have been coated with the bleach and organisms in the air will be carried down in the spray into a layer of bleach on the floor of the box. The sleeves and gloves must also be well sprayed, preferably at both the start and end of the sterilizing. My experience is that by the time this progress is completed, the box is essentially sterile. Wait a further five minutes or so to be absolutely sure.
All materials, tools etc. needed for the flasking session must be put into the box before the spraying begins. Tools should be laid in a shallow dish full of the bleach solution before closing the lid. All flasks or bottles that are going to be worked with before starting, together with a flask of sterilised water, capped with foil. All must be thoroughly sprayed during the sterilising procedure. If some essential item is forgotten it will be necessary to do the whole procedure over again, SO have a check list and consult it before sterilising!
Access to Sigma/Aldrich or some other reliable supplier of Chemicals and apparatus.
[In the UK this firm is probably the largest and most comprehensive one engaged in this field, and I have found them to very efficient and helpful. Free catalogues - very well produced.]
These firms can provide. almost everything needed, and thousands of other things, too!
UK Addresses and Phone Numbers:-
Telephone:- Orders :- 0800 373731 Customer Service :- 0800447788
The company appears to be active in most other parts of the world, but I'm sure there are
alternatives in most places.
(Approx UK prices shown (1998) - subject to change)
Medium:- Phytamax Orchid Maintenance Medium with charcoal and banana, P1056. 1 Litre size = £1.60 - Phytamax P0931 is without charcoal.
Agar:- A 1296, 500g - £47.20; 100g. -£14.30
Calcium hypochlorite - Fluka 21182, 250g @ £5.10; lkg = £14.30
(This is 65% to 70% strength, 3.5g per 100ml for five minutes is probably sufficient for seed sterilisation).
Suncaps (flask closures with filter vent)
C 6795- foil with 3.00 mm.. filter; 500 £ 43.30
Domestic bleach - Domestos or similar, hand sprayer to use for spraying Domestos 1 :4 solution in glove box.
Can all be obtained from Sigma but most can be obtained from other sources.
Measuring jug, 500 ml., pyrex or similar. - kitchen departments of department stores.
Glass funnel for filtering.
Filter paper - Whatman 185mm. Grade 1 or 4 (faster)
A few 5OOml glass flasks, with rubber bungs, are useful for making and filtering the hypochlorite solution, water, and so on.
Large bag or elastic bands for temporary securing of Suncaps.
Micropore tape for final securing of Suncaps.
Shallow oblong dishes for holding flasking tools (see below) for sterilising (inside glove box)
Supply of suitable bottles - jam jars, honey jars, baby food bottles (Sigma) etc.
Labels for bottles. Small self adhesive ones made for labelling preserves/jams etc.
Small stoppered bottle to hold seed during sterilisation.
Pressure cooker high enough to hold 500mL flasks.
Scales to read down to 0.05 gm. (0.1gm. divisions). (For making up the majority of solutions needed for flasking, colchicine use CSMColchicine.htm chromosome counting, etc. this should be adequate. To make up one's own medium really requires 0.01 gm. divisions. )
A microwave cooker can be used instead of a pressure cooker but, on balance, the pressure cooker is quicker and more convenient. For good results from sterilising in a microwave oven it is necessary to include about one litre of water in bottles with each batch of flasks. This acts as a thermal ballast so that enough time can be given to ensure sterility without boiling over of flasks or loss of their seals.
Reference:- Microwave Sterilization of Plant Tissue Culture Media; BrentTisserat, Danny Jones, and Paul D. Galleta. Hortscience 27 (4): 358 - 361 1992.)
Sigma medium is clearly labelled with the weight required to make one litre. I usually make batches of 500mL. This is sufficient for ten to a dozen bottles. I usually prepare a few bottles with a deeper layer of medium for use when re-plating, others more shallow for seed sowing. Do not be tempted to make the layer of medium very thin, however, as it will soon become too dry and stiff!
Fluorescent tubes 4 foot - daylight, one or two ( at the most) for each shelf. Doesn't seem to be any marked advantage using UV lights at this stage.
Plastic trays to place flasks on -- 9 inches from lights gives good results. Some cool growing genera are prone to produce protocorms with little or no chlorophyll if light to heat ratio is too low. Bringing flasks to lower (cooler) level results in some or all of these 'greening up'
Time clock to give 12 or 14 hour days. This timing seems to be optimum for Oncidiinae genera.
This can be ripe/dry seed or 'green pod' - (unripe capsule! )
Keep seed as clean and dry as possible before use. Some people say to dry the seed in a dessicator - I haven't found this to be a good thing. Sow as soon after harvest as possible - but will keep for several weeks if dry. Presumably gradually loses viability.
I use a 50 ml. bottle or flask with screw cap or tight fitting bung. Make a 'solution' of Calcium Hypochlorite (60-70 % available chlorine) using 7.0 or 3.5 gm. of Hypochlorite in 100ml water .
(Traditionally 7 gm. of calcium Hypochlorite in 100 ml. of water has been used. Recent articles by Fred Bergman suggest that this is too strong. I've recently tried batches of the same seed at 7gm., 3.5 gm., and 1.75 gm., in 100 ml. All have germinated - better germination in weaker bleach, one batch at 1.75/100ml. showed contamination. All batches at 3.5gm/100ml. are O.K. I'll use 3.5 gm. routinely now, continuing to try out some at 1.75 gm/100 ml.)
Filter into a flask. Place seed in the 50ml bottle/tube and add about 15ml Hypochlorite and ONE drop of kitchen detergent (Squeezie). Shake well and after 5 minutes filter the contents (Cover the funnel top with clean sheet of filter paper.) Rinse twice with STERILE water- keeping the funnel and the open bottle of water covered with filter paper during transfer to the funnel. The covered funnel is then transferred to the glove box. Use a spatula (sterilised in Domestos 1 part,. water 4 parts) to transfer seed to flasks. After everything has been sterilised by spraying as described above, carefully remove the foil top from one of the bottles of medium, placing the top, top surface up, on the floor of the box. The bleach solution will prevent contamination of the inside of the foil top and it can be replaced on the bottle after sowing. The foil will have turned down edges and corners from being secured to the bottle during and since sterilising. It will sit on these turned down corners so that the inside of this lid will remain clean and dry (in most cases! ) .
Capsules sometimes abort before they are ripe. These can often be salvaged. Also, some seeds will not germinate after being sterilised but will germinate if sown from green pod.
Soak and brush the capsule in 1 :4 Domestos solution. Give good 10 minutes in the bleach.
Make sure that a suitable scalpel blade or small knife is in the glove box before starting! It is best to put the capsule, spatula and blade together into a dish or bleach inside the box, together with flasks, bottles etc. needed for sowing, before spraying as above.
Open the capsule inside sterile glove box (after sterilising as described above), and scrape seeds with the end of the spatula into flasks.
Check your flasks/bottles every week or so. Germination often occurs quite quickly so that tiny green protocorms can be seen on top of the medium, Some seed takes a long time to germinate, so do not be too hasty to throw out seed that does this, I have heard of seed germinating a year or more after being sown!
Any contamination will also usually be apparent in the first few weeks. It is wise to sow at least two bottles of each lot of seed so that throwing out the contents of a contaminated bottle does not negate the whole sowing. If a very precious batch of seed does become contaminated.. you could try treating this with 1:4 Domestos. IN THE STERILE BOX open the flask, apply the bleach to the patches of contaminant and an area around them. Reseal the flask and hope. If caught early and the contamination is slight, this does work, but not always.
Replating is usually beneficial Even with charcoal in the medium waste products will accumulate. Also the medium dries out due to air exchange through the filter and uptake of water by the seedlings. This is an opportunity for picking out the most vigorous seedlings and giving them more room
I replate whenever seedlings seem to have slowed their progress, grown too large for the bottle, or if the medium seems to have become too stiff. I also tend to replate bought-in flasks unless they are growing very well.
I use the same type of medium for sowing and replating.
If you are sowing or replating Paphiopedilums, Phragmipediums or terrestrial orchids you will need to modify these methods, very likely needing a different medium. I have no personal experience of these types of orchid. You might be able to obtain advice from Kew regarding terrestrial genera.
Doing one's own flasking allows one to de-flask at optimum times and also allows replating of any smaller backward seedlings. Thus the yield is usually much higher.
The process is essentially simple. Have a large bowl of clean tepid water to hand. Remove the foil cap from the bottle or flask and use a wire hook to gently ease the seedlings out into the bowl. If you use flasks rather than bottles, this can be difficult as the seedlings jam in the narrow neck. For this reason I much prefer bottles.
It helps to fill the flask with tepid water and allow this to melt the agar. It is usually then possible to use the wire hook to tease out one or two seedlings initially, followed gradually by more until the flask is empty. I have no doubt that de-flasking from narrow necked containers causes more damage to the seedlings, If you have very precious seedlings in such a flask, it may be worthwhile carefully breaking the flask before attempting removal.
We plant seedlings out into community pots rather than individual containers. We plant them in perlite as per the instructions in these pages.
After planting the seedling, ensure the compost is moist, cover the container with a bell-jar type plastic lid, Or a plastic bag. Ensure that the seedlings do not become too dry. During the dark English winter, at least, we keep community pots under lights for some (It is useful to have some spare shelf space in the flask cabinet for this purpose. We find that this suits most seedlings very well.)
Unless there are signs of contamination this does not seem to be needed .
I use two types of tool, both home made.
Both are made from eighth or quarter inch brass rod (wire,) from a toy/model shop. Very cheap and very easy to work.
For the spatula, a 12 inch length of quarter inch rod has one inch of one end flattened by hammering with a light hammer - very easy - don't hit too hard!
The flattened area is then gently bent to a suitable curve with pliers.
For the fork, use either quarter inch or eighth inch rod - depending on size fork required Use a fret-saw with fine blade to make a cut centrally in one end of the rod for a depth of half an inch, Pliers and a fine metal file are used to make points, separate the ends and produce a suitable curve.
It is useful to have two sizes or fork, one for small seedlings, the other for the large ones.
Stainless steel is mentioned by most authors, but I have had no problems with brass. It stands up to bleach solutions well, and does not appear to have any bad effect on seedlings. It is cheaper and easier to obtain as well as being much easier to work.