NL1012394C2 - Method and device for simultaneously and independently sampling frozen cultures of microorganisms and / or eukaryotic cells. - Google Patents

Description

Title: Method and device for simultaneously and independently sampling frozen cultures of microorganisms and / or eukaryotic cells.

The invention relates to a method for sampling frozen cultures of microorganisms, eukaryotic cells, and the like, which cultures are housed in separated compartments in a common container, a sample being taken by contacting a culture transfer with a transfer member, as well as to a device for carrying out such a method.

Many different types of microorganisms and eukaryotic cells are known today and more are still being discovered. Many of said microorganisms and eukaryotic cells can be cultured and / or propagated in microbiological laboratories. In addition, many of the aforementioned microorganisms and eukaryotic cells are genetically engineered so that they differ in one or more properties from the starting material. In order to access all these different organisms and cells, 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, usually in the presence of 15 or 20% glycerol, at a temperature below -70 ° C is a widely used and also simple method for long term (multi-year) storage. In particular, the method is often used to store pure cultures of cellular and viral micro-organisms. Ten! To obtain growing cultures from these stored pure cultures, a small amount of the frozen pure culture is scraped off the frozen surface, for example using a sterile spatula or needle, and then transferred to a growing medium (for example, an agar medium). After this, the growth medium can be incubated at a suitable temperature in order to effect multiplication of the relevant microorganism. Although this technique is satisfactory for standard laboratory work, the method proves to be so labor-intensive that it is an obstacle to scaling up to larger numbers of cultures. In several research areas it is desirable to have several thousands of growing cultures of microorganisms and / or eukaryotic cells available quickly and simultaneously. In those cases, progress is limited by the time, manpower and equipment required to reproduce or multiply the available frozen cultures. to cultivate.

Therefore, it is often not or not entirely possible to handle large numbers of cultures at the same time.

The object of the invention is to considerably reduce the time and / or manpower required for cultivating large numbers of different micro-organisms and / or eukaryotic cells.

The object is achieved according to the present invention in a method of the type described in the preamble, by taking several samples at the same time by bringing each sampled culture into contact with a transfer member in each case, the transfer members movable independently of one another are carried by a common carrier, which is operated in such a manner and for so long that each culture to be sampled is brought into contact with a transfer member by mutual displacement of the various transfer members. By using a carrier with multiple transfer means, it is possible to sample multiple frozen cultures simultaneously and a significant reduction in time and / or manpower can be obtained when culturing frozen cultures. However, in practice the situation frequently arises that the heights of the surfaces of the frozen cultures are not at one, equal height for each culture. For example, it is quite normal that not all containers of a container with. the same amount of liquid. Also, the shape of the frozen surface can vary in an unpredictable manner, so that the height of the surface of one frozen culture is variable. This problem is now obviated in the method according to the invention by having the various transfer members move resiliently relative to each other. This makes it possible to ensure that a sample is taken from all the cultures simultaneously with a single operation, after which all the samples can be simultaneously transferred to and deposited on, for example, a growth medium.

As the transfer member, various well-known elements can be used, such as spatulas, etc.

In accordance with further embodiments of the invention, however, it is preferred that elongated pins are used as transfer elements, which are arranged mutually substantially parallel in the common carrier such that each pin is resiliently movable at least in its longitudinal direction relative to the carrier. The advantages of such a displacement are that relatively large differences in distance 30 can be bridged. Alternatively, the pins could also be embedded in a resilient carrier; Pins which are resiliently movable with respect to the carrier are however preferred because of their better handling.

In order to bring all pins into contact with the various cultures with the same contact force, it is further preferred that each pin is resiliently mounted in the common carrier so that a substantially constant resilience is generated when the said pin is resiliently displaced. An essentially constant resilience has the advantage that it is possible to draw nearly identical samples from the frozen cultures.

Preferably, each transfer member for contacting with a frozen culture is brought to such a temperature that a small portion of said frozen culture is thawed by contact with said transfer member. In this preferred method, the surface of the frozen culture that contacts the pin is liquefied by heat transfer from the pin to the surface of the frozen culture. After withdrawal of the pin, part of the liquid, containing part of the frozen culture of microorganisms and / or eukaryotic cells, then adheres to the end of the pin. To promote heat transfer, at least the end of the pin can be made of a material with a relatively large heat conducting capacity. For further improvement of the heat transfer, the end of the pin which is brought into contact with the culture is preferably made in a blunt and preferably slightly rounded shape.

When transferring microorganisms and / or eukaryotic cells, it is important to note that indeed a sample of the frozen surface has been taken from all frozen cultures; this while the view is obstructed by the pins with carrier extending into the trays. In this connection, according to a further embodiment of the invention, it is preferable that each transmission member is provided with an indication mechanism, which becomes active when said transmission member is resiliently displaced.

1012394 »5

The present invention also relates to a device for use in a method of the invention, which device is provided with at least one transfer member with a handling part. In accordance with the invention, it is provided that the handling part comprises a carrier which carries at least two resiliently movable transfer members, so that the various transfer members 10 can be brought into contact independently with a frozen culture. With such a device, the time and / or manpower required for sampling large quantities of frozen cultures is considerably reduced.

If the transfer members consist of pins, which are arranged mutually substantially resiliently relative to the carrier made of a relatively rigid material, the device can be realized relatively simply and inexpensively.

Preferably, the pins are longitudinally displaceably disposed in the carrier along a path defined by rams, the pins each being pressed by spring force to an end position defined by a runner. An unambiguous starting position with all pin ends in the same plane can thus be obtained. A more or less the same pin end face is particularly advantageous when the device is placed on the container with the frozen cultures.

If the carrier is provided with an inner space in which there is at least one spring for each transfer member, the various springs are housed in an enclosed space in a maintenance-friendly manner. In addition, the housing of the springs in an enclosed space is advantageous in sterilizing the device.

Another enclosed embodiment is available if a pin is telescopically resiliently received in a sleeve part which is fixed in the carrier.

i- You? / 7 '··' 6

Furthermore, it may be preferred that the pins contact the frozen culture in phases with regard to the contact force. In that case, provision may be made for at least 5 for each transfer member. two series-connected springs with mutually different spring constants. The advantage of this is that the same device can be used under two or more different operating pressures allowing different sampling modes. In addition, in this way (by increasing the pressure required for pressing), a control mechanism for pushing through the transfer members can be built in, for instance for preventing pushing through of agar bottoms.

If printing alone is not sufficient to sample or transfer certain cultures, it is preferable according to a further embodiment of the invention that each pin is fixed in such a way that at least the start of a longitudinal displacement from said end position is accompanied by a rotational movement. A more scraping contact of the transfer members with the surface to be touched is achieved with a rotary movement.

In order to promote an even contact between the pins and the cultures, it is preferred that the handling part is provided with a guiding device in which the carrier is slidable in only one direction, which direction will usually be the longitudinal direction of the pins. It may furthermore be preferred that the handling part is provided with positioning means for aligning the device with respect to a holder with a number of box-shaped compartments for receiving a number of frozen cultures mutually separated.

In order to be able to detect on the side of the carrier facing away from the pins that each pin has been brought into contact with a culture, a further one may be carried out. :., "I ί! In the embodiment of the invention, it is provided that each pin is provided with an end with an end surface which in said end position forms part of a larger surface and exits from said larger surface 5 at a longitudinal displacement of said pin. Preferably, the larger surface is a surface of the carrier which is visible when the device is used.

When seeding microorganisms and / or eukaryotic cells, it is generally important not to inoculate other microorganisms and / or eukaryotic cells than those intended. To achieve this, work must be done sterile. This means that it must be possible to sterilize at least the end of the pins that is brought into contact with said cultures. Therefore, at least the end of the pins that can be brought into contact with said cultures is made of a sterilizable material. Sterilization can be done in various ways, such as, for example, by heat, immersion in a disinfectant and / or by killing radiation such as ultraviolet light or radioactive radiation. Preferably, at least the end of the pins that can be contacted with said cultures 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 brought into contact with the said cultures is made of a plastic material. The entire device can also be made of plastic. One of the advantages of a plastic version is that the device can be manufactured at such a low cost that single use for most applications does not encounter financial concerns from the end user.

It is clear that a device of the invention is not only suitable for sampling frozen cultures. It is also extremely suitable for sampling freeze-dried cultures. The device of the invention can also be used for transferring liquid cultures and cultures growing on a soft culture medium such as agar.

With reference to exemplary embodiments shown in the figures, the method and device according to the present invention will now be further elucidated, by way of example only. Thereby 10 shows:

Fig. 1 a front view of a first embodiment of the device according to the invention;

Fig. 2 is a top view of FIG. 1;

Fig. 3 the use of a device according to the invention;

Fig. 4 a front view of a second embodiment of the device according to the invention; .

Fig. 5 an enlarged alternative embodiment of a pin-carrier connection; and FIG. 6 on a larger scale a further variant of a pin-carrier connection.

In FIG. 1 and 2, a sampling device is shown which is provided with a carrier 1 with a handle 2. The carrier 1 is provided with a number of bores 25 through which transfer elements in the form of pins 3 extend in the longitudinal direction. Each pin 3 is provided with a head 4 at one end and at its end

V

other end of a rounded tip 5. In the position shown in FIG. 1, the rest position of the device shown, all heads 30 rest on the top side of the carrier 1 and are held in that position by springs 6, which on the one hand take support against the underside of the carrier 1 and on the other hand against a ring 7 fixed on each pin 3 The carrier 1 is further provided near its four corners with a guide pin 8 rigidly fixed therein, with a locating point 9, which extends beyond the rounded points 5 of the pins 3. As shown in Fig.

9 2, the carrier is provided with 96 pins 3 and 4 guide pins 8.

In FIG. 3 is the sampling of cultures 10 using an apparatus according to FIG. 1 and 2 are shown.

The cultures 10 to be sampled are accommodated in a cross-section holder 11, which is adapted to the device, that is to say a holder with 96 mutually separated compartments 12 in a pattern corresponding to the arrangement of the pins 3 and of guide bores. 13 in a pattern corresponding to the arrangement of the guide pins 8.

For placing the device on the holder 11, the device is brought above the device with the aid of the handle 2, so that the guide pins 8 are in line with the guide bores 13. When the device is subsequently lowered, the search points 9 ensure that the guide pins 8 can slide smoothly into the guide bores 13, whereby the pins 3 are positioned relative to the compartments 12 such that one pin 3 is located above each compartment 12. When the device is lowered further by means of the handle 2, the pins 3 each reach into a compartment 12. As shown in FIG. 3, the level of the various cultures 10 in the various compartments 12 is different.

In the case shown in FIG. 3, the level in the third compartment from the left is highest and the level in the right-most compartment is lowest. Since the guide pins 8 ensure that the carrier 1 maintains and maintains a horizontal position during the downward movement, the pin 3 in the third compartment from the left with its rounded point 5 will first touch the surface of a frozen culture bounce. At that time, therefore, the other points 5 of the other pins 3 are still at a distance from the surfaces of the various cultures.

Continued downward movement of the carrier 1 is possible because the pin 3 in the third compartment of 1012394¾ 10 can move resiliently to the left with respect to the carrier 1, ie remain stationary while the carrier 1 is moved further down. Thus, all rounded tips 5 of all pins 3 can be successively brought into contact with the surface of the associated culture 10. This is now the situation shown in Fig.

3, the rounded tip 5 of the pin 3 in the rightmost compartment just making contact with the surface of the culture 10 in that compartment.

It is noted that in the situation of FIG. 3 all coupling 4 except one have come loose from the top of the carrier 1. This is a sign that the relevant points 5, which are no longer visible from above, have come into contact with a culture 10. Due to the downward movement 15 of to continue the carrier 1 until the last head 4 has also come off the top of the carrier 1, it is ensured that the tips 5 of all pins 3 are in contact with the surfaces of the various cultures 10. Moreover, the spring action ensures that the contact 20 between the various pins 3 and cultures 10 takes place with a force which can be preset by the springs 6, so that at each compartment 12 when the carrier 1 moves upwards again, causing the pins 3 to separate from the cultures 10, the desired amount of culture 10 can be extracted and transferred to a culture medium or the like.

In FIG. 4 shows a device according to the invention which is provided with a carrier 21, which is composed of a top plate 22, a side edge 23 and a bottom plate 24. Thus, an enclosed space 25 is formed through which a number of pins 26 extend. Each pin 26 carries a head 27 at one end and is provided at the other end with a sharply protruded end 28. The top plate 22 and the bottom plate 24 are provided with aligned bores for the conductive sliding fit of a pin 26. The top plate 22 is further provided with recesses 29, 11 of which have a height equal to that of a head 27 and can accommodate all the heads in such a way that they lie with their top surfaces in the top surface of the top plate 22. A spring 5 is arranged around each pin 26 in the enclosed space, which on the one hand supports against the underside of the top plate 22 and on the other hand rests against the top of the bottom plate 24 via a ring 31 fixedly connected to the pin. The springs 30 and the arrangement of the rings 31 on the pins 26 are selected such that in the rest position of the device, as shown in FIG. 4, the heads 27 are located in the recesses 29 and the top surfaces of those heads 27 are flush with the top surface of the top plate 22. The latter is provided with projecting edge portions 22a, which serve as handles. The operation of this device in contacting all ends 28 of the pins 26 with the surfaces of cultures not shown further is the same as in FIG. 3, so that this operation will not be described further.

It should be noted that choosing pin ends with sharp protrusions, as shown in FIG. 4, in some cases may be preferable to rounded ends, as shown in FIG. 1. Furthermore, the heads 27 may have a colored side peripheral edge, so that it is better 25 and easier to detect when a pin 26 is shifted, and thus its tip 28 has made contact with the surface of a culture. After all, the colored side peripheral edge of the head 27 will exit the recess 29 and thus give a clear indication of the movement of the pin 26.

A further possible embodiment of a pin-carrier connection is shown in Fig. 5. A plate-shaped support (partly shown) is herein provided to which a number of guide sleeves 42 are attached. The end of the guide sleeve 42 remote from the bearing 41 is narrowed to a passage for sliding pin 43 through sliding. At its upper end, the pin 43 terminates in a boss 44 which is slidable in the guide sleeve 42 is. Starting from the top of the thickening 44, an indicator pin 45 extends 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 in the guide sleeve 42 around the indicator pin 45, which tries to press the thickening 44 against the narrowed lower end of the guide sleeve 42.

In FIG. 5 shows the situation where the lower end of the pin 43, not shown, has been brought into contact with the surface of a culture, after which downward movement of the carrier 41, for reasons as discussed above, has continued. Before the intended contact was made, the action of the spring 47 caused the bead 44 to rest against the narrowed end of the guide sleeve 42 and the upper end of the indicator pin 45 to be located in the top surface of the carrier 41.

The displacement of the pin 43 can thus be detected quickly and reliably by the protruding indicator pin 45, as shown in Figs. 5.

Fig. 6 shows yet another embodiment for 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 support 53. The pin 51 can now move

V

with respect to the carrier 53 by a resilient deflection of the block 52. To facilitate the resilient deflection, the block 52 can be provided with two grooves as shown in Figs. 6 is indicated by a thin line 54.

It goes without saying that many modifications and variants are possible within the scope of the invention, as laid down in the appended claims. Thus, in the embodiment of FIG. 4, the carrier is guided with respect to a 101 239 4 * 13 culture container, whether or not in the manner as shown in FIG. 3. The points can also be designed in any suitable or desired manner and the number of pins per carrier can be chosen as desired. Furthermore, provisions 5 may be present which also cause a pin to make a rotation during a longitudinal shift, for example by the thickening 44 in Fig. 5 to give the shape of a worm gear and to provide the guide sleeve 42 with a corresponding screw thread.

10

Onion twerking example:

An apparatus of the invention and a procedure for storage and growth of aerobic bacteria has been tested for a collection of 48 different soil isolates. The 15 different bacterial strains have been isolated from sixteen different soil samples from different parts of the Netherlands and subsequently characterized using 16SRNA analysis. The master microtiter plate was prepared as follows: 500 µl of sterile nutrient broth agar (8 g / 1 nutrient broth, 2 g / 1 bacto agar) was added to all 96 wells of a sterile deepwell 20 microtiter plate. After solidification of the nutrient broth agar, 48 wells were seeded, each containing one of the 48 strains (streaked 3 days previously on nutrient broth agar plates).

25 One well was inoculated and one well not inoculated. This created a pattern where each inoculated well is surrounded by 2-4 non-inoculated wells. After two days of growth, 500 μΐ liquid nutrient broth medium (8 g / l) was added to all 96 wells. The 30 microtiter plate was then covered with a sterile lid consisting of a 96-hole perforated rubber layer and a cotton wool layer on top. The whole was clamped together and shaken on an orbital shaker (300 rpm) for two days at 25 ° C. After these two 35 days of growth, 150 µl of a sterile glycerol: water (60:40 v / v) mixture was added to all wells and mixed. The microtiter plate was then frozen at -70 ° C.

14

After a few days, this master microtiter plate was taken out of the freezer for the shortest possible time and using a device of the invention, a small amount (approximately 0.3 μΐ) was taken from each well and transferred to a sterile microtiter plate filled with 150 μ gevuld sterile potassium phosphate buffer (50 mM, pH 7.0). The contents of all 96 wells from this plate were plated at nutrient broth plates in various dilutions and after three days of incubation the colonies were counted and checked for purity. From this data, it was calculated how many colony-forming units were taken up from each of the wells of the master microtiter plate (see Table 1). The number of colony-forming units grafted by the device varied between 180 and 50,000 for the different strains (see Table 1). Colony-forming units were not found in any of the 48 wells of the master microtiter plate that had not been seeded. This indicates that during the described procedure, the sterile wells were not infected by the inoculated wells. In a.

In the second experiment, the device was similarly used to record a small volume from the master microtiter plate. The device was then transferred directly to a nutrient broth agar plate. After two days, a regular pattern of colonies formed was seen on this agar plate, fully consistent with the pattern of the inoculated wells in the master microtiter plate. Then, part of the cellular material from the individual colonies on this agar plate could be transferred to a deep well plate filled with a liquid growth medium (750 µl / well) using a device of the invention. After incubation on an orbital shaker for 2 days (as described above), cell densities on the order of 2-8 g / l were achieved, which is sufficient for most applications (eg tests for the presence of a specific enzyme).

15

Table 1.

Number of colony-forming units taken up from the master microtiter plate, broken down by bacterial species.

number of colony forming units included '* by the device number of lowest average standard strains deviation

Pseudomonas viridiflava 1 5000 5000

Pseudomonas syringae 6 6400 39400 35290

Pseudomonas stutzeri 3 10000 26667 20817

Pseudomonas putida 5 180 4132 4028.

Pseudomonas mendocina 1 20000 20000

Pseudomonas marginalis 6 10000 50000 40988

Pseudomonas fulva 2 20000 20000 0

Pseudomonas fluorescence 2 20000 35000 21213

Pseudomonas cepacia · 1 50000 50000

Pseudomonas azotoformans 2 20000 35000 21213

Rhodococcus opacus 6 513 2679 2124

Rhodococcus globerulus 5 500 7560 7807

Rhodococcus erythropolis 2 15000 57500 60104

Alcaligenes sp. 3 8000 9333 1414

Achromobacter xylosoxydans 1 10000 10000 -

Variovorax sp. 1 50000 50000

V

101 2394a

Claims (16)

1. Method for sampling frozen. cultures of microorganisms, eukaryotic cells and the like, which cultures are housed in separated compartments in a common container, a sample being taken by contacting a culture with a transfer member, characterized in that several samples are simultaneously be taken in that each culture to be sampled is brought into contact with a transfer member in each case, the transfer members being independently movable with respect to each other, supported by a common carrier, which is operated such and so long that by mutual displacement of the various transfer means each culture to be sampled is contacted with a transfer means. 2. A method according to claim 1, characterized in that elongated pins are used as transfer elements, which are arranged in the common carrier such that each pin is resiliently movable at least in its longitudinal direction relative to the carrier. 3. Method according to claim 2, characterized in that each pin is resiliently mounted in the common carrier 25 such that a substantially constant spring force is generated when said pin is resiliently displaced. 4. A method according to any one of the preceding claims, characterized in that each transfer member for contacting with a frozen culture is brought to such a temperature that a small part of said frozen culture is thawed by contact with said transfer member. 5. A method according to any one of the preceding claims, characterized in that each transfer member is provided with a:. indicator mechanism, which becomes active when the said transfer member has been resiliently displaced. 6. Device for use in a method according to any one of the preceding claims, and provided with at least one transfer member with a handling part, characterized in that the handling part comprises a carrier, which carries at least two transfer members which are movable relative to each other . 7. Device as claimed in claim 6, characterized in that the transfer members consist of pins which are resiliently mounted relative to the carrier made of a relatively stiff material. 8. Device as claimed in claim 7, characterized in that the pins are longitudinally displaceable in the carrier over a path determined by rams, wherein the pins are each pressed by spring force to a final position determined by a runner. 9. Device as claimed in any of the claims 6-8, characterized in that the carrier is provided with an inner space 20 in which at least one spring is located for each transfer member. 10. Device as claimed in any of the claims 6-8, characterized in that a pin is slidably telescopically accommodated in a sleeve part fixed in the carrier. 11. Device as claimed in any of the claims 6-10, characterized in that for each transfer member there are at least two springs connected in v series with mutually different spring constants. 12. Device as claimed in any of the claims 6-11, characterized in that the handling part is provided with a guiding device in which the carrier is slidable in only one direction. * o s - - -. 12. Device as claimed in any of the claims 8-10, characterized in that each pin is fastened such that at least the beginning of a longitudinal displacement from said end position is accompanied by a rotational movement. 13. Device as claimed in claim 12, characterized in that the handling part is provided with positioning means for aligning the device relative to a container with a number of box-shaped compartments for receiving a number of frozen cultures mutually separated. 14. Device as claimed in any of the claims 6-13, characterized in that each pin is provided with an end with an end surface which, in said end position, forms part of a larger surface and exits from said larger surface during a longitudinal displacement of said pin. Device as claimed in claim 14, characterized in that said larger surface is a surface of the carrier which is visible when the device is used. Device according to any one of claims 6-15, characterized in that at least the end of the pins which can be brought into contact with said cultures is made of a sterilizable material. v 101 239 4¾.