Method for isolating microorganisms on a culture medium, and related device

Abstract

Method for isolating microorganism from a sample likely contaminated by microorganism, including: (a) device for isolating microorganisms including a bottom waterproof layer, a nutritional layer, which is placed on the bottom layer and includes a dehydrated culture medium, an isolation layer which is pervious to elements included in the nutritional layer and is capable of retaining the bacteria on the surface and covering all or part of the nutritional layer, and a top protective layer; (b) depositing a volume of the sample on the isolation layer; (c) isolating the microorganisms by impoverishing or layering the sample using an isolating device; (d) incubating the device for an amount of time at a temperature to enable growth of microorganisms, method including at least one step of rehydrating the culture medium using a volume of liquid before or with step b) and/or c) and/or d), before or simultaneously with step b) and/or c).

Claims

1. A device for culture of microorganisms comprising: a bottom layer impermeable to water; a calendered nutrient layer, arranged on the bottom layer, comprising a support impregnated with a dehydrated culture medium; an isolating layer permeable to the elements comprised in the nutrient layer, able to retain the microorganisms on its surface and covering the whole or a portion of the nutrient layer; and a protective top layer.

2. The device as claimed in claim 1, further comprising at least one reservoir integrated with the device and/or channels allowing rehydration of the nutrient layer.

3. The device as claimed in claim 1, wherein the isolating layer is a porous membrane.

4. The device as claimed in claim 2, wherein the isolating layer is a porous membrane.

5. The device as claimed in claim 1, wherein the isolating layer contains hydrophobic and/or hydrophilic units.

6. The device as claimed in claim 2, wherein the isolating layer contains hydrophobic and/or hydrophilic units.

7. The device as claimed in claim 3, wherein the isolating layer contains hydrophobic and/or hydrophilic units.

8. The device as claimed in claim 4, wherein the isolating layer contains hydrophobic and/or hydrophilic units.

9. A method comprising: depositing a portion of a sample on the isolating layer of the device as claimed in claim 1; and isolating at least one microorganism from the sample.

10. A method of obtaining the device as claimed in claim 1, said method comprising: pouring a predetermined volume of liquid onto the bottom layer impermeable to water, arranging the isolating layer on the nutrient layer, the whole being placed on the bottom layer impermeable to water that has previously received said predetermined volume of liquid in order to allow instantaneous and homogeneous rehydration of said dehydrated culture medium, and then superposing the protective top layer.

11. A device obtainable by the method as claimed in claim 10.

Description

(1) The invention, its functionality, its applications as well as its advantages will be better understood on reading the present description, referring to the figures, in which:

(2) FIGS. 1A and 1B are schematic representations of the device according to the invention. The device 10 comprises a bottom layer impermeable to water 14, a nutrient layer 12, arranged on the bottom layer, comprising a dehydrated culture medium, an isolating layer 20 permeable to the elements comprised in the nutrient layer, able to retain the bacteria on its surface and covering the whole or a portion of the nutrient layer, a protective top layer 15. This device also comprises hydrophilic/hydrophobic zones 13, a reservoir 19 and channels 18 allowing rehydration of the bottom part or lateral part of the nutrient layer. The device may have separating means 16 allowing the top layer 15 not to be in contact with the colonies.

(3) FIG. 2A is a photograph of the device according to the invention that has a useful isolation area of 25 cm.sup.2;

(4) FIG. 2B shows an enlargement of the central portion of the device. The device was seeded with a sample of Escherichia coli at a concentration of 10.sup.8 CFU/mL

(5) FIG. 3 shows the contents of a Petri dish seeded with a strain of Klebsiella pneumoniae in the presence of a UriSelect 4 culture medium with an isolating layer consisting of a Macherey Nagel cellulose nitrate filtering membrane, as an example;

(6) FIG. 4 shows the contents of a Petri dish seeded with a strain of Klebsiella pneumoniae in the presence of a UriSelect 4 culture medium with an isolating layer consisting of a Sartorius nitrate filtering membrane, as an example;

(7) FIG. 5 shows the contents of a Petri dish seeded with a strain of Klebsiella pneumoniae in the presence of a UriSelect 4 culture medium with an isolating layer consisting of a cellulose acetate filtering membrane, as an example;

(8) FIG. 6 shows the contents of a Petri dish seeded with a strain of Klebsiella pneumoniae in the presence of a UriSelect 4 culture medium with an isolating layer consisting of a polyester filtering membrane;

(9) FIG. 7 shows the contents of a Petri dish seeded with a strain of Escherichia coli in the presence of a UriSelect 4 culture medium with an isolating layer consisting of a Macherey Nagel cellulose nitrate filtering membrane, as an example;

(10) FIG. 8 shows the contents of a Petri dish seeded with a strain of Escherichia coli in the presence of a UriSelect 4 culture medium with an isolating layer consisting of a polyester filtering membrane, as an example;

(11) FIG. 9 shows the contents of a Petri dish seeded with a strain of Escherichia coli in the presence of a UriSelect 4 culture medium with an isolating layer consisting of a cellulose acetate filtering membrane, as an example;

(12) FIG. 10 shows the contents of a Petri dish seeded with a strain of Escherichia coli in the presence of a UriSelect 4 culture medium with an isolating layer consisting of a polyester filtering membrane, as an example;

(13) FIG. 11 shows the contents of four Petri dishes in the presence of a UriSelect 4 agar culture medium and of a dehydrated UriSelect 4 culture medium impregnated on a nutrient layer comprising a nonwoven support of the Glatfelter type, Airlaid 150 g/m.sup.2 in the presence of a sample of a strain of Escherichia coli, as an example;

(14) FIG. 12 shows the contents of four Petri dishes in the presence of a UriSelect 4 agar culture medium, of a dehydrated UriSelect 4 culture medium impregnated in a nutrient layer comprising a nonwoven support of the Glatfelter type, Airlaid 150 g/m.sup.2 in the presence of a sample of a strain of Enterobacter cloacae, as an example;

(15) FIG. 13 shows details of FIG. 16, showing the contents of two Petri dishes, as an example;

(16) FIG. 14 shows the contents of four Petri dishes in the presence of an impregnated UriSelect 4 culture medium in a nutrient layer comprising a nonwoven support of the Glatfelter type, Airlaid 150 g/m.sup.2 in the presence of an isolating layer such as a cellulose acetate filtering membrane and in the presence of a sample of a strain of Clostridium freundii, as an example;

(17) FIG. 15 shows the contents of a Petri dish comprising a nutrient layer containing a nonwoven support impregnated with an agar culture medium of the Chrom ID CPS ID3 type in the presence of a sample of Enterococcus faecalis, as an example;

(18) FIG. 16 shows the contents of a Petri dish comprising a nutrient layer containing a nonwoven support of the type Airlaid MH 100 137 impregnated with a culture medium of the Chrom ID CPS3 type, dehydrated and without agar;

(19) FIG. 17 shows the contents of a Petri dish in the presence of a culture medium of the Chrom ID CPS ID3 agar medium type and of a nutrient layer comprising a nonwoven support impregnated with a medium of the ChromID CPS3 type, dehydrated and without agar, in the presence of an isolating layer such as a polyester filtering membrane and a sample of a strain of Enterobacter cloacae, as an example;

(20) FIG. 18 shows details of a nutrient layer comprising a nonwoven support of the Airlaid MH 100 137 type impregnated with a culture medium of the Chrom ID CPS ID3 type without agar and in the presence of an isolating layer such as a polyester membrane;

(21) FIG. 19 shows another example of support according to FIG. 18.

EXAMPLES

Example 1: Obtaining Isolated Colonies from a Heavily Contaminated Solution on a Petrifilm Rehydrated Medium

(22) From a solution calibrated at a theoretical bacterial load of 10.sup.8 CFU/ml, 1000 l of solutions loaded with Escherichia coli at different concentrations obtained by successive dilutions by a factor of 10 are deposited at the center of the bottom film of Petrifilm. The top film of Petrifilm is lowered onto the sample. A plastic diffuser, concave face downwards, is placed at the center of the Petrifilm assay. The sample is uniformly distributed by exerting light pressure at the center of the plastic diffuser. The inoculum is thus distributed over the entire growth zone before the gel forms.

(23) TABLE-US-00001 TABLE 1 Dilution CFU/ml 10.sup.8 10.sup.7 10.sup.6 10.sup.5 10.sup.4 10.sup.3 10.sup.2 10 Colonies 300 300 300 300 300 300 78 7 Not Not Not Not Not Not countable countable countable countable countable countable

(24) The results show that six dilutions are necessary in order to obtain isolated and usable colonies.

Example 2: Obtaining Isolated Colonies from a Heavily Contaminated Solution on a Rehydrated Medium According to the Resent Invention

(25) Starting from the same solution calibrated at a theoretical bacterial load of 10.sup.8 CFU/ml used for inoculation of the Petrifilm, mechanical inoculation by the dial method is carried out on the device according to the invention, allowing isolated colonies to be obtained on a limited area (25 cm.sup.2) of the device.

(26) Thus, the device according to the invention was seeded with 10 l (contents of one loop) of a solution calibrated at a theoretical bacterial load of 10.sup.8 CFU/ml loaded with Escherichia coli and deposited on the 1st dial 21 of the isolation surface of the device whose useful isolation area is 25 cm.sup.2. The second dial 22 is seeded with a new loop, drawing several streaks starting from dial 21. The third dial 23 is seeded like the second without changing the loop. The 4th dial 24 is seeded with streaks not drawn starting from dial 22.

(27) The device is formed by an isolating layer with hydrophobic/hydrophilic units 28 on which isolation is performed. The hydrophobic/hydrophilic units make it possible to improve isolation, notably on a small area (25 cm.sup.2) by spatially delimiting the growth of the microorganisms.

(28) A layer containing the rehydrated culture medium 25. A bottom layer impermeable to water 26 and a translucent top layer sealing the device 27.

(29) FIGS. 2a and 2b show that the mechanical isolation on the device according to the invention allows formation of isolated colonies. Isolation is thus possible using a single device without prior dilution.

Example 3: Obtaining Isolated Colonies on an Isolating Layer of the Filtering Membrane Type, Arranged on a Nutrient Layer Consisting of a Nonwoven Support Impregnated with a Dehydrated Nutrient Medium

(30) The aim of this example is to compare the morphotypes and the growth time of colonies developing on a porous and/or filtering membrane (preferably filtering) positioned on an agar culture medium or on a nutrient layer impregnated with dehydrated culture medium.

(31) The size and color of the colonies obtained from different bacterial species seeded on these porous and/or filtering membranes are evaluated by the operator.

(32) 3.1 Materials

(33) The experiments described below notably relate to strains of Escherichia coli, Clostridium freundii, Enterococcus faecalis, Klebsiella pneumoniae, and Enterobacter cloacae.

(34) The isolating layers tested for the present example comprise: a polyester filtering membrane (Macherey Nagel Polyester) comprising pores with a diameter of 0.2 m, 0.4 m, 1 m and 5 m (trade reference: PORAFIL PE), a cellulose nitrate filtering membrane (Macherey Nagel Polyester) comprising pores with a diameter of 0.2 m, 0.4 m, 1 m and 5 m (trade reference: PORAFIL NC). a cellulose acetate filtering membrane (Macherey Nagel Polyester) comprising pores with a diameter of 0.2 m, 0.4 m, 1 m and 5 m (trade reference: PORAFIL CA), a filtering membrane of cellulose mixed esters (Macherey Nagel Polyester) comprising pores with a diameter of 0.2 m, 0.4 m, 1 m and 5 m (trade reference: PORAFIL CM), a cellulose nitrate filtering membrane (Sartorius stedim Biotech) comprising pores with a diameter of 0.45 m.

(35) For the purposes of the present experiments, the following nonwoven supports are used: Glatfelter, Airlaid 100 g/m.sup.2, Glatfelter, Airlaid concert 150 g/m.sup.2, PDI supports 60 g/m.sup.2.

(36) The culture media used for impregnating the nonwoven support in the present experiments are: a Trypcase soybean broth (TSB-D), a culture medium of the UriSelect type 4 (trade reference: BioRad), or a culture medium of the Chrom ID CPS 3 type without agar.

(37) The agar culture media used in the present experiments are as follows: UriSelect4 (trade reference BioRad) and Chrom ID CPS 3.

(38) 3.2 Experimental Protocol

(39) Firstly, the various filtering membranes are tested on an agar medium of the UriSelect 4 type (cf. section 3.3.1 below).

(40) Secondly, the UriSelect 4 agar culture medium is replaced with the various nonwoven supports impregnated with a culture medium mentioned above (cf. section 3.3.2 below).

(41) The nonwoven supports, impregnated with the culture medium, are rehydrated using a predetermined volume of sterile water and a bacterial inoculum at the moment of performing the analysis. The volume/amount of sterile water necessary for rehydration of the nonwoven support, impregnated with the culture medium, varies as a function of the nature of the nonwoven support and the size of the latter. This information can easily be determined by a person skilled in the art based on his general knowledge, and routine tests if necessary.

(42) The assembly of filtering membrane and impregnated nonwoven support or filtering membrane and agar medium is incubated at a temperature of 37 C. Visual reading of the results for determining the morphotype of the colonies and quality of isolation on the surface of the porous and/or filtering membrane is carried out firstly after an incubation time of 24 h and then secondly after a total incubation time of 48 h.

(43) 3.3 Results

(44) 3.3.1 Isolation of Various Microorganism Different Types of Filtering Membranes, in the Presence of an Agar Culture Medium

(45) FIGS. 3, 4, 5 and 6 show isolation of a strain of Klebsiella pneumoniae in the presence of a UriSelect 4 agar culture medium, after an incubation time of 24 h.

(46) More precisely, the filtering membrane in FIG. 3 is of cellulose nitrate (Machcrey Nagel).

(47) The filtering membrane in FIG. 4 is of Sartorius cellulose nitrate (Sartorius).

(48) The filtering membrane in FIG. 5 is of cellulose acetate.

(49) The filtering membrane in FIG. 6 is of polyester.

(50) In these FIGS. 3, 4, 5 and 6, the presence of well individualized colonies that are directly usable (CDU) is noted.

(51) FIGS. 7, 8, 9 and 10 show the presence of Escherichia coli in the presence of a UriSelect 4 agar culture medium, after an incubation time of 24 h.

(52) More precisely, the filtering membrane in FIG. 7 is of Macherey Nagel cellulose nitrate.

(53) The filtering membrane in FIG. 8 is of polyester.

(54) The filtering membrane in FIG. 9 is of cellulose acetate.

(55) The filtering membrane in FIG. 10 is of polyester.

(56) In these FIGS. 7, 8, 9 and 10, the presence of well individualized colonies that are directly usable (CDU) is noted. The bacterial growth is optimal, the morphotypes and growth of the colonies obtained comply with what is expected of microbial growth directly on agar medium. Isolation performed on a porous and/or filtering membrane leads to morphotypes of colonies and a quality of isolation that are obtained conventionally with isolation carried out directly on agar medium.

(57) 3.3.2 Isolation and Count/Counting of Microorganisms on Filtering Membranes Deposited on Nonwoven Supports Impregnated with a Dehydrated Culture Medium

(58) The present experiments bring an impregnated nonwoven support into contact with a dehydrated culture medium. While the analysis is carried out, the nonwoven support is impregnated with water in order to rehydrate the culture medium.

(59) As shown in FIG. 11, the impregnation support used is of the Glatfelter type, Airlaid 150 g/m.sup.2. The latter is impregnated with UriSelect 4 dehydrated culture medium.

(60) The contents of the Petri dishes shown in FIG. 11 provide evidence of growth of the Escherichia coli bacteria and the presence of well individualized colonies that are directly usable (CDU).

(61) FIG. 12 shows similar results in the presence of Enterobacter cloacae bacteria

(62) FIG. 13 shows results similar to those in FIGS. 11 and 12, in the presence of an agar medium of the UriSelect 4 type.

(63) Similar results can also be seen for the contents of the Petri dishes shown in FIG. 14 in the presence of a UriSelect 4 culture medium impregnated dry in a nonwoven support of the Glatfelter type, Airlaid 150 g/m.sup.2 in the presence of a cellulose acetate filtering membrane.

(64) 3.3.3 Impregnation with a Culture Medium of the Chrom ID CPS IDS Dehydrated Type

(65) The present experiments relate to a sample of Enterococcus faecalis in the presence of a Chrom ID CPS ID3 agar culture medium (cf. FIG. 15) and a dehydrated Chrom ID CPS3 medium without agar impregnated in the nonwoven support in the presence of a polyester filtering membrane (cf. FIG. 16).

(66) Another experiment relates to a sample of Enterobacter cloacae in the presence of an agar culture medium of the Chrom ID CPS ID3 type and a culture medium impregnated with the Chrom ID CPS3 dehydrated type without agar in the presence of a polyester filtering membrane (cf. FIG. 17). In FIG. 17, the presence of well individualized colonies that are directly usable (CDU) is noted.

(67) FIGS. 18 and 19 show the results of an experiment bringing a Chrom ID CPS ID3 culture medium without agar impregnated on an Airlaid MH 100 137 nonwoven support in the presence of a polyester filtering membrane.

(68) As shown in FIGS. 18 and 19, the presence of well individualized colonies that are directly usable (CDU) is noted. Although isolation was carried out on filtering membranes positioned on dehydrated culture mediaand not on agar mediathis did not affect bacterial growth, which proved to be optimal. Moreover, the morphotypes of the colonies obtained are similar to those obtained with isolation on porous and/or filtering membranes positioned on agar medium.

(69) 3.4 Conclusions

(70) The results of the experiments relating to example 3 indicate that it is possible to perform isolations of microorganisms on an isolating layer of the filtering membrane type. It should be noted that these filtering membranes are not used for the action of filtration of liquid, which is their primary use, but for carrying out isolation of a sample that may be heavily laden with microorganisms, which requires them to have the same surface qualities as those that are obtained on agar media. Moreover, isolation did not generate deformations of the filtering membrane, the latter remaining as if glued to the underlying nutrient layer (nutrient support) without requiring any physical or chemical bond between the filtering membrane and the nutrient layer. This intimate proximity of the filtering membrane with the nutrient layer after isolation is verified when we examine the integrity and continuity of the isolation path through the arrangement of the bacterial colonies.

(71) Besides compatibility of the porous and/or filtering membranes with the operation of microbial isolation, the applicant has demonstrated that the superposition of the isolating layer of the filtering membrane type and the nutrient layer of the nonwoven support type impregnated with dehydrated culture medium allows optimal growth of the microorganisms on the isolating layer, as evidenced by the morphotypes of the bacterial colonies obtained.

(72) It also appears that the nonwoven support impregnated with a dehydrated culture medium represents a valid alternative to culturing microorganisms in the presence of a gelose culture medium containing agar. In fact, the nutrient layer allows exchanges of nutrients with the microorganisms located on the isolating layer in order to allow quality microbial growth. Thus, the presence of an isolating layer arranged above a nutrient layer impregnated with a dehydrated culture medium makes it possible to obtain isolated colonies of microorganisms on the surface of the isolating layer. The porosity of the filtration membrane allows retention of the microorganisms on its surface and the transfer of the dissolved nutrients present in the nutrient support to the surface of the filtration membrane. Example 3 clearly demonstrates that such transfers are optimal as no delay of growth suggested notably by a reduced size of the colonies was observed. Note once again that this transfer is optimal in the absence of bonding means or binder between filtration membrane and nutrient layer.

(73) In general, the exchanges of nutrients and of water between the nutrient layer and the filtering membrane allow optimal microbial growth. The impregnated culture medium is rehydratable or may be rehydrated a short time before or simultaneously with microbial isolation.

(74) Example 3 notably demonstrates that the device according to the invention is compatible with isolation and microbial growth.