Method of sampling andlor depositing a sample of biological
matter and device implementing such a method
The aim of the present invention is a method of sampling and a
method of depositing a sample of biological matter, crude or enriched cultured
5 through contact with a culture medium, as well as a device implementing
these methods.
The sampling of a sample of microorganisms (bacteria, moulds,
yeasts or the like) cultured on an agar culture medium in a Petri dish, or on
any other support, is currently accomplished with the aid of single-use tools
10 such as oeses, sticks, tubes or cones.
However, these consumables do not make it possible to certainly
and efficiently sample all types of microorganisms because the latter can have
very different forms, sizes, consistencies, structures or appearances.
On the other hand, these consumables do make it possible to
15 optimally deposit the biological matter on analysis supports such as plates,
nor to easily re-suspend said biological matter.
Furthermore, it is important to be able to allow sampling of a
bacterial colony or of a fraction of this colony without sampling the culture
medium situated under the colony: this could vitiate the analysis results.
20 The quality of the analysis results also depends on the
concentration of the deposit of biological matter formed from the sampled
sample, and on its homogeneity on the support on which it is deposited.
The present invention aims to remedy all or some of the
disadvantages mentioned above.
25 To this end, the object of the present invention is a method of
sampling all or part of a sample of biological matter, crude, enriched or
cultured through contact with a culture medium, possibly agar, using a probe
equipped with a terminal end, said sampling method comprising the steps of
cooling the terminal end of the probe, sticking all or part of the sample of
30 biological matter to be sampled through contact of the terminal end onto the
sample of biological matter, or by applying a pressure exerted by the terminal
end onto the sample of biological matter, and sampling all or part of the
sample of biological matter so as to separate the sample of biological
matter from its support, such as a culture medium.
This method makes it possible to sample any type of sample of
biological matter cultured in vitro regardless of form and consistency.
Furthermore, with this sampling method, there is no possible contamination
5 of the probe because its end, in contact with the sample, can be sterilised
after each use, or is never in direct contact with the sample.
Furthermore, with' this sampling method, there is not necessarily
any need to use a single-use terminal end, which reduces the cost of use
whilst allowing faster use cycles (better productivity of the device).
10 According to one implementation means, the probe is a cryogenic
probe comprising means for being cooled.
According to one implementation of the sampling method, prior to
the sticking step, a step of wetting the terminal end of the probe in a liquid
solution such as distilled water is carried out, in order to form a layer of ice on
15 the terminal end.
This arrangement allows a consumable to be made
extemporaneously which allows the sample to be stuck in order to sample it.
Furthermore, with this implementation of the sampling method, the sample is
never directly in contact with the terminal end of the probe.
20 According to one implementation of the method, prior to the
sticking step, the sample of biological matter to be sampled is covered with a
liquid solution, in order to form a layer of ice around the sample during the
step of sticking all or part of said sample to the terminal end of the probe.
According to one implementation of the method, the liquid
25 solution is taken from the group comprising water, a saline solution, a buffer,
a liquid culture medium or a matrix commonly used for ionising the sample of
biological matter with a view to analysing it using a measuring device such as
a mass spectrometer. A buffer may for example a carbonate buffer (1 0 to 100
mmolIL, ideally 25 mmolIL)
3 0 This arrangement makes it possible to reduce by one step the
sample mass spectrometer analysis method, the matrix having the double
function of being used for the sticking step during sampling and for a sample
preparation step with a view to its mass spectrometer analysis.
Alternatively, the liquid solution can be a microorganism culture
35 medium.
According to one implementation of the sampling method, the step
of wetting the terminal end of the probe or the sample of biological matter to
be sampled in a liquid solution is repeated successively at least twice, in order
to form overlaying layers of ice, like a stalactite.
This arrangement makes it possible to enlarge the sticking surface
5 of the stalactite created in this way and thus to be able to sample larger
samples.
According to one implementation of the sampling method, the
terminal end is removable.
This arrangement makes it possible to considerably increase the
10 useful sticking surface of the terminal end.
According to one implementation of the sampling method, the
terminal end possesses ferromagnetic properties.
According to one implementation of the sampling method, the step
of sampling all or part of the sample of biological matter consists in applying a
15 magnetic field, for example using an electromagnet, adjacent to the
ferromagnetic terminal end so as to attract the terminal end and thus recover
it.
This arrangement makes it possible to automate the sampling
without risking the contamination of the sampled sample.
20 Another object of the present invention is a method of depositing
into a container or onto an analysis plate all or part of a sample of biological
matter stuck onto a frosted terminal end of a probe, said depositing method
including a step of separating the terminal end of the probe from all or part of
the sample of biological matter stuck onto said terminal end.
25 This arrangement makes it possible to employ all or part of the
sample.
According to one implementation of the depositing method, the
separation step is carried out without the sample of biological matter stuck on
the frosted terminal end coming into contact with the container or the analysis
30 plate.
In this case, the step of separating the terminal end from all or part
of the sample of biological matter stuck on said terminal end is carried out by
applying a mechanical shock onto the terminal end.
This arrangement makes it possible to immediately collect all of
35 the sampled sample in a frozen state.
According to one implementation of the depositing method, the
step of separating the terminal end from all or part of the sample of biological
matter stuck on the frosted terminal end is carried out by a heating means or
in ambient air.
This arrangement makes it possible to collect the sample in a
5 liquid state and makes it possible to distribute several parts of the sample on
several different supports.
According to one implementation of the depositing method, the
separation step is achieved by sequential contact of the sample of biological
matter with the container or the analysis plate, this contact bringing about the
10 melting of a superficial layer of ice and the depositing of biological matter.
This arrangement makes it possible to obtain control of the
quantity of sample deposited and of the choice of depositing support, and the
melting of the superficial layer makes it possible to obtain a homogeneous
deposit, both in terms of distribution of biological matter and the depth of the
15 deposited layer.
According to one implementation of the depositing method, all or
part of the sample of biological matter is distributed on an analysis plate so as
to make several distinct deposits of parts of the same sample of biological
matter, which is stuck on the terminal end.
20 This arrangement makes it possible to take several measurements
on the deposits from the same sample, each of these deposits being
homogeneous, which makes it possible to improve the quality of certain
measurement spectra, in particular for mass spectrometry measurements
where a low degree of homogeneity in the deposit leads to spectra comprising
25 less intense peaks with a signal which includes a lot of noise.
According to one implementation of the depositing method, the
depositing is effected through continuous contact, for example in the form of
lines, of all or part of the sample of biological matter stuck on the terminal end
with the container or the analysis plate.
30 This arrangement makes it possible to achieve a biological matter
concentration gradient on the deposit support, and then to focus the
measures on the gradient zone which makes it possible to obtain the best
results, for example for mass spectrometry measurements.
Another object of the present invention is a device for sampling
35 and depositing all or part of a sample of biological matter, crude, enriched or
cultured through contact with a culture medium and intended to be deposited
into a container or an analysis plate, characterised in that it comprises: a
probe equipped with a terminal end, a cooling means intended for frosting the
terminal end, driving means intended to exert a pressure from the probe onto
the sample so as to freeze all or part of the water contained in the sample in
5 order to stick it to the terminal end, to separate all or part of the sample from
its support, such as a culture medium, to bring all or part of the sample to the
container or the analysis plate.
This arrangement provides an automated and reusable device
which makes it possible to carry out several sampling and deposition in a
10 minimum amount of time.
According to one embodiment, the device comprises a heating
means intended for detaching the sample from the terminal end.
According to a particular embodiment, the heating means is
intended for sterilising the terminal end. Such a sterilisation is carrying out
15 before any sampling.
Advantageously, the cooling means and the heating means are
composed of at least one Peltier element. Advantageously, several overlaid
Peltier elements are used.
This arrangement allows the sample to pass quicker to liquid state
20 and facilitates its re-suspension and homogenisation.
According to one embodiment, the terminal end is metallic or
mineral.
This arrangement makes it possible to absorb more quickly the
heat contained in the water of the sample, and thus freeze it more quickly.
25 Advantageously, the terminal end is at least partially covered with
a hydrophobic coating or treatment. Such a coating enables easier and
optimised drainage of the liquid and/or the sample present on the terminal end
when it is unfrozen.
According to one embodiment, the terminal end is removable.
3 0 This arrangement makes it possible to separate the frosted
terminal end, in order to stick it directly to the sample of biological matter to be
sampled.
It furthermore makes it possible to store the terminal end in a
refrigerated environment, which can be particularly advantageous if it is
35 desired to carry out an extemporaneous analysis.
According to one embodiment, the terminal end has a shape
which optimises the sticking of all or part of the sample of biological matter to
be sampled or of liquid solution. According to a particular embodiment, the
terminal end comprises a pointed end. Advantageously, the shape of the
terminal end can be tapered. Such a shape greatly facilitates the sticking of
5 the sample or of the liquid solution during wetting.
This arrangement makes it possible to precisely sample the
sample, but also to sample very small samples such as bacterial
microcolonies.
According to one embodiment, the device comprises at least one
10 sensor which controls the pressure exerted by the driving means via the
probe in contact with the sample of biological matter to be sampled which is
cultured on the culture medium, and does this in order to halt this pressure.
This arrangement makes it possible to prevent any sticking of the
sample support, such as an agar culture medium.
15 According to one embodiment, the sensor is a pressure or force
sensor, which can be placed under a Petri dish support for example.
This arrangement makes it possible to control the quantity of
biological matter, in the manner of an electronic scale.
According to one embodiment, the device comprises a sensor
20 which detects the contact between the terminal end and the biological matter
in order to avoid any pressure.
According to one embodiment, the sensor is a binary electric
sensor.
This arrangement makes it possible to prevent any sticking of the
25 sample support, such as an agar culture medium, at lower cost.
Another object of the present invention is a kit comprising a device
as described previously, which includes a plurality of interchangeable terminal
ends.
These terminal ends may or may not be single-use. They may be
30 pointed, loop-shaped, or cylindrical, and of different sizes. This arrangement
makes it possible to have terminal ends of different shapes and sizes for the
same device, in order to adapt to the size of the sample to be sampled.
Another object of the present invention is a biological analysis
apparatus which contains a device or a kit such as described previously.
35 In any case, the invention will be better understood with the aid of
the following description, with reference to the attached schematic drawings
which show, in a non-limiting manner, a device which implements the steps of
a method according to the invention.
Figure 1 shows the overview of a device according to the
invention.
5 Figure 2 shows some steps of a sampling method according to a
first implementation according to the invention.
Figure 3 shows some steps of a sampling method according to a
second implementation according to the invention.
Figure 4 depicts several examples of sampling and depositing all
10 or part of a sample.
Figure 5 depicts an embodiment of a cooling means for the
terminal end using Peltier elements.
Figure 6 shows a mass spectrum obtained after sampling and
depositing a colony of Staphylococcus aureus using the methods according to
15 a first embodiment of the invention.
Figure 7 shows a mass spectrum obtained after sampling and
depositing a colony of Escherichia coli using the methods according to the
first embodiment of the invention.
Figure 8 shows a mass spectrum obtained after sampling and
20 depositing a colony of Escherichia coli using the methods according to a
second embodiment of the invention.
As depicted in figure 1, a sampling and depositing apparatus 1
according to the invention comprises a sampling and depositing device 2.
This sampling and depositing device 2 contains a probe 3, as well
25 as driving means 5 intended for spatial movement of the probe 3.
This driving means 5 may be constituted by automated articulated
' arms, or any other equivalent means known to the person skilled in the art.
The probe 3 comprises a metal, removable, pointed terminal end
4, which can be replaced by a terminal end 4 with a different-sized point which
30 makes it possible to carry out precise samplings on the biological matter 7.
The probe 3, by the action of the driving means 5, moves above a
culture of biological matter 7, constituted here by bacterial colonies cultured
on an agar culture medium 8 in a Petri dish 6.
The composition of the biological matter 7 essentially comprises
35 liquid water.
The probe 3 also comprises a cooling means (not shown) for
frosting its terminal end 4. This means can for example be constituted by
liquid nitrogen routed to the terminal end 4 by a conduit situated in the probe 3
or by depressurising a refrigerant gas in a volume which is in contact with the
terminal end 4.
5 It can also be constituted by one or more Peltier elements in
contact with the terminal end 4. In particular, figure 5 shows an embodiment in
which the cooling means comprise two stages 17a and 17b of Peltier
elements on which the terminal end 4 is mounted.
These elements have the advantage of being used for both
10 cooling and heating the terminal end 4, by reversing their electric supply
voltage.
In order to prevent heat exchange with the ambient air, it can be
advantageous to insulate the upper part of the terminal end 4, which does not
come into contact with the sample. This insulation makes it possible to
15 optimise the thermal action of the terminal end 4.
It is in fact possible to envisage having more than two overlaid
Peltier elements. The greater the number of elements, the greater the
extreme temperatures obtained.
Thus, the cooling and heating of the terminal end 4 will be
20 quicker. It may be possible to envisage using this construction to sterilise the
terminal end by heating to a high temperature. This is particularly
advantageous in preventing contamination between the different samples of
biological matter sampled successively.
The cycle of samplingldepositing biological matter 7 can thus be
25 substantially accelerated and controlled.
The implementation of a sampling method makes it possible to
stick to the terminal end 4 of the probe 3 all or part of the sample 11 of the
biological matter 7 to be sampled; in this present case, a bacterial colony
cultured on the culture medium 8 in a Petri dish 6.
30 In a first embodiment of the sampling method, a film of ice is
deposited on the terminal end 4 by means of the cooling means.
The formation of the film of ice 13 is enabled by the presence of
the water contained in the ambient air.
To this end, the device 2 can directly contain a cryogenic source
35 intended for cooling the probe 3 and the terminal end 4.
As shown in figure 2, the probe 3 with its terminal end 4, via the
driving means 5, is brought into contact with the colony 11 of bacteria 7 which
has developed on the agar 8 contained in the Petri dish 6.
These driving means 5, via the terminal end 4, apply a pressure
on the colony 11 to be sampled. This pressure associated with the low
5 temperature of the film of ice 13 which covers the terminal end 4 makes it
possible to freeze the water contained in the bacterial colony 11.
This pressure is measured by a sensor (not shown) which is
situated below the Petri dish 6 and which controls the descent of the driving
means 5, stopping this descent beyond a specified pressure threshold.
10 Alternatively, the terminal end 4 can simply come into contact with
the colony 11, without exerting pressure on said colony. To do this, it is useful
to have a contact sensor which stops the descent of the driving means 5
once contact is made. Such a sensor may for example be a binary electric
sensor.
15 When it freezes, the water contained in the colony 11 forms a
solid block with the layer of ice 13 situated on the surface of the terminal end
4 of the probe 3. All or part of the colony 11 to be sampled is included in the
ice, and so is stuck onto the terminal end 4.
The pressure or contact exertion time can be advantageously
20 controlled by the device 2.
In a second implementation of the sampling method, depicted in
figure 3, the terminal end 4 of the probe 3 is wetted in a liquid solution 12,
such as liquid water or a matrix commonly used for ionising the sample 11 of
biological matter 7 with a view to analysing it by a measuring device such as
25 a mass spectrometer.
The liquid solution 12 then forms a layer of ice 13 generally in the -
form of a drop 16 at the end of the terminal end 4. The drop 16 freezes under
the action of the cooling means to form an adhesion surface which comes
into contact, via the driving means 5, with the bacterial colony 11 to be
30 sampled.
In a variant of this second implementation of the sampling method
also illustrated in figure 3, the terminal end 4 of the probe 3 is wetted in the
liquid solution 12 several times in succession in order to increase the size of
the layer of ice 13 used for the adhesion of the colonies 11 to be sampled.
3 5 It is thus possible to adjust the size of this layer of ice 13 to the
dimensions of the colony 11 to be sampled.
In these implementations of the sampling method, it can be
advantageous to have a terminal end 4 of the probe 3 which has a shape
capable of facilitating the attachment of the drop(s) of liquid solution 12 on
said end. This shape can be tapered. The end can also have a groove, for
5 example a horizontal groove, on the edge of the end in order to optimise the
attachment of the drop of liquid solution 12.
According to a particular embodiment, the container containing
the liquid solution can also have a particular shape. Firstly, it is advantageous
to have a receptacle with a small volume in line with the volume of the
10 drops(s) to be formed. Secondly, the interior of the container can
advantageously have a specified shape correspond to that which the drop
must have. Thus, the interior of the receptacle can be tapered. It is moreover
beneficial for the container to be deep, insofar as it makes it possible to obtain
a relatively long frozen drop and thus limit the risks of contamination of the
15 terminal end 4 by the sample of biological matter.
According to another particular embodiment, the container can
have its own cooling means. In that case, the liquid solution 12 contained in
the container is cooled. By keeping said liquid solution at a temperature close
to its freezing point, it is possible to obtain quicker formation of the frozen drop
20 when the terminal end is dipped into the liquid solution.
In a third implementation of the sampling method (not shown), a
drop of liquid solution 12 is deposited onto the colony 11 to be sampled, such
that the latter is totally or partially covered in liquid. The terminal end 4 of the
probe 3 is then applied onto the drop of liquid solution 12 such that the latter is
25 frozen, trapping all or part of the colony 11 to be sampled and in turn freezing
the latter.
In a subsequent step, the driving means 5 separate all or part of
the colony 11 from the agar layer 8, thus accomplishing the sampling step.
The driving means 5 make it possible to move the sampled
30 colony 11 above a container 9, such as a test tube 9 or an analysis plate 14,
such as an analysis plate used in mass spectrometry.
The implementation of a depositing method subsequently makes
it possible to separate the frosted terminal end 4 of the probe 3 from all or part
of the colony 11 stuck onto it.
3 5 In this first implementation of this depositing method, the
separation between the colony 11 and the terminal end 4 is accomplished
without contact with any support in ambient air after interruption of the action
of the cooling means. Indeed, once the action of the cooling means is
interrupted, the ice formed on the terminal end 4 of the probe 3 melts, thus
freeing the colony which is stuck to it. It then forms a drop, with a more or
5 less substantial volume depending on the quantity of ice formed initially, in
which the bacteria are suspended.
In a variant of this first implementation of the depositing method
according to the invention, a heating means 10 contributes to making the ice
holding the colony 11 against the terminal end 4 melt more quickly.
10 This heating means 10 can equally well be constituted by a flame,
a hot air welding unit, convergent light rays, the joule effect produced by
passing a current into a resistive electrical component, or any other equivalent
heat-producing means.
This heating means 10 can also be used subsequently as a
15 sterilising means to sterilise the terminal end 4 of the probe 3.
In the same manner as for the first implementation of the
depositing method described above, the colony 11 sampled from the second
implementation of the sampling method described above is also separated
from the terminal end 4, by means of a heating means 10.
20 In this implementation of the depositing method, the colony 11 can
also be separated from the terminal end 4 of the probe 3 by applying a
mechanical shock onto the layer of ice 13.
This mechanical shock causes the layer of ice 13 to break. This is
then collected at the same time as the colony 11 sampled in a test tube 9.
25 Such a tube can then be employed for example by an automated analysis
system such as the V I T E 2~ a utomated system, or an analysis plate 14.
According to a second implementation of the depositing method,
all or part of the colony 11 is deposited through contact of all or part of the
colony 11 onto the analysis plate 14, with this contact progressively melting
30 the superficial layer of ice 13 of the sample by the action on the layer of ice 13
of the surface tension of the surface of the container 9 or of the analysis plate
14.
This contact may be sequential in the form of spots distributed on
the analysis plate 14, or continuous in the form of lines.
35 Depositing in the form of spots makes it possible to obtain a
plurality of different deposits from the same sample of sampled biological
matter (colony 11). These spots can then be employed for example by a mass
spectrometer.
Depositing in the form of continuous lines makes it possible to
obtain a concentration gradient of biological matter 7 with stronger
5 concentrations at the start of depositing than at the end of depositing. It is
thus possible to take measurements on different zones of the concentration
gradient, in order to locate the zone where the analysis results are best, when
the mass spectrometer is acquiring measurements.
Figure 4 illustrates some means of implementing the sampling and
10 depositing methods.
In an application 1A corresponding to the sampling method, the
entirety or a part of the bacterial colony 11 is directly frozen and then sampled
in its Petri dish 6 by the probe 3 to then be deposited in a test tube 9 in which
the bacteria will be in suspension after liquefaction of the icicle formed by the
15 probe 3 around its terminal end 4, with a view to undergoing a conventional
analysis.
In an application IB, part of the bacterial suspension contained in
the test tube 9 is sampled to be deposited onto the analysis plate 14.
In an application IC, part of the bacterial suspension contained in
20 the test tube 9 is sampled to be deposited onto another support with a view to
a second conventional analysis, for example a test for sensitivity to antibiotics.
In an application 2A, the bacterial colony 11 or a part of this colony
is directly frozen and then sampled in the Petri dish 6.
A part of this sampling is deposited directly onto the analysis plate
25 14 of a mass spectrometer, whilst another part, in an application ZB, is
deposited onto another support with a view to a second conventional analysis,
for example a test for sensitivity to antibiotics.
The sampling can also be preserved in frozen form on the probe 3
in order to defer the second analysis. This is facilitated if the terminal end 4 is
30 removable. In fact, the terminal end 4 can be preserved in frozen form by
depositing this end inside a freezer.
The analysis plate 14 used to accomplish the mass spectrometry
analysis of all or part of the colony 11 contains a matrix 15 used to ionise the
sample.
3 5 In a particular implementation, the matrix 15 is in the form of a
dried deposit, after distribution in the form of spots on the analysis plate 14.
The liquefaction of the frozen deposit of all or part of the colony 11
in contact with the analysis plate 14 makes it possible to re-suspend the
elements of the matrix 15 in a homogeneous liquid mixture also containing the
bacteria.
5 Another solution, illustrated by the application 3A, consists in
sampling the colony 11 in the Petri dish 6 after wetting the terminal end 4 of
the probe 3 directly into the matrix 15 which is in its normal form in liquid
state, forming an icicle of matrix 15 around the terminal end 4 of the probe 3.
The liquefaction of the frozen deposit in contact with the analysis
10 plate 14, composed of all or part of the colony 11 sampled by the probe 3 and
of the matrix 15, will re-suspend the elements of the matrix 15 in a
homogeneous liquid mixture also containing the bacteria.
In these two latter embodiments it is important to ensure in
advance that the concentration in elements of ionisation matrix will be
15 sufficient with regard to the number of bacteria present in the mixture.
Furthermore, the colonies 11 sampled by these methods, using a
non-toxic liquid solution such as water, and reseeded on a Petri dish 6 grow
again and are therefore not killed by these sampling and depositing methods.
Examples of industrial applications are suggested below.
20 EXAMPLES
Example I :
In this first example, an ice tip is used to sample an unknown
microorganism colony, and to deposit it on a mass spectrometer target in
order to identify the colony of unknown microorganisms using a mass
25 spectrometer in accordance with the following protocol:
- sample the colony on blood agar (bioMerieux ref. ) with an ice
,needle,
- with the ice tip, deposit the colony on a 384-position mass
spectrometer target (Bruker). The deposit is obtained by briefly applying the
30 ice tip onto the target at ambient temperature. A slight film of water and
microorganisms is thus deposited onto the surface of the target.
- deposit 2 vl of matrix (alpha-cyano acid dissolved to 10 mglml in
a 50150 solution of acetone and water) at the surface of the sample.
- introduce the target into a MALDI-TOF (Ultraflex II, Bruker)
mass spectrometer,
- adjust the mass spectrometer to optimise the acquisition of
masses between 2000 and 20000 Da. The person skilled in the art is
especially accustomed to adjusting the tension and the laser power of the
5 instrument.
- calibrate the mass spectrometer with the benchmark proteins
(ProteinMixte, Bruker).
- analyse the sample using 20 series of 50 laser shots. The mass
spectrum obtained for each series is summed to obtain the mass spectrum of
10 the microorganism. The number of shots can be adjusted by the person skilled
in the art to obtain the most informative spectrum possible, i.e. with the most
possible and the best defined mass peaks,
- determine the masses observed on the mass spectrum of the
microorganism using the Flex Analysis software (Bruker),
15 - compare the masses observed on the mass spectrum of the
microorganism with the masses contained in a database. Several databases
exist for this purpose: the applicant has its own database, and the databases
of the companies Bruker (Biotyper) or Anagnostec (Saramis) can also be
used.
20 - identify the microorganism which has the masses closest to
those observed on the mass spectrum of the unknown microorganism.
For this example, this protocol has been applied to two different
colonies and has made it possible to obtain the mass spectra visible on
figures 6 and 7.
25 The mass spectrum of figure 6 has made it possible to identify the
colony as being a Staphylococcus aureus colony.
The mass spectrum of figure 7 has made it possible to identify the
colony as being an Escherichia coli colony.
This method is advantageous because it makes it possible to
30 accomplish the sampling and depositing very quickly with an excellent rate of
success. From the first attempt, all of the colonies, regardless of their type,
are successfully sampled and then deposited by the ice needle.
Furthermore, since the target is at ambient temperature, the ice
needle melts very slightly when it touches the surface. The slight stream of
35 water which results from this carries the sample and provides a fine and
homogeneous deposit of microorganisms.
This latter point is particularly advantageous because a thick
deposit causes signal suppression in mass spectrometry. This phenomenon,
which is well known, is due to the excess of salts and a proportion of matrix
which is unsuited to the quantity of sample. A heterogeneous deposit is also
5 disadvantageous, as the signal becomes heterogeneous, which makes it
difficult to adjust the mass spectrometer.
Example 2:
In a second example, the same protocol is implemented as in
10 example 1, with the difference being that in step 2 the deposit is obtained by
rubbing the ice tip on the surface of the mass spectrometer target.
The spectrum figuring in figure 8 is obtained. This spectrum leads
to the identification of Escherichia coli.
This protocol has the same advantages as example 1. It furthermore makes it
15 possible to deposit over a larger surface, which can be useful for
accomplishing several successive acquisitions of the same sample, or for
successively acquiring several mass spectra with different parameters.
Example 3:
20 In a third example, 25 strains of known species are cultured in a
Petri dish with a COS (Columbia sheep blood medium, bioMerieux, reference
43041) or SDA (Sabouraud glucose medium, bioMerieux, reference 43555)
culture medium in accordance with the indications figuring in table 1 below:
25 Table 1:
Culture
medium
COS
COS
COS
COS
COS
COS
COS
BioMerieux
culture medium
reference
43041
43041
43041
43041
43041
43041
43041
Microorganism species
Burkholderia multivorans
Proteus vulgaris
Pseudomonas putida
Streptococcus pseudopneumoniae
Bacillus lich eniformis
Micrococcus luteus
Staphylococcus haemolyticus
BioMerieux
microorganism
reference
0301 039
0509142
05091 08
05071 05
060801 6
0602045
0704061
- After 16 hours of culturing, for each Petri dish, a colony is
sampled with an ice needle and deposited onto a MALDI-TOF target according
to the method of the invention. This operation is repeated to obtain two
5 independent deposits for each species studied. The samplings and the
deposits are carried out in accordance with the following operating mode. .
- A metallic tip (terminal end), which is cylindrical with a tapered
base, kept at -10°C by Peltier effect (metallic tip in contact with two Peltier
elements in accordance with figure 5). It is then dipped for 15 seconds into a
10 receptacle of water kept at 9°C.
- The metallic tip is removed from the water. A water drop
naturally sticks to the metallic tip. This water drop freezes in 15 seconds by
transfer of cold from the metallic tip at -10°C. An ice needle is thus formed.
- The needle is positioned on a colony of microorganisms and is
15 immediately withdrawn. The colony of microorganisms instantly adheres to the
ice needle and remains stuck on the ice needle. The colony of microorganisms
is thus sampled by the ice needle. Conversely, the agar of the culture medium
COS
COS
COS
COS
COS
COS
COS
COS
COS
COS
COS
COS
COS
COS
COS
COS
COS
SDA
43041
43041
43041
43041
43041
43041
43041
43041
43041
43041
43041
43041
43041
43041
43041
43041
43041
431 01
Proteus mira bilis
Enterococcus raffinosus
Pseudomonas aeruginosa
Escherichia coli
Bacteroides fragilis
Shigella flexneri
Streptococcus pyogenes
Streptococcus constellatus ssp constella
Bacillus megaterium
Chryseobacterium indologenes
Vibrio parahaemolyticus
Citrobacter farmeri
Aeromonas hydrophila
Salmonella ser. Gallinarum (pullovorum)
Corynebacterium jeikeium
Pseudomonas oryzihabitans
Enterococcus casseliflavus
Geotrichum capita tum
0805068
0903030
1006028
I 006021
1009220
7709005
7701 086
781 1150
8004066
81 05051
8305091
8608073
60601 9)
8703202
9203007
951 01 57
9710016
9409060
is not picked up.
- The colony of microorganisms is deposited onto a disposable
48-position Fleximass DS target from Shimadzu. Each depositing position is a
circle with a diameter of 3 mm. Depositing is carried out by rubbing the ice
5 point on the surface of one of the 48 deposit positions of the target with a
spiral motion. This motion is initiated at the centre of the depositing position
and completed after 4 circuits so as to cover the entire depositing position with
a slight film of water and microorganisms.
- The sample of microorganisms is dried in open air for several
10 minutes.
- 1 1.rL of matrix (alpha-cyano-4-hydroxycinnamic acid ready to be
used in solution (bioMerieux, reference 41 1071) is deposited onto the surface
of the sample.
- The sample of microorganisms and the matrix are dried in open
15 air for several minutes.
- The target is introduced into a MALDI-TOF mass spectrometer
(Axima Assurance, Shimadzu) with a FLEXIMASS DS target support
(Shimadzu).
- The mass spectrometer is adjusted to optimise the acquisition
20 of masses between 2000 and 20000 Da. The person skilled in the art is
especially accustomed to adjusting the tension and the laser power of the
instrument.
- The mass spectrometer is calibrated with a deposit of E. coli
(ATCC 8739 strain).
25 - The sample is analysed using 100 series of 5 laser shots. The
mass spectrum obtained for each series is summed to obtain the mass
spectrum of the microorganism. The number of shots can be adjusted by the
person skilled in the art to obtain the most informative spectrum possible, i.e.
with the most possible and the best defined mass peaks.
30 - the masses observed on the mass spectrum of the
microorganism are determined with the aid of the Launchpad version 2.8
software (Shimadzu),
- The masses observed on the mass spectrum of the
microorganism are compared with the masses contained in the Saramis
35 (bioMerieux) database using the Spectral ID version 1.1.0 interface
(bioMerieux), and the microorganism is identified by comparison with the mass
spectra of microorganisms present in the database.
For this example, the protocol has made it possible to identify the
deposits according to the results set out in table 2 below:
5 Table 2:
Expected
microorganism
species
Burkholderia
multivorans
Proteus vulgaris
Pseudomonas
putida
Streptococcus
pseudopneumoniae
Bacillus
lichenifomis
Micrococcus luteus
Staphylococcus
haemolyticus
Proteus mirabilis
Enterococcus
raffinosus
Pseudomonas
aeruginosa
Escherichia coli
Bacteroides fragilis
Shigella flexneri
Streptococcus
pyogenes
Streptococcus
constellatus ssp
constella
Bacillus megateriurn
Chryseobacterium
indologenes
Deposit No.
Species identified with
the protocol from
example 3
BurWlol. multivorans
Proteus vulgaris
Ps.putida
Str.pseudopneurnoniae
6. lichenifomis
Mic. luteudylae
Staph. haemolyticus
Proteus mirabilis
Entero. rattinosus
Ps.aeruginosa
Esch. coli
Bac. fragilis
Esch. coli
Str. pyogenes
no identification
B.megaterium
Chryse.indologenes
2
Probability
of
identification
(%)
100
99.99
99.99
65.67
99.99
100
99.99
100
100
99.99
99.99
99.99
99.99
99.99
-
99.99
99.99
Deposit No.
Species identified with
the protocol from
example 3
Burkhol. multivorans
Proteus vulgaris
Ps.putida
no identification
B.lichenifomis
Mic. luteudylae
Staph. haemolyticus
Proteus rnirabilis
Entero. rattinosus
Ps. aeruginosa
Esch. coli
Bac. fragilis
Esch. coli
Str. pyogenes
Str.constellatus
Bmegaterium
Chryse.indologenes
1
Probability
of
identification
100
100
99.99
100
99.99
99.99
100
99.99
99.99
99.99
99.99
99.99
100
99.9
99.99
99.99
The species identified corresponds exactly to the species
expected, taking into account the comments below:
- Spectral ID provides the abbreviated name of
5 the species. The person skilled in the art is accustomed to these
abbreviations. By way of example, it is clear to him that Gxapitatum
means Geotrichum capitatum, that Entero.casseliflavus means
Enterococcus casseliflavus, and so on.
- Micrococcus luteus is identified as
10 Mic.luteusAylae, i.e. as possibly being Micrococcus luteus or Micrococcus
lylae. It is not possible to differentiate between these two species using
MALDI-TOF analysis and the Saramis database.
- Shigella flexneri is identified as Esch. coli, i.e.
Escherichia coli. As above, Shigella flexneri and Escherichia coli are two
15 species which are too close to be distinguishable by MALDI-TOF.
Spectral ID and Saramis class these two species as Escherichia coli.
- Aeromonas hydrophilia is identified as
Aer. salm. salmonicida or Aerhydro./caviae. The 3 species are very close
and are difficult to differentiate by MALDI-TOF.
20 - Salmonella ser. Gallinarum (pullovorum) is
identified as Salmonella group. MALDI-TOF analysis does not have
99.99
99.99
99.99 or
98.16
78.15
100
99.99
100
V.parahaemolyticus
Citro. farmeri
Aer.salm.salrnonicida or
Aer. hydro./caviae
Salmonella group
Coryn.jeikeium
Ps. oryzihabitans
No identification
G.capitatum
Vibrio
parahaemolyticus
Citrobacter famen
Aeromonas
hydrophila
Salmonella ser.
Gallinarum
(pullovorum)
Corynebacterium
jeikeium
Pseudomonas
oryzihabitans
Enterococcus
casseliflavus
Geotrichum
capitatum
V.parahaemolyticus
Citro. farmeri
Aer.salm.salmonicida
or Aer.hydro./caviae
Salmonella group
Coryn. jeikeium
Ps. oryzihabitans
Entero. casseliflavus
G. capitatum
100
99.99
99.99 or
99.73
78.25
100
99.99
100
100
sufficient resolution to unequivocally distinguish serovar Gallinarum
(pullovorum) from the other serovars. The Spectral ID software and
Saramis therefore give only one identification at the level of the
Salmonella genus.
5
The protocol has thus made it possible to identify all of the
analysed species. 72 deposits out of 75 have been identified, which
represents 97.3% correct identification. This identification rate is very
good, indeed even better than the person skilled in the art is
10 accustomed to obtain with manual sampling and deposition followed by
a MALDI-TOF analysis.
This method is advantageous because it makes it possible to
accomplish the sampling and depositing very quickly with an excellent rate of
15 success and for any type of microorganism (bacteria or yeast (G.capitatum)). In
particular, this method makes it possible to sample, and deposit with equal
effectiveness, microorganisms which have very different forms and
consistencies (Proteus, Bacillus, coliforms, etc.).
20 Although the invention has been described in connection with
particular implementation examples and embodiment examples of the
invention, it is clear that it is by no means limited by these and that it
encompasses all of the equivalent techniques of the steps and means
described, and combinations thereof.

CLAIMS
1. A method of sampling all or part of a sample (11) of
biological matter (7), which is crude, enriched or cultured through contact with a
culture medium (8), possibly an agar culture medium, using a probe (3)
5 equipped with a terminal end (4), said sampling method comprising the steps
of:
cooling of the terminal end (4) of the probe (3),
sticking all or part of the sample (11) of biological matter
(7) to be sampled through contact of the terminal end (4) onto the sample (11)
10 of biological matter (7), or by applying a pressure exerted by the terminal end
(4) onto the sample (11) of biological matter (7), and
sampling all or part of the sample (11) of biological
matter (7) so as to separate the sample (11) of biological matter (7) from its
support, such as a culture medium (8).
15
2. The sampling method according to claim 1, wherein prior
to the sticking step, a step of wetting the terminal end (4) of the probe (3) in a
liquid solution (12) is carried out, in order to form a layer of Ice (13) on the
20 terminal end (4).
3. The sampling method according to one of claims 1 to 2,
wherein prior to the sticking step, the sample (11) of biological matter (7) to be
sampled is covered with a liquid solution (12), in order to form a layer of ice
25 (13) around the sample (11) during the step of sticking all or part of said
sample (11) to the terminal end (4) of the probe (3).
4. The sampling method according to one of claims 2 to 3,
wherein the liquid solution (12) is taken from the group comprising water, a
30 saline solution, a buffer, a liquid culture medium or a matrix commonly used for
ionising the sample (11) of biological matter (7) with a view to analysing it
using a measuring device such as a mass spectrometer.
5. The sampling method according to one of claims 2 to 4,
35 wherein the step of wetting the terminal end (4) of the probe (3) or the sample
(11) of biological matter (7) to be sampled in a liquid solution (12) is repeated
22
successively at least twice in order to form overlaying layers of ice (13).
6. The sampling method according to one of claims 1 to 5,
wherein the terminal end (4) is removable.
5
7. The sampling method according to claim 6, wherein the
terminal end (4) possesses ferromagnetic properties.
8. The sampling method according to claim 7, wherein the
10 step of sampling all or part of the sample (11) of biological matter (7) consists
in applying a magnetic field, for example using an electromagnet, adjacent to
the ferromagnetic terminal end (4) so as to attract the terminal end (4) and
thus recover it.
15 9. A method of depositing into a container (9) or onto an
analysis plate (14) all or part of a sample (11) of biological matter (7) stuck
onto a frosted terminal end (4) of a probe, said depositing method including a
step of separating the terminal end (4) of the probe (3) from all or part of the
sample (11) of biological matter (7) stuck onto said terminal end (4).
20
10. The depositing method according to claim 9, wherein the
separation step is carried out without the sample (11) of biological matter (7)
stuck on the frosted terminal end (4) coming into contact with the container (9)
or the analysis plate (14).
25
11. The depositing method according to claim 9, wherein the
step of separating the terminal end (4) from all or part of the sample (11) of
biological matter (7) stuck on said terminal end (4) is carried out by applying a
mechanical shock onto the terminal end (4).
30
12. The depositing method according to claim 10, wherein
the step of separating the terminal end (4) from all or part of the sample (11) of
biological matter (7) stuck on the frosted terminal end (4) is carried out by a
heating means (10) or in ambient air.
35
13. The depositing method according to claim 9, wherein the
23
separation step is actiieved by sequential contact of the sample (11) of
biological matter (7) with the container (9) or with the analysis plate (14), which
thereby melts a superficial layer of ice (13) and deposits biological matter (7).
5 14. The depositing method according to claim 13, wherein all
or part of the sample (11) of biological matter (7) is distributed on an analysis
plate (14) so as to accomplish several distinct deposits of parts of the same
sample (11) of biological matter (7), which is stuck on the terminal end (4).
10 15. The depositing method according to claim 9, wherein the
depositing is effected through continuous contact, for example in the form of
lines, of all or part of the sample (11) of biological matter (7) stuck on the
terminal end (4) with the container (9) or the analysis plate (14).
15 16. A device for sampling and depositing (2) all or part of a
sample (11) of biological matter (7), which is crude, enriched or cultured
through contact with a culture medium (8), and intended to be deposited into a
container (9) or onto an analysis plate (14), characterised in that it comprises:
- a probe (3) equipped with a terminal end (4),
20 - a cooling means intended for frosting the terminal end (4),
- driving means (5) intended:
• for exerting a pressure from the probe (3) onto the
sample (11) so as to freeze ail or part of the water
contained in the sample (11) in order to stick it to the
25 terminal end (4),
• for separating all or part of the sample (11) from its
support, such as a culture medium (8),
- to bring all or part of the sample (11) to the container (9)
or the analysis plate (14).
30
17. The device (2) according to claim 16, comprising a
heating means (10) intended for detaching the sample (11) from the terminal
end (4).
35 18. The device (2) according to claim 17, wherein the heating
means (10) is also intended for sterilising the terminal end (4).
24
19. The device (2) according to one of claims 16 to 18,
wlierein the cooling means and the heating means are composed of at least
one Peltier element.
5
20. The device (2) according to one of claims 16 to 19,
wherein the terminal end (4) is metallic or mineral.
21. The device (2) according to one of claims 16 to 20,
10 wherein the terminal end (4) is at least partially covered with a hydrophobic
coating or treatment.
22. The device (2) according to one of claims 16 to 21,
wherein the terminal end (4) is removable.
15
23. The device (2) according to one of claims 16 to 22,
wherein the terminal end has a shape which optimises the sticking of all or part
of the sample (11) of biological matter (7) to be sampled or of the liquid solution
(12).
20
24. The device (2) according to one of claims 16 to 23,
wherein the terminal end (4) comprises a pointed end.
25. The device (2) according to one of claims 16 to 24,
25 comprising at least one sensor which controls the pressure exerted by the
driving means (5) via the probe (3) in contact with the sample (11) of biological
matter (7) to be sampled which is cultured on the culture medium (8), and does
this in order to halt this pressure.
30 26. The device (2) according to claim 25, wherein the sensor
is a pressure or force sensor.
27. The device (2) according to one of claims 16 to 26,
comprising a sensor which detects the contact between the terminal (4) and the
35 biological matter (7) in order to avoid any pressure.
•
25
28. The device (2) according to one of claims 26 to 27,
wherein the sensor is a binary electric sensor.
29. A kit comprising a device according to one of claims 16 to
5 28 including a plurality of interchangeable terminal ends (4).
30. A medical analysis apparatus (1) containing a device
according to one of claims 16 to 28, or a kit according to claim 29.