US 3846247 A
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United States Patent 3,846,247 MOISTURE BARRIER Donald P. Kronish, Rockaway, Lee S. Zuritr", Parsippany,
William D. Young, Montclair, and Paul R. Lewandowski, Parsippany, N.J., assignors to Warner-Lambert Company, Morris Plains, NJ. No Drawing. Filed Sept. 27, 1972, Ser. No. 292,701 Int. Cl. 'C12k l /00 US. Cl. 195-127 3 Claims ABSTRACT OF THE DISCLOSURE The present invention relates to an improved moisture barrier which is particularly suited for use in the manufacture of diagnostic strips used in the identification of bacteria.
Diagnostic strips for the detection and identification of bacteria made of bibulous materials on which are sup ported in various zones or area substrates and reagents are well known. Strips of this type are described and claimed in US. Pats. Ser. Nos. 3,341,427 and 3,359,180 granted to Evans et al.
Distinguishing characteristics of various species of bacteria are detectable by use of a series of reagent impregnated strips. Identification of the Enterobacteriaceae is accomplished by the use of a series of ten such strips. The identifying tests are directed to the detection of the products of a specific enzyme activity of a particular organism. The results of the test are interpreted using a combination of a flow chart and checkerboard table.
Reagent impregnated test strips are not only convenient, easily handled, and more stable than so called classical tests, but they lend themselves to obtaining results more rapidly with an accuracy equivalent to the classical wet tests which were preferred previously. A further advantage is that the test reactions are completed in no more than four hours and therefore do not require aseptic working conditions.
Test strips currently used for rapid identification of the Enterobacteriaceae include those which test for:
H S detection Lysine decarboxylase Voges-proskauer Malonate utilization Esculin hydrolysis It is to be noted that several of these tests are useful for purposes other than the rapid identification of the Euterobacteriaceae such as the simple determination of the presence of nitrate radicals or sulfide ions.
A high grade bibulous paper such as Eaton-Dykeman No. 623 is the usual material which is impregnated with the substrates and reagents required to produce the desired test strip. Other bibulous materials may be used instead of paper such as felt, porous ceramic strips, woven or matter glass fibers and the like.
There are certain considerations which obtain in most of the test procedures. One is that the colonies of bacteria tested are usually suspended in an isotonic saline solution. This suspension is placed in an incubation tube in a rather small amount, usually in the order of 0.3 ml. The suspension must contact the part of the strip bearing an initial substrate or reagent but must not be dissipated by capillarity. This is important since some reactions are read by color changes in the saline solution, and other tests depend upon the reagent zones being contacted only after incubation or upon contacting various zones in a specified sequence. Premature mixing of reagents could invalidate a test result.
The bibulous nature of the strip is necessary for impregnation to be carried out successfully. The same characteristic then requires that a reliable moisture barrier be applied to one or more area to prevent dissipation of the saline by absorption into the strip. This also insures that reagents will not be prematurely contacted by the solution or mixed in those tests in which the order in which they are used is significant.
What can be done is incubate a suspension of unknown bacteria in saline with a suitable substrate. The products of the bacterias enzyme metabolism accumulate in the saline after a period of time. The reagent or indicator zone is contacted by the incubated cell suspension by tilting the incubation tube. The result is determined by reading the color developed on the reagent zone.
The limiting of the inoculum containing saline to contact certain zones for predetermined periods has been accomplished by placing various moisture barriers on and in the bibulous strip. One such barrier was formed by impregnating a chrome complex, hydrophobic material available under the trade name Quilon described in US. Pat. 3,341,427. Quilon is a chrome complex coordination compound of the formula:
wherein R represents a C to C fatty acid radical.
Experience with this moisture barrier material has shown it to be extremely effective for this purpose. It suffers, however, from two significant drawbacks. This chrome complex produces a sufliciently acidic environment to affect some of the more delicate tests and the presence of chrome ions can prevent the bacterial growth which is necessary to produce the metabolites which these tests detect.
A substitute barrier material was then utilized which was a colorless ethyl methacrylate and methyl acrylate copolymer in a toluene solvent and known as Krylon 150 Crystal Clear. Krylon solution was advantageously modified by further diluting it with compatible thinners such as toluene or alower aliphatic alcohol to improve its ease of application. The range of such dilution when suitable is from 3 to 33% volume of added solvent per volume of acrylic resin solution. Where the test strip is made from a suitable filter paper such as Eaton-Dykman No. 623 the preferred combination on a volume/volume basis is Krylon and 15% ethyl alcohol U.S.P.
The barrier provided by Krylon alone was found to become impaired when the rolls of paper which have had the various zones impregnated with reagents applied by rollers Were cut to the final strip form by a conventional cutter. One run of several rolls was found to have produced percentages of strips which had faulty moisture barriers ranging from 3% to greater than It is not clear how this failure occurred. It could have been a change in one of the variables over which control is difficult or impractical. This could include the paper itself, the moisture content of the paper, the coating solutions, the conditions or mechanics of the process.
The present invention relates to an improved moisture barrier which stops the migration of various solutions up the test strip and which continues to maintain its functional integrity after cutting. The moisture barrier of the present invention comprises an initial impregnation of the bibulous material with Quilon followed by a second impregnation over the first and extending over either side of it with Krylon. Experience with this moisture barrier has shown only negligible leakers despite rigid examination of randomly sampled test strips for purposes of quality control.
It must be taken into consideration that the preferred way of making reagent impregnated test strips is by using a roll of suitable bibulous paper as long as 300-400 feet and rolling on to it, lengthwise, at discrete intervals, the required substrates, reagents and moisture barriers. This is usually accomplished by concurrent or successive passing of the paper between rollers which pick up the impregnauts from reservoirs and deposit them on the paper as it passes over them. The amounts deposited are determined by several factors including the physical characteristics of the impregnant such as viscosity, flowability and wettability, the width of the applicator wheel and the pressure of the applicator wheel on the paper.
It is obvious that more than one pass may be made by the paper through the impregnating process and that both sides of the paper may have the same or different materials applied to the same zonal level. The moisture barriers are applied on both sides, at the same level, to insure complete penetration into the body of the paper.
The following example of providing a moisture barrier in accordance is given by way of illustration and not by way of limitation:
A 10 meter length of Eaton-Dykman No. 623 filter paper 83 millimeters wide was used as the bibulous material for impregnation. Two compositions were prepared for combined use as a moisture barrier.
Quilon S, a chrome coordination complex which is made with stearic acid is supplied in a solution which is about 70% isopropyl alcohol and 30% of the chrome complex. The preferred dilution for the impregnant is prepared by taking 5 ml. of Quilon S as received, adding 20 ml. isopropyl alcohol and 50 ml. of distilled (deionized) water. The apparent pH is adjusted to 3.5 using 0.05 Na'OI-I. This usually results in a final volume of about 100 ml. This solution is placed in a reservoir from which it is fed to coat the surface of an applicator wheel having a width of 5 mm. The clearance between the applicator wheel and the opposing surface is such that the rotating wheel both applies the impregnating solution to the paper and simultaneously advances the paper. A preferred rate of advance is about 20-40 feet/min.
The second moisture barrier material is applied over the first and preferably extending on either side thereof, and preferably so that the width of the area of the ethyl methacrylate and methyl acrylate copolymer superim posed impregnation is about three times that of the chrome complex coordination compound area of impregnation and extends about equidistantly from it on either side. This is done by using a 15 mm. wide applicator wheel centered over the same portion of the paper as the first. The two moisture barrier compositions are completely compatible. It is not necessary to wait for the first to dry before applying the second.
The Krylon solution is supplied containing about 22 to 26% acrylic resin solids in toluene and diluted with ethyl alcohol U.S.P. The impregnating composition was prepared by placing 85 ml. of Krylon solution in a graduate cylinder and adding a sufljcie nt quantity of ethyl.
4 alcohol U.S.P. to bring the volume to ml. The solutions are completely miscible and therefore require no special mixing. It was applied as previously described. Krylon drys at room temperature and low humidity in about 4-8 hours, and the Quilon S complex is cured in about one day.
The roll of impregnated test paper is then cut into strips 6.3 mm. wide. The strips thus produced were tested for moisture barrier integrity by partially immersing the portion next to the barrier in isotonic saline solution containing a dye. The integrity of the barrier is judged as having been maintained when the zone above the barrier remains free of dye. None out of 200 samples tested failed after four days. The tests were not continued beyond this since the reagent impregnated test strip would be exposed to the possibility of moisture barrier failure not more than four hours:
What is claimed is:
1. In a strip of bibulous material, capable of absorbing fluid by capillary action, said strip being divided into several zones which may be impregnated with various materials, the improvement of which comprises said strip having at least one moisture barrier zone area formed by having impregnated therein a chrome complex coordination compound of Cir- References Cited UNITED STATES PATENTS 3,341,427 9/1967 Evans et al 103.5 R 3,359,180 12/1967 Evans et al 195103.5 R
ALVIN E. TANENHOLTZ, Primary Examiner R. J. WARDEN, Assistant Examiner US. Cl. X.R.