CA1077814A - Storage stable antibiotic susceptibility test kit - Google Patents

Abstract

TITLE: STORAGE STABLE ANTIBIOTIC SUSCEPTIBILITY TEST KIT

INVENTORS: ERIC JOHN MESSNER
27 Surrey Court Pearl River, New York 10965 ALBERT CARL DORNBUSH
145 Forest Avenue Pearl River, New York 10965 ABSTRACT OF THE DISCLOSURE
The susceptibility of microorganisms to various antibiotics is determined by propagating microorganisms in the presence of different concentrations of various antibio-tics in a plurality of test chambers, using a serial dilution technique. Microgram quantities of each antibiotic are retain-ed in a graduated series of concentrations in each of a ser-ies of test chambers by poly(vinyl pyrrolidone) as a water--soluble bulking agent and carrier, in frozen-dried form.

Description

1~7~8~4 A related U.S. patent of another inventor, but common assignment, No. 3,992,265 - ANTIBIOTIC SUSCEPTIBILITY TEST, Lloyd Frank Hansen, which issued on November 16, 1976 shows test wells or chambers for holding anti-biotics for use with serial dilution techniques, and have claims to specific test cell devices.
This invention relates to antibiot;c susceptibility testing and more particularly test kits having a series of various concentrations of various antibiotics or therapeutic control agents which are used to ascertain the interaction between such control agents and various microorganisms, lO usually pathogenic.
IVitll the proliferation of antibiotics and other drugs both in the hospital and in the laboratory as well as educational institutions there is an increasing demand for information concerning the susceptibility or sensitivity of a particular microorganism to various antibiotics or drugs, as well as information on the assay of particular constituents in blood, or other biological liquids.
The use of automated analytical procedures has become of increasing importance. For both chemical and biological procedures the number of samples to be run has been increasing exponentially as new procedures are 20 developed, and existing procedures are adapted to large quantlty requirements.
U.S. Patent 3,272,719 - METHOD AND APPARATUS FOR DETERMINING THE
SENSITIVITY OF BODY FLUID INFECTANTS TO DRUGS, Avakian, September 18, 1966 shows rectangular or square compartments, with a common wall between adjacent rows and files.

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A notch is provided for a string saturated with infected I`lu-id to sag into compartments containing a sterlle nutrient and known concentrations of drugs under test.
U. S. Patent 3,301,065 - LI~UID SAMPLE SUPPLY AP-'j PARATUS, Fahrenbach, Bell and Sandage, January 31, 1967, shows an automatic sampling system in which a series o~ cups contain-ing samples are fed serially into an analytical system. The s~mples mfly be in CUp6 on a spiral in a plate, or may be fed as a series of individual pallets locked together and fed ~-long a belt. Locking the pallets together insures coordination in feedlng separate pallets.
BelgLum Patent 691,532 9 February 28, 1967, shows lyoph~lized (freeze-dried) antibiotics or chemotherapeutic a-gents ~n various concentrations, including a blank, in separate cells arranged in columns and rows in a tray, for testing the resistance of microorganisms to antibiotics or ~gents. Retain-~ng sppendices pro~ect from the base of the culture cells to retain the lyophilized material in the individual cells. Ident-1fying covers cooperate with each cell to close, and identify the contents of, each cell. A culture medium and/or indicator m~y be present in the lyophilized state in the test cells The cells and the covers are essentially transparent to permlt observatlon of the cultures U. S. Patent ~,453,180 - TEST ARTICLE, Fraser and AtkLnson, July 1, 1969 shows ~ bibulous test strip for deter~
mLnlng glucose levels in urine having as an impregnant the drLed residue of a liquid mixture which comprises liquid glu-cose ox~dase, peroxidase, citric acid-sodium citrate buffer system o-tolidine hydrochloride, poly(vinyl pyrrolidone), an ~0 ac~d and/or partial ester derivative of an interpolymer of methyl vinyl ether and maleic anhydride9 sodium lauroyl sarco-slnate, and a long~chain polymer of ~,6-anhydro-D-galactose and sulfated D-g~lactose residues.

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U. S. Patent 3,533,744 - METHOD AND APPARATUS FOR
PERFORMING ANALYTICAL OPEMTIONS, Uneer, October 13, 1970, In Fi~ure 2 shows a s~mple carrier for automatically analyz-Lng blood, which carrier is in the form of an inte~ral rectan-gular slide, h~vLng thirty elongated ~uxtaposed shallow recept-acles for receiving fractions of one single sample. The plas-tLc sample carriers are disposable and of transparent plastic m~terisl U. S. Patent 3,546,131 - STABILIZED CYANMETHEMO-GLOBIN REAGENT CONTAINING FERRICYANIDE, CYANIDE AND POLYVINYL-PYRROLIDONE, Stern and Reardon, December 8, 1970, shows a dry ~ormulated reagent containing ferricyanide and cyanide for use in the photometric determination o~ hemoglobin in blood, com-prising fl water soluble macromolecular desiccant such as poly-vinylpyrrolidone, the polyacrylamides, gelatinJ dextrin, the alkall metal salts of cellulose, and hydrolyzed polyvinyl alco-hol derivatives, to enhance resistance to light, heat and cold, and aglng.
U. S. Patent 3,578,412 - AUTOMATED TRANSPORT SYS-TEM, Martin, May 11, 1971, shows an automated chemical analyzer havlng Lndividu~l sample capsules, with several chambers in each sample capsule.
U. S. Patent 3,649,464 - ASSAY AND CULTURE TRAY, Freeman, March 1~, 1972, shows a transparent tray having rows ~nd column~ of upstanding cups or wells, which ~re spaced apart to avold cross-contamination. A peripheral wall around the tray permits stacklng of a set of trays. A strip having a ser-ies of well seals is shown th seal off an individual row of wells.
U. S. Patent 3,713,985 - DEVICE AND METHOD FOR TEST~
ING POTENCY OF BIOLOGICAL CONTROL REAGENTS, Astle, Janu~ry 30, 1973, ~hows s serie3 of biologic~l reagents in a series of cup~, In a strip, or pallet, with the strip having dovetails : ~ 3 ~

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to longitudinally l~ck a group of the strips together to form a tray. A
foil cover to protect lyophilized contents during storage is disclosed, with reconstitution of the contents at time of use. The culture medium and the test organism are added sequentially and separately at time of use, so that the control reagent is first redispersed. It is then inoculated, and incubated.
U.S. Patent 3,890,201 - MULTI-CHAMBER IMPEDANCE MEASURING MODULE-CAP COMBINATION, Cady, June 17, 1975, shows rows and columns of upstanding cylinders on a flat base, forming cells, with electrically conductive strips in each cell to permit imp~dance measurement of the cell contents. The impedance in the cell is a function of microorganism growth. Separate caps are provided for each cell to permit gas flow into the individual cells during incubation.
S~ ~ RY OF THE INVENTION
It has now been found that the reliability and ease of use of an antibiotic susceptibility test kit is markedly improved by using poly~vinyl pyrrolidone) as a water-soluble bulking agent and antibiotic retaining carrier for an antibiotic in test chambers.
According to the present invention, there is pTovided a test kit for measuring the antibiotic susceptibility of a microorganism comprising a plurality of integral test chambers, with at least some of said chambers having therein a graduated series of concentrations of an antibiotic and a uniform quantity of poly(vinyl pyrrolidone) as a wa~er-soluble bul~ing agent and antibiotic retaining carrier, with ~he antibiotic in dispersed and reconstitutable frozen-dried form in the poly(vinyl pyrrolidone).
. Preferably the antibiotic is present in a two-fold series of ; dilutions including at least part of the range of 1 to 10 micrograms per - milliliter.
In another aspect, the invention provides a method of testing the antibiotic susceptibility of a microorganism by a serial dilution technique `- comprising: forming a series of concentrations of solutions in water of an -~

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antibiotic in a uniform concentration of poly(vinyl pyrrolidone) as a wa~er-soluble bulking agent and antibiotic retaining carrier> loading uniform quantities of said series of concentrations in a series of integral test chambers, freezing and freeze-drying said series of concentrations, storing until time of use, and reconstituting the antibiotic in the poly(vinyl pyrrolidone) by adding a culture medium inoculated with a test microorganism, incubating, and then reading the test microorganism growth.
The quantity of antibiotic present for inhibition of the growth of pathogenic mîcroorganisms is of~en in the range of about 0.1 to about 250 micrograms per milliliter. With 0.2 ml of solution being a convenient size test sample, the quantity of antibiotic in a series of chambers is between about 0.02 and 50 micrograms. These quantities are so small that the unaided eye would not be able to confirm thPir presence in the test chambers~
By using poly~vinyl pyrrolidone) as a water-soluble bulking agent and antibiotic retaining carrier, particularly when the polyt~inyl pyrrolidone) and antibiotic are loaded into test chambers in aqueous solution, and then ; frozen and .

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dried; the antlbiotic is bulked into the poly(vinyl pyrrolidone) foam, and both adhesively held in the test chamber, and con-clusively shown to be present in the test chamber by vi8~al ob-servation at time of use. The visibility of the foam gives phys1cal confirmation of the presence of the antibiotic, and a psychological enhancement to the user.
The dried poly(vinyl pyrrolidone) aids in solution of the flntibiotic, as it keeps the antibiotic finely subdivided and ~s soluble itself. Poly(vinyl pyrrolidone) is biological-ly inert. It has been used as a blood extender, and in many biologic~l environments. It is listed in the U. S. Pharmaco~
poeia as "Povidone", and biological uses are well known. It Ls so inert that a ~uantity from less than 250 to at least lO,OOO micrograms per milliliter is satisfactory for forming a sponge to hold the antibiotic. A preferred concentration Or

2,000 micrograms per milliliter is convenient. This gives 400 micrograms per 0.2 milliliter test container--which is conven-ient both from the point of constitution and reconstitution, and also Ls ready visible as a foam to confirm the presence of both tne poly(vinyl pyrrolidone~ and the antibiotic.
Preferably the same concentration of poly(vinyl pyr-rolidone) is used in each test chamber to reduce the number of var~able8, and permlt visual observation to confirm that eflch .; .
well is uniformly filled.
Often a well is used whlch h~s no flntibiotic to con-firm growth characteristics in the absence of any inhibition, or to confirm sterility of the test kit. This test chamber may optionally contain poly(vinyl pyrrolidone! as a blank.
Obviously the test kit should be sterile so that on-ly the test organism is cultured. Sterile techniques are used ln filling ~nd drying. Some antibiotic or growth controlling substances in the nature of, and acting as a mlcroorganism in-h1bit1ng agent may be ~terilized in sltu by ethylene oxide, -7~8~
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r~diatLon, heat or other ~gent compatible with the growth controlling substance. Certaln o~ the sulfa drugs are very st~ble under sterilizing processes.
While not limited thereto, one test plate useful w~th the present invention is a molded transparent plate of a blologically inert plastic such as a polymer of methyl metha-crylate, or a vinyl resin, but which may be of any biologically Lnert transparent or nearly transparent plastic, which plate h~s a row of approximately rectangular wells. The wells pre-ferably have a slight taper, which permits molding in simpli-fled molds. ~y having about 1/2 to 43 draft or molding taper on all vertlcal sur~aces, a two piece mold can be used to eco-nomically produce the test plates. A slight taper permits the use of a mechanical light source, and optical reader or scan-ner, w~th ne~ligible inaccuracies from the taper. Parallel s~des which are more optically true can be used, and while opti-cally more desirable, increase the cost of production of the test pl~tes. Vlsual inspection or "eyeball" reading is often used to detect inhibition of growth in the several wells.
; 20 For Incubation a well cover protects from chance contamination. A flexible plastic such as polyethylene is low ln cost, and readily molded. The well cover is shaped to fit an entire plate row of wells, of`ten 8 or 10, and has a rim to f~t into each well, thus closing the well and positioning the well cover. A lifting flap on the well cover permits the well cover to be readily lifted from the test plate. The well cov-er ls revers~ble, so that in one position the lifting flap is flat aga1nst the test plate, and when rotated 180, extends outwsrdly as an indexlng flap. The flap may be treated to improve the adhesion of an ink or label.
By having a number of test plates with wells in each, a separate antlbiotic can be used in each test plate. Diff`er-ent pat~ents in a hospital may have different spectra of 778~L4 antibiotlcs to be tested.
By being stack~ble, a stack of 5 or 10 can be hand-led as ~ unit ~n incubation ~nd storage. A plurality of test plates, 5 or lO, are conveniently stacked with a dehydrating agent in a bag until time of use, A foil bag may be used to give m~ximum protectlon from moisture.
DRAWINGS
FIGURE 1 is a sectional view of a culture test plate w~th a pl~stic snap cover closing the individual wells, show-Lng the antibiotic in a poly(vinyl pyrrolidone) foam, FIGURE 2 ~s a view of a plastic snap cover.
FIGURE 3 ls a sectional view of a well filled with a liquid solution of antibiotic and poly(vinyl pyrrolidone).
FIGURE 4 i~ a sectional view of the well after the liquld has been frozen and dried to form a foflm.
FIGURE 5 shows a sectional view of the well with the snap cover in place.
FIGURE 6 shows the contents of a well being recon-stituted by the addition of a liquid.
~o FIGURE 7 shows a view in section of a well with the snap cover in place, showing the clear liquid either before the growth of any microorganisms or with the growth having been Lnhibited.
FIGURE 8 shows a single well with the snap cover in place with a cloudy or turbid liquid resulting from the propa-gation of microorganisms therein.
FIGIJRE 9 is a pictoria 1 view showing a group of test plates ln eflch of two plastic bflgs which are sealed inside of a moisture proof foil envelope.
~o FIGURE 10 is a pictorial view of a single culture test pl~te.
FIGuRE 11 ls a slide elev~tlon of a single culture test plate.

, 1a~778 ~L91 FIGURE 12 is an end view of a c~lture test plat~.
FIGURE 1~ is an end view of a stack of 5 culture test pl~tes stacked for shipment or h~ndling.
~s shown in FIGURE 1 and 10 the biological culture r) test plate 21 consists of a flat platform 22 having therein a serles of rectangular wells 2~. Each rectangular well has a flst bottom 24 and approximately rectangular walls 25, By uslng approximately parallel walls, light can be p~ssed through two approximately parallel walls wlth minimum distortion or hendin~; which permits either inspection by eye or a mech~ni-c~1 opt~cal device to me~sure the turbidity of materials with-in the well, It ~s desirable that the well have a slight taper within the range of about l/23 to 4 as such taper per-mits the molding of wells, and the withdrawal of the molding mandrel, Tf there is no taper, it is more difficult to with-draw the mandrel; ~nd if the taper is more than about 4 the well starts to become somewhat prismatic in its action on 1ight.
As shown in FIGURE 1 for the specific embodiment shown, there is a series of 10 wells, Obviously, the number of wells may vary but 10 is a convenient number for most test purposes.
At the front of the flat platform 22 is a dependent or downw0rdly extending-~ont skirt 26. The front skirt gi.ves addittonal rigldity, and also has therein a group of indexin~
notches 27. Each indexing notch is in a coordinated spacial relatlonship wlth a well. Conveniently the notches are cen~
tered wlth respect to each well and serve the purpose of in-dex~ng the test plate with respect to a reading device when ~o a mechan~cal reed system is used in connection with an electro-optlcal density readin~ system, Conveniently, but not necessarily, at the back of the fl~t platform is a rear skirt 28, Also ad~scent the 77~

rear of the platform is a stiffening rib 29, This rib is sl~ghtly tapered for convenience in molding and extends down-wardly from the flat platform a sufficient distance that the test plate rests horizontally on a horizontal flat surf~ce.
Preferably, the stiffening rib and the wells have a common bottom plane. This provides for the culture test plates to re~t flat on a work surface during filling and culturing, and also that permits the test plates to be stacked without tip-ping.
In- the ends of the test plate are stacking handles 30. These handles are interiorly hollow and tapered where-by the handles nest when test plates are stacked. The front and rear of the handles conveniently are extensions of the ; front sktrt and rear skirt 26 and 28 and have a rise member L5 ~1 and a flat top 32 on each end. The skirt extensions are at such an angle that when stacked, the assembly nests with-out blnding but without undue free motion.
`` The top of the flat platform above the stiffening rib may have spacing buttons 33. These spacing buttons 33 are such size that when well snap covers 34, referred to be-low, flre placed in the wells the stiffening rib contacts the spscing buttons and gives uniform vertical stacking.
For shipment, incubation, and storage, the we ls are closed and the contents protected by a well snflp cover 34 The well snap cover is of a thin sheet 35 of flexible plastic.
It is sllghtly larger than the wells to be covered and has depending therefrom a series of rectangular well seals 36.
As shown in FIGURE 2 and 5 each well seal has a `::
rectangular configuration and such size as to fit into a ~0 rectangular well 23. The spacing between them corresponds to the spaclng in the series of rectangular wells 23. The well seals are conveniently hollow and extend part way into - the well ln assembled position. The rims 37 conveniently _ 9 .

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are an easy press fit into the rectangular well 13 so that the snap cover 34 may be easily removed and replaced, and when placed in position will not fall out under shipping and handl-ing stresses, Also on the snap cover is a lifting flap 38.
The lift~ng flap has in part a textured area 39. The tex-tured area is formed in molding by texturizing the mold so that the texturized area is roughened and accepts ink or a label more readily than the smooth surface of the snap cover.
The lifting flap conveniently extends from the snap cover a-bout the w~dth of the snap cover and when placed inwardly fits ,` against the cultured test pla~e so that the lifting flap 38 can be picked up with a fingernail; but can be rotated 180 upon a vertical axis so that the lifting flap extends out-W8 rdly as an identificatlon tab.
The textured area on the lifting flap of the snap cover permits the identification of a particular culture test plate in a stack. Conveniently but not necessarily, on - the face of the culture test plate 21 is a label 40. Conven-lently the label includes the name of the antibiotic or active agent, identification as to batch number, dates and origin and has room for the name of the patient, the date of the test and other information at the time of use.
The ends o~ the handles 30 may have a molded legend 4l therein. It is convenient ~or a trademark or name of the manufacturer to be molded into the surface of the handle for ~dentification.
The use o~ the culture test plate is shown in FIGURE

3 throu~h 8, The culture test plate is molded with 10 rectan-gular wells. As shown in FlGURE 3 the well is filled with a liquid antlbiotic solution 42 containing poly(vinyl pyrrolidone) ~s a water soluble bulking agent and antibiotic retaining carrler. Poly(vinyl pyrrolidone) is bLologically inert and has no effect on the antibiotic, the culture medium or the , .
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' microorganlsms; and yet when frozen and dried, fills the well ; w~th g sponge which resembles cotton candy in texture whichholds the antlbiotic in pl~ce and prevents migration of the antibiotic As shown in FIGURE 4 the liquid filled into the wells is frozen and dried to form a dried antibiotic in the poly-(vinyl pyrrolidone) 43. Conveniently, a group of the culture test pl~tes are stacked with the well snap covers removed, as for exflmple as shown in FIGURE 13, and a group of such stacks are placed on the shelves of a freezing chamber, the .
contents frozen, the chamber evacuated and using conventional lyopi:~ilizing techniques dried to a sponge. The dryness of the sponge Ls protected by replacing the well snap covers and storing in a dry environment until time for use.
FIGURE 5 shows the dried sponge with the well ~snap cover in position At time of use, as shown in FIGURE 6, a liquid di-luent is added to the dry sponge.
Usually, using tube dilution practice, the liquid dlluent is an appropriate culture medium 44 which has been Lnoculated wlth a test organism. Preferably the test organism is at a standard concentratlon so that the test plate results are quantltative as well as qualitative.
Theoretically, the culture medium itself may be mixed with the antibiotic and dried down and retained in storage so that only the test organism and in an inert dilu-ent, namely water, need be added at the time of use, It is preferred that t~e culture medium be added with the test organ-Lsm (1) because the ~st organism can be addèd to the culture medium before it ls added, to avoid a double addition, (2) a culture medlum can be chosen which is particularly appropriate for s speciflc test organi6m, and (3~ the test organism con-centration Ls uniform for all tests. Many dehydrated culture 778~L

; medis are hygroscopic, and by their attraction of moisture could hasten the destruction of some antibiotics. Different laboratories prefer different culture med~a for different orgsnisms or even the s~me organism. By adding the organism being tested in the culture medium there is additional flex-ibllity in selecting the culture medium. Also, ~ithout the culture medium, there is a minimum risk of having a sys~em pr~sent which could support bacterial growth during storage.
In FIGURE 7 is shown a well having a clear solution 45 therein. After the sponge and antibiotic have dissolved ln the culture medium, the solution is clear. If there is sufficlent antibiotic to inhibit growth of the test organism, the solution rem~ins clear, if not, the microorganism grows and causes the solution to become cloudy 46 as shown in FIG-URE 8.
A clear solution 45 shows no bacterial growth. The cloudy solution 46 shows bacterial growth.
The reading of the solutions to determine bacterial growth may be either by inspection with the human eye, in effect an "eyeball" readingJ or it msy be done by electro-optLcal equipment such as a photosensitive reader and a con-- stant ~ntenslty light. The light may have a selected wave length or color depending upon the solution. Separate read-ers may be used for each cell or the same reader may be used for a group of 10 cells in a culture test plate serially.
For shipment and storage a group of 5 test Dlates in stack are convenlently enclosed in a platic envelope 47 as shown 1n FIGURE 9. A dessicant may also be placed in the en-velope to lnsure dryness. Two or more such plastic envelopes msy then be placed in a foil outer envelope 48, The ~il out-~ er envelope may have additional dessicant envelopes in it and Is sealed so ~s to protect its contents from ambient molsture for an extended length of time.

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When so se~led, the present culture test plRtes m~intain essentially their original labeled potency for sev~
er~l months, and it can be expected that the test plates will be satisfactory for at least several years, The size of the wells is not critical but a well size of 8 x 10 mm. at the top and 5 x 8 mm.at the bottom with 9 mm. depth permits working with 0.2 ml. of fluid with the wells about half full, and permits a convenient working size .
with the consumption of a minimum quantity of reagents and materials. A thickness of the cells themselvesJ the test plate) and the h~ndles through out, of about o.8 mmO (1/32 lnch) gives good results. Such a thickness is a compromise between having the parts thick enough for strength and thin enough to require a minimum of material. With such a thick-ness the culture test plates are strong enough to be reused if desired but are sufficiently inexpensive that it is usually cheaper to consider the test plates as disposable, The basis of the culture test plate method is the broth or tube dilution technique. Details o~ standardized methods and recommendations for use of this procedure are found in (1) Ericson, H.M. and Sherris, J.C. 9 "Antibiotic Sen-sitivity Testing, Report of an International Collaborative Study~" Act~ Path. Microbiol. Scand. Sect. B, Suppl. 217 (1971) and (2) Manual of Clinical Microbiology, Second Edition, Len-nette, E.H., Spaulding, E.~.~ and Truant, J.P., Eds. American Society for Microbiology (1974).
EXAMPLE
The antibiotic to be tested is provided in the two-fold dilution range of interest by starting with 10 four liter flasks into the f~rst of which w~s added 6 g. of poly-(vinyl pyrrolidone) (Povidone USP) and 3 liters of triple distilled water. In each of the other nine flasks was added 3 g. of poly(vinyl pyrrolidone) and 1~500 ml. of triple dis-- :~3D778~4 . .
tilled w~ter, To the first flask was added 206,25 mg, of tetra-c~ycllne hydrochloride, after which the flask was shaken un-t~l the contents were dissolved and uniformly distributed, One half of the contents of the first flask was then added to the second flask and the contents of the second flask shaken unt~l uniform, One half of the contents of the second flask was then added to the third flask, etc.~ and the series con-tlnued until the serial two-fold dilution was obtained in the ninth flask, The excess diluted solution in t~e ninth flask w~s d~scarded, The tenth flask hed only poly(vinyl pyrroli-done) and triple distilled water, It may be left empty.
The contents of each flask was sterile filtered into two liter reagent bottles which were capped and kept in an ice water bath for filling by sterile techniques, Fill-~ng should not be unduly delayed. The solutlons normally remain stable and without change for at least 24 hours lf kept cold, but it is preffrable that they be filled immediately to guarantee against loss of potency.
2/10 ml. of the contents of each of the flasks were f1lled into the respective wells of a single test plate.
` The wells in a total of five thousand test plates were filled, the test plates stacked and placed in racks in cold chamber. The cold chamber was pre-chilled with the ?5 cold chamber being maintained at colder than -40C. with shelf cooling being maintained until the contents of all of the wells in all of the test plates were frozen solid, This should occur in less than 12 hours. After freezing, the cold chamber was evacuated to less than 100 mlcrons total pressure, after ; 30 whlch the shelf temperature was raised to about 10C. and malntalned at this temperature until temperature probes in the assembly lndicate that the temperature within the wells was within about 5-10C. of the temperature of the shelves - 14 _ ~77~3~4 themselves, The shelves were then warmed up to about ~onc., ~nd after the test plates had warmed up appropriately, the temperature was increased to 40C, and the chamber held for

4 hours. ~t this point the contents of each well were thoroughly dry, While continuing the use of sterile techniques, well snap covers were placed over the dried test plate wells and a set of 5 test plates were stacked for convenience and placed in a polyethylene plastic envelope. ~ 5 g. silica gel packet was placed ln each of the plastic envelopes to aid in main-taining dryness. Two such envelopes containing 5 test plates each were then placed in an outer foil pouch which foil outer envelope is essentially impermeable to moisture and maintains dryness of the test plates for an extended period of at least months and predictably for at least several years, lf not indefinltely, The culture test plates have a label on each which ; ind1cates the particular antibioticland its concentration in each of the wells, with space for identifica~ion data as to date, the patient and test conditions under which the culture test plate is used, A number of foil envelopes are packaged in a shipping container, the number being based on the usuage of customers, When filled in this fashion, the wells contain:

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- TABLE I

WellTetr~cyciine No.hydrochloride_ _ _ 1 12.5 mcg~ 10~ excess plus 400 mcg. providone 2 6.25 mcg. " "
3~.125 mcg. " "
4 1.56 mcg. " "

5 o.78 mcg.

6 0.39 mcg. " "
70.195 mcg.
8o . og8 mcg. " "
90.049 mcg. " "
10 0.00 mcg. - "

Culture test plates conveniently are used for any antiblotic or therapeutic control agent such as penicillin, amplcillln, clindamycin, erythromycin, methicillin, tetra-cycline, demethylchlortetracycline, 7-dimethylamino-6-demethyl--6-deoxytetracycline, minocycline, ceph~lothin, gentamycin, collstin, carbenicillin, chloramphenicol, kanamycin and any ``! of the sulfonamides.
Other antibiotics, either those known or those yet to be discovered may be used--any if the antibiotics require a range other than that listed, the concentration may be modi-fled--but wlth the wide range covered by the nine dilutions in the cavities, the proper dosage of most antibiot~cs will be obtained.
The tenth cup has no antibiotic and hence if inocu-lated with the test microorganism, shows the growth of the microorganlsm under uninhibited conditions; or if not inocu-lated is used to show that no contaminants are present.
The number of culture test plates and choice of culture test plates, e~ch with a difference antibiotic, de-- 16 ~

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pends upon the preferences of the medical staff of the usLng f~cility, At the time of use, aliquots of broth inoculated with the bacterial culture to be tested are pipetted into each plate well following which the plates are incubated for spec~fic time at an indicated temperature, The test is read by visual inspection for growth as indicated by turbid-lty or no growth as shown by a non-turbid suspension. The endpoint is defined as the well containing the lowest con-c~ntration of antibiotic with no detectable microbial growth.
The control well on each plate, having no antimicrobial agent present, serves as a measure of the uninhibited growth of the bacterial culture.
PROCEDURE
Specimens obtained in the laboratory from clinical sources are cultured on primary agar plates. Isolated colon-ies of the organism suspected of being implicated in an in-fectious process should be selected, Ideally, identification procedures should be per-formed concurrently with susceptibility testing.
Mixtures of different types of organisms (mixed cul-tures) should not be used for susceptibility testing unless there ~s a clinical emergency. In these instances, or in cir-cumstances where testing is done directly from clinical spec-~5 iments, susceptibility tests should be repeated using a pure culture.
Aerobic, facultatively aerobic and clinically sign-ificant obli~ative anaerobic bacteria may be used for suscept-ibility testing. Anaerobic bacteria shguld be suspended in `~o freshly boiled medium, dispensed into plate wells and incu-b~ted under anaerobic conditions within 15 minutes.
Three to five colonies of the organism to be test-ed are suspended in 4 to 5 ml. of sterile trypticase soy :~77~

broth The tube containing this inoculum is covered and plac-ed ~n a water bath for incubation at 34-46C. ~or two or three hours or until a turb~d suspension is produced.
The bacterial density of the inoculum is preferably stsnd~rdized at 1 x 105 Colony Forming Units (CFU) per milli-liter prior to use.
The required 1 x 10 CFU/ml. suspension of bacteria can be standardized by preferably ad~usting to 105 CFU/ml. with the use of a standardized nephelometer.
Alternatively a BaS04 standard as used for the Kirby-~auer disc diffusion test can be employed.
` 0.5 ml. o~ o.o84 M BaCl2 or 1.17~ (w/v) BaClz 2H20 is added to 99.5 ml. of 0.36~ v/v) H2S04. This suspension is equivalent to approxim2tely 10 Enterobacteriaceae per ml.
?he Ba S 04 turbidity standard should be dispensed`
into tubes of the same si~e used to grow the broth inoculum ~nd stored in the dark for no longer than six months at 20-- 25C. These turbidity standards must be vigorously mixed prior to use.
The inoculum, when standardized by visual comparison against the BaS04 turbidity standard (108 CFU/ml.), should be then diluted 1:1000 by mixing 0.1 ml. of the bacterial sus pension with 99.9 ml. of fresh sterile broth. The diluted suspension now contains 10 CFU/ml.
Broth media used for dilution of the initial bacter-ifll suspenslon is Mueller-Hinton broth, except when testing with organisms such as streptococci, neisseriaJ hemophiline rods, and certaln other fastidious organisms. Trypticase soy broth may be used for streptococcal cultures. Supplementa-~ tlon of medla for growth of fastidious species should be done accord~ng to recommendatlons of the International Collaborative Study, supra.

.

~(:977i!~

The plastic snap covers are removed from each cul-ture test plate and are placed on a clean surface, preferably ` tn an tnverted position.
Each well of the plate is then filled with 0,2 ml, '1 (200 ~1) of broth standardized to contain 105 CFU/ml, of the microorgan~sm under test.
A repetitive pipetting device may be used to intro-duce the inoculated broth into the plate wells providing the dellvery at the indicated volume is within +5~ and the de-vlce can be sterilized prior to use without affecting the de-l~very, or a manual pipette may be used.
The plastic covers are then snapped back into place using the s~me covers orlginally removed from the plate and the plates put in an incubator at 35-36C.
Grossly discernible growth is observed in most in-stances in four to six hours with rapidly growing bacterie.
A Minimum Inhlbitory Concentration tMIC) endpoint taken at th~s time has been shown to be equivalent to endpoints taken after 18 hours of incubation in tests done with microorganisms ,~ 20 whose growth is rapid, In addition, tests with bacteria ex-hibiting a slower rate of growth indicate that preliminary MIC's can be found after four to six hours of incubation al-though plates should be incubated for the full 18 hours to ob-tflin the flnal result.
The endpoint is defined as the well containing that concentration of ~ntimicrobial agent where there is no detect able mlcrobial growth estimated visually as confluent turbidity or reasonable amounts of flocculation or clusters of bacteri~.
A slight haze or a small number of particles seen

7~0 at the bottom of a well does not constitute growth.
The MIC in mcg. or units per ml, is obtained by multlplytng the corresponding content figure imprinted next to the well showing no growth by 5.

1~7~7~3i4 In addition to determination of MIC, a Minimum Bac-terlcidal Concentration (MBC) can be determined by removal of a loopful of organisms Prom two or more wells on either side of the MIC bre~kpoint, plating them out on a solid medium and observing ror viable organisms after an incubation period.
In use is a laboratory or hospital it is customary to have a plurality of test plates such as above described and in which have been placed a group of different antibiotics or chemotherapeutic agents and the various test plates contain-ing antibiotics to be tested are cultured together and read ~; either by visual inspection, a so called "eyeball" test, or by using a radiation source such as an electric light and a suitable r~diation detector with the test well placed between the radiatlon source and the detector. For small operatlons it is convenient to use a single electro-optical reader. For 1srger installations a group of lO so that all ten of the wells ; ~n a test plate may be read simultaneously is convenient.
The report of the minimum inhibitory concentration ~ of each antibiotic or chemotherapeutic agent is reported so - 20 the attending physician can select an antibiotic which is most effective for a particular patient.
The use of the poly(vinyl pyrrolidone) of this in-vention overcomes two ma~or problems found in the preparation and use of prior art test kits First, since the amount of antibiotic ~n the wells ~s ~n minute (microgram) quantities and is serially diluted and frozen-dried in the succession of chambers, it is diffi-cult to v~sually determine from a quality control standpoint, whether a given chamber contains any antibiotic. The addition of poly(vinyl pyrrolidone) as a bulking agent makes visu~l de-tectlon easier Secondly, the use of poly(vlnyl pyrrolidone) in con-~unctlon with an antibiotic produces a freeze-dried foam plug .

~778~
of ~ conslstency not unlike cotton candy, which fits snugly into ~nd adheres to the bottom and sides of the chamber.
This combination is much less likely to be dislodged from the well during the sealing and unsealing ofthe plates, and shipment and storsge, Poly(vinyl pyrrolidone) is readily put into aqueous 801ution with the antibiotic and later inoculated with the aqueous nutrlent broth. Further, poly(vinyl pyrrolidone) does not react with the antibiotic, nor affect its potency, nor the growth characteristics of pathogenic organism3.

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~5 : 30

Claims (7)

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

1. A test kit for measuring the antibiotic sus-ceptibility of a microorganism comprising a plurality of integral test chambers with at least some of said chambers having therein a graduated series of concentrations of an antibiotic and a uniform quantity of poly(vinyl pyrrolidone) as a water-soluble bulking agent find antibiotic retaining carrier, with the anti-biotic in dispersed and reconstitutable frozen-dried form in the poly(vinyl pyrrolidone),

2. The test kit of Claim 1 in which the antibiotic 19 present in a two fold series of dilutions including at least part of the range of 1 to 10 micrograms per milliliter.

3. The test kit of Claim 2 in which the test cham-bers hold about 0.2 milliliters of solution and the range of antibiotic is about 0.05 to 12.5 micrograms per chamber.

4. The test kit of Claim 3 packaged in sterile dry conditions in a moistureproof container.

5. A method of testing the antibiotic suscepti-bility of a microorganism by a serial dilution technique com-prising: forming a series of concentrations of solutions in water of an antibiotic in a uniform concentration of poly(vinyl pyrrolidone) as a water-soluble bulking agent and antibiotic retaining carrier, loading uniform quantities of said series of concentrations in a series of integral test chambers, freezing and freeze-drying said series of concentrations, storing until time of use, and reconstituting the antibiotic in the poly(vinyl pyrrolidone) by adding a culture medium inoculated with a test microorganism, incubating, and then reading the test microorgan-ism growth.

6 The method of Claim 5 in which the antibiotic is present in a two-fold series of dilutions including at least part of the range of 1 to 10 microorganism per milliliter

7. The method of Claim 6 in which the test cham-bers hold about 0.2 milliliters of solution and the range of concentrations of antibiotic is about 0.05 to 12.5 micrograms per chamber.