METHOD FOR DETECTING AND DIRECTLY IDENTIFYING A
MICROORGANISM IN A BIOLOGICAL SAMPLE BY AN OPTICAL ROUTE
The present invention relates generally to the field of analysis, for example
5 biological analysis. More specifically, the present invention relates to a method for
detecting and directly identifying at least one microorganism by an optical route in an,
optio~lallye nriched, biological sample.
Microbiological analysis requires accurate metl~odsi, n wllich the time to obtain the
10 result must be as short as possible.
In the medical field, it is necessary to predict and diagnose the risk of infection: the
quicker and more accurate thc diagnosis, the more effective the management of the
patients, the risk of transmission being minimized. The approach is similar for animal
health.
15 There are identical problems in the food indust~yT. here, however, a distinction is
made between: . pathogenic ~ilicroorganistns and their toxins, where research applies to the
raw materials, intermediates, and marketed finished products, . non-pathogenic microorganisms, used as indicators of quality of the
20 production process, from the raw materials to the finished products,
tlvoughout the chain, and . bacteria of technological interest such as ferments.
Rapid and precise detection of suspected contatninants makes it possible to control
them and thus apply corrective measures.
25
Technically, microbiological analysis call e~llploy one or more steps of preenrichi~
enat nd/or em'ichment, one or more steps of detection, and one or more steps of
counting the microorganisms. For particular applications such as microbiological control
in the food industry, a cot~fir~natiostie p may also be required, in order to comply xvith the
30 standards in force in this field.
At present, no method exists for detecting a target microorganism in a large initial
amount of sample, without employing an enrichment step.
The elxiclunent step employs selective or non-selective culture media, which aim to
5 promote growth of the target microorganisms in biological or enviromnental samples,
while limiting the growth of the non-target flora. The media are often used in containers
of the sterile plastic bag type, in which they are brought into contact with the food
samples or environnlental samples, for purposes of resuspension and enrichment of the
microorganistns being sought. This step is necessary in order to meet the requirement of
10 detecting the potential initial presence of at least one target microorganism in an amount
of sample that is very variable and optionally is very large, e.g. 25 grams (g) to 375 g
diluted in 225 to 3375 millilitres (mL) in the culture medium. At the end of this
enrichment step, an aliquot (from 5 microlitres (~1t)o 5 mL) is taken for carrying out the
step of detecting the target microorganisms. Now, it is necessary for this aliquot to
15 contain a sufficient amount of target microorganisms to ensure that they are
systematically detected. A step of secondary enrichment or subculture may then be
necessary.
The detection step is based historically on culturing the microorganisms on agar
20 media, for detecting the metabolic characters of the microorganisms being sought.
Conventionally, specific enzymatic substrates are used. These enzymatic substrates
generally consist of two parts, a first part specific to the enzyme activity to be detected,
also called the target part, and a second part acting as a marker, called the marker part,
generally consisting of a chromophore or a fluorophore. Based on the choice of these
25 substrates, depending on whether there is reaction or not, it is possible to characterize the
nature of a microorganism or distinguish between different groups of microorganisms.
Tlms, appearance or disappearance of coloration or of fluorescence will be the signature
of a genus or of a type of microorganism. In this respect, the use of cllromogenic media
allows simultaneous detection and identification of the microbes being sought. It
30 simplifies the process and greatly reduces the time to obtain the result. We may mention,
as a concrete example, the applicant's CluomIDB media. These cluonlogenic media are
based on detection of specific metabolic characters of the nlicrobes being sought, for
exa~npleb eta-glucuronidase enzyme activity for Esclierichin coli.
Immune-assays constitute another of the technologies used for detection testing.
They make use of the immunogenic cliaracteristics of the microorganisms being sought.
5 Nan-exhaustively, we may ~nention the techniques of imnunofluorescence, the ELISA
(Enzyme-Linked ImmunoSorbent Assay) techniques, competitive or of the sandwich type.
These techniques en~ploy a step of so-called indirect detection that employs a secondary
antibody conjugated with an enzyme for subsequent detection via a substrate specific to
tlie latter.
10 Document EP-B-1440316 describes for example a device for detecting
microorganisms. This device consists of a solid substrate, on which capture partners
specific to the target microorganisms, such as antibodies, are fixed. The capture substrate
is then placed in various containers con~prisingt he sample to be analysed and the various
reagents for carrying out an ELISA reaction.
15 This step of so-called indirect detection then involves (following the enrichment
step) the execution of various treatment steps (taking the sample, heating, centrifugation,
washing, etc.) of the sample before the screeninddetection step, which consequently
make the operating protocol more complex, make the analysis less convenient and
increase the time to supply the results.
20 Finally, the techniques of molecular biology, based on the genomic characters of the
microorganisms being sought, are also employed for detecting and identifying the target
microorganis~ns. We may mention, as exanlples, the conventional techniques of
amplification such as PCR (Polymerase Chain Reaction) and NASBA (Nucleic Acid
Sequence Based Atnplification), which can be coupled to techniques for real-time
25 detection known by a person skilled in the art. Neve~-thelesst,h ese techniques require an
arduous step of preparation of the sanlples, consisting of isolating the microorganis~ns,
lysing them in order to release the nucleic acids, and finally purifying the latter. This also
has a direct effect on the conlplexity of the operating protocol, making tlie analysis less
convenient and increasing the time to supply the results.
30
Regarding the confirmation step, it is more pasticularly associated with
microbiological atlalysis in the food industry. hi fact, when the result of the methods
developed previously is positive, it is necessary to confirm the presence of the pathogen
being sought. This requires an additional test and the use of a principle of detection
different from that used in the first analysis. The techniques described above are used at
leisure for confir~nation.
The eonlplete and accurate identification of a microorganistn in a sample therefore
requires several successive steps: emiclunent, optionally subculture, detection and
confirmation. Standardization of the tests used routinely has allowed automation of the
methods of detection, but they still take a long time. A drawback of the prior art is in fact
10 that these steps are carried out seqnentially and require a large number of time-consuming
manipulations, thus having an impact on the time taken to supply the results.
In view of the technical problems raised by the prior art considered above, one of
the essential aims of the present invention is to provide a simplified method for the
15 detection, identification and confirmation of the microorganisms present in samples,
especially food samples.
Another aim of the present invention is to provide a tnethod for detecting and
identifying the mic~oorganismsw, hich makes it possible to reduce the necessary time and
20 cost for analysis of the sample.
These aims, among others, are achieved by the present invention, which relates
firstly to a method for detecting at least one microorganism, said method essentially
comprising the following steps:
a) in a first container, bringing said sample into contact with at least one culture
medium,
b) placing said first container in suitable conditions to permit growth of the
microorganism or microorganisnls,
c) bringing some or all of the mixture consisting of the sample and the culture
medium into contact with a reaction mixture and a substrate for capturing said
microorganism(s) in said first container or in a second container, said reaction
~nixturec omprising means for detecting said mieroorganism(s);
d) detecting, within said first or second container, the presence of the
microorganism or n~icroorganis~ndse tected by the detecting means and fixed
on the capture substrate.
5 According to a particular enlbodinlent, the method according to the invention
comprises an intermediate step c') consisting of placing the first or second container in
suitable conditions to permit growth of the microorganism or microorganisms.
According to another particular embodiment, the method according to the
10 invention comprises an additional step e) consisting of confirming detection of the
n~icroorganismo r microorganisms detected. Preferably, the confirmation step e) is carried
out using a detecting means identical to or different fro111 the detecting means used for
detection.
15 Advantageously, at least one specific or non-specific binding partner of the
microorganism or microorganisms is fixed on the capture substrate. According to a
preferred embodiment of the invention, the specific binding partner is taken from the
group comprising: antibodies, Fab fragments, Fab' fragments, aptamers, recombinant or
non-recombinant phage proteins, phages or any other ligand well known by a person
20 skilled in the art.
The capture substrate can be any suitable substrate allowing detection of the
n~icroorganismsW. e tnay notably mention particulate substrates, optionally magnetic, or
single-piece substrates, optionally porous. It can be quite simply an inert substrate, such
25 as a plate made of plastic or glass fibre. The capture substrate can advantageously be
sensitized with a binding partner, optionally specific. The capture substrate can also be a
compressible single-piece substrate.
Advantageously, the substrate can be protected by a protective filtn.
According to another particular embodiment, it is possible to execute detection
30 and confirmation with the same technology.
Preferably, detection of the ~nicroorganism or microorganisms is carried out in
real time. However, as an alternative, detection of the lnicroorganisnl or tnicroorganisms
can be carried out, at the end point, at the end of a step of growth of said
microorganism(s).
According to a particular embodiment of the metl~od according to the invention,
the first and/or the second container is a homogenizing bag. They can also be rigid
containers such as flasks, bottles or tablet containers.
10 According to another particular embodiment of the method according to the
invention, the detection step can be carried out using a reader. Such a reader can consist
for example of a catnera pointing at the capture substrate, for recording or analysis of
images of said support.
15 The aims and advantages of the present invention will be better understood in light
of the detailed description given below, in conjunction with the drawings, where:
- Fig. 1 is a schematic representation of the different steps of the method
according to a first embodiment of the invention.
- Fig. 2 is a schematic representation of a sensitized capture substrate.
- Fig. 3 is a schematic representation of the sensitized capture substrate shown in
Fig. 2, after analysis with a positive result.
According to a first e~nbodimenot f the present invention, the method for detecting
25 or for identifying n~icroorganism(s)c onsists of employing a sterile plastic homogenizing
bag, conventionally called Stomacher@ bag. Such a bag is referenced 10 in Fig. 1A. This
bag 10 consists of two roughly rectangular sheets of plastic, joined together on three
sides, so as to define an internal space intended to receive the culture medium and the
sample to be analysed. It additionally coniprises a filter 12 of roughly rectangular shape,
30 joined to the sheets on one side, separating the internal space into two.
During step A, the closed homogenizing bag 10 is incubated with a food sample
14, consisting in this case of a sanlple of unpasteurized milk cheese. This food sample 14
is immersed in an enriclulient medium 16, which can be selective or non-selective.
Incubation can be carried out at temperatures between 25 and 44°C for 6 to 48h.
A fiaction of the enrichment mediurn is then taken in tlie homogenizing bag 10
and transferred during step B to a secondary container, consisting here of a tube 20. This
5 tube 20 comprises a reaction mixture 22. Said reaction ~ilixture2 2 can consist of a diluent
(e.g. tryptone salt broth) suitable for maintaining integrity of the target microorganisms,
and at least one detecting means. The detecting means can be a dye that is able to stain the
microorganisms present in the fiaction of enrichment medium transferred, obtained fiom
the food sample 14. The detecting means can also be a fluorescent compound that makes
10 the ~nicroorganis~nflsu orescent. When we wish to perform a subculture in tube 20, tlie
reaction mixture can contain, in addition to the nutrients, a selective system allowing the
population of target bacteria to increase.
According to a particular example, the detecting means is based on the reduction
of triphenyl 2-3-5-tetrazolium chloride (TTC) by the n~icroorganisms. Sitnultaneously
15 with growth of the niicroorganisn~s, TTC (colourless in its non-reduced form) is
internalized by said n~icroorganisms,a nd then reduced within the cytoplasu~b y the latter
to triphenyl-formazan (red), thus staining said ~nicroorganis~nresd so that they can then
be detected on the substrate. Other tetrazolium salts can be used (CTC, MTT, etc.).
Moreover, colnpounds for speeding up the reaction of reduction of the tetrazoliu~n salts
20 can be added to the reaction mixture.
It is also conceivable to use membrane stains, such as gentian violet or fnchsin.
In the case when the detecting nieans is a fluorescent compound, it can be acridine
orange or fluorescein diacetate.
25 In step C, a sensitized capture substrate 24 is put in tube 20 and is kept immersed
in the reaction mixtnre 22 by any suitable means. The sensitized capture substrate 24 is
functionalized with at least one specific binding partner of a target microorganism to be
detected. The capture substrate can consist of any substrate suitable for fixation of
specific binding partners and well known by a person skilled in the art. As a non-litniting
30 example, a suitable capture substrate can be made of irradiated polystyrene, such as that
marketed by the company Nunc/Thern~o Scientific (Cat. No. 472230). A capture substrate
of this kind is shown schelnatically in Fig. 2, with the reference 24. According to a
preferred embodiment, the lower portion can advantageously be divided into two. The
zone referenced 241 can be sensitized with a solution of binding partners (polyclonal
antibodies, monoclonal antibodies, Fab' or FabZ fragn~ents, aptamers, phage proteins),
~vllereasth e upper portion 242 remains free from any binding partner and thus plays a role
5 of negative control. The techniq~~efso r sensitizing substrates with specific binding
partners are well known by a person skilled in the art.
According to one alternative of the method according to the invention, it may be
advatltageous to carry out a step of subculture, once the capture substrate 24 is inlmersed
10 in the reaction mixture 22. This subculture consists of incubating tube 20 for 1 to 18h, at
temperatures between 25 and 44°C. According to this alternative, the intensity of staining
of the microorganisms increases simultaneously with their growth owing to the detecting
means contained in the reaction mixture. Analysis can then be carried out in real time.
15 Once there is effective capture of a certain amount of stained or fluorescent target
nlicroorganisms (in the case of a positive satnple), there is a change in the optical
properties of the substrate tlxough appearance of coloration or fluorescence on the latter
(i.e. transduction of the biological signal). This coloration or fluorescence of the capture
substrate is then detectable by eye or can be measured using an auto~naticr eader such as a
20 camera. The capture substrate is sllown schenlatically in Fig. 3, after analysis with a
positive result. As can be seen, zone 241 appears coloured owing to fixation of the target
nlicroorganislns on the specific binding partners. For its part, zone 242, performing the
role of negative control, still has the initial colour of the capture substrate.
25 To facilitate reading, the sensitized captnre substrate should preferably no longer
be in contact wit11 the reaction mixture. For this purpose, it can be envisaged for example
to remove the capture substrate 24 from said reaction mixture by any suitable means, as is
clearly s11ov.n in step D, in Fig. 1. As explained above, the reading can be taken at the end
point, as dotted lines or in real time.
30
According to another alter~lative of the method according to the invention, the
capture substrate consists of sensitized particles, namely bearing a specific or non-specific
binding partner of the microorganis~no r microorganisms to be detected. Detection is then
preferably indicated by appearance of a coloration or fluorescence of the initially
colourless sensitized particles, due to binding of the target n~icroorganisnis to the latter,
during the reaction.
5 According to a particular embodiment, the sensitized particles can be magaetic
particles. Reading can then be done manually and visually, using a nlagnetizing system
that will allow collection of the magnetic particles on which the stained or fluorescent
tnicroorganisnls are captured, in the form of clusters against the container wall.
Movement of the ~nagnetizings ystem vel-tically upwards makes it possible to remove the
10 cluster of magnetic particles from the reaction mixture and thus facilitate analysis. It can
also be envisaged to immerse tlie magnetizing system directly in the container, in order to
collect tlie magnetic particles on which the ~nicroorganisms are fixed. In this case the
magnetic particles will become fixed directly on the magnetizing system, w11ich then
becomes coloured or fluorescent.
According to another embodiment of the method according to the invention, the
reaction mixture and the capture substrate, as described above, are added directly to the
first container at the end of the e~nichn~esntte p. The detection step is therefore canied out
in said first container without transferring some or all of the mixture consisting of the
20 culture ~nediu~ann d the sample to be analysed to a second container. Prior to this
detection step, a s~~bculturcea n optionally be carried out in order to increase the
population of target microorganisms.
It sliould be noted that, advantageously, the capture substrate can be protected
25 using a protective film. The purpose of this film is to prevent fouling of the capture
su~bstrate. In fact, sucli fouling is likely to impair the capture performance of said
substrate. Such a fihn can be placed pernlanently on the capture substrate. Alternatively, it
can be a film that can dissolve after a certain time of contact with tlie liquid culture
medium.
30
The method according to the invention is particularly advantageous because at the
end of the analysis, only the containers identified as positive by the detecting system
(described below) are opened in order to carry out additional analyses for confirming the
presumptive result obtained. The confirnlation step can be carried out by means of
tecl~nologyd ifferent from that employed in the method according to the invention.
5 The aim of the exanlples presented below is to present different emboditnents of
the ~netllod according to the invetltion and the results obtained. They do not limit the
invention in any way.
EXAMPLES
10 Era~iiple 1: Optical detection of S(i1tinotielln Napoli, by lneans of a sensitized
substrate, in a food satnple in subculture in a reaction mixture
The aim of this experiment is direct detection of the presence of the target
bacterium Saltnoi~ella Napoli in a food satnple in subculttire in a reaction mixture, by
15 means of a sensitized substrate made of irradiated polystyrene, ~narketedb y the company
N~~~~c/TheSrcrineont ific (Cat. No. 472230) and shown in Figs. 2 and 3.
As detailed below, detection is performed during the reaction step by immersing
the capture substrate sensitized with a recombinant phage protein specific to Sn1111ot?e//o
in a tube that contains the enriched sa~npled, iluted to 11100th in the reaction mixture.
Protocol:
Step 1: Resuspending the sa~npleisn tile pprimarv enxichment medium
Two samples are prepared as follows:
Sample A: In a l~omogenizing bag, 25g of unpasteurized milk cheese
25 contaminated with 5 colony fornling units (CFU) of Srrl~l~otiellNrra poli is resuspended in
225 mL of Buffered Peptone Water (BPW) (bioM&rieux, Ref. 42043), supplemented with
1 ml of Supplement SPT (bioMQieux, Ref. 42650);
Sample B: In a homogenizing bag, 25g of unpasteurized milk cl~eese not
30 contaminated with S(rltilotzel/rr Napoli is resuspended in 225 mL of BPW (bioMerieux,
Ref. 42043) supplenlented with 1 mL of Supplenlent SPT (bioM&rieux, Ref. 42650).
Step 2: After 16h of incubation, transfer of a 0.1-mL ali~uot from the
homogenizing bag to the reaction tube
0.1 mL from the Sample A homogenizing bag is transferred to tlie reaction tube
containing 10 mL of SX2 @ioMkrieux, Ref. 42121) and 1.6 g/L of TTC (bioMirieux, Cat.
5 No. 04568088). This gives Sample A'.
A similar operation is carried out for Sample B.
Step 3: Immersion of the sensitized substrates in the reaction tubes before
subculture and reaction
10 The sensitized capture substrate is placed in each tube (Samples A' and B'). The
tubes are then closed again and incubated in a stove at 37OC for 6 h.
Step 4: Reading the capture substrates at the end of the incubation period
At the end of incubation (6h at 37OC) and following non-specific reduction of TTC
15 by all of the bacteria present in the sample (i.e. belonging to the additional flora and the
target flora), the reaction mixture has turned red. Tl~usi, n order to obselve the capture
substrate, revealing whether the sample analysed is positive or negative, the tubes are
slanted so as to isolate said capture substrate from the reaction mixture.
20 In accordance with the experimental design, the capture substrate placed in sample
A' appears coloured red, confirming that saniple A' is positive, whereas the capture
substrate placed in sample B' remains colourless, confirnling that sample B' is negative.
Analysis of these same samples by the VIDASB SPT method, marketed by tlie applicant
(ref. 30707), led to similar results, thus confirming the resnlts obtained by optical reading
25 of the sensitized capture substrate.
Example 2: Optical detection of Snlrrtonelln Napoli in an enriched biological sample,
by liieans of a sensitized substrate ilninersed in a reactioii niixture
30
The aim of this experiment is direct detection of the presence of the target
bacterium Srrl~t~o~~Nelalpno li in an enriched food sample, by means of a sensitized
substrate made of irradiated polystyrene, marketed by the company NuncIThernlo
Scientific (Cat. No. 472230) and shown in Figs. 2 and 3.
As detailed below, detection is performed during the reaction step by immersing
5 the sensitized capture substrate with an anti-Sa11itonella recombinant phage protein in a
tube containing the enriched sample, diluted to 112 in the reaction mixture.
Protocol:
Step 1 : Resuspending the san~pleisn the primary enrichment medium
Two samples are prepared as follows:
Sample A: In a homogenizing bag, 25g of minced steak contaminated with
Sal1i1onelln Napoli is resuspended in 225 mL of BPW (bioM&ieux, Ref. 42043)
supplemented with 1 lnL of Supplement SPT (bioMkrieux, Ref. 42650);
15
Sample B: In a homogenizing bag, 25g of minced steak not contaminated with
Srr~l~iionellNo apoli is resuspended in 225 nlL of BPW (bioM&rieux, Ref. 42043)
supplemented with 1 mL of Suppletnent SPT (bioMkrieux, Ref. 42650);
20 Step 2: After 16h of incubation, transfer of a 1-mL aliquot from the honlogenizing
bag to the reaction tube
1 mL from the Sample A homogenizing bag is transferred to the reaction tube
containing 1 mL of tryptone salt (bioMerieux, Ref. 42076) supplemented with 10 pL of
25 gentian violet (bioMirieux, Ref. 55545). This gives Sample A'.
A similar operation is carried out for Satnple B.
Step 3: Inlmersion of the sensitized substrates in the reaction tubes before reaction
30 The sensitized capture substrate is placed in each tube (Samples A' and B'), as
described below. The tubes are then closed again during the reaction period.
Step 4: Reading the capture substrates at the end of the reaction period
At the end of the reaction (40 min at room temperature), all of the bacteria present
in the sanlple (i.e. belonging to the additional flora and the target flora) are stained violet.
5 Thus, in order to be able to observe the capture substrate, revealing whether the sample
analysed is positive or negative, the tubes are slanted so as to isolate said capture substrate
from the reaction mixture.
In accordance with the experimental design, the capture substrate placed in sample
10 A' appears coloured violet, confirming that sample A' is positive, whereas the capture
substrate placed in sample B' remains colourless, confirnling that sample B' is negative.
Analysis of these same samples by the VIDAS@ SPT method, marketed by the applicant
(ref. 30707), led to similar results, thus confirlning the results obtained by reading the
sensitized capture substrate by eye.
15
CLAIMS
1. Method for detecting at least one n~icroorganis~pnr esent i11 a sample, said method
esse~ltiallyc omprising the followi~igs teps:
a) in a first container, bringing said sample into contact with at least one culture
medium,
b) placing said first container in suitable conditions to permit growth of the
microorganis~no r microorganisms,
c) bringing some or all of the mixture consisting of the sample and the culture
medium into contact with a reaction mixture and a substrate for capturing said
microorganism(s) in said first container or in a second container, said reaction
mixture comprising means for detecting said ~i~icroorganism(s);
d) detecting, within said first or second container, the presence of the
microorganism or microorganisn~s detected by the detecting means and fixed
on the capture substrate.
2. Method according to the preceding claim, comprising an intermediate step c')
consisting of placing the first or second container in suitable conditions to permit
growth of the inicroorganistn or microorga~lisms.
3. Method according to one of the preceding claims, comprising an additional step e)
consisting of confirming detection of the microorganism or microorganisms detected.
25 4. Method according to Claim 3, in which the confimiation step e) is carried out using a
detecting means identical to or different from the detecting means used for detection.
5. Method of detection according to one of the preceding claims, in wliich at least one
specific or non-specific binding partner of the n~icroorganism(s) is fixed on the
30 capture substrate.
6. Metl~od of detection according to tlie preceding claini, in which the specific binding
partner is taken from the group conll~rising: antibodies, Fab fragments, Fab'
fiagnlents, aptamers, rcconlbinant or non-reconlbinant phage proteins, phages.
5 7. Method according to one of tlie preceding clauns, in which detection of tlie
nlicroorganism or nlicroorganis~nsi s canied out in real tinie.
8. Method of detection according to one of the preceding claims, in wl~iclld etection of
the tnicroorganisn~o r ~nicroorganislnsi s carried ouf at the end of the step of gro\vtIi of
10 said microorga~~is~~l(s).
9. Method of detection according to one of the preceding claims, in which the first
and/or the second container is a l~omoge~uzinbga g, a flask, a bottle or a tablet
container.
15
10. Metllod according to one of the preceding claims, in which tlie capture substrate is a
single-piece or particulate substrate, optionally porous.
11. Method according to CIainl 10, in which tlie cal~ture substrate is protected by a
20 protective film.
12. Method according to Claini 10 or 11, in m~lliclI~h e particulate support consists of
sensitized particles.
25 13. Metllod according to the preceding claim, in ~vl~ictlliie sensitized particles are
magnetic.
I
14. Method according to Clai~il1 0 or 1I , in \vl~icllt he single-piece capture substrate is a
co~npressibles ubstrate.
30
Dated this June 24 201 4
' (RANJNA MEIITA-DU'IT)
OF REMFRY & SAGAR
ATTORNEY FOR THE APPLICANT[S]
ABSTRACT
METHOD FOR DETECTING AND DIRECTLY IDENTIFYING A
MICROORGANISM IN A BIOLOGICAL SAMPLE BY AN OPTICAL ROUTE
The present invention relates generally to the field of analysis, for example biological
10 analysis. More specifically, the present invention relates to a method for detecting at least
one microorganism present in a sample, said method essentially comprising the following
steps:
a) in a first container, bringing said sample into contact with at least one culture
medium,
b) placing said first container in suitable conditions to permit growtli of the
microorganism or microorganisms,
c) bringing some or all of the mixture consisting of the sample and the culture
medium into contact with a reaction mixture and a substrate for capturing said
niicroorga~iism(s) in said first container or in a second container, said reaction
mixture co~nprisingm eans for detecting said microorganism(s);
d) detecting, within said first or second container, the presence of the
~ilicroorganismo r microorganisms detected by the detecting means and fixed
on the capture substrate.