Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N35/1065—Multiple transfer devices
  • G01N2035/1076—Multiple transfer devices plurality or independently movable heads

Definitions

• This invention relates to a method for sampling frozen cultures of microorganisms, eukaryotic cells, and the like, which cultures are accommodated in mutually separate compartments in a common holder, wherein a sample is taken by contacting a culture with a transfer element, and to a device for carrying out such method.
• microorganisms and eukaryotic cells are known and more and more are being discovered. Many of such microorganisms and eukaryotic cells can be cultured and/or amplified in microbiological laboratories. In addition, many of these microorganisms and eukaryotic cells are genetically engineered, so that they differ from the starting material in one or more properties. To have all these different organisms and cells available, storage methods have been developed for long-term preservation in viable form. Often, cultures of microorganisms and/or eukaryotic cells are frozen in the presence of 15-20% glycerol. The microorganisms and/or eukaryotic cells can then be stored at temperatures below — 20 ° C.
• Freezing such cultures mostly in the presence of 15 or 20% glycerol, at a temperature lower than -70 ° C is a widely used and also simple method for long-term (several years') storage.
• the method is often used in particular for the storage of pure cultures of cellular and viral microorganisms.
• a small quantity of the frozen pure culture is scraped off the frozen surface, for instance by means of a sterile spatula or needle, and subsequently transferred to a growth medium (for instance an agar nutrient medium). After this, the growth medium can be incubated at a suitable temperature in order to effect replication of the respective microorganism.
• the object of the invention is to considerably reduce the time and/or manpower needed for culturing large numbers of different microorganisms and/or eukaryotic cells.
• elongated pins are used, which are disposed in the common carrier in mutually substantially parallel relation, such that each pin is springingly displaceable, at least in its longitudinal direction, with respect to the carrier.
• the advantages of such a displacement is that relatively large differences in distance can be bridged.
• the pins could also be embedded in a springing carrier; however, pins springingly displaceable with respect to the carrier are preferred because of their better handling properties.
• each pin is spring-mounted in the common carrier such that upon springing displacement of the pin a spring force is generated which remains substantially the same.
• a spring action which remains substantially the same has as an advantage that basically equal samples can be drawn from the frozen cultures.
• each transfer element prior to being contacted with a frozen culture, is brought to a temperature such that a small portion of the frozen culture is thawed by contact with the transfer element.
• the surface of the frozen culture coming into contact with the pin is liquefied through heat transfer from the pin to the surface of the frozen culture. After retraction of the pin, a portion of the liquid having therein a portion of the frozen culture of microorganisms and/or eukaryotic cells then sticks to the end of the pin.
• at least the end of the pin can be designed in a material of a relatively large heat conducting capacity.
• the end of the pin that is contacted with the culture is preferably designed in a blunt and preferably slightly rounded form.
• each transfer element is provided with an indication mechanism, which becomes active when the transfer element has been springingly displaced.
• the present invention further relates to a device for applying a method of the invention, which device comprises at least one transfer element with a handling part.
• the handling part comprises a carrier which carries at least two transfer elements springingly movable with respect to each other, thereby enabling the various transfer elements to be contacted with a frozen culture independently of each other.
• the transfer elements consist of pins which are so arranged with respect to the carrier manufactured from relatively stiff material, as to spring substantially parallel to each other, the device is relatively easy and inexpensive to realize.
• the pins are arranged in the carrier so as to be movable in the longitudinal direction over a path defined by stop elements, with the pins each being pressed by spring force to an end position defined by a stop element.
• a uniform starting position with all pin ends in the same plane can be obtained.
• a more or less the same plane of the pin ends is advantageous in particular when placing the device on the holder with the frozen cultures.
• the carrier is provided with an interior space in which at least one spring for each transfer element is accommodated, the various springs are maintenance-friendly accommodated in an enclosed space.
• the accommodation of the springs in an enclosed space is advantageous in sterilizing the device.
• Another enclosed design can be obtained if in a sleeve part secured in the carrier a pin is received for telescopic springing movement.
• the pins come into contact with a frozen culture in a phased manner.
• the advantage of this is that the same device can be used under two or more different operating pressures, thereby enabling different sampling methods.
• a control mechanism for pushing through the transfer elements can be incorporated, for instance for preventing agar media from being pushed through.
• each pin is mounted such that at least the beginning of a longitudinal displacement from the end position referred to is accompanied by a rotational movement. With a rotational movement a more scraping contact of the transfer elements with the surface to be touched is effected.
• the handling part comprises a guiding device in which the carrier is slidable in one direction only, which direction will typically be the longitudinal direction of the pins. Further, it may then be preferred that the handling part comprises positioning means for directing the device with respect to a holder with a number of trough-like compartments for receiving a number of frozen cultures in mutual separation.
• each pin comprises an end having an end face which, in the end position mentioned, forms part of a larger surface and upon a longitudinal displacement of the pin mentioned moves out of that greater surface.
• the larger surface referred to is a surface of the carrier which lies within view in the use of the device.
• At least the end of the pins that can be contacted with the cultures mentioned is made of a material that can be sterilized several times.
• a material that can be sterilized several times is stainless steel.
• at least the end of the pins that can be contacted with the cultures is made of a plastic material.
• the whole device may be made of plastic.
• a device of the invention is suitable not only for sampling frozen cultures. It is also eminently suitable for sampling freeze-dried cultures. Also, the device of the invention can be used for transferring liquid cultures and cultures growing on a solid nutrient medium such as agar.
• Fig.l shows a first embodiment of the device according to the invention in front view
• Fig. 2 shows a top plan view of Fig. 1;
• Fig. 3 shows the use of a device according to the invention
• Fig. 4 shows a second embodiment of the device according to the invention in front view
• Fig. 5 shows on an enlarged scale an alternative embodiment of a pin-carrier connection
• Fig. 6 shows on an enlarged scale a further variant of a pin-carrier connection.
• a sampling device which comprises a carrier 1 with a handle 2.
• the carrier 1 is provided with a number of bores through which extend transfer elements in the form of pins 3, so as to be movable in the longitudinal direction.
• Each pin 3 is provided at one end with a head 4 and at its other end with a rounded point 5.
• springs 6, which are supported on one side against the underside of the carrier 1 and on the other side against a ring 7 fixed on each pin 3.
• the carrier 1 is provided with a guide pin 8, rigidly mounted therein, having a pilot point 9 which reaches beyond the rounded points 5 of the pins 3.
• the carrier is provided with 96 pins 3 and four guide pins 8.
• FIG. 3 the sampling of cultures 10 by means of the device according to Figs. 1 and 2 is represented.
• the cultures 10 to be sampled are accommodated in a holder 11, represented in cross section, which is tailored to the device, that is, a holder with 96 mutually separate compartments 12 in a pattern corresponding to the arrangement of the pins 3, and is provided with 4 guide bores 13 in a pattern which corresponds to the arrangement of the guiding pins 8.
• the device For placing the device on the holder 11, the device is brought above the holder 11 using the handle 2, such that the guide pins 8 are aligned with the guide bores 13.
• the pilot points 9 provide that the guide pins 8 can slide smoothly into the guide bores 13, so that the pins 3 are positioned with respect to compartments 12, such that above each compartment 12 one pin 3 is disposed.
• the pins 3 each reach into a compartment 12.
• the level of the various cultures 10 in the various compartments 12 is different. In the situation represented in Fig. 3, the level in the third compartment from the left is highest, and lowest in the rightmost compartment.
• the pin 3 in the third compartment from the left will be the first to butt by way of its rounded point 5 against the surface of a frozen culture. At that moment, therefore, the other points 5 of the other pins 3 are still spaced from the surfaces of the various cultures.
• the pin 3 in the third compartment from the left can move springingly with respect to the carrier 1, that is, stand still while the carrier 1 is moved further down.
• all rounded points 5 of all pins 3 can be contacted with the surface of the associated culture 10. Now, this is the situation shown in Fig. 3, where the rounded point 5 of the pin 3 in the rightmost compartment just contacts the surface of the culture 10 in that compartment.
• a device according to the invention comprising a carrier 21, which is composed of an upper plate 22, a side edge
• the upper plate 22 and the lower plate 24 are provided with bores located in line for guidingly receiving a pin 26 with a sliding fit.
• the upper plate 22 further comprises recesses 29 which have a height equal to that of a head 27 and can receive all the heads 27, such that they have their top surfaces located in the top surface of the upper plate 22.
• a spring 30 is arranged around each pin 26, which spring 30 is supported on one side against the underside of the upper plate 22 and on the other side, by way of a ring 31 fixedly connected to the pin, rests against the top surface of the lower plate 24.
• the springs 30 and the arrangement of the rings 31 on the pins 26 is selected such that in the rest position of the device, as shown in Fig. 4, the heads 27 are disposed in the recesses 29 and the top surfaces of those heads 27 are located in one plane along with the top surface of the upper plate 22. This latter is provided with projecting edge portions 22a, which serve as grips.
• the operation of this device as regards contacting all ends 28 of the pins 26 with the surfaces of cultures, not shown, is the same as that according to Fig. 3, so that this operation will not be further described here.
• the heads 27 can have a colored lateral circumferential edge, which makes it easier to see when a pin 26 has shifted and hence its point 28 has contacted the surface of a culture, since in that case the colored lateral circumferential edge of the head 27 will leave the recess 29 and thus give a clear indication of the displacement of the pin 26.
• a further possible embodiment of a pin-carrier connection is represented in Fig. 5. There is provided a platelike carrier (shown in part) to which a number of guide sleeves 42 have been secured.
• the end of the guide sleeve 42 remote from the carrier 41 has been narrowed to a passage for passing a pin 43 therethrough with a sliding fit.
• the pin 43 terminates in a thickening 44 which is displaceable with a sliding fit within the guide sleeve 42.
• an indication pin 45 which reaches through a bore 46 provided in the carrier 41.
• a spring 47 is arranged around the indication pin 45 in the guide sleeve 42, which virges the thickening 44 against the narrowed lower end of the guide sleeve 42.
• Fig. 5 the situation is represented where the lower end (not shown) of the pin 43 has been contacted with the surface of a culture, whereafter downward displacement of the carrier 41, for reasons as discussed above, has been continued.
• the thickening 44 through the action of the spring 47, rested against the narrowed end of the guide sleeve 42 and the upper end of the indication pin 45 was disposed in the top surface of the carrier 41.
• the displacement of the pin 43 can thus be rapidly and reliably perceived by way of the projecting indication pin 45, as shown in Fig. 5.
• Fig. 6 shows yet another variant embodiment of the pin-carrier connection.
• the pin 51 is secured in a block 52 of resilient or elastic material, which block in turn is secured in a carrier 53.
• the pin 51 can now move with respect to the carrier 53 through a resilient deflection of the block 52.
• the block 52 may be provided with two grooves as indicated in Fig. 6 with a thin line 54.
• a guiding provision for the carrier with respect to a culture holder which may or may not be in the fashion as shown in Fig. 3.
• the points can be designed in any suitable or desired manner, and the number of pins per carrier may be chosen as desired.
• a device of the invention and a procedure for storage and growth of aerobic bacteria was tested for a collection of 48 different soil isolates.
• the different bacterial strains were isolated from 16 different soil samples from different parts of the Netherlands and subsequently characterized by means of 16SRNA analysis.
• the mother microtitre plate was prepared as follows: to all 96 wells of a sterile deepwell microtitre plate were added 500 ul of sterile nutrient broth agar (8 g/1 nutrient broth, 2 g/1 bacto-agar). After solidification of the nutrient broth agar, 48 wells were inoculated, each with one of the 48 strains (which had been streaked on nutrient broth agar plates 3 days earlier).
• the microtitre plate was frozen at -70 ° C. After a number of days, this mother microtitre plate was taken from the freezer for as short a time as possible and with the aid of a device of the invention a small amount (approximately 0.3 ⁇ l) was taken from each well and transferred to a sterile mictotitre plate filled with 150 ⁇ l of sterile potassium phosphate buffer (50 mM, pH 7.0). The contents of all 96 wells of this plate was plated out in different dilutions on nutrient broth plates and after three days of incubation, the colonies were counted and checked for purity.

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• Physics & Mathematics (AREA)
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• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
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• Apparatus Associated With Microorganisms And Enzymes (AREA)

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• 1999
  • 1999-06-21 NL NL1012394A patent/NL1012394C2/nl not_active IP Right Cessation
• 2000
  • 2000-06-20 WO PCT/NL2000/000426 patent/WO2000079237A1/en active Application Filing
  • 2000-06-20 AU AU57154/00A patent/AU5715400A/en not_active Abandoned
  • 2000-06-20 EP EP00942549A patent/EP1192438A1/en not_active Withdrawn

Non-Patent Citations (1)

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