DE202008003950U1 - Device and use of the device as a breeding vessel - Google Patents

Abstract

contraption and use of the apparatus for growing Calliphorid larvae in hard-walled or partially flexible vessels on suitable nutrient media under sterile conditions a gas-permeable membrane embedded in the lid or in the walls, characterized in that they have a barrier layer which prevents access of the larvae to the gas-permeable membrane.

Description

Summary

sterile Calliphorid larvae can be easily and efficiently in Cell culture flasks are grown when these are gas permeable Own membrane. This is particularly efficient when the membrane protected by a barrier layer from the larvae.

description

Heavy Healing wounds heal only when the dead tissue is removed from them becomes (debridement). Larvae of calliphorids will be in larval stage 2 therefore, used to selectively target dead tissue from living tissue to separate and thus this medically desired effect to achieve. Wound healing processes are through by larvae debrided tissue significantly accelerated.

State of the art

Larvae that are bred for this purpose must be sterile. For this purpose, appropriately prepared eggs of calliphores can be placed on special nutrient medium containing the necessary nutrients and prevent the drowning of the larvae by adding agar or other measures. The devices used for this purpose are themselves sterile. Devices describing such a breed are in WO 2006/029778 and cited literature there as well as in EP 1,102,531 played.

task

Become the larvae kept on such media, the device surrounding them must make sure that the animals can not escape and that no bacteria can penetrate from the outside.

• • weder die Larven daran hinderte, die Petrischale zu verlassen noch
• • Bakterienkontamination verhinderte, die durch die teils zurückkriechenden Larven eingeschleppt wurden.

It was necessary to find a device that ensures these two tasks. In first experiments, Petri dishes filled with special agar medium were used, which were sealed with adhesive tape or parafilm. This device proved unsuitable because it

• • neither prevented the larvae from leaving the petri dish nor
• • Prevented bacterial contamination that was introduced by the partly crawling larvae.

Cell culture bottles, which are equipped with a rotating lid, are better here suitable, but they do not prevent the escape of the larvae sure, since the remaining gap between the neck and lid is insufficient can be set exactly. In this case, the problems are identical to those of the Petri dishes.

Troubleshooting

Easy and conveniently the problem was solved by using cell culture bottles were used for the rearing of the larvae, the fest can be closed and on the lid or in the Wall through special gaspermeable but bacteria-proof membranes Make sure the larvae do not escape or microorganisms can penetrate. In addition to cell culture bottles can also other hard-walled or in whole or in part with flexible walls configured devices are used as long as a part the outer surface by a special gaspermeable but bacterial density membranes is represented.

These Solution is not really satisfactory when high stocking densities of the larvae are reached on the medium should or if the larvae spend a long time on the medium should be fed to a certain size to reach. If the animals are kept for 12 h or longer on medium it is not uncommon to that the animals inexplicably become motionless without a reason for this would be apparent. If parallel Cultures that do not differ from each other It is possible that one or more of these with larvae occupied devices contain motionless larvae, while others not and therefore further growth and development of larvae different good.

It it was now surprisingly found that when the larvae it be prevented from reaching the lid of the tissue culture flasks, Devices with motionless larvae are not observed.

In order to the larvae do not reach the lid, are different measures possible. So can substances on the inside of the Neck of the device that repel the larvae. Or gauzes or dense tissues can be introduced which prevent penetration of the larvae to the lid. These Gauzes or fabrics can act as a layer, as it were Protective wall can be fed or they can be rolled in or other compact form stuck in the neck of the device then, in addition and subsequently the lid is applied with the gas-permeable membrane. Ultimately was surprisingly observed that must be ensured be that the larvae do not reach the lid.

Depending on the moisture content of the medium, it is equally important that the gauze or fabric material Repels water or does not absorb it in an excessive way because it stops the supply of oxygen and allows the larvae to die.

When Wadding (wound wadding or hydrophobized cotton wool) is suitable Glass wool, rock wool, barrel made of plastic fibers and the like Materials proved through a dense composite of fibers stand out and be deformed so that they are firmly in the throat sit the device and do not let the larvae pass.

Foam has proven to be very suitable, which safely prevents the penetration and / or penetration of larvae in its pore size and which can be prepared so that it securely and firmly fitting as a stopper protects the lid of the device from the larvae. For this purpose, the foam may be made of the usual materials that are used for foams. The foams may be hydrophobic or hydrophilic, with hydrophobic materials being preferred. To be exemplary but not limiting:
Polyethylene (PE, EPE), polypropylene (PP, EPP), styrene (PS, EPS), polyethylene terephthalate (PET), acrylic butadiene (NBR), polyurethane (PU), both as thermoplastic or elastomeric foams.

It You can also combine several materials and techniques be used as long as the goal is achieved that the larvae Do not reach the gas permeable membrane.

Of the Foam should be made so that it does not seal acts, but that the cells / bubbles with continuous Channels connected, and gas exchange ensured is.

Very foams that are sufficiently suitable are suitable Flexibility for easy handling excel and the have a high restoring force to firmly against the wall to be attached and in addition to the gas permeability also by thermal Procedure can be sterilized using other methods the sterilization of the material are also possible (eg. B. chemically with ethylene oxide or formaldehyde or physically Irradiation or heating).

There the foam now safely the escape of the larvae from the device prevented, can be dispensed with the tight completion of the lid and instead a not completely closed lid, if necessary, also without gas-permeable membrane. Will, however dispensed with a lid, is through the plastic foam, the gauze, the cloth or other o. g. Not closures ensures that bacteria or other microorganisms invade can, what does not fulfill the purpose of the device.

The barrier layer is in no case designed so that it completely encloses the larvae and therefore an escape of the larvae is excluded from this barrier. Rather, the barrier layer only prevents contact with the lid and / or the gas-permeable membrane on the wall and, moreover, allows full freedom of movement of the larvae. This distinguishes this device from the in EP 1,102,531 implicitly mentioned.

Examples

• A. State of the art: about 200 eggs from Lucilia sericata were prepared on a Petri dish with blood agar according to the rules of microbiology, incubated for 96 h. The bowl was closed with parafilm between lid and bottom. The well-developing larvae are also outside the Watch the shell. The agar soon shows contamination of bacteria at. This device is unsuitable for the rearing of sterile larvae.
• B. State of the art: about 200 eggs of Lucilia sericata were in a cell culture bottle (manufacturer: TPP, gaspermeable rotatable lid) with an amount and area of blood agar similar to A, manufactured according to the regulations of microbiology, for Incubated for 96 h. The larvae develop well, a contamination by bacteria, as well as the escape of the larvae is observed. From 10 bottles are 0 to 9 bottles at the end of the breeding season with motionless Larvae occupied. This device is for the rearing of sterile larvae not very suitable because there is no contamination with microorganisms can be found, but the yield varies greatly and often low is.
• C. According to the invention: about 200 eggs of Lucilia sericata were stored in a cell culture flask (TPP, gas-permeable rotary lid) with an amount and area of blood agar similar to A, manufactured according to the regulations of microbiology, for Incubated for 96 h. It is in the neck of the bottle a sterile Inserted cotton plug, which sits firmly on the neck. The larvae develop Well, bacterial contamination will be just as little the escape of the larvae is observed. From 10 bottles are 0 to 1 at the end of the breeding season with motionless larvae. This device is suitable for the rearing of sterile larvae, because no contamination can be detected with microorganisms.
• D. According to the invention: about 200 eggs of Lucilia sericata were incubated in a cell culture flask (TPP, gas-permeable rotary lid) with an amount comparable to A of blood agar prepared according to the specifications of microbiology for 96 hours. In the process, a sterile foam plug is inserted in the neck of the bottle, which sits firmly on the neck. The larvae develop well, no contamination by bacteria is observed, nor is the escape of the larvae. Out of 10 bottles 0 are occupied at the end of the breeding season with motionless larvae. This device is optimally suited for the rearing of sterile larvae, since no contamination with microorganisms can be detected and all bottles can be used.
• E. According to the invention: about 200 eggs from Lucilia sericata were stored in a cell culture flask (TPP, gas-permeable rotary lid) with an amount and area of blood agar similar to A, manufactured according to the regulations of microbiology, for Incubated for 96 h. It is in the neck of the bottle a sterile Foam plug introduced, which sits firmly on the neck. The lid will not be locked tight. The larvae develop well, one Bacterial contamination is as important as escaping the larvae are observed. Out of 10 bottles are 0 at the end of the breeding season motionless larvae occupied. This device is for rearing sterile larvae, since very rarely a contamination can be detected with microorganisms.
• F. Deposition example: about 200 eggs of Lucilia sericata were in a cell culture flask (TPP without lid) with a similar to A. Quantity and area of blood agar prepared according to regulations of microbiology, incubated for 96 h. It will a sterile foam plug is inserted in the neck of the bottle, which sits firmly on the neck. The larvae develop well, a contamination by bacteria, as well as the escape of the larvae is observed. Out of 10 bottles are 0 at the end of the breeding season with motionless larvae occupied. This device is unsuitable for the rearing of sterile larvae, occasionally found a contamination with microorganisms can be.

A

cover with gas permeable membrane

B

Vessel, optionally with nutrient medium

C

airspace above the nutrient medium

D

Plug in the neck of the device

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - WO 2006/029778 [0003]
• - EP 1102531 [0003, 0018]

Claims (11)

Device and use of the device for growing Calliphorid larvae in hard-walled or partially flexible vessels on suitable nutrient media under sterile conditions with a gas-permeable membrane embedded in the lid or in the walls, characterized in that they have a barrier which prevents access of the larvae Gas permeable membrane prevented. Device according to claim 1, characterized characterized in that they have a barrier layer in the neck of the vessel possess that prevents the access of the larvae to the lid. Device according to one of the preceding claims, characterized in that the barrier layer comprises a gauze, a cloth, is a foam or a fibrous layer that prevents the Larvae they can pass. Apparatus according to claim 1 or 2, characterized in that the barrier layer is a gauze, a Cloth, a foam or a fibrous layer that prevents that the larvae can pass them, but at the same time does not hinder gas exchange. Device according to one of the preceding claims, characterized in that the barrier layer is a flexible plastic foam which prevents the larvae from penetrating or passing through it can. Apparatus according to claim 5, characterized characterized in that the barrier layer is a plastic foam, which is sterilized by steam and / or heat and the gaspermeable is. Device according to claim 6, characterized characterized in that the barrier layer is a hydrophobic plastic foam is. Device according to one of the claims 1 to 4, characterized in that the barrier layer is a cotton plug which prevents the larvae from penetrating or passing through it can. Apparatus according to claim 8, characterized characterized in that the barrier layer is a hydrophobized cotton wool is. Device according to one of the preceding claims, characterized in that the barrier layer prevents the Larvae can penetrate him or pass and that therefore after partial opening of the lid on a gas-permeable membrane can be dispensed with in the lid. Use of the device from one of the aforementioned Claims for the rearing of Calliphorid larvae for wound treatment.