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.

At present, a great many different kinds of 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 amplifie 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. In order to revive these stored pure cultures to growing cultures, 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.

Although this technique is quite satisfactory for standard laboratory work, the method is found to be so labor intensive as to constitute an impediment to upscaling to larger number of cultures. In various fields of research, the ready and simultaneous availability of several thousands of growing cultures of microorganisms and/or eukaryotic cells is desirable. In those cases, progress is limited by the time, manpower and equipment needed to revive the available frozen cultures to replication again or culture them.

Therefore it is often not properly possible to handle large numbers of cultures simultaneously.

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.

This object is achieved, according to the present invention, in a method of the type described in the opening paragraph hereof, by taking a plurality of samples simultaneously in that each culture to be sampled is contacted with a respective transfer element, the transfer elements being carried by a common carrier so as to be springingly movable independently with respect to each other, the carrier being operated in such a manner and so long that through relative displacement of the various transfer elements each culture to be sampled has been brought into contact with a transfer element. By using a carrier with a plurality of transfer elements, it is possible to sample a plurality of frozen cultures at the same time, and a considerable reduction in time and/or manpower can be obtained when taking frozen cultures into culture. However, in practice, often a situation arises where the levels of the surfaces of the frozen cultures are not at one level that is the same for each culture. Thus, it is quite normal that not all wells of a holder are filled with the same amount of liquid. Also, the shape of the frozen surface may vary in a non-predictable way, so that the level of the surface of an individual frozen culture is variable. This problem is now overcome in the method according to the invention by enabling the various transfer elements to move springingly and independently with respect to

each other. Thus, a single operation can cause a sample to be taken simultaneously from all the cultures, whereafter all the samples can be transferred simultaneously to and deposited on, for instance, a growth medium.

As transfer element, various well-known elements can be used, such as spatulas and the like. According to further embodiments of the invention, however, it is preferred that as transfer elements, 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. Alternatively, 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.

To contact all pins with the various cultures with the same pressure force, it is further preferred that 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.

Preferably, 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. In this preferred method, 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. To promote heat transfer, at least the end of the pin can be designed in a material of a relatively large heat conducting capacity. For a further improvement of the heat transfer, the

end of the pin that is contacted with the culture is preferably designed in a blunt and preferably slightly rounded form.

When inoculating microorganisms and/or eukaryotic cells, it is of importance to be able to verify that a sample of the frozen surface has in effect been taken from all frozen cultures; this while the view is obstructed by the pins extending into the wells and the carrier. In this connection, it is preferred, according to a further embodiment of the invention, that 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. According to the invention, there is provided that 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. With such a device, the time and/or the manpower needed for sampling large amounts of frozen cultures is reduced considerably.

If 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.

Preferably, 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. Thus, 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.

When 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. In addition, 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.

Further, it may be preferred that the pins, as regards the press-on force, come into contact with a frozen culture in a phased manner. In that case, it may be provided that for each transfer element at least two series-connected springs of mutually different spring constants are present.

The advantage of this is that the same device can be used under two or more different operating pressures, thereby enabling different sampling methods. In addition, in this way (owing to the increase of the pressure needed for compression) a control mechanism for pushing through the transfer elements can be incorporated, for instance for preventing agar media from being pushed through.

Should mere pushing be insufficient to sample or transfer particular cultures, it is preferred, according to a further embodiment of the invention, that 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.

For promoting a uniform contact between the pins and the cultures, it is preferred that 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.

To be able to see, on the side of the carrier remote from the pins, that each pin has been brought into contact with a culture, it may be

provided, according to a further embodiment of the invention, that 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. Preferably, the larger surface referred to is a surface of the carrier which lies within view in the use of the device.

When transferring microorganisms and/or eukaryotic cells, it is generally of importance not to transfer any other cells than the microorganisms and/or eukaryotic cells in question. To accomplish this, work must be done aseptically. This means that it must be possible to sterilize at least the end of the pins that is contacted with the cultures mentioned. Therefore at least the end of the pins that can be contacted with the cultures is manufactured from a sterilizable material. Sterilization can be done in different ways, such as, for instance, by heat, immersion in a disinfectant and/or by lethal radiation such as UV light or radioactive light.

Preferably, 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. An example of a material that can be sterilized several times is stainless steel.

In another embodiment of a device of the invention, at least the end of the pins that can be contacted with the cultures is made of a plastic material. Also, the whole device may be made of plastic. One of the advantages of a design in plastic is that the device can be fabricated so inexpensively that single use for most applications does not meet with financial objections on the part of the end user.

It is clear that 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.

Referring to the exemplary embodiments represented in the drawings, by way of example only, the method and device according to the present invention will presently be clarified in more detail. In the drawings: Fig. 1 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; and Fig. 6 shows on an enlarged scale a further variant of a pin-carrier connection.

In Figs. 1 and 2, a sampling device is represented 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. In the rest position of the device represented in Fig. 1, all heads 4 rest on the upper side of the carrier 1 and are held in that position by 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. Further, adjacent its four corners, 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. As appears from Fig. 2, the carrier is provided with 96 pins 3 and four guide pins 8.

In 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.

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. Upon subsequent lowering of the device, 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. Upon further lowering of the device by means of the handle 2, the pins 3 each reach into a compartment 12. As appears from Fig. 3, 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. Because the guide pins 8 ensure that the carrier 1 has, and continues to have, a horizontal position during lowering, 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. Continued downward movement of the carrier 1 is possible in that 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.

Thus, successively, 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.

It is noted that in the situation of Fig. 3, all heads 4 but one have come clear of the upper side of the carrier 1. This is a sign that the respective points 5, which are no longer visible from above, have come into contact with a culture 10. By continuing the downward movement of the carrier 1 until the last head 4 has also come clear of the upper side of the carrier 1, it is ascertained that the points 5 of all pins 3 are in contact with

the surfaces of the various cultures 10. Moreover, through the spring action, it is ensured that the contact between the various pins 3 and cultures 10 occurs with a force which can be pre-set by the springs, so that from each compartment 12, as the carrier 1 is moved up again whereby the pins come clear of the cultures 10, the desired amount of culture 10 can be extracted and transferred to a nutrient medium or the like.

In Fig. 4 a device according to the invention is represented, comprising a carrier 21, which is composed of an upper plate 22, a side edge 23 and a lower plate 24. Thus an enclosed space 25 is formed, through which extend a number of pins 26. Each pin 26 carries a head 27 at one end, and at the other end comprises an end 28 provided with sharp projections.

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. In the enclosed space, 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.

It is noted that choosing the pin ends with sharp projections, as represented in Fig. 4, in some cases may be preferable to rounded ends, as represented in Fig. 1. Further, 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. At its upper end the pin 43 terminates in a thickening 44 which is displaceable with a sliding fit within the guide sleeve 42. Starting from the upper end of the thickening 44 extends an indication pin 45 which reaches through a bore 46 provided in the carrier 41. Between the thickening 44 and the underside of the carrier 41, a spring 47 is arranged around the indication pin 45 in the guide sleeve 42, which urges the thickening 44 against the narrowed lower end of the guide sleeve 42.

In 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 continue. Before the contact referred to came about, 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. To facilitate the resilient deflection, the block 52 may be provided with two grooves as indicated in Fig. 6 with a thin line 54.

It will be clear that within the framework of the invention as set forth in the appended claims, many modifications and variants are possible.

Thus, in the embodiment of Fig. 4, there may be provided 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. Also, the points can be designed in any suitable or desired manner, and the number of pins per carrier may be chosen as desired. Further, there may be provisions present which cause a pin during a longitudinal displacement to make a turn at the same time, for instance by giving the thickening 44 in Fig. 5 the shape of a worm wheel and to provide the guide sleeve 42 with a corresponding screw thread.

Practical example: 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). Alternately, one well was inoculated and one was not. This yielded a pattern where each inoculated well is surrounded by 2-4 non-inoculated wells. After two days of growth, to all 96 wells 500 pl of liquid nutrient broth medium (8 g/1) were added. The microtitre plate was then covered with a sterile cover consisting of a 96-hole perforated rubber layer and a layer of cotton wool above it. The whole was clamped together and shaken for two days at 25°C on an orbital shaker (300 revolutions per minute). After these two days of growth, to all wells 150 gel of a sterile glycerol : water (60: 40 v/v) mixture were added, followed by mixing.

Subsequently, 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 pl) was taken from each well and transferred to a sterile mictotitre plate filled with 150 al 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. From these data it was calculated how many colony-forming units had been taken up from each of the wells of the mother microtitre plate (see Table 1). The number of colony-forming units transferred by the device varied for the different strains between 180 and 50,000 (see Table 1). In none of the 48 wells of the mother microtitre plate that had not been inoculated were any colony-forming units found. This indicates that during the procedure described, the sterile wells were not infected by the inoculated wells. In a second experiment, the device was used in the same manner to take up a small volume from the mother microtitre plate. The device was subsequently transferred directly to a nutrient broth agar plate. After two days, a regular pattern of colonies which had formed was to be seen on this agar plate, entirely in agreement with the pattern of the inoculated wells in the mother microtitre plate. Subsequently, a part of the cell material of the individual colonies on this agar plate could be transferred with the aid of a device of the invention to a deepwell plate filled with a liquid growth medium (750 ul/well). After incubation on an orbital shaker for 2 days (as described above), cell densities of the order of 2-8 g/1 were achieved, which is sufficient for most applications (for instance screens for the presence of a specific enzyme).

Table 1.

Number of colony-forming units taken up from the mother microtitre plate, listed according to bacterial species. numberofcolony-forming unitspickedup bythedevice number of standard loweat average deviation Pseudomonasviridiflava1 5000 5000 Pseudomonassyringe5 6400 3940035290 Pseudomonasstutzeri3 10000 26667 20817 Pseudomonasputida5 180 4132 4028 Pseudomonas mendocina 1 20000 20000 Pseudomonasmarginais6 10000 5000040988 Pseudomonasfulva2 20000 200000 Pseudomonasfluorescence2 20000 3500021213 Pseudomonascepacia1 50000 50000 Pseudomonasazotoformans2 20000 3500021213 Rhodococcusopacus 6 513 26792124 Rhodococcusgloberulus5 500 7560 7807 Rhodococcuserythropolis2 15000 5750060104 Alcaligenessp.3 8000 93331414 Achromobacterxylosoxydans1 10000 10000 Variovorax sp. 50000 50000