The laminar flow hood is arguably one of the most important pieces of equipment for flasking and re-plating orchid seeds and seedlings. It allows you to work in a sterile space provided by a steady stream of pre-filtered sterile air delivered by a blower through a HEPA filter (High Efficiency Particulate Air filter). Initially I used a sterile transfer hood to do all my flasking and re-plating and I have had good success with this chamber containing a germicidal UV lamp but contamination still creeps in from time to time and losses as a result could be avoided by upgrading to a full laminar flow hood. In addition, my existing sterile transfer chamber has a relatively short lifespan because of the degrading affect of UV on the acrylic walls.
Of course cost is always a factor and by no means are HEPA filters cheap, nor are they easy to find in Cape Town. HEPAs are classed according to their accuracy and ability to remove particles from the air. For flasking, a HEPA with an H14 class which is rated to remove 99.95% of all airborne microbiological contaminants is standard.
There are two main types of design for laminar flow hoods, a horizontal laminar flow which provides a horizontal flow of sterile air through the working chamber towards the operator, and a vertical laminar flow hood which delivers sterile air from the top of the chamber towards the base which usually has escape channels in it, or the air simply escapes through the front working access. I chose to design a horizontal laminar flow for my requirements. The concept of sterility is often not easy to grasp unless you have actually had experience working in a truly sterile area. I am fortunate enough to have been trained in molecular techniques in a DNA laboratory working with various techniques including PCR. Here, sterility is essential to avoid contamination and you begin to realise just how unsterile everything can be. Two of the most important contamination sources include the air and YOU. Imagine briefly how many suspended particles are in the air at any given point. Often some of these particles can be seen when the light from the sun shines through a window in the early morning. It is nearly impossible to avoid contamination in flasks when opening them or during transfer without first sterilising the air. The laminar flow hood provides this steady flow of sterile air into the working chamber at a higher pressure than ambient so no contaminated air can get into the working area when switched on. Of couse, all equipment must also be completely sterile when used in the working area and I also find that spraying the outside of flasks down with a sodium hypochlorite solution before placing them into the working area also reduced contamination risk when re-plating. I also get into the habbit of ensuring as few as possible movements for any single action or sets of actions. Everything is well timed and well planned. Anything that accidentally comes into contact with my tools during an operation is deemed to be a potential contamination risk and I either change my tool immediately or re-sterilise that tool by ethanol-flaming. After each completed action, tools are ethanol-flamed.
My laminar flow hood will be constructed from waterproof ply (12mm or 16mm thick depending on cost) and will be epoxy coted for longevity and for ease of cleaning with either ethanol or oxidising agents like sodium hypochlorite or hydrogen peroxide. The design is "L" shaped and will be free-standing. The HEPA chamber will have an access panel at the back for the initial HEPA installation and future replacement and a second chamber will house twin blowers delivering the required amount of air needed to produce a laminar flow of 0.5m/second filtered air. The HEPA I have ordered measures 610mm X 305mm (H14 class) and will be attached to the inner frame with a gasket secured with bolts and wing nuts for easy removal.
Another important feature of the laminar flow hood is the blower. These need to be able to deliver the correct flow of air through the HEPA at the required filter's static pressure. To avoid using a blower that is too powerful and that may damage the HEPA, I have chosen to use two smaller twin units mounted horizontally one above the other. A third blower is kept as a replacement unit in case one of the two fails during operation. Both blowers are easily accessible through the side chamber which will be fitted with a verically sliding and fully removable pre-filter.
|Laminar flow plans|
I will follow up with the progress on the laminar flow hood construction as I get there. The HEPA will take about 4 weeks to arrive from Germany so in the mean time I will start working on the case.