There are two types of PTFE membrane suitable for aseptic venting namely standard membranes and microporous membranes. During manufacture standard PTFE membranes are stretched to reach the desired thickness and this allows the formation of a range of hole sizes according to the degree of stretch. Microporous PTFE does not rely on this stretching process but starts with granules of PTFE all of the same size and is constructed from these resulting in even sized holes being formed. Simple comparisons with a natural sponge and a synthetic sponge (polyurethane foam) illustrate this well. The natural sponge with variable holes represents the standard PTFE membrane available as Flasking Patches, while the synthetic sponge with even holes represents the microporous PTFE membrane available as Adhesive Microfiltration Discs. These two manufacturing differences have a significant effect on the resultant membrane affecting their efficiency as aseptic filters.
The standard PTFE, of Flasking Patches has a range of hole sizes (all of which are small enough to stop contamination) and relies on enmeshment of particles. Air molecules carrying contamination have to travel a tortuous route before they become entangled in the micro-fibril PTFE network. This means some particles can travel to the inside of the PTFE and become lodged in the interior,
With the microporous PTFE of the Adhesive Microfiltration Discs, the range of hole size (all of which are small enough to stop contamination) is strictly controlled during the manufacturing process. Air molecules and contamination find it very difficult to enter the PTFE membrane as the hole size stops them almost immediately. Any that pass through the initial pores are quickly engulfed by the layers or entrapping micro-fibrils. The result is a very efficient filtering systems that makes passage of contamination and particles impossible and has provided a technological leap forward in filtering efficiencies.
Microbial contamination can come from a number of sources with bacteria, yeasts, fungal spores, dust particles and moisture ingress probably the most common. During the in-use life of a Flasking Patch or Adhesive Microfiltration Discs, these particles will become trapped in the PTFE membranes, thereby avoiding contamination and loss of cultures and help maintain the aseptic integrity of the cultures. The accumulated contamination particles could be regarded as the filters 'contamination load' which will increase over time and is related to the levels of background infection sources. Cultures held in high specification growing rooms with HEPA filtration, or clean rooms will have a lower 'contamination load' compared to cultures held in greenhouses subject to water splashes or condensation drips!
The filters 'contamination load' will affect airflow rates. Over time the airflow rates are likely to be reduced though we do not have measurements for this. However filtering efficiency with regard contamination in not reduced.
Quality control in the air exchange rates is one of the reasons why we do not recommend the reuse of our Flasking Patches or Adhesive Microfiltration Discs. Using a new filter with each new flask will establish a base line for air flow for the life of each new vented culture vessel, and contribute to achieving optimal results.
Uniform air flow rates and uniform aseptic venting is a key to producing quality control in the aseptic environment leading to uniform plant production. By ensuring the venting is the same from culture to culture a variable is eliminated (number of air exchanges in the life of an in-vitro generation) and more uniform crops can be produced. Poorly vented flasks or differently vented flasks of orchids will grow at different rates - this is possibly related to the number of air exchanges the plants are exposed to, among other factors.
Developed to ensure the correct air flow and the number of air exchanges allowing better plant production. Although this is a function of the PTFE construction and ventilation hole size our products will allow the correct balance to be achieved. Preventing too rapid air exchange and possible dehydration problems due to the wrong type of PTFE vent, incorrect venting and water loss. Although in-vitro flask volume should be taken into account the critical factor is the number of air exchanges plants are exposed to during their culture life. This can also be affected by plant density and external factors such as air flow over the top of the venting system and temperature regimes. Successful use of PTFE venting depends critically on ensuring parameters are correct to allow good aeration without dehydration. Temperatures suitable for actively growing tropical plants to cold temperature germplasm storage of hardy crops are at the two extremes, but work has shown cultures are known to all benefit from such AMD and FP venting. For non-conventional and conventional approaches or crops we are always happy to advise or help if we can.
During the normal photoperiodic growth of plants in-vitro, the internal vessel temperatures rises and falls according to the light or dark cycle. With lights on the warming effect of fluorescent light irradiation may allow an internal rise of +3-4 degrees Centigrade. In an unvented sealed and flexible container such as our Plant Culture Containers (PCC) this can be seen as expansion of the sides. On entering the dark cycle the container will return to its normal size and internal air pressure. This gives a visual indication of the possible air exchanges that could go on 'passively' if the PCC is subsequently vented. Should in-vitro cultures be vented, the cooling cycle sets up a flow of air into the container due to the temperature mediated pressure drop of a partial vacuum. When a venting system such as a Adhesive Microfiltration Disc or Flasking Patch is used this air exchange can go on freely and without contamination ensuring good air exchange and in many cases improve plant growth and growth rates. The in and out flow of air relies on good quality filtration systems and should these fail this is one common cause of contamination and loss of cultures. Such problems are often associated with traditional cotton wool venting.
Conventional in-vitro produce plants are sometimes describes as growing photomixotrophically where the carbon uptake for growth is from media added sugars and carbon dioxide. Carbon dioxide gas exchange can be considerably increased by the use of a good quality venting system.
Photomixotrophic plants in-vitro start absorbing carbon dioxide primarily through stomata at the beginning of the photoperiod. The carbon dioxide concentration in an airtight culture vessel decreases sharply within one to two hours nearly to the carbon dioxide compensation point, (K.Fujiwara, T Kozai, I Watanabe in J.Agric. Meteoreol. Tokyo, 43, 21-30 (1987) (J Pospilova, J Katsky, J Solarova, I Ticha, in Biol Plant, 29, 415-421 (1987) The carbon dioxide compensation point is a carbon dioxide concentration at which the net photosynthetic rate of plants is balanced to be zero (gross photosynthesis rate = respiration rate) even at optimum photosynthetic photon fluxes and temperatures.(T.Kozai, SMA Zaboyad, Acclimatizaton.)
By the use of PTFE venting carbon dioxide concentrations can be maintained and depletion be avoided or reduced.
In conventional micropropagation, relative humidity in the culture vessel is always higher than about 95% (T.Kozai, K Tanaka, B R Jeong and K. Fujiwara, in J.Jpn Soc. Hortic.Sci. 62, 413-417 (1993) because the culture vessel, containing liquid water, is sealed, and the temperature is approximately constant with time. However the purpose of sealing the culture vessel at the multiplication and rooting stages is not to keep the relative humidity high. The culture vessel is sealed to prevent microbes from entering it causing contamination and loss of plants. Thus the high relative humidity is an adverse side effect resulting from sealing the culture vessels for the prevention of contamination. If the relative humidity can be reduced without contamination nonhyperhydrated plants with normal stomata and cuticular wax layers can be obtained. (B.R.Jeong, K.Fujiwara and K Kozai, in Hortic. Rev. 17 125-172 (1995) and (T.Kozai, SMA Zaboyad, Acclimatizaton.)
PTFE venting achieves such an improvement in relative humidity and prevents microbial contamination problems.
Conventional venting solutions to the raising in-vitro plants in the past have had to rely on cotton wool plugging. However there are problems associated with such techniques. If insufficient cotton wool is used contamination risks are high. It is often necessary to have at least 1 inch (2.5cms) cotton wool to provide a long and tortuous enough route for entrapment and blocking contamination. To achieve this often involves the use of glass tubes filled with cotton wool wadding. This often is not sufficient enough barrier and soaking cotton wool with solutions of anti-contamination and hazardous or toxic chemicals is another route. The cotton wool venting approach also fails when it comes to quality control of air exchanges. Sometimes the flasks are tightly plugged - lack of air flow, sometimes loosely plugged - good air flow but suffer from contamination risk, loss of humidity and dehydration of the media. Cotton wool, both standard and the preferred non-absorbent type can allow the passage of water and microbial contamination from drips, accidental splashes or high-humidity environments. External contamination is a common route but also contamination from a moisture wicking effect can occur from internal seepage of 'aseptic liquid' through the bung after inversion. A conical flask, knocked over, or even stored under lights on its side ( a common approach to maximize an agar growing surface) can allow liquid to accumulate around the cotton wool venting plug. Seepage to the outside provides an easy route back into the flask for bacterial and fungal contamination from the air. Growth can be surprisingly rapid in the sugar rich liquid. In addition it puts at risk the internal flask neck region where colonies can grow and provide a threat on opening the flask for saving the cultures or during normal transflasking of an 'apparently contamination free' flask! Wetted cotton wool provides poor passage for normal gas venting. Cotton wool plugging does not provide a barrier to mites - see later section.
It has been claimed elsewhere on the www that non-absorbent cotton wool prevents seepage of liquids and the associated contamination risks. However in our experience this is not the case, unless the utmost care is taken never to allow inflask spillage. If the cottonwool is 'forced tightly' into bungs this may reduce this problem but this does not lead to efficient gas exchanges (in fact it maybe little different to using sealed containers)! Chemical treatment with anti-contaminants may help.
Hydrophobic PTFE vents do not suffer from this problem as the level of hydrophobicity is too high to allow such a process. This issue has been specially addressed at manufacture in the specifications of the PTFE used in both Flasking Patches and Adhesive Microfiltration Discs. Although moisture and liquid is know to pass though these vents under experimental conditions, the rate is very low under normal atmospheric pressure. Experimentally forced water passage under increased pressure was very low, and due to the high filtering capacities of the PTFE any contamination could not be carried back through the membranes due the sub-micron retention of particles. (Indeed PTFE filters are used to filter liquids and produce aseptic solutions as a standard laboratory procedure).
Cotton wool vents can sometimes suffer from establishment of contamination after autoclaving. Should the sugar rich media boil over or enter the cotton wool plug the integrity of the cotton wool may be put at risk. Excess steam passage may also aggravate this problem by wetting the plug.
PTFE venting being highly hydrophobic does not suffer from this problem as water or condensation is repelled in the flask post autoclaving.
Dehydration and water loss form in-vitro cultures can be a problem with some venting systems. However as our Flasking Patches and Adhesive Microfiltration discs have been manufactured with high levels of hydrophobicity these problems can be overcome allowing reducing water loss problems even in long term cultures. As the PTFE is highly hydrophobic the ability of water vapor to pass through the membrane is reduced making it possible to achieve very high air exchange rates in-vitro. Advice to customers is offered on placing orders.
A good venting system needs to work filtering the air continuously from the second a flask leaves the autoclave or is put into use. The shelf life of the flask may only be a few weeks, or extend to many months. At Tissue Quick Plant Laboratories many cultures have to be vented long term, due to the slow rate of growth of plants such as orchids. Aseptic integrity can be readily achieved for periods of 18-24 months. We have also found that the Adhesive Microfiltration Discs are suitable for the shipping by air travel of aseptic plants in-vitro where they are of necessity exposed to fluctuations in air pressure. Aseptic integrity and contamination free cultures can be achieved post air travel, This is especially useful where plants need to spend some additional time growing in another laboratories' growing rooms or used as starter cultures for further plant production.
Not all PTFE venting systems are the same, Indeed our products have been manufactured according to our specifications, working closely with the manufacturer, based on our experience in raising plants in-vitro. If you are using the wrong form of PTFE or inferior products you are likely to have a range of problems.
We were the original and first in the UK (and probably worldwide) to introduce multilayered PTFE adhesive microfiltration discs in early 1995 to improve the quality of orchid plants for the commercial industry.
The incorporation of an autoclavable polypropylene mesh into the PTFE membrane give the Adhesive Micorfiltraiton Discs additional strength and integrity. This is especially important in the day to day laboratory production of plants for nursery use where greenhouse handling of cultures requires products to be of a robust nature.
Alternatives and limitations.
Mite Control. Contamination and culture loss due to mites can be a problem in some plant and fungal cultures. The genera Tyroglyphus and Tarsonemus are the usual cause. These feed on almost any organic material including cultures. They are often brought into the laboratory on plant material but can live around the laboratory. Infection is detected usually by trails of bacteria contamination as the mites walk across the agar surface. It is known that cotton wool plugs and even 'tight cotton wool' does not provide a total barrier to this contamination route. This is an important reason for switching away from such a venting system to a more secure system such as PTFE venting.
Other PTFE venting discs - we have tried most of the products in the marketplace and some are clearly more robust than others. In our opinion some of these products are not suitable for use and would have severe limitations in the nursery industry. Some have not been manufactured with aseptic venting for in-vitro raising of plants as their primary usage. They have different air flow rates and particle enmeshment capabilities to the products we use and sell for the raising of plants.
TQPL / Brookside / Southern Lane / New Milton / Hampshire / BH257JE / United Kingdom Tel/Fax/Ans: 01425 616 608 / Int. +44 1425 616 608 / e-mail to: firstname.lastname@example.org
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