METHOD FOR ISOLATING MICROORGANISMS ON A CULTURE MEDIUM,
AND RELATED DEVICE
5 The present invention relates generally to the field of n~icrobiological analysis. More
particularly, it relates to a method for isolating at least one microorganism obtained
fiom a contaminated sample.
Isolation of microorganisms on a gelled culture medium, starting from a sample to be
10 analyzed or a suspension of microorganisms, is often an indispensable step in many
methods of microbiological analysis. This step is notably used for performing
identifications, verifying the microbial purity of a sample or for performing a bacterial
count by counting the isolated colonies thus obtained.
15 The aim of the isolation techniques is to obtain, on the surface of a gelled nutrient
medium, colonies that are directly usable (CDU) for identifying and determining
sensitivity to antibiotics. They are well known by a person skilled in the art, the streak
technique being the reference technique. The latter consists of depositing the inoculum
by streaking on a surface with an equal probability per unit of area traversed. The
20 distributed local density decreases approximately exponentially during passage of the
tool. Thus, several spreading zones are prepared from a single inoculum, with or
without overlapping of the zones, in order to obtain the suitable effect of distribution
and a thinning out of bacteria in the next spreading segment. At the end of spreading,
the cells are sufficiently isolated from one another so that microbial developments in the
25 form of CDU (visible colonies or microcolonies) are not superposed, even partially.
This techtlique may also be carried out by a single continuous inoculation in a spiral by
means of a rotating plate or by a magnetic bead driven by a device producing
continuous inoculation that is not overlapping or optionally is partially overlapping.
30 Another widely used technique cotisists of isolation on a dish of gelled medium by
spreading on the surface. In this case, a mixtare of cells at a low cellular concentration
allowing 30 to 300 cells to be put in culture is spread on the surface of the gel in a Petri
dish with a diameter of 9 cm, each cell developing into an isolated colony. When fewer
than 30 cells are brought into contact with the nutrient gel, statistical problems affect the
accuracy of the count.
When the number counted is above 300 cells, counting errors arise owing to
5 overlapping of the surfaces of the colonies. Spreading is usually effected xvith a tool
co~nprisingfo r exa~nplea linear part that is in contact with the gel or using beads a few
tnilli~netersi n diameter rolling randomly on the surface in disordered cnovement. This
technique is only suitable for a slightly contaminated or diluted sample as a high
number of cells increases the probability of confluence of the colonies as a result of
10 growth.
It is also possible to perfonn isolation on a dish by deep inoculation. The initial sample
is diluted several times so as to reduce the microbial population sufficiently and obtain
separate colonies. Small volu~neso f each of the diluted sa~nplesa re then mixed with a
15 liquid gel, usually of agar supercooled to about 45°C. The inixtt~resa re immediately
poured into sterile culture dishes and after gelling and incubation, each cell is
immobilized and forms a colony.
Certain mauual methods have been auto~nated owing to the developnlent of devices.
20 This is so for example in the case of docoment EP-0 242 114, which describes
equipment and a method for inoculation of a culture medium with a sample. The
method consists of performing several segnlents ofs preading from one inoculu~nT. hese
segments are in the fonn of circular arcs and are performed by means of four different
spreading heads. An effect of dilution of the sample is obtained by partial overlapping
25 of the subsequent segments. The method described in the document is in fact very
similar to the reference method of manual isolation.
More recently, new methods of isolation have been developed, allowing improvement
of bacterial exhaustion by using an optimized applicator as described in document WO-
30 A-200507'1055. This applies notably to the method of inoculation etnployed in the
auto~naticd evice marketed by the applicant under reference P R E V II~so~la.
However, these isolation techniques are only effective on gelled culture media.
111 the areas of clinical diagnostics and of industrial tnicmbiological control ia the food,
phannaceatical or cosmetics industry, gelled culture media in a Petri dish, most often
5 agar, have constituted, since the end of tlte 19th century, an indispensable tool for the
detectiou and identification of pathogenic microorganisms.
However, a great tnany products have been developed for replacing the Petri dish. For
these products, rehydration is carried out in situ, i.e. directly in the space serving for
10 iuoculation and for incubation. One of these, the petrifilntn1 system, comprising
rehydratable nutrients, is vely widely used. Another system developed by the company
Nissai Pharmaceutical, Coutpact Dry, also consists of a dehydrated medium. These
culture media have the advantage that they keep longer than a ready-to-use agar culture
medium. They may also, like petrifilmmf, be compact and thus use a sn~all incubation
15 space. The surface available for culture is limited and the nutrients available per cell are
of suitable concentration to give colonies of smaller diatneter than on conventional
gelled medium, and the form of tlte colonies tnay also be modified owing to the low
solidity of the gel used or high coefficients of diffusion ofthe bacteria.
20 Nevertheless, isolation of colonies on these media is only possible by inclusiou in the
gel formed during rehydration, and therefore front an initial sample with low level of
cot~tamiuation or that has undergone a series of dilutions. The final concentration to be
deposited on the medium 111i1sbt e uttder 300 CFUIIIII (CFU: colony forming unit), these
conventional data depending on the size of the colony.
2s
Moreover, these media cannot withstand the mechanical stress of a means of isolation
by exhaustion without undergoing deterioration. Thus, one of the major problems of
these culture media that are rehydratable in situ, i.e. directly in the space serving for
inoculation and incubation, is that they are not compatible with mauual or automated
30 mechanical isolation of microorganisms. When the initial sample is heavily
contaminated (above 300 CFUImI), they require a series of dilutions to be carried out,
meaning taking a larger sample, loss of time and the consumption of a large number of
reagents (culture inedium, tubes of diluents, etc.), generating a large volulne of waste
(autoclaving, cost of treatment). Moreover, if a large number of dilutions is carried out,
there is a risk of losing, by the dilution effect, the target pathogenic tnicroorganistn, if
the latter is present in a small amount relative to the total microflora.
5
It is clear from the prior art considered that tlo tnethod of isolation is available that is
sitnple to carry out starting from a satnple to be aualyzed or a bacterial suspension, on a
culture medium rehydratable in sito, which allows isolated colotlies to be obtained.
10 A first aitn of the present itlvetltion is therefore to supply a tnethod for isolating
microorganisms and a related device that offer better performance and are simpler to
apply than the methods aud devices of the prior art.
A second aitn of the present invention is to supply a device and a method for isolating
15 tnicroorgauisms that can be used on a culture medium that is rehydratable, or rehydrated
just before or si~nultaneouslyw ith microbial isolation, directly in the space serving for
inoculation and incubation.
A third aim of the present invention is to supply a method for isolating microorganisms
20 from a sample having a high iuitial microbial concentration.
A fourth airu of the present invention is to supply a method of isolation that is also
cotnpatible with counting of the microorganisms.
A fifth aim of the present invention is to supply a device and method of isolation
cotnpatible with the use of chro~nogenicm edia.
25
These aims, among others, are achieved by the present invention, which proposes a
method for isolating at least one microorganistn from a satnple that may be
contaminated with said microorganistn, cotnprising the following steps:
(a) supplyitlg a device for isolation of microorganisms comprising
30 o a bottom layer impermeable to water
o a nutrient layer, arranged on the bottotn layer, cotnprising a dehydrated
culture medium
o an isolating layer permeable to the ele~iients comprised in the nutrient
layer, able to retain the bacteria on its surface and covering tlie whole or
a portion ofthe nutrient layer
o a protective top layer
5 (b) depositing a defined volume ofthe satiiple on the isolating layer
(c) isolating the riiicroorganisms by exhaustion or by coating the sample using
isolating means,
(d) incubating the device for a predetermined time and at a predetermined
temperature allowing growth of tlie microorganisms
10 said ~ilethoda lso conlprising at least one step of rehydration of tlie culture medium with
a predetermined volume of liquid before or si~liultaneouslyw ith step b) and/or c) and/or
d), preferably before or si~iiultaneouslyw ith step b) and/or c).
According to a preferred embodiment, the device mentioned in step a) is obtained by the
15 method comprising the following steps:
- pouring said predetennined volume of liquid onto the layer impemieable to wvatet;
- arranging the isolating layer on the nutrient layer; the wiiole being placed on tlie layer
impermeable to water that previously received said predeterniined volume of liquid in
order to allow instantaneous and honiogeneous rehydration of said dehydrated culture
20 mediam, and
-then superposing the protective top layer.
In other words, said device is advantageously obtained by successive superposition of
tlie following layers: 1) layer impemieable to water (preferably having received said
25 predetermined volume of liquid), 2) nutrient layer, 3) isolating layer and 4) protective
top layer, at least tlie nutrient layer 2) and isolating layer 3) being mechanically
independent of one another, so that they can easily be separated. This notably offers the
advantage that each of the two layers mentioned above can be sold and/or stored
separately. They can then easily be superposed by the user. Preferably, all the layers are
30 mechanically independent of one another, so that they can easily be separated from one
another.
"Layers mechanically independent of one another" means layers that are not joined
together by at least one bonding means, said bonding means that may notably be of a:
- physical nature, for example one or more clamps, or
- chemical nature, such as glue, melting of some or all of the two layers under the action
5 of a solvent to give rise to a compositelhybrid layer (also called "bonding layer"), or
else a gel or a gelling agent that is interposed at least partially between said layers
(namely at least partially at the interface behveen said layers).
Besides the aforementioned advantage (separate selling and/or storage of the nutrient
10 and isolating layers), the applicant discovered, against all expectations, that omitting a
bonding means fornling an interface between the nutrient layer and the isolating layer
makes it possible, by reducing the distance between these two layers, to obtain better
growth and/or snrvival of the nlicmorganisms deposited on the isolating layer. In fact, a
bonding means of this kind is of such a nature that it slows the passage of the nutrients
15 from the rehydrated nutrient layer to the microorganisms present on the isolating layer,
thus reducing the growth and/or the chances of survival of these microorganisms.
Sample means a small portion or small amount separated from an entity by a subtractive
act usually called sampling, for purposes of analysis.
20
The sample rnay be of biological, human, ani~nal, vegetable or environnlental origin. It
may relate to a product in the course of an industrial process or a finished product, for
exatnple food. It niay therefore correspond to a sample of biological fluid (whole blood,
serum, plasma, urine, cerebrospinal fluid, organic secretion), a tissue sample or sample
25 of isolated cells. It may be of industrial origin, i.e., according to a nonexhaustive list, a
sa~npleo f air, a sa~npleo f water, sampling from a surface, a piece or a product in the
course of treatment or manufacture, a product of food origin. Among samples of food
origin, we may mention nonexhaustively a sample from milk products (yoghurts,
cheeses, etc.), meat, fish, eggs, fruits, vegetables, water, drinks (milk, fruit juices, soda,
30 etc.) and the constituent or ancillary products of the finished product. A food sample
may finally be obtained fkom feed intended for animals, such as notably meal as animal
feed. Before analysis, this sample may undergo preparation such as enrichment,
extraction, concentration, purification, by lnetllods known by a person skilled in the att.
According to a preferred embodiment, the satnple voluole deposited on the culture
lnediunl is behveen 10 and 1000p1.
5
In the sense of the present invention, the tertn microorganism covers Gram-positive or
Gram-negative bacteria, yeasts, molds, amoebae and, more generally, unicellular
organisms, invisible to the naked eye, which can be manipolated and multiplied in the
laboratory.
10
According to a preferred embodiment of the invention, the tnicroorganistn is a Gramnegative
or Gram-positive bacteriutn, or a yeast.
As Gram-positive bacteria, we may mention the bacteria of the following genera:
15 Enterococcris, Streptococcris, Lnctobricilltis, Bifirobr~cteriurr~S,t rq~l~j~lococcBriusc, illris,
Li.steria, Clostridirtrrr, A@cobncteria, Nocar&, Coq~rnebncteriri, Micrococctis and
Deirlococc~is.
As yeasts, we may mention the yeasts of the following genera: Caiidida, Cr~ptococczis,
20 Sacch~~ronryceasn d Pichosporoi~.
As molds, we tnay mention the molds of the following genera: Aspergilltis, Pe~ticillitmr,
~ / C ~ ~ O S ~ J O ~ ~ ~ ~ I ! I .
25 The present invention also relates to a device for calt~treo ft nicroorganistns comprising
o a bottom layer itnperlneable to water
o a nutrient layel; arranged on the bottotu layer, comprising a dehydrated
culture medium
o an isolating layer permeable to the elements comprised in the nutrient
layer, able to retain the bacteria on its surface atid covering the whole or
a portion of the tlutrient layer
o a protective top layer.
Preferably, the nutrient layer and the isolating layer are mechanically independent of
oue anothel; as already mentioned.
5 In the sense of the present invention, the nutrient layer colnprises a support containing a
dehydrated culture medium. The support may be based on various absorbent
compounds with very strong water retentivity such as rayon, cotton, natural or
chemically-modified cellulose fibers such as carboxymethylcellulose, absorbent or
super-absorbent chemical polymers such as polyacrylate salts, acrylate/acrylamide
10 copolymer. This support may be impregnated with a culture medium in liquid form and
then dehydrated, i.e, having an Aw (activity of water) incompatible with tnicrobial
development. Alter~~ativeliyt ,i s covered or impregnated dry with a culture medium or
its constituents in the form of powder. Alternatively, liquid impregnation may, after
dehydration, be suppletnented by adding powder according to the means described
15 above.
Culture medium means a medium comprising all the elements necessary for the survival
and/or growth of microorganisms. In practice, a person skilled in the art will select the
culture medium as a function of the target tnicroorganistns, according to criteria
20 perfectly known and within the capability of the person skilled in the art.
The nutrient layer according to the invention may contaitl optional additives, for
example: peptones, one or more growth factors, carbohydrates, one or more selective
agents, buffers, dyes, one or more gelling agents, hydrogels, viscous agent, etc.
25
Preferably, the medium contailis little or no cold gelling agents, such as, for example,
agar, agarose, poloxamers, guar gum, xanthan, etc. Generally, the nutrient layer may in
addition contain a substrate allowing detection of at1 enzymatic or tnetabolic activity of
the target microorganisms based on a sigual detectable directly or indirectly. For direct
30 detection, this substrate may be bouud to a part sewing as fluorescent or chromogenic
marker. For indirect detection, the nutrient layer according to the inventiou tnay in
addition comprise a pH indicator, sensitive to the pH change induced by the
cons~unption of the substrate and revealing growth of the target microorganisms. Said
pH indicator may be a chromophorc or a fluorophore. As examples of chro~nophores,
we may mention neutral red, aniline blue, bromocresol blue. The fluorophores comprise
for example 4-methylun~belliferone, liydroxycoutnarin derivatives or resorufin
5 derivatives. Thas, the PC-PLC fluorescent substrate preferably used for carrying out the
method according to the invention corresponds to 4-methyl-umbelliferyl-choline
phosphate (4 MU-CP).
According to a preferred elllboditnent of the invention, after dry impregnation of the
10 llutrieut layer with the dehydrated culture medium, the latter undergoes a calendering
operation. Calendering, by the pressure and heating temperature generated, allows
retention and stable nlaintenat~ce over time of the dehydrated culture medium in the
nutrient layer, ensuring retention of the nutrients in the nutrient layer. It also tnakes it
possible to obtain a rigorously smooth and flat surface of the nutrient layer. This is
15 particularly advantageous for ensuring good cohesion behveen the nutrient layer and the
isolating layer when the latter is arranged on the nutrient layer. Besides accelerating the
rehydration of the nutrient layer relative to an uncalendered nutrient layer, calendering
allows compression of the fibers constituting the nutrient layer. This compression,
combined with the presence of the dehydrated medium within the nutrieut layer,
20 generates a large increase in capillary capacity of the latter, causing its allnost
itlstantaneous rehydration and generating a phenomenon of aspiration of the isolating
layer arranged on its surface. The isolating layer is then flattened against the nutrient
layer (while being lllechanically independent of the latter), thus ensuring absence of
space behveetl the two layers and in consequence optitnal microbial growth and/or
25 survival on the entire surface of the isolating layer. There is thus 110 need to have
recourse to a bonding uleaus (for example a bonding layer) between the isolating layer
atld the nutrient layer. This represents a significant advantage, in that said bonding
rneatls slows passage of the nutrients from the rehydrated nutrient layer to the
microorganisms present on the isolating layel; thus reducing growth and/or the chances
30 of sulvival of these microorganisms.
As noted above, tlie possibility of separating the nutrierit layer and tlie isolating layer,
thus allowving the two layers to be sold and/or stored separately, represents a real
industrial bonus. Such a technical effect then allows the nutrient and isolating layers to
be considered independetitly of one another, including from a comniercial and/or
5 logistic (storage) standpoint. This represents a significant advantage for tlie user, who is
notably able to combine at will nutrient and isolating layers with different properties,
suitable for the microorganisms to be detected.
Isolating layer means a layer whose main constituent is a material which by its nature,
10 its size, and its steric arrangement, retains the microorganisms on its surface while being
permeable to the elements comprised in the nutrient layer located beneath tlie isolating
layer.
The isolating layer may be based on one or Inore materials or derivative of these
15 materials such as latex, polytetrafluoroethylene, poly(viny1idene) fluoride,
polycarbonate, polystyrene, polyamide, polysulfone, polyether-sulfone, cellulose, a
mixture of celluloses and nitrocellulose. Advantageously, the isolating layer is porous,
preferably it is a porous tilembrane permeable to the elements comprised in the nutrient
layel; able to retain the tnicroorganisms on its surface and covering tlie whole or a
20 portion of the nutrient layer. The applicant discovered that the me~nbranes for
~nicrofiltration of water (and generally of liquids) currently marketed generally have the
properties required for use as the isolating layer. They make it possible to obtain very
good resistance to tearing during manipulation, controlled porosity, a perfectly smooth
charactel; small thickness and most of the time are highly hydrophilic. Their color,
25 generally white, ~ilakesit possible to opti~iiized ifferentiation of stained colonies on their
surface. The applicant discovered, surprisingly, that the use of these filtration
membranes not for a function of "traditional" filtration of liquids (as is done for water or
generally for liquids) but as a support that is smooth and strong for isolating tlie test
sample on their surface was particularly suitable for carrying out the present invention.
30 Regarding the filtration capacity and the hydropliilicity of the filtratiotl rnetiibrane, they
are utilized in order to allow and optimize passage of the liquid nutrients present in the
tmtrient layer (after rehydration thereof) to the isolating layer while preventing the
bacteria, yeasts and tilolds seeded on tlie surface of the isolating layer from migrating in
the opposite direction.
For tlie purposes of the present application, the aforesaid filtration tnetnbranes are
5 designated either as "filtratiotl metnbraties", or "microfiltratiotl membranes" or else
"filtering membranes", these expressions being synonyms of one another. These
filtration tnetnbranes are comprised in the group consisting of the porous membranes.
In one embodiment of tlie invention, the isolatirlg layer is active. It may thus contain
10 specific sites that are receptors ofbacteria such as antibodies or fragments of antibodies,
bacteriophages or fragments of bacteriophages, aptatners or any specific ligands that
may be fixed covalently or indirectly by sitnilar biochemical elements, at least one of
which is fixed covalently, for example a system using streptavidin and biotin.
15 Alternatively, the isolatitig layer is active, containing cotnpounds that inhibit or destroy
bacteria such as bacteriophages, antibacterial peptides, antibiotics, which inay or tnay
not be fixed in or on the isolating layer. The compounds tiiay be fixed covalently or
indirectly by related biochemical elements. Moreover, in another embodiment of the
invention, the isolating layer is active, containing elements that stimulate or accelerate
20 growth of the microorganisms. As an example, a viscous agent soch as polyethylene
glycol is arranged on tlie isolating layer.
Isolation may be carried out on some or all of tlie isolating layer, defining the usefitl
surface for isolation.
25
The isolating layer consists of a surface allowing passage of the eletnetlts froin the
nutrient medium and selective or reactive agents.
Advantageously, the isolating layer comprises pores whose diameter is between 0.01
30 and O.8p111 and preferably behveen 0.2 pm and 0.6 11111 so as to retain the bacteria,
yeasts and tnolds on its surface. According to a particular embodiment, the isolating
layer coniprises pores whose diameter is between 0.25 lun and 0.6 pm, for exa~iiple
between 0.3 pm and 0.6 [un, or else between 0.4 pni and 0.6 pm.
Alternatively, it may be a layer that does tiot have measurable pores, such as a dialysis
5 membrane, a cellulose tnenlbrane permeable to water and to chemical compounds and
defined by its capacity for retention of compounds with a molecular size above a
threshold. Advantageously tlie tiietnbrane will allow retention of chemical tnolecules
with a niolecular weight greater than 500 000 dalton (or between 5000 and 500 000
dalton).
10
Advantageously, the isolating layer comprises confined zones that make it possible to
facilitate isolation of the microorganisms. They may also make it possible to reduce the
growing time and therefore obtain the result Inore quickly.
15 In one embodiment, the isolating layer has hydrophobic andlor hydrophilic units
spatially delimiting the growth of tlie bacteria. The size of hydrophilic unit is between
10 and 500 pm, preferably between 300 and 500 pm, namely a diameter greater than the
resolving power of the eye. When it is a question of classic microbiology and therefore
visual observatioti.
20
In another embodi~nent, the isolating layer contains nanostructured onits, incompatible
with tiiicrobial growth, delimiting confined zones allowing growth of the
microorganism.
25 The hydrophobic onits are arranged in the for111 of a surface pattern which is a regular or
irregular grid, an assenibly of regular or irregular hexagons or according to a mixtare of
the two patterns, or optionally of other fonns.
The desirable materials for making this hydrophobic unit include wax, hydrophobic ink,
30 epoxy resins, silicone oil, silicone wax, polystyrene resin, alumina, polyfluoroethylene,
etc.
The isolating layer covers the whole or a portion of the nutrient layer. The isolating
layer is positioned on the notrient layer, partly covering it in order to allow rehydration
of the medium by a liquid. In a preferred embodiment, the nutrient layer nlay be
covered by the isolating layer completely.
5
The device may contain at least one rehydrating tneans in contact with the nutrient layer
such as a rese~voir attached to the device or included in the latter andlor channels
allowing rehydration of the bottom part or lateral part of the nutrient layel; preferably of
the bottom part of the latter.
10
The useful surface for isolation may be surrounded by a zone that does not allow
microbial develop~nent. It may be a zone tnade of an impermeable material or a
hydrophobic zone. Advantageously, said zone may be structured for guiding manual
use.
15
"Isolating means" is to be onderstood as a tneans allowing the microorganisms to be
brought into contact with the culture medium by moving over the latter in order to
deposit the sample. As the sample becomes i~npoverished as rubbing takes place
between the sample and/or the isolating means and the isolating layer, isolated cells are
20 deposited, so that after growth, isolated colonies lnay be obtained.
The isolating means has one or more surface(s) of contact with said culture tnedium.
Such a rneans tnay be used manually, for example a loop, a platinum loop, a pipette, a
ground rod, a rod comprising a tertnillal ball, a swab. It may also be beads.
25
Advantageously, the method according to the invention may be itnpiemented by means
of an automated system. A particularly suitable system is the PREVI~' Isola system
such as is protected it1 patent applicatiot~ WO-A-2005071055 and is marketed by the
applicant.
30
According to the invention, the culture lnediu~n is rehydrated with a predetermined
volutne of liquid.
A suitable volume of liquid is propagated in the culture medium. In practice, a person
skilled in the att will select the appropriate volume of liquid as a function of the
viscosity of the liquid, the diameter of the nutrient layel; it1 order to rehydrate the
5 medium and allow growth of the microorganisms. The rehydration step takes place
before or simultaneously with step b) and/or c) and/or d), preferably before or
simultaneously with step b) and/or c). 111 a particular embodiment, the liquid is added
manually using a pipette, or automatically. In another embodiment, the liquid is
contained in at least one reservoir integrated with the device and/or channels allowitlg
10 rehydration of the nutrient layer. The liquid then spreads in the nutrient layer by simple
pressure of the reservoir. An advantage of rehydration via the bottom and/or lateral part,
preferably via the bottotli part, is that it allo\vs uniform hydration of the entire nutrient
layer. This embodiment notably prevents the nutrients of the nutrient layer being
entrained by the liquid into the bottom part of the device.
15
According to one embodiment of the invention, the liquid used for reconstituting the
culture medium is an aqueous solution. According to a particular embodiment, the
liquid contains rnicroparticles with lipid envelopes, and preferably the liquid contains
red blood cells.
20
The liquid may also be a buffer or a solution containing supplements of the coltore
medium, such as antibiotics or substrates.
According to the invention, the device comprises on either side of the nutrient layer and
25 the isolating layer
a bottom layer impermeable to water
a protective top layer.
The top layer may be translucent or transparent so that the colonies are visible through
30 this layer. Preferably, the protective top layer is separated f'rom the colonies by a
separating means. The separating means may be a side wall or any other means
allowing the top layer not to be in contact with the colonies.
Advantageously, tlie top layer will be able to rest oti the patts of the isolating layer that
do not allow microbial developn~ent, such as the peripheral zones or the hydrophobic
itt~its.
5
The top layer tnay be attached by one of tlie sides to one of the other layers of the
device by any means, natnely for example an adhesive or mechanical means.
It also tnakes it possible to prevent contamination during incubation. It is itnpertneable
10 to bacteria and limits the loss of water vapor. In fact, the device is incubated for a
predetermined time and at a predetermined temperature allowing growth of the
microorganisms independently of the ambient hutnidity conditions. Thus, the nature of
the top layer is selected so as to allow the gaseous exchanges necessary for growth of
the tnicroorganistns while allowing local hydration. The bottotn layer is impermeable to
15 water. Preferably, this bottotn layer is rigid, allowing better grip of the device in the
hand. It is manofactuted from cornpounds such as polyester, polypropylenes,
polystyrene. Preferably, it is manufactured from cellulose. It may be cardboard or paper
combined with a film itnpertneable to water. It tnay contain thermoformed channels that
will serve for proper rehydration of the nutrient layer.
20
Advantageoasly, the various layers of the device are tnade frotn recyclable materials.
According to a patticular embodiment of the inventioti, tlie bottotn layer and/or the
nutrient layer and/or the isolating layer is/are translucent or transparent.
25
According to a patticular etnboditilent of the invention, the device also cotnprises an
identification code such as barcodes or RFID tags.
A device accordit~gt o the invention tnay be of conventional shape, natnely of round
30 shape. It tnay nevertheless be of a different shape and notably of square or rectangular
shape. According to another variant, the various layers may be of different shapes.
Thus, for exatnple, the top, bottotn and nutrient layers tnay be of square shape and the
isolating layer atidlor surface useful for isolation of mund shape. The various layers
rnay be of different colors, facilitating discrimination of the suspected bacteria.
The invention also relates to the use of a device according to the invention.
5
The invention, its functionality, its applications as well as its advantages will be better
utiderstood on reading the present description, referring to the figures, in which:
- Figs. 1A and 1B are schematic representations of the device according to the
10 invention. The device 10 cotnprises a bottorn layer impertneable to water 14, a
nutrient layer 12, arranged on the bottom layer, co~nprisinga dehydrated culture
tnediam, an isolating layer 20 permeable to the elements comprised in the
nutrient layel; able to retain the bacteria on its surface and covering the whole or
a pottion of the nutrient layer, a protective top layer 15. This device also
15 comprises hydrophilic/hydrophobic zocies 13, a reservoir 19 and channels 18
allowing rehydtation of the bottom part or lateral part of the nutrient layer.
The device rnay have separating means 16 allowing the top layer 15 not to be in
contact with the colonies.
20 - Fig. 2A is a photograph of the device according to the invetition that has a
usefill isolation area of 25 cm2;
- Fig. 2B shows an enlargement of the central portion of the device. The device
was seeded with a sample ofEsclte~~iclc~oilai at a concentration of 10' CFUItnl.
25
- Fig. 3 shows the contents of a Petri dish seeded with a strain of Klebsiellc/
prrettrirorriae in the presence of a UriSelectTM4 cultc~rem edium with an isolating
layer consisting of a Macherey Nagel cellulose nitrate filtering membrane, as an
example;
30
- Fig. 4 shows the conteuts of a Petri dish seeded with a strain of KlebsieNrr
~~te~rnroninio teh e presence of a UriSelectTM4 ccultore medium wit11 an isolating
layer consisting of a Sartorius nitrate filtering tnembratie, as an example;
5 - Fig. 5 shows the contents of a Petri dish seeded with a strain of Klebsiello
p~teunloltirrei n the presence of a UriSelectTM4 culture tnediutn with an isolating
layer consisting of a cellalose acetate filtering membrane, as an example;
- Fig. 6 shows the contents of a Petri dish seeded with a strain of Klebsiello
10 pi7errttlortioe in the presenceof a UriSelectThl 4 culture medium with an isolating
layer consisting of a polyester filtering membrane;
- Fig. 7 shows the contents of a Petri dish seeded with a strain of Escl7erichio coli
in the presence of a UriSelcctThf 4 ccultore medium with an isolating layer
15 consisting of a Macherey Nagel cellulose nitrate filtering membrane, as an
example;
- Fig. 8 shows the contents of a Petri dish seeded with a strain of Eschericltic~c oli
in the presence of a UriSelectT" 4 culture inediu~n with an isolating layer
20 consisting of a polyester filtering membrane, as an example;
- Fig. 9 shows the contents of a Petri dish seeded with a strain of Escherichio coli
in the presence of a UriSelectThf 4 culture medium with an isolatiug layer
consisting of a cellolose acetate filtering membrane, as an example;
25
- Fig. 10 shows the contents of a Petri dish seeded with a strain of Eschericliio
coli in the presence of a UriSelectT" 4 culture medium with an isolating layer
consisting of a polyester filtering ~ne~nbranaes, an example;
30 - Fig. I I shows the contents of four Petri dishes in the presence of a UriSelectT" 4
agar cultore medium and of a dehydrated UriSelectTM 4 culture tnediutn
i~npregnated on a nutrient layer comprisiug a nonwoven support of the Glatfelter
type, Airlaid 150g/111i~n the presence of a sample of a strain of Eschericl7io o l i ,
as an example;
- Fig. 12 sliows the contents of four Petri dishes in the presence of a UriSelectThl 4
5 agar culture medium, of a dehydrated UriSelectTM 4 culture medium
itnpregnated in a nutrient layer comprising a nonwovetl support of the Glatfelter
type, Airlaid 150g/m2 in tlie presence of a satnple of a strain of Enterobnctei.
clorrcrre, as an example;
10 - Fig. 13 shows details of Fig. 16, showing the contents of hvo Petri dishes, as an
example;
- Fig. 14 shows the contents of four Petri dishes it1 the presence of an impregnated
UriSelectT" 4 culture tnediutil in a nutrient layer comprising a nonwoven
15 support of the Glatfelter type, Airlaid 150g/m2 it1 the presence of an isolating
layer such as a cellulose acetate filtering tnetnbtane and in the presence of a
satnple of a strain of Clostridizrnr~ez117diais, an example;
- Fig. 15 shows the contents of a Petri dish comprising a nutrient layer containing
20 a nonwoven support impregnated with an agar culture tnedium of the Clirotn ID
CPS ID3 type in the presence of a satnple of E~ite~'ococctfrrsr ecnlis, as ail
example;
- Fig. 16 sliows the contents of a Petri dish comprising a nutrient layer containing
25 a nonwoven support of the type Airlaid MN 100 137 impregnated with a culture
lnedittm of the Chrotn ID CPS3 type, dehydrated and without agar;
- Fig. 17 shows the contents of a Petri dish in the presence of a culture medium of
the Chrom ID CPS ID3 agar mediotn type and of a nutrient layer comprising a
30 nonwoven support impregnated with a tnedi~ttn of the ChrotnlD CPS3 type,
dehydrated and without agal; 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;
- Fig. 18 shows details of a nutrient layer comprising a nonwoven support of the
5 Airlaid MH 100 137 type impregnated with a culture medium of the Chla~nT D
CPS ID3 type without agar and in the presence of an isolati~igl ayer such as a
polyester tnenibratie;
- Fig. 19 shows another exa~npleo f support according to Fig. 18.
10
EXAMPLES
Example 1: Obtaining isolated colonies fiotn a heavilv contaminated solution on a
15 Petrifilm rehvdrated medium
From a solution calibrated at a theoretical bacterial load of 10' CFUItnl, 1000 p1 of
solutions loaded with Escliericl~ia coli at different concentrations obtained by
successive dilutions by a factor of 10 are deposited at the center of the bottom film of
PetrifilmB. The top film of PetrifiltnB is lowered onto the sample. A plastic diffuser,
20 concave face do\vnwards, is placed at the center of tlie Petrifilm@ assay. The satiiple 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 fortns.
Table 1:
Dilution
CFUInil
Colonies
The results show that six dilutions are necessary in order to obtain isolated and usable
Example 2: Obtaining isolated colonies fi.otn a heavily contatninated solution on a
rehvdrated medium according to the present invention
Starting fi.0111 the sacne solution calibrated at a theoretical bacterial load of 10' CFUhl11
5 used for inoculation of the Petrifilm@, mechanical inoculation by the "dial" method is
carried ont on the device according to the invention, allowing isolated colonies to be
obtained on a limited area (25 cm2) ofthe device.
Thus, the device according to tlie invention was seeded with 10 p1 (contents of one
10 loop) of a solution calibrated at a theoretical bacterial load of 10' CFU/ml loaded with
Escltericl~inc oli and deposited on tlie 1st dial 21 of the isolation surface of the device
whose useful isolation area is 25 cm2. The second dial 22 is seeded with a new loop,
drawing several streaks starting fi.om 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
15 from dial 22.
The device is formed by an isolating layer with hydrophobic/hydrophilic units 28 on
which isolation is perfornied. The hydrophobic/hydrophilic units make it possible to
improve isolation, notably on a small area (25 cfn2) by spatially delimiting the growth of
20 the microorganisms.
A layer containing tlie rehydrated cultt~re medium 25. A bottom layer impermeable to
water 26 and a translacent top layer sealing the device 27.
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
25 device without prior dilution.
Example 3: Obtaining isolated colonies on an isolatinv laver of tlie filtering membrane
type. arranged on a nutrient layer consisting of a nonwoven supoort impregnated with a
30 dehvdrated nutrient medium
The aim of this exatnple is to compare the morphotypes and the gmwth titile of colonies
developing on a porous andlor filtering tnetnbrane (preferably filtering) positioned on
an agar culture tnediutn or on a nutrient layer impregoated with dehydrated coltore
medium.
5 The size and color of the colonies obtained fiotn different bacterial species seeded on
these porous andlor filtering membranes are evaluated by the operator.
3.1 Materials
10 The experiments described below notably relate to strains of Esckerichin coli,
Clostrirlirii~+~e triidii, Enterococclrs fnecrrlis, Klebsielln p17errnlonine, and Enterobncter
cloncae.
The isolating layers tested for the present exatnple comprise:
15 - a polyester filtering membrane (Macherey Nagel Polyester) cotilprising pores
with a diameter of 0.2 pm, 0.4 pm, 1 pm and 5 ptn (wade reference: PORAFILtC
PE),
- a celli~losen itrate filtering membrane (Macherey Nagel Polyester) comprising
pores with a diameter of 0.2 pm, 0.4 pm, 1 ptn and 5 pm (trade reference:
20 PORAFIL@ NC),
- a cellulose acetate filtering tnembrane (Macherey Nagel Polyester) colnprising
pores with a diameter of 0.2 him, 0.4 pm, 1 pm and 5 pm (trade reference:
PORAFILO CA),
- a filtering membrane of cellulose mixed esters (Macherey Nagel Polyester)
25 comprising pores with a diameter of 0.2 pm, 0.4 pm, 1 ptn and 5 pm (trade
reference: PORAFILO CM),
- a cellulose nitrate filtering membrane (Sartorius steditn Biotech) comprising
pores with a diameter of 0.45 pm.
30 For the purposes of the present experiments, the following nonwoven supports are used:
- Glatfelter, Airlaid 100g/m2,
- Glatfelter, Airlaid concert 150g/tn2,
PDI supports 6og/m2.
The culture tnedia used for impregnating the nonwoven support in the present
experiments are: a Trypcase soybean broth (TSB-D), a culture medium of the
5 UriSelectB type 4 (trade reference: BioRad), or a culture n~ediutn of the Chrom ID CPS
3 type without agar.
The agar culture media used in the present experiments are as follows: UriSelectB4
(trade reference BioRad) and Chrom ID CPS 3.
10
3.2 Experimental protocol
Firstly, the various filtering membranes are tested on an agar medium of the UriSelectO
4 type (cf. section 3.3.1 below).
15
Secondly, the UriSeleet@ 4 agar culture medium is replaced with the various nonwoven
suppolls impregnated with a culture ~nediurnm entioned above (cf. section 3.3.2 below).
The nonwoven supports, impregnated with the culture medium, are rehydrated using a
20 predetermined volume of sterile water and a bacterial inoculum at the moment of
performing the analysis. The volurne/a~nout~otf sterile water necessary for rehydration
of the nonwvoven support, impregnated with the culture medium, varies as a fi~netion of
the nature of the nonm70veu support and the size of the latter. This inforniation can
easily be determined by a person skilled in the art based on his general knowledge, and
25 routine tests ifnecessary.
The assembly of filtering membrane and impregnated nonwoven support or filtering
membrane and agar mediu~n 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
30 the surface of the porous andlor filtering membrane is carried out firstly after an
incubation time of24h and then secondly after a total incubation time of 48h.
3.3.1 lsolation of various tnicroorganisnts on different types of filtering
mentbranes, in the oresence of an agar culture medium
5
Figs. 3,4, 5 and 6 show isolation of a strain ofKlebsieN~rp17e1m1or~iinr rteh e presence of
a UriSelectT" 4 agar culture tnedium, after at1 incubation time of 24h.
More precisely, tlte filtering metnbrane in Fig. 3 is of cellnlose nitrate (Macherey
10 Nagel).
The filtering metnbratte in Fig. 4 is of Sartorius cellulose nitrate (Sartorius).
The filtering tne~nbratlein Fig. 5 is of cellulose acetate.
15
The filtering membrane in Fig. 6 is of polyester.
In these Figs. 3,4, 5 and 6, the presence of well individualized colonies that are directly
usable (CDU) is noted.
20
Figs. 7, 8, 9 and 10 show the presence of Escltericl?in coli in the presence of a
UriSelectT" 4 agar culture medium, after an incubation time of 24h.
More precisely, the filtering metnbrane in Fig. 7 is of Macherey Nagel cellulose nitrate.
25
The filtering membrane in Fig. 8 is of polyester.
The filtering membrane in Fig. 9 is of cellulose acetate.
30 The filtering membrane in Fig. 10 is of polyester.
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 co~nply with what is expected of tnicrobial growth
directly on agar medium. Isolation performed on a porous and/or filtering membrane
5 leads to morphotypes of colonies and a quality of isolation that are obtained
conventionally with isolation carried out directly on agar medium.
3.3.2 Isolation and coi~nt/countino~f microor~anisms on filtering
tnembranes deposited on nonwoven s u ~ ~ oi~mplrseg nated with a dehydrated
10 culture niediunl
The present experitnents 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 mediutn.
15
As shown in Fig. 1 1, the impregttation suppot-t used is of the Glatfelter type, Airlaid
150g/m2. The latter is itnpregnated with UriSelectT" 4 dehydrated culture medium.
The contents of the Petri dishes shown in Fig. 11 provide evidence of growth of the
20 Escher.icl7in coli bacteria and the presence of well individualized colonies that are
directly usable (CDU).
Fig. 12 shows similar results in the presence of Entervbocfer cloncne bacteria.
25 Fig. 13 shows results similar to those in Figs. 11 and 12, in the presence of an agar
medium of the UriSelectTM4 type.
Similar results can also be seen for the cotltet~tso f the Petri dishes shown in Fig. 14 in
the presence of a UriSelectTM4 culture medium impregnated dry in a nonwoven support
30 of the Glatfelter type, Airlaid 150g/m2 in the presence of a cellulose acetate filterit~g
membrane.
3.3.3 Imureenation with a culture medium ofthe Chrom ID CPS ID3
dehydrated tyi~e
The present experi~i~enrtesl ate to a sample of Eitferococctrsfrreccrlis in the presence of a
5 Chrom ID CPS ID3 agar culture mediotn (cf. Fig. 15) and a dehydrated Chrom ID CPS3
~nediom withoot agar impregnated in tlie nonwoven support in the presence of a
polyester filtering membrane (cf. Fig. 16).
Another experiment relates to a sample of Eittembrrcter clorrcrre in the presence of an
10 agar culture niedium of tlie Chro~nI D CPS ID3 type and a cult~cren ledinm 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.
15 Figs. 18 and 19 show the results of an experiment bringing a Chro~nI D CPS ID3 culture
medium without agar impregnated on an Airlaid MH 100 137 nonwoven support in the
presence of a polyester filtering metnbrat~e.
As shown in Figs. 18 and 19, the presence of well individualized colonies that are
20 directly nsable (CDU) is noted. Although isolation was carried out on filtering
membranes positioned on dehydrated culture media - and not on agar media - this did
not affect bacterial growth, which proved to be optitnal. Moreover, the morphotypes of
the colonies obtained are similar to those obtained with isolation on porous and/or
filtering membranes positioned on agar ~nedinni.
25
3.4 Conclusions
The results of the experitnents relating to example 3 indicate that it is possible to
perform isolations of microorganisms on an isolating layer of the filtering membrane
30 type. It should be noted that these filtering membranes are not nsed for the action of
filtration of liquid, which is their primary ose, but for carrying out isolation of a satnple
that may be heavily laden with microorganisms, which requires them to have the same
surface qualities as those that are obtained on agar tnedia. Moreovel; isolation did uot
generate deformations of tlie filtering metnbrane, 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
5 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.
Besides compatibility of the porous andlor filtering membranes with the operation of
10 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 nlorphotypes of the bacterial
colonies obtained.
15
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
20 nlicrobial growth. Thus, the presence of an isolating layer arranged above a nutrient
layer impregnated with a dehydrated culture medium rnakes it possible to obtain
isolated colonies of microorganisms on the sorface 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 nutrieut support to the surface of the
25 filtration membrane. Example 3 clearly detnonstrates 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 tnetnbraue aud nutrient layer.
30 In general, the exchanges of nutrients and of water between the nutrient layer aud the
filtering membrane allow optimal microbial growth. The impregnated culture tnediutn is
rehydratable or ]nay be rehydrated a short time before or simultaneously with microbial
isolation.
Example 3 notably detllonstrates that the device according to the invention is
5 cotnpatible with isolation and microbial growth.
CLAlMS
1 . A method for isolating at least one microorganism fiom a sample that may be
5 contaminated with said microorganism, comprising the following steps:
(a) supplying a device for isolation of microorganistns comprising
o a bottom layer impenileable to water
o a nutrient layel; arranged on the bottotn layer, comprising a dehydrated
culture medium
o an isolating layer permeable to the elements comprised in the nutrient
layer, able to retain the bacteria on its surface and coveritig the whole or
a portion ofthe nutrient layer
o a protective top layer
(b) depositing a defined volume of the sample on the isolating layer
15 (c) isolating the microorganisms by exhaustion or by coating the sample using
isolating means
(d) incubating the device for a predetermined time and at a predetermined
temperature allowing growth of the microorganisms
said method also comprising at least one step of rehydration of the culture tnediutn with
20 a predetermined volume of liquid before or simultaneously with step b) andlor c) andlor
d), preferably before or simultaneously with step b) andlor c).
2. The method as claimed in claim 1, wherein the isolating layer has hydrophilic andlor
hydrophobic units.
25
3. The tilethod as claimed in claim 1 or 2, wherein the device also comprises an
identification code st~cli as barcodes or WID tags.
4. The tnethod as claimed in one of claims 1 to 3, wherein the liquid used for
30 rehydrating the culture medium is all aqueous solution and/or a liquid containing
~nicroparticlesw ith lipid envelopes.
5. The method as claimed in one of claims 1 to 4, wherein at least the nutrient layer and
the isolati~igla yer are ineclianically independent of one another within said device.
6. The method as claimed in one of claims 1 to 5, wherein said rtutrient layer co~nprises
5 a support contailling a de11ydrated culture medium, said nutrient layer being obtained by
impregnation, preferably d~yo,f said support with the dehydrated culture medium, then
calendering; said suppol2 being for example a nonwoven support.
7. A device for culture of microorganis~nsc omprising:
10 o a bottom layer impermeable to water
o a nutrient layer, arranged on the bottom layer, comprising a dehydrated
culture medium
o an isolating layer pernieable to the elen~entsc o~nprisedi n the nutrient
layel; able to retain the bacteria on its surface and covering the whole or
a portion of the nutrient layer
o a protective top layer,
in which at least the nutrient layer and the isolating layer are
mechanically independent of one another.
20 8. The device as claimed in claini 7, said device co~nprising at least one reservoir
integrated with the device andlor channels allowi~~regh ydration of the nutrient layer.
9. The device as claimed in clai~n 7 or 8, wherein the isolating layer is a porous
membrane, preferably a membrane for n~icrofiltration of a liquid.
25
10. The device as claimed in one of claims 7 to 9, wherein the isolating layer contains
hydrophobic andlor liydropliilic units.
11. The device as claimed in one of clainls 7 to 10, wherein said nutrient layer
30 co~nprisesa support containing a dehydrated culture inedium, said nutrient layer being
obtained by impregnation, preferably dry, of said support \vith the dehydrated culture
medium, then calendering; said support being for example a nonwoven support.
12. The use of a device as claimed in one of claims 7 to 11 for isolating at least one
microorganism.
5 13. A method of obtaining a device as claimed in one of claims 7 to 11, said method
comprising the following steps:
- pouring said predetermined volurne of liquid onto the layer impermeable to water,
- arrauging the isolating layer on the nutrient layel; the whole being placed on the layer
impermeable to water that has previously received said predetermined volu~neo f liquid
10 in order to allow instantaneous and homogeneous rehydration of said dehydrated culture
medium, and
- then superposing the protective top layer.
14. A device obtainable by the method as claimed in claim 13.