FR2550546A1 - Application of a new membrane for micro- and ultrafiltration, of screen type, in the biology of the pro- and of the eukaryotes, in particular in microbiology and in virology - Google Patents

Abstract

These membranes consist essentially of a polymeric material cast on a support, said material being chemically inert and accepted without any interference by living cells in contact with it, its structure exhibiting a complete impregnation neutrality at the same time in the case of the substances capable of exerting deactivating effects, static or mortal inhibitors and, in the case of the various molecules ensuring exchanges through it, its selectivity being exercised solely in respect of the size of the filtered particles in relation to the pore opening. The polymeric material is slightly hydrophobic, its resistance to strong and weak acids and bases is good at low and medium concentrations, at normal temperature, and it is compatible with most organic solvents, except for chlorinated or aprotic solvents which, at low temperature, are absorbed and cause swelling and, at high temperature, dissolve the polymer.

Description

APPLICATION IN BIOLOGY OF PRO- AND EUKARYOTES,
IN PARTICULAR IN MICROBIOLOGY, AND IN VIROLOGY,
A NEW MEMBRANE OF MICRO AND ULTRAFILTRATION
SCREEN TYPE
The present invention relates to the applications and uses in biology of the pro- and eukaryotes, in particular in microbiology, and in virology, of a novel screen-type micro and ultrafiltration membrane.

 The cell culture culture of eukaryotes as well as prokaryotes is a primordial basic element that makes it possible to satisfy the requirements of one or more individuals forming a more or less dense population, without alteration or modification, for the purpose of maintaining them, or to make them multiply abundantly, or even to obtain future hosts susceptible to parasites, especially the strict parasites that are viruses.

The cultivation methods are numerous; most of them can be grouped together, in a general way, as follows:
- culture in liquid nutrient medium. This results in either a film of cells on the surface, or a proliferation in depth, or even with adhesion to the wall of the container or on particles in suspension, a more or less homogeneous suspension throughout the nutrient medium, possibly accompanied by a deposit at the bottom of the receptacle; an enumeration in a liquid medium (by a "Most Probable Number" NPP-type method) is considered to be approximate, because empirically developed by statistical results;
- culture in a semi-solid nutrient medium (by adding 4% O agar-agar) :: this technique has a very interesting advantage compared to the culture in a liquid medium, because it allows to obtain individualized and isolated colonies , by formation of clones occupying a small volume around the starting individual from which they are derived; hence a very attractive possibility because it is quick and accurate to count living individuals;
- culture in or on a solid medium. The cells are deposited on the surface of a suitable culture medium made solid or "solid" by addition of 2% agar agar. It is also possible to cultivate anaerobic cells, strict or optional, directly in bulk in this nutrient medium. the same advantages are found in a count as in the case of a culture model in a semi-solid medium.

 The determination of the. The adequate physico-chemical presence of culture media has posed problems for biologists; they have also endeavored to simplify, to improve the results according to the various imperatives of these methods and especially to make compatible disparate samples, both because of their very nature and their size (heterogeneous environments, impure cultures , solid samples, liquid samples in large volumes ...). Membrane filtration was widely welcomed for this purpose thirty years ago.

This is now a common technique, extremely flexible, fast, and that advances in the technicality of membranes have led to promote.

 The application, in biology, of this membrane filtration technique, however, comes up against certain problems; it is in fact dependent on the compatibility with the living world, the quality, behavior and properties of the components of these membranes, as well as the size of their pores for screen-type membranes (the diameter of these membranes, at the level of porous film exerting the selection of the filter membrane, must indeed always be strictly less than the size of the particle, molecule, virus or cell, that we want to retain).

 The incompatibilities presented during the use of the membrane, for example having adsorption or absorption phenomena to various particulate, molecular, ionic and / or hydrophobic substances, and / or behaving as a more or less important barrier to the cell-culture medium interaction, and thus making it impossible to perform tests, whether cytological, physicochemical, biochemical, immunological or any other tests. It has also been found that, in the case of dense populations, membranes known to be usable without limitation do not allow the quantitative realization of the volume of exchange which would have been necessary in order to satisfy all the individuals present in growth; the phenomenon of cavitation illustrates the problems encountered in bacteriology.

 The inventor, in the course of the many researches he has carried out in this field, has surprisingly discovered a class of membranes compatible with the living world of pro- and eukaryotes, and behaving with exceptional neutrality, without any disturbing phenomena. , showing the cell colonies in a manner that is obtained during a conventional process from a culture performed directly within or on the surface of a nutrient medium, and whose intrinsic performance allow important exchanges for particular populations or dense suspensions, both in quality and quantity.

 Thus, the subject of the invention is the application in biology of proprugs and eukaryotes of composite membrane filter membranes of the screen type, which can be used in micro and ultrafiltration, said polymer being cast on a support which is chemically inert. and accepted without disturbance by cells living in contact with it, its structure exhibiting no phenomenon of absorption, adsorption, or selective retention (therefore of a total impregnation neutrality) for both substances likely to exert inactivating effects, static inhibitors, or lethal and for the various molecules ensuring exchanges through it. Thus, they satisfy, whatever the density or the nature of the population, the consumption of the individuals present. In other words, its selection is exerted only at the level of the size of the particles filtered compared to the opening of the pores.

 Knowing that it is indeed admitted that the importance of exchanges that can be considered through a "theoretical" (that is to say perfect) screen-type membrane between the filter face, porous film side calibrated, and the The underside of said membrane (thus its opposite surface) is a function of both the pore diameter of the film side (directly selecting the particles according to their size) and the number of pores per unit area: this is particularly surprising. Moreover, as evidenced by scanning electron microscopy, the number of pores per unit area is quite lower, at worst equal to that found on conventional commercial membranes (such as polycarbonate membranes). or in cellulose acetate which, as will be demonstrated later in the examples, do not in any way allow to obtain comparable results), that the porosity used is less than 0.2 μm and that the polymer is by its nature slightly hydrophobic . Its resistance to acids and bases, strong or weak, is good, in the low or average concentrations, at normal temperature.

The materials are compatible with most organic solvents, except chlorinated or aprotic solvents (type DMAC dimethylacetamide DMSO dimethylsulfoxide) which, at low temperature, are absorbed (and cause swelling of the membrane). At high temperature, they solubilize the polymer.

 The membrane withstands up to 1200C (in both dry and wet heat) and has little response to the exposure of ionizing radiation (at a dose of 2.5 megarads).

Its water flow rate, which is higher than that of other membranes, in particular membranes of the same category, is around 20,000 liters / day / m 2, under ultrafiltration conditions, this flow being able to be almost ten times under conditions microfiltration
In short, the phenomenon of cavitation is not observed with the membranes according to the invention.

 Finally, it must be emphasized that the membranes according to the invention are thin (about 0.3 mm) and particularly flexible, while being removable to tearing, folding and stretching, to pressure and that they are not ionic; they do not therefore exhibit electrostatic phenomena in contact with metal surfaces.

 A particular example of the membranes according to the invention consists of membranes shortly marketed under the name CPMO2.

The present invention will be better understood and its advantages will emerge from the following examples, made with the membrane shortly marketed under the reference Cl'M02
In all the examples described below, the membrane according to the invention is cut into disks 50 mm in diameter. These discs are then sterilized either in small groups or individually with a little deionized-distilled water. The sterilizing factor is either autoclaving (the container being then a glass Petri dish) carried out for 20 minutes at 1200C (in moist heat), or radiation sterilization by exposure to ionizing radiation Zf, in heat-sealed polyethylene bags, and minimum dose of 2.5 Mrad.

 The discs are kept at ordinary temperature. Each disc is then placed at a sterile post of a standard vacuum filtration boom with the smooth side of the membrane facing up (calibrated microporous polymer side).

 The desired sample is then filtered commonly, with a vacuum ranging from 0.4 to 1 bar.

 The disc is then taken up with sterile forceps and transferred to the surface of a culture medium agar plate Petri dish, the smooth face is always turned upwards; it is ensured, in a conventional manner, not to leave air bubbles between the membrane and the agar.

 In the particular case of the culture of anaerobic germs, 10 ml of culture medium maintained supercooled on the membrane that has just been deposited. This mass is then expected to set.

 The incubation, inverted Petri dish, is then carried out at the appropriate temperature for the necessary time.

EXAMPLE 1

 Escherichia coli on Hektoen agar. Incubation 24 hrs at 37 ° C.

 The smooth, curved, shiny colonies, with regular borders, have a beautiful macroscopic appearance. Their yellow coloration is noted, due to the turn of bromothymol blue (colored indicator of pH initially contained in the medium) because of the acidification, following the degradation by fermentation of lactose provided by the nutrient medium.

EXAMPLE 2

Mixture of Escherichia coli and Salmonella kedougou on agar
Hektoën 24 h to 37pu.

We also note the beautiful macroscopic appearance of the colonies
- yellow colonies (due to the turn of bromothymol blue), shiny, smooth, curved and on board regular Eschericha coli
- colorless colonies, with black centers, convex, shiny, regular edge of Salmonella. The black center results from the production of hydrogen sulphide by the germs (positive H2S) which, thanks to a ferrous salt dissolved in the agar, precipitates in the form of a black iron sulphide.

 Daylight transparency makes it easier to distinguish colorless Salmonella colonies from black centers.

EXAMPLE 3

Rhône water at dilution IO 3, taken at random. Middle
EMB (eosin, methylene blue). Incubation 48 h at 370C.

From a microbiologically unknown medium, a very diverse shoot, composed of
- very developed colonies, convex, shiny, with regular, smooth, dark brown edge;
- shiny, smooth, round colonies, with an orange domed center, bearing a flat, colorless collar, with straight but irregular borders;
- smooth, shiny bulging colonies, with regular edges, of various colors: orange, pink, red, brown ...;
- flat, whitish colony, formed of two concentric circles, the peripheral circle being smooth and shining, the inner circle being matt and rough in appearance, the center of which is hollow;
- colony flat, shiny, ovoid, with a straight edge, granular, dark brown in color;
- brown colonies, "umbilicated" (that is to say, at center collapsed with respect to the periphery), shiny, smooth.

 They seem to be colonies of fungi, yeasts and bacteria.

 It may be noted that some colonies have grown on a part of the membrane which has never been in direct contact with the culture medium.

EXAMPLE 4

 Unmarketed wine diluted 10 times. Middle Sabouraud. Incubation 24 h at 370C.

 There are large white, rough, regular-edged colonies, which are yeasts whose growth is electively favored by the composition of the culture medium. The smaller colonies are bacterial colonies or undeveloped yeast clones.

EXAMPLE 5

 Protein on regular nutrient agar. 24 hours at 370C.

 It is well observed, by comparing with a horny part of the membrane which is thus always detached from the culture medium, the characteristic invasion of the colonies of proteus, forming a particular drawing, rough aspect sometimes filamentous.

 There is also an intermediate zone between the peripheral right part of the culture medium and the deeper part entirely covered by the undifferentiated colonies: it is a band about 2 cm wide. At this level, the membrane is also well removed from the culture medium but there is a very thin film of sprouting of the germs that still try to invade the surface of the non-adherent part of the membrane (this is the characteristic veil of the proteases ).

EXAMPLE 6

 Same unmarketed wine (diluted 10 times) as example # 4.

 Schaedler medium pH = 4.5. Incubation 24 hrs at 37 ° C.

The middle Schaedler is chosen here to favor electively
the growth of wine bacteria. We observe the large amount of
colonies that have invaded the membrane to overtake and grow
directly on the agar plate. These are very mobile bacillus colonies
A close up view gives a very good account of the macromorphology of bacillus colonies: either white of powdery appearance, indeterminate form; either brown of ovoid shape or forming convolutions with an elevated peripheral limit; or cerebrally-And this at all stages of their growth.

EXAMPLE 7

 Staphylococcus aureus. Middle of Chapman. 48 h to 37 C.

 The colonies round, white, shiny, curved, are well individualized and take their small characteristic size because of the hypersalced medium (7.5% O NaCl).

EXAMPLE 8

 Serratia rubidea on regular nutrient agar. 48 h to 30 C.

 On a close-up photograph, only illuminated in light by the light of day, we distinguish, from the same pure culture, pigmented colonies, some in the course of loss or acquisition of pigmentation, others finally completely depigmented .

EXAMPLE 9

 The tests are made on the water of the Rhône, taken at random.

Dilution at 10,000th. Middle of Wilson Blair. 24 h to 37 "C.

 The colonies growing in the gelose between the membrane and the additional layer of added culture medium are perfectly distinguishable in order to create the anaerobiosis.

 Examination of the petri dish, taken in transparency by under-lighting, shows the well-defined appearance of the outline of the colonies.

EXAMPLE 10

 Escherichia coli on EMB medium. 24 hours at 370C.

 The membrane according to the invention makes it possible to obtain the famous metallic green appearance "in back of beetle", as if the colonies were grown directly on the agar. Macromorphologically, the colonies are well punctuated, bulging, shiny. If conventional cellulose acetate membranes (Schleicher and Schüll, calibrated 0.45) are used, the opposition is very clear; from the same strain, cultivated exactly under the same conditions, the aspect "back of beetle" is never obtained with more than 30 trials. On the other hand, there is a heterogeneous series of colorations ranging from blue to a series of concentric circles respectively, from the center to the periphery, pink-blue purplish to orange sometimes.

 The macromorphological appearance of the colonies is much better defined with the membrane according to the invention than with a conventional membrane made of cellulose acetate and the colonies are much more specific.

EXAMPLE II

 Escherichia coli on standard agar + 1 calibrated ampicillin disc. Incubation 24 hrs at 37 ° C.

 The paper disk soaked in a calibrated manner with an antibiotic is placed on the surface of the agar, then slightly pressed so that it does not cause separation of the deposited membrane afterwards with respect to the known culture medium. the diffusion antibiogram technique in an agar medium, we observe the central zone corresponding to a clear halo where the Escherichia coli shoot is inhibited by the antibiotic contained in the disc and diffuses; in this case it is ampicillin. Halo well visible, classic, easy diameter measurement.

 EXAMPLE 12

 Streptococcus pyogenes (group A) on sheep blood agar.

Incubation 24 h at 370C.

 The halo of lysis of hemoglobin is very easily identifiable, its diameter is greater than that of the membrane, which shows that the haemolysin secreted by the germs diffuse well and in perfect conformational state.

EXAMPLE 13

 Escherichia coli on Hektoen agar. 24 hours at 370C.

 With a suspension of high cell density, the results are as follows: On a view taken from below the bottom of the Petri dish, the colonies stained yellow (by bromothymol blue) appear significantly darker.

- On a view from above (side agar surface) colonies, although very numerous, are all well separated and the counting is perfectly possible with a simple magnifying glass.

QUANTITATIVE STUDY.

 From a known control suspension in physiological saline of Staphylococcus aureus of correct density, a series of continuous filtrations was carried out in order to obtain count values of the seeds in this suspension with the membrane of the invention and in parallel in order to be able to compare with a standard cellulose acetate membrane calibrated at 0.45 p (Schleicher and Schüll).

The results are as follows

<tb> Type <SEP> of <SEP> membrane <SEP> Invention <SEP> Acetate
<tb> Number <SEP> of samples <SEP> 15 <SEP> 15
<tb> plus <SEP> small <SEP> value <SEP> of <SEP> 28 <SEP> 25
<tb> count <SEP> obtained
<tb> plus <SEP> large <SEP> value <SEP> of <SEP> 58 <SEP> 46
<tb> count <SEP> obtained
<tb> average <SEP> of <SEP> values <SEP> 41.5 <SEP> 35.1
<tb> difference <SEP> type <SEP> of <SEP> values <SEP> 9.7 <SEQ> 6.8
<Tb>
Culture on ordinary nutrient agar. Incubation 24 h at 370C.

DISCUSSION OF RESULTS.

 1. Qualitative aspect.

- macroscopic morphology of colonies.

All the colonies take an identical appearance to that observed during a direct growth on the surface of an agar culture medium, whether in prokaryotes: Salmonella colonies (Example 2), Escherichia coli (Examples 1, 2, 10 and 13), Proteus (Example 5), Bacillus (Example 6), various unidentified colonies (Example 3) or in lower eukaryotes: yeasts (Example 4).

- reading - biochemical tests (first-rate information) related to the metabolic activity of germs
colonies of Salmonella H2S positive (Example 2)
- Escherichia coli colonies yellow because of the change in the colored indicator of pH (examples 1 and 2);
colonies of Escherichia coli "back of beetle" (Example 10).

- respect for growing conditions.

- Streptococcus pyogenes shoot, fragile and capricious seed, demanding (Example 12);
enantant colonies of protein (Example 5);
- grows anaerobic sprouts (Example 9);
pigmentation of Serratia (Example 8); - perfect exchanges between the colonies and the culture medium and vice versa.The membrane behaves with extreme neutrality, respecting not only the physicochemical conditions rise of all the molecules necessary for cell growth; impregnation and dilution of the waste by the mass of the agar; medium of the medium of Chapman (example 7)], the transit of the small molecules (identification by chemical reactions (examples 1 and 2), but also all the passages and the interactions of the biological molecules.) It is at this stage interesting to underline the Theoretical and conceptual consequences of Examples 11 and 12:
Example 11 Escherichia coli Simulation of the antibiogram by diffusion in agar. The membrane passes the antibiotic culture medium without disturbing either its bactericidal action or its concentration gradient due to its diffusion from the calibrated soaked disc.

 Example 12 Hemolytic Streptococcus on Sheep Blood Agar The haemolysins released into the external medium by the germs pass through the membrane in large quantities without modifying their biological property, namely the lysis of hemoglobin from erythro- cytes of sheep blood. At the structural and thus biochemical level, it is fundamental to note that the passage of a protein with enzymatic character through the physical structure, in contact with the constitutive or residual chemical groups (ions, solvents, various impregnated molecules, ligands) of the membrane is not limited and does not alter its conformational state.

- Impregnation of the membrane.

 - During filtration, the impregnation of water is perfect and homogeneous over the entire surface of the membrane according to the invention, which is not the case for cellulose acetate membranes which often have small areas which do not hydrate and thus do not fulfill their role during filtration. With Escherichia coli, which gives non-invasive colonies, this phenomenon is highlighted: the white areas in the center and which go out to the periphery represent the places that remain dry, seemingly very little hydrophilic and do not imbibe the filtered sample (Example 10).

 - When culturing on the nutrient medium, Example 7 (Staphylococcus on Chapman's medium) thanks to the colored pH indicator that it contains (phenol red taking the color red if pH> 6.8) shows that the entire medium impregnates the entire membrane which is in contact with the agar. On the other hand, examples 3 (Rhone water) and 5 (Proteus) confirm this and specify that the impregnation of the membrane by the culture medium extends even where the membrane which is cornea does not rest on the surface agar.

- transparency of the membrane.

 The possibility of observation by transparency reveals the permeability of the photon membrane (Examples 2, 8, 9, 12 and 13). This provides certain advantages, such as under-lighting for observation with the naked eye, magnifying glass, - under the microscope, and compatibility with automatic counting machines previously reserved for mass enumeration (with or without without lighting above).

 2. Quantitative aspect.

The membrane according to the invention has two important advantages:
- easier counting with the naked eye, making the result more accurate for enumeration, especially with colorless colonies;
- in an unfortunate case in the laboratory where the dilution range for an unknown product is not large enough, we can "recover" the result thanks to the possibility of counting more than 300 colonies (300 is the maximum number of colonies countable in a box according to the official texts) (example 13).

 In conclusion of the foregoing, it may be emphasized once again the interest of the membranes according to the invention, applied in microbiology and virology, which are autoclavable and sterilizable by ionizing radiation, also possess a water flow rate. sufficient to be adapted without any modification to conventional devices such as filtering rail or filter module.

Claims (5)

 1. Applications and uses in biology of proteic and eukaryotic filter membrane membranes, usable in micro and ultrafiltration, consisting essentially of a polymeric material flows on a support, said material being chemically inert and accepted without disturbance any by the cells living in contact with it, its structure exhibiting a total impregnation neutrality both for the substances likely to exert inactivating, static inhibitory, or deadly effects and for the various molecules providing exchanges through it, its power selecting only at the level of the size of the filtered particles with respect to the opening of the pores.  2. Filter membranes according to claim 1, characterized in that the polymeric material is slightly hydrophobic, in that its resistance to acids and bases, strong and weak, is good in low or medium concentrations, at normal temperature, and in that that it is compatible with most organic solvents, except chlorinated or aprotic solvents which, at low temperature, become absorbed and cause swelling, and at high temperature solubilize the polymer.  3. Filter membranes according to claim 1 and claim 2, characterized in that they resist up to 1200C (in dry heat as wet) and have little reaction to the radiation exposure (at a dose of 2.5 megarads).  4. Filter membranes according to any one of claims 1 to 3, characterized in that their flow rate to water is around 20,000 liters / day / m 2 under ultrafiltration conditions, this flow rate can be almost tenfold in microfiltration conditions.  5. Membrane filter according to any one of claims 1 to 4, characterized in that they have a cast light transparency and in particular natural light.