CA1158529A - Method for improved microbiological testing - Google Patents

Abstract

ABSTRACT

An improved method for microbiological testing of a liquid sample in a device which comprises a casing having a vertical axis and containing a central chamber and transparent cells containing growth media distributed around the chamber. Each cell is connected to the chamber by a closure cup. A capillary constriction separates each pocket from the corresponding cell. The improvement comprises adding polyvinylpyrollidone to the transparent cells to stimulate a homogeneous bacterial growth.

Description

l~S~S29 The present invention relates to improvements in the operation and function-ing of devices for measuring predetermined volumes of a liquid sample and possibly subjecting said volumes to analytical operations. Such devices are described in United States Letters Patent Nos. 3,986,534 and 4,070,248.

It has been found that in using such devices for determination of bacterial growth, such as in antibiotic susceptibility testing and identification of micro-organisms, bacterial growth of some species tends to concentrate near surfaces and hence accurate automated photocolorimeter reading through the bacterial growth medium is rendered difficult.

It has been further found that the analytical results can more accurately be determined, particularly when using a photocolorimeter operating automatically, by supplementing the bacterial growth medium with polyvinylpyrrolidone.

DETAILED DESCRIPIION OF THE INVENTION

United States Letters Patent Nos. 3,986,534 and 4,070,248 describe devices for carrying out the method of this invention. Such a device comprises an axially vertical casing having an axis which is kept vertical in operation, an axial upwardly 20 open chamber arranged to receive said liquid sample and a plurality of test cells having transparent walls distributed around said chamber, and a plurality of pockets each having an end communicating with said chamber and located to be filled by liquid flowing from said chamber and another end communicating with a respectiveone of said cells via a capillary construction, and closure means arr~nged to separate 25 said chamber from said pockets and to retain said volumes in said pockets when inserted into said casing.

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~3- 1158S'~9 The term "capillary constriction" is used to mean a passage which is of such size as to prevent the sample liquid from flowing therethrough when subjected to a hydrostatic pressure corresponding to a liquid head of a few centimeters. On the other hand, the constriction should have dimensions such that the sample liquid can be spun out into the cel]s when the ]iquid is subjected to an acceleration 5 exceeding about 10 times the gravitational acceleration. The flow path of the ]iquid through the constriction should be such that the centrifugal acceleration, when the device is rotated, has a component which tends to drive the liquid out of the pockets. The flow path will typically be app~oximately radial with respect to the axis of the device.
Advantageously, each pocket is laterally bounded by vertical walls which are substantially parallel and radially directed and are at a distance notexceeding a few millimeters (1 to 5 mm in most cases). The top wall of each pocket can be flat, horizontal or slightly sloping, to prevent the capture of bubbles which 15 would result in inaccuracies of measurement. The lower wall is typically concave towards the top, over most of its extent at least.

The closure means may be cup-shaped and formed to be suitable as a vessel to supply the sample to be divided into fractions. The side wall of the 20 closure means may be shaped to cooperate with the lateral wall of the cylindrical central chamber, either by forcefitting or by a threaded connection. The contents of the pockets can be isolated by forcing the edge of the cup wall against the lower wan of the chamber. The terminal edge of the side wall can be convex or knife-edged and can engage in a circular groove at the bottom of the chamber for 25 sealingness. Alternatively, the side wall of the chamber can be provided withaccess apertures to the pockets, in which case the side wall of the cup may also be provided with apertures adapted to be placed opposite the access apertures, depending on the angular pasition given to the cup.

1~5~529 The device has numerous applications, more particularly in medicine and biochemistry and, more generally, when the volumes have to be analysed using different reagents. The reagents, in dried or lyophilised form if necessary, can be placed before hand in the cells. In the case, for example, where antibiograms bydilution in a liquid medium are to be obtained, the reagent may be a culture medium s containing the antibiotic whose effect is to be measured, and a colour indicator, e.g.
a pH indicator. A similar approach may be used for identifying strains of micro-organisms.
For example, similar test reagents to those described in U.S. Patent 3,936,356 can be employed.
Since the cells have transparent walls, the analytical results can be deter-mined visually or, more accurately, using a photocolorimeter which can operate automatically. Photocolorimeters of known type can be used whenever a positive reaction is shown by a colour change in the body of liquid in the cell. If the cells 15 have parallel surfaces, it is simply necessary to convey each cell in turn between a suitable source of light (for instance yellow light at 380 nm wave length in the case of antibiograms) and a suitable detector, which is disposed behind an optical filter if necessary. If a positive reaction is shown only be turbidity, the latter can be detected by absorption of light at a longer wavelength, e.g. approx. 650 nm.
Also see the system described and claimed in Canadian application 352,261, filed May 20,1980 filed in the names of Thomas L. Kraft, Howard A. Vick and Miles Gerald Hossom, and issued as Canadian Patent 1,128,338 on July 27,1982.
The improvement of this invention comprises supplementing the bacterial growth medium with polyvinylpyrrolidone during the incubation period such that the 25 bacterial growth is rendered more homogeneous throughout the growth medium.
Such more homogeneous bacterial growth in the cell is conducive to more accuratephotocolorimeter determinations. The polyvinylpyrrolidone can be added to the cell 15, as shown in the drawings of U.S. Patents 3,986,534 and 4,070,248, in variousway8. ~or example, it can be added to the cell with the growth medium, with the 30 growth indicator, with the antibiotic or it can be added separately to the cell prior to joining together bottom plate 18 and top plate 19 or with the liquid sample to be tested and introduced through - .;
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~8529 compartment 14 and pockets 13. The amount of polyvinylpyrrolidone added to the cell is an amount sufficient to provide a solution in the cell, when the liquid to be tested is present in the cell, containing about 0.3 to about 3.0%, preferably about 1%, weight by volume of polyvinylpyrrolidone having an average molecular weight - greater than about 40,000 and less than about 400,000 preferably about 360,000.

Polyvinylpyrrolidone (PVP) as a well known commercial product produced commercially as a series of 6 products having mean molec~ar weights ranging from about 10,000 to 700,000. Generally available commercial grades have average molecular weights in the range of 10,000 to 360,000, for example, General Aniline and Film Corporation (GAF) markets at least four viscosity grades available as K-15, K-30, K-60, and K-90 which have average molecular weights of about 10,000, 40,000, 160,000 and 360,000, respectively.

K-values are derived from viscosity measurements and calculated accord-ing to Fikentscher's formula (Kline, G.M. Modern Plastics 137 No.1945):

log rel = 75Ko2 + Ko c 1+1.5Koc K= lOOOKo where c= concentration in g/lOOml solution rel= viscosity of solution compared with solvent The molecular weight of PYP samples has been determined by osmometry, in the ultracentrifuge, by lightscattering measurements, thermodiffusion methods, sedimentation constants, turbidity titrations, and viscosity techniques.

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1'~L58S29 The particular fraction of PVP useful in the present invention is that fraction having an average molecular weight greater than about 40,000 and less than about400,000, preferably an average molecular weight of about 360,000. Particularly useful is viscosity grade K-90 marketed by GAP having an average molecular weight of about 360,000. The manufacture of this viscosity grade is disclosed in U.S. Patent 5 Nos. 2,265,450 and 2,335,454. A similar commercial product is available from BASF-Wyandotte.

The invention will be better understood from the following description of embodiments thereof, which are given by way of non-limitative examples.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure l is a simplified elevation view of the device, partly in cross-section along a vertical plane, with the closure means removed.
Figure 2 shows the device of Figure 1 after fractions of a sample have been transferred into analytical cells, the device being disposed on a centrifuge used for spinning the fractions into the cells of the pockets.

Figure 3 is similar to Figure l and shows a modified embodiment;

Figure 4 is a view on an enlarged scale showing a cell and the accompanying components of a device according to a modification of the device in Figure l, before assembly;

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~58s2g DESCRIPTION OF PREFERRED EMBODIMENTS

Referring to the drawings, Figures 1 and 2 show diagrammatic representation of a device for obtaining fractions of a liquid sample; the device comprises a casing 10, consisting of several assembled components and removable closure means 11. The casing has a substantial rotational symmetry around an axis which is located vertically during use. The casing contains a central chamber which opens upwardly and has a capacity varying from a few milliliters to a few tens of milliliters. The chamber is connected via lateral apertures 12 to a number of pockets 13 formed in the casing, regularly distributed around the central chamber and extending substantially radially. Each pocket is associated with an analysis cell 15 in the form of a test-tube having a transparent side wall.
A constriction 16 is provided between each pocket 13 and the corresponding cell 15, the transverse dimensions of each constriction being such that it is capillary ior the liquid to be divided into fractions (of course the liquids can have greatly variable surface tensions). The connecting passage 17 provided between each constriction 16 and the corresponding cell 15 is flared so as to prevent the liquid in pockets 13 form seeping along the wall to cells 15.

The casing 10 in Figure 1 comprises a bottom plate 18 and a top plate 19 - force-fit into one another. The bottom plate 18 forms the bottom of chamber 14, and the bottoms and side wal]s of pockets I3 and cells 15 (24 cells being provided in the embodiment illustrated).
Since the cells walls must be transparent, all of plate 18 is advantageously made of a plastic material which is transparent over a wide range of optical freguencies, which is rigid and which can be shaped by moulding. It can be made of crystal polystyrene, which withstands most conventional chemical reagents.
The moldable plastic material sold under the TM DIACON and comprising methylmethacrylate and polystyrene may also be used.

The top plate 19 forms the side wall of chamber 14, the top walls of pockets 19 and the covers of cells 15. It can be made of the same material as the bottom plate or of a material which is more flexible than that of the bottom plate, so as to facilitate force fitting. It may be made inter alia of . ~
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5~3529 polypropylene or polyethylene. The plstes may be shaped so that the casings can be stored by stacking, as indicated by the chain-dotted lines in Figure 1.

The casing shown in Figure 1 contains a layer of reagent 20 at the bottom of each cell 15. In the case of a device for obtaining antibiograms, the reagentmay be e.g. a culture medium containing a specific antibiotic and a colour indicator, e.g. a pH indicator.

Advantageously, a label 21 is disposed opposite each cell so as to identify each antibiotic. All the labels can be carried by a single flexible ring 22 secured between plates 18 and 19. In addition, an identification notch 23 can be formed in the lower skirt of plate 18 so that it can be mounted in only one angular position on a data-reading device.

A narrow slot 26 connects each cell to atmosphere, so that air can escape from it and the liquid can flow into it.

Advantageously, each pocket is nat in the vertical direction; to this end, the bottom plate has slits having vertical, parallel and substantially radial walls and spaced apart by 1 to 5 mm. The bottom 24 of each slot curves smoothly and is advantageously concave along its first part from the central chamber. The topwall 25 of the pockets may be nat and horizontal or slightly conical downwards or upwards, so as not to trap bubbles. In the resulting casing, all the pockets can have the same volume. Preferably, the two plates are not secured by gluing at the constrictions, since drops of glue may block the constrictions or reduce their cross-section.

The device also comprises a closure member 11 (Figure 2) which can be made of moulded material, e.g. the same material as plate 19. In the embodiment shown in Figure 2, the side wall of the closure means 11 has beads 27which are forced into the side wall of the cell, so that when the closure memberhas been completely pressed down it is retained in position. The terminal edge of the side wall of means 11 is rounded and bears against the bottom of a groove 28formed at the bottom of the chamber so as to separate the interior thereof from pockets 13.

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s~sza Closure member 11, which is cup-shaped, can be given a sufficient capacity for use as a storage vessel for containing the liquid sample and transferring it to the casing.

A way of operating the device according to the invention will now be described, in the case where antibio-grams are prepared.

The clo6ure means 11 shown in Figure 2 has a side wall with a rounded terminal edge. In the modified embodiment illustrated in Figure 3, on the other hand, cl~ure means lla is a cup having a knifeblade terminal edge which engages in a correspondingly shaped groove 28a formed in the bottom plate 18a. For 10 increasing the surface traversed by the light for colorimetry, the cells 15a are typically located adjacent each other, without any gap between successive cells,and of substantially rectangular horizontal cross section.

The device can be used inter alia to prepare a wide variety of antibiograms from a small-volume sample, each pocket usually having a capacity 15 less than 100 microliters and each circular row being easily capable of containing 36 cells with an overall diameter of 8cm. The cell can contain antibiotics at different concentrations and different combinations of antibiotics. A plurality of devices can be provided and used in succession, a first device being used to determine the antibiotics towards which the strain is active or resistant, and a20 second device (whose cells contain different concentrations of the same antibiotics) being used to determine the minimum inhibiting concentration (MIC) of the active antibiotics.

Referring to Figure 4 (where, for simplicity, like elements bear like references to Figure 1) show a modified embodiment which differs mainly in that 25 each cell lSb (or at least some of the cells) is provided with a compartment 31 containing a reagent 32. Referring to Figure 4, there is shown a bottom plate 18b and a top plate l9b before assembly. Plate l9b has ribs 33 which are force-fitted in correspondingly-shaped grooves leaving a constricted passage 16b, the width of which usually varies from l/lOth to a few tenths of a millimeter.

- ` ~3 S8S29 Compartments 31 are formed in capsllles 34 made of plastic which is deformable but highly resistant to tearing. Capsules 34 are secured, e.g. by gluing, to a thin plastics or metal strip 35 which tears when pressure is exerted on the top wall of a capsule. Strip 35 and the capsules are held in position by stubs 36 which are distributed around each cell 15b and engage in corresponding apertures formed in strip 35 and the capsule strip. The stubs may also engage inthe apertures of a strip 21b bearing labels.

The device shown in Figure 4 is of particular interest for chemical measurements, more particularly for detecting abnormal proportions of constituents in organic liquids such as blood or urine, for detecting enzymes orthe like. In such cases it is frequently necessary to use two reagents which cannot be stored together. One is then placed in cell 15b and the other in compartment 31. It may also be necessary to add an additionPl reagent for detection: it is again stored in compartment 31. The additional reagent may e.g.be necessary to inhibit or indicate the reaction; it may be an accelerator to beintroduced at the last moment; it may be a light density solvent for collecting coloured products just under the liquid free level, etc.
Many other modified embodiments of expendable devices according to the invention are possible. When used for medical purpose, the device is used once only and then destroyed. In an embodiment for chemical use, all cells 15 may contain a same reagent and the closure means having a single clo6able lateral aperture and a lower end wall. The closure means has been inserted into the casing so that its aperture registers with an aperture in the casing. Operation is then the same as before, the pocket being used as a pipette for storing a predetermined volume and transfering it to the corresponding cell. Next, the cl~;ure means is removed and replaced by a second closure means which is positioned opposite another aperture. In this manner, the same reaction can be performed on samples coming from different patients, e.g. for quantitative analysis of urea. Each closure means may have not one but two or three apertures, which are located in coincidence with pockets corresponding to cells containing two or three different reagents, e.g. for determining urea and cholesterol.

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~L58S:~9 Finally, the casing may comprise cells disposed in a number of concentric rows, in which case of course the pockets connecting the cells in the outer row are connected thereto by ducts comprising non-radial portions.

A liquid sample, is prepared, comprising a dilute solution of bacteria, the sensitivity of which is to be determined against various antibiotics in the device.

The volume of sample need not be precisely determined, provided that it is sufficient to fill all the pockets. The sample is poured into the central chamber 14, from where it flows into the pockets, which it fills up to the constrictions 16. Next, the closure member 11 is positioned so as to separate the contents of pockets 13 (which form a corresponding number of pipettes) from the liquid remaining in chamber 14. When the closure means is in position, the dilute solution of bacteries cannot contaminate the environment. Furthermore, if the closure means is used as a conveying cup, there is no additional contaminated vessel to be discarded and destroyed.

Next, the device is placed on the rotating part of a centrifuge, which can be manual or driven by a motor at a given speed of rotation. Figure 2 diagrammatically shows the device on top of the rotating part 19 of a centrifuge, the outline of which is shown by broken lines. The centrifuge frame has an arm bearing on closure member 11, the arm being sufficiently heavy to prevent the device from moving during centrifuging. A conventional centrifuge can be used.
The electric ~otor is energized by a timing device so that the centrifuging conditions are reproducible. If the device has a diameter of approx. lOcm, a speed of a few tens of r.p.m. is sufficient.
After the contents of each pocket 13 has been transf erred into the corresponding cell 15, the device is placed in an incubator. It is shaped so that it can easily be placed horizontally. After a certain period, usually about one day, the data are read, either visually or, advantageously on an automatic photocolorimeter which may be conventional and comprises a light source (e.g. a light emitting diode) which directs a radial light beam f (Figure 2) onto a suitable detector.

~.~L5~3529 The photocolorimeter can operate stepwise, bringing each cell in turn between the source and the detector and holding it there for the necessary time.Alternatively, the number of detectors can be equal to the number of cells, although the latter method is expensive. Still other methods may be used.

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' 1~585Z9 EXAM PLE

In order to demonstrate the effectiveness of the polyvinylpyrrolidone in the method of this invention, the following test work was performed.

A supply of filled rotors, i.e. with test reagent, were received from the manufacturer. These rotors were of the type described in Figures 3 and 4 of U.S.Patent 4,070,248 and particularly at column 4, line 67 through column 6, line 31. The rotors were made from crystal polysterene and were sized to contain one-tenth milliliter of liquid in each of pockets 13 for discharge into cells 15. The rotors were prepared with Eugon (a Difco product) broth for testing antibiotic susceptibility of various streptococcus species to various antibiotics including ampicillin, penicillin, 10 nitrofurantoin, clindamycin, erythromycin, vancomycin, chloramphenicol and tetracycline.

Twenty three strains of Streptococci representing four species were tested by the standard Kirby/Baur and micro-broth dilution MIC tests, which results are 15 recorded in the first two columns respectively of Table I below. Duplicate tests using rotors were then carried out and the results appear in the second two columns of Table I, the only difference being that polyvinylpyrrolidone was used in the tests reported in the fourth column.

Thus in the third column tests, suspensions of the microorganisms were prepared in distilled water to yield approximately 1.5 x 108 colony-forming units/milliliter and used as inoculum whereas in the fourth column tests, suspensions o~ the microorganisms were prepared in a 1% weight by volume solution of poly-vinylpyrollidone in distilled water and used as inoculum. The polyvinylpyrrolidone 25 solution was previously prepared by stirring 60 grams of polyvinylpyrrolidone having an average molecular weight of 360,000 (Aldrich Chemical Company Catalog No. 85-647-9) in 6 liters of distilled water, i.e. 1% weight by volume polyvinylpyrrolidone and then was sterilized by filtration.

i8529 --l 4--The inoculum in each instance was introduced into central chamber !4, the clcsure means 11 inserted into proper position, and each rotor was placed on a centrifuge which was operated at a peak 3000 RPM for 15 seconds.

The rotors were then placed into an incubator for 18-24 hours and were read on an automatic photocolorimeter. Results are recorded as R= resistant to the drug, S=sensitive to the drug and I=intermediate response to the drug. The results shown in Table I indicate that the addition of polyvinylpyrrolidone to broth based susceptability tests results in a superior growth medium and the ease and reliability of the test is greatly increased.

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T A BLE

Kirby/ Without With Organism Ba~er MIC PVP PVP
-Cli damycin (cc) 1014 S. bovis S S S S
1030 S. faecalis R R S R
1026 S. faecalis R R S R
1148 S. ~urans R R S R
1032 S. faecalis ,R R I R
1182 S. faecalis ~ R R S
-1144 S. faecum R R S R
1094 Enterococcus R R S R
1073 Enterococcus R R R R
14 Strep. strains S S S S

_rythrom~ (E) 1073 Enterococcus R R S R
1040 Enterococcus S I S
21 Strep. strains S ' S S S

Vancomycin (Va) 23 Strep. strains S S S S

Chlorampheni,col (C) 23 Strep. strains S S S S

Tetracycline (Te) 1014 S. bovis R R I R
1160 S. durans S S S S
__ . ,,_ .. _ _ _ _ 1038 S. faecalis S S S S
. _ _ . _ ___ ._ , 1182 S. _aecal_s S S S S
19 Strep. strains R R S R

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Kirby/ Without With Organ sm Bauer MIC ~VP _ P _ Ampicillin (~m) 23 Strep s~rains 5 S S

Peni_illin (P) 1026 S, faecalis I S S
1028 S. faecalis I S S
1032 S. faecalis I S S
Remainder of the 20 strains behaved similarly ~ (SXT) 1148 S. durans R R S 1~ -1030 _. faecalis R R S R
1160 S. ~urans S S S S
-1149 S. bovis S S S S
19 Strep. strains R R S R

Nitro~urantoin (F/M) . . .
23 Strep. strains S S S S

Improved results have also been obtained with gram-negative and Staphylococcus microorganisms.

~s~sz9 Although I do not wish to be bound by any theory as to the mechanism of action of the polyvinylpyrrolidone in the method of this invention, it appears that bacterial growth is enhanced or that suspension of the bacterial growth is enhanced, i.e. that adherence of the bacteria to the plastic or to each other was diminished. This phenomenon was peculiar to polyvinylpyrrolidone in the method of this invention since similar res~ts were not obtained using TWEEN 20, TWEEN
8D, potassium nitrate, collagen (a colloidal protein) or polyvinylchloride.

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Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. In a method for testing susceptibility of bacteria in a liquid sample to a plurality of different antibioties, comprising the steps of:
providing a rotatable casing defining an upwardly open central chamber concentric with the rotational axis of said casing, a plurality of radial pockets communicating at the inner ends thereof with said central chamber for gravity flow of liquid from said chamber into each of said pockets, the outer ends of each of said pockets communicating by means of a flow restriction with each of the plurality of vented test cells formed in the rotatable casing, some at least of said cells containing a bacteria test medium and an antibiotic in lyophylised or other dried form, filling said central chamber with said liquid until the pockets are filled up tosaid constrictions by gravity flow from said chamber, inserting a closure means to isolate said predetermined volumes of liquid in said pockets, rotating said casing about said axis to create centrifugal forces sufficient to overcome said capillary forces and to cause transfer of said volumes from said pockets into said test tubes, incubating said bacteria for a predetermined time period, and optically determining the growth of bacteria in each said cell, the improvement which comprises rendering the bacterial growth more homogenous throughout the growth medium by incubating said bacteria in said liquid sample in said cells in the presence of from about 0.3 to about 3.0%
weight by volume of polyvinylpyrrolidone having an average molecular weight greater than about 40,000 and less than about 400,000.

2. The method of claim 1 wherein the average molecular weight of the polyvinylpyrrolidone is about 360,000 and its concentration in the liquid sample is about 1%.

3. In a method for the identification of bacteria in a liquid sample by subjecting the liquid sample to a plurality of different test media comprising the steps of: providing a rotable casing defining an upwardly open central chamber concentric with the rotational axis of said casing, a plurality of radial pockets communicating at the inner ends thereof with said central chamber for gravity flow of liquid from said chamber into each of said pockets, the outer ends of said pockets communicating by means of a flow restriction with each of the plurality of vented test cells formed in the rotatable casing, some at least of said cells containing a bacteria test medium and indicator in lyophylised or other dried form, filling said central chamber with said liquid sample until the pockets are filled up to said constrictions by gravity flow from said chamber, inserting a closure means to isolate said predetermined volumes of liquid in said pockets, rotating said casing about said axis to create centrifugal forces sufficient to overcome said capillary forces and to cause transfer of said volumes from said pockets into said test tubes, incubating said bacteria for the predetermined time period, and optically determining the chemical change produced by the growth of bacteria in each said cell, the improvement which comprises rendering the bacterial growth more homogenous throughout the growth medium by incubating said bacteria in said liquid sample in said cells in the presence of from about 0.3 to about 3.0%
weight by volume of polyvinylpyrrolidone having an average molecular weight greater than about 40,000 and less than about 400,000.

4. The method of claim 3 wherein the average molecular weight of the polyvinylpyrrolidone is about 360,000 and its concentration in the liquid sample is about 1%.