CA1140051A - Boric acid complexes and their use as functional preservation agents - Google Patents
Boric acid complexes and their use as functional preservation agentsInfo
- Publication number
- CA1140051A CA1140051A CA000321844A CA321844A CA1140051A CA 1140051 A CA1140051 A CA 1140051A CA 000321844 A CA000321844 A CA 000321844A CA 321844 A CA321844 A CA 321844A CA 1140051 A CA1140051 A CA 1140051A
- Authority
- CA
- Canada
- Prior art keywords
- boric acid
- complex
- urine
- samples
- bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical class OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 title claims abstract description 95
- 238000004321 preservation Methods 0.000 title claims description 14
- 239000003795 chemical substances by application Substances 0.000 title description 7
- 239000004327 boric acid Substances 0.000 claims abstract description 85
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 54
- 238000000034 method Methods 0.000 claims abstract description 36
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 17
- 239000000203 mixture Substances 0.000 claims abstract description 16
- -1 polyoxy Polymers 0.000 claims abstract description 10
- 239000005715 Fructose Substances 0.000 claims abstract description 9
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 9
- 229930091371 Fructose Natural products 0.000 claims abstract description 9
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 8
- 229930195725 Mannitol Natural products 0.000 claims abstract description 8
- 239000000594 mannitol Substances 0.000 claims abstract description 8
- 235000010355 mannitol Nutrition 0.000 claims abstract description 8
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims abstract description 7
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims abstract description 7
- 239000000600 sorbitol Substances 0.000 claims abstract description 7
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 claims abstract description 4
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims abstract description 4
- 235000013772 propylene glycol Nutrition 0.000 claims abstract description 4
- 230000000087 stabilizing effect Effects 0.000 claims abstract description 4
- 239000003381 stabilizer Substances 0.000 claims abstract 2
- 210000002700 urine Anatomy 0.000 claims description 58
- 239000000654 additive Substances 0.000 claims description 15
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims description 12
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 230000000996 additive effect Effects 0.000 claims description 7
- 238000004040 coloring Methods 0.000 claims 3
- 239000000126 substance Substances 0.000 claims 2
- 241000894006 Bacteria Species 0.000 abstract description 42
- 150000001875 compounds Chemical class 0.000 abstract description 17
- 238000002360 preparation method Methods 0.000 abstract description 2
- 229960002645 boric acid Drugs 0.000 description 65
- 235000010338 boric acid Nutrition 0.000 description 65
- 238000012360 testing method Methods 0.000 description 23
- 239000000523 sample Substances 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 230000032258 transport Effects 0.000 description 13
- 230000000721 bacterilogical effect Effects 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000005057 refrigeration Methods 0.000 description 9
- 239000003755 preservative agent Substances 0.000 description 8
- 230000002335 preservative effect Effects 0.000 description 7
- 241000588748 Klebsiella Species 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 241000588769 Proteus <enterobacteria> Species 0.000 description 5
- PEEKVIHQOHJITP-UHFFFAOYSA-N boric acid;propane-1,2,3-triol Chemical compound OB(O)O.OCC(O)CO PEEKVIHQOHJITP-UHFFFAOYSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 241000589516 Pseudomonas Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000003086 colorant Substances 0.000 description 4
- 229940093476 ethylene glycol Drugs 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 230000006641 stabilisation Effects 0.000 description 4
- 238000011105 stabilization Methods 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000000376 reactant Substances 0.000 description 3
- 241000790917 Dioxys <bee> Species 0.000 description 2
- 241000219470 Mirabilis Species 0.000 description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000013100 final test Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000009533 lab test Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 244000293889 Bombax malabaricum Species 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 241000375722 Cyperus eriocauloides Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000012868 Overgrowth Diseases 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000191963 Staphylococcus epidermidis Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000005576 amination reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000006161 blood agar Substances 0.000 description 1
- 125000005619 boric acid group Chemical group 0.000 description 1
- RERVRRLGFUNERS-UHFFFAOYSA-N boron;propane-1,2,3-triol Chemical compound [B].OCC(O)CO RERVRRLGFUNERS-UHFFFAOYSA-N 0.000 description 1
- 208000037516 chromosome inversion disease Diseases 0.000 description 1
- 229940000425 combination drug Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000000881 depressing effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000020200 pasteurised milk Nutrition 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000008279 sol Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/04—Esters of boric acids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Ceramic Products (AREA)
- Amplifiers (AREA)
- Chemical And Physical Treatments For Wood And The Like (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Novel boric acid complexes of the formulae
Novel boric acid complexes of the formulae
Description
This invention relates to novel compounds, a process for the preparation of such compounds, and to compositions con taining such compounds, as well as a method of their use~
More particularly, the compounds of the present invention possess bacterial preservation properties which are particularly suitable for the stabilization of urine samples for subsequent bacteriological testing.
The use of boric acid for the preservation and stabiliza-tion of bacteriological samples is well known in the art. Thus, for example, boric acid has been used as a stabilization agent in bacteria tests on pasteurized milk where a 1% acid solution was used as a "transport" medium for transporting the samples to bacteriological laboratories where the tests were carried out as in the case of production-hygiene tests. In such tests, it was established that the tubercular bacteria were not destroyed in the milk samples and that the 1% boric acid solution, used as a preser-vation additive, had made it possible to transport the samples over a longer period of time than would have normally been possible, and further, that the 1% boric acid solution did not interfere with the examination of the samples in the laboratory testing.
The possibility of facilitating the bacterioloyical laboratory wor]c for bacteria analyzing of urine samples with the aid of boric acid additives, had, however, still to be accepted by the end of the 1920's. The importance of immediate bacteriological e~amination of urine samples in cases of suspected infection of the urine tract, in order to avoid additional or overgrowth of the bacteria in the samples, has been scressed very strongly despite the fact that boric acid can be used to aid in the preservation of the samples without interfering with the measurements or the parameters . .
for the subsequent laboratory testsr Thus, the possibility of 0~531 using boric acid, in the ~orm o~ a 1% solution, for transportlng urine samples ~or subsequent labora-tory examination, for e.g., determination of brusella, aboxtus, where the boric acid functions as a preservation agent, has been known.
soric acid has also been used for a number o~ studies, both in huma~s and animals, for bacteria growth and retardant functions in different vehicles. Despite this, it has been pointed out in several scientific publications that bacteriological examina-tion of urine samples must be carried out immediately, and evan under refrigerated conditions, e.g. 4C, ~he studies must be carried out within 48 hours.
~ otwithstanding the above, the problems o* additional growth or ovel~rowth of bactel~ia in the samples, during slow or delayed transportation of the samples to bacteriological labora-tories, remains a problem. In a study at the end o~ the 1960~s, it was surprisingly pointed out that the use of boric acid as a pre-servative for urine during its transportation had not been inves-tigated or considered. Yet, in another study, it was also found that the optimum concentration for boric acid is in the order of 1.8%
solution for proper functioning as a preservative for urine trans-port but that the sample should not be stored for more than 4 days prior to testing. Despite the scientific medical work, and in spite of the fact that boric acid has been known for a long period of time, as a functional preservative with respect to bacteria growth, the knowledge and methodology has been very limited in bacteriological laboratory work on the international point of view, and has not been used to any extent. There are several reasons ~or the non-adaptation of boric acid as a preservative even though it has many advantageous features over the use of refrigeration for ~d ~0 the same purpose. On the other hand, there are many practical L 4l00S~
di~iculties relative to the use of boric ac-id - for example, normally one has to measure very small quantities o~ a powder (the form in which boric acid is normally used~, it has ~o be transported in very small packages or tu~es and, boric acid has a very low relative solubility in urine.
Additionally, if boric acid powder is dissolved in water to facilitate measurement in exact volumes, too large volumes of boric acid solution in urine are created to make it of practical usage for transportation. In addition, its use as a preservative agent in urine samples involves an additional work-step in the laboratory to distinguish between those urine samples with boric acid and those without. This latter point is somewhat disturbing for practical reasons since a centralized laboratory diagnostic facility utilizes as few as possible work-steps due to the large number of samples being analyzed daily - which may t~pically be in the order of 500 per day for a large laboratory. The known solu-bility of boric acid in water is as follows: At zero degress Celsi
More particularly, the compounds of the present invention possess bacterial preservation properties which are particularly suitable for the stabilization of urine samples for subsequent bacteriological testing.
The use of boric acid for the preservation and stabiliza-tion of bacteriological samples is well known in the art. Thus, for example, boric acid has been used as a stabilization agent in bacteria tests on pasteurized milk where a 1% acid solution was used as a "transport" medium for transporting the samples to bacteriological laboratories where the tests were carried out as in the case of production-hygiene tests. In such tests, it was established that the tubercular bacteria were not destroyed in the milk samples and that the 1% boric acid solution, used as a preser-vation additive, had made it possible to transport the samples over a longer period of time than would have normally been possible, and further, that the 1% boric acid solution did not interfere with the examination of the samples in the laboratory testing.
The possibility of facilitating the bacterioloyical laboratory wor]c for bacteria analyzing of urine samples with the aid of boric acid additives, had, however, still to be accepted by the end of the 1920's. The importance of immediate bacteriological e~amination of urine samples in cases of suspected infection of the urine tract, in order to avoid additional or overgrowth of the bacteria in the samples, has been scressed very strongly despite the fact that boric acid can be used to aid in the preservation of the samples without interfering with the measurements or the parameters . .
for the subsequent laboratory testsr Thus, the possibility of 0~531 using boric acid, in the ~orm o~ a 1% solution, for transportlng urine samples ~or subsequent labora-tory examination, for e.g., determination of brusella, aboxtus, where the boric acid functions as a preservation agent, has been known.
soric acid has also been used for a number o~ studies, both in huma~s and animals, for bacteria growth and retardant functions in different vehicles. Despite this, it has been pointed out in several scientific publications that bacteriological examina-tion of urine samples must be carried out immediately, and evan under refrigerated conditions, e.g. 4C, ~he studies must be carried out within 48 hours.
~ otwithstanding the above, the problems o* additional growth or ovel~rowth of bactel~ia in the samples, during slow or delayed transportation of the samples to bacteriological labora-tories, remains a problem. In a study at the end o~ the 1960~s, it was surprisingly pointed out that the use of boric acid as a pre-servative for urine during its transportation had not been inves-tigated or considered. Yet, in another study, it was also found that the optimum concentration for boric acid is in the order of 1.8%
solution for proper functioning as a preservative for urine trans-port but that the sample should not be stored for more than 4 days prior to testing. Despite the scientific medical work, and in spite of the fact that boric acid has been known for a long period of time, as a functional preservative with respect to bacteria growth, the knowledge and methodology has been very limited in bacteriological laboratory work on the international point of view, and has not been used to any extent. There are several reasons ~or the non-adaptation of boric acid as a preservative even though it has many advantageous features over the use of refrigeration for ~d ~0 the same purpose. On the other hand, there are many practical L 4l00S~
di~iculties relative to the use of boric ac-id - for example, normally one has to measure very small quantities o~ a powder (the form in which boric acid is normally used~, it has ~o be transported in very small packages or tu~es and, boric acid has a very low relative solubility in urine.
Additionally, if boric acid powder is dissolved in water to facilitate measurement in exact volumes, too large volumes of boric acid solution in urine are created to make it of practical usage for transportation. In addition, its use as a preservative agent in urine samples involves an additional work-step in the laboratory to distinguish between those urine samples with boric acid and those without. This latter point is somewhat disturbing for practical reasons since a centralized laboratory diagnostic facility utilizes as few as possible work-steps due to the large number of samples being analyzed daily - which may t~pically be in the order of 500 per day for a large laboratory. The known solu-bility of boric acid in water is as follows: At zero degress Celsi
2.66 gr/100 gr. water and at 20C, 4~9 gr~100 g water.
From the above, it will be seen that in practice, one cannot work with a solution higher than a 3% concentration o~ boric acid to avoid crystallization during temperature changes. Addition-ally, for economic reasons, it is also desirable to use a dosage unit which is as small as possible for transportation purposes, and to to this end, standardized transport containers with a volume of 10 ml are the most common ones in usage.
h~hen using a 3% boric acid solution, 13 rnilliliters must be added to 9 ml of urine to achieve a concentration of 1.8% boric acid which is necessary for the boric acid to act as a preservative.
This means that a large amount of water is added to the sample and ~0 that the resulting volume increases to more than double.
. _.
s~
With this invention, applicant has developed a novel series of compounds which have preservation properties for urine samples, and in addition, a method of preparing such compounds~
Still ~urther, there is also provided a method of using such compounds to stabilize urine samples during transpor-tation to laboratories and as well, a composition containing a functional preservation agent for urine samples which contain the novel com~
pounds.
More particularly, in accordance with the present inven-tion, there are provided novel boric acid complexes of the formula [ \ / \ ~ [ ~ ~ \ / R ¦ H
wherein the group R represents a member selected from ethyleneglycol; propanediol-1,2; glycerol fructose; mannitol; sorbitol;
and as well, other water-soluble dioxy or polyoxy complexes of the above formula. In accordance with a still fur-ther aspect o~ the invention, there is pxovided a method of preparing the above bor.ic acid complexes which comprises heating boric acid with the afo.re-mentioned dioxy or polyoxy compounds including ethylene glycol,propanediol-1,2; glycerol, fructose; mannitol; or sorbitol for a relatively short period of time and until 1 to 2 moles of reaction water have been driven off, whereby water-soluble complexes of the above formu~a are obtained.
The above compounds have been Found to achîeve a more efficient and effective preservation of the bacteria in urine samples and may be employed in relatively low amounts compared to boric acid per se. More particularly, in accordance with a further aspect of this invention, it has been found that the above ~ .~
compounds function as an effective preservative for the bacteria ` _, !
in urine samples employing between about .5 to about .8% boric acid utilized in the form of the aforementioned boric acid com-plexesO In the compounds of the present invention, the concentra-tion of boric acid is relatively high and the addition of between about .4 to about 1.4 ml to 9 ml of urine has been -~ound to be sufficient. Thus, in accordance with the present invention, aompared to samples which contain no preservation or stabili~ing agent, the volume of the urine samples utilizing the complexes of this invention are only increased in a very limited manner so that standardized transport tubes or containers may be utilized for this purpose. Thus, in brief summary of the latter aspect of the invention, there is provided a method o~ preserving samples, par-ticularly urine samples, on which bacteriological laboratory tests are to be subsequently carried out, characterized in that a boric acid complex of the above formula is employed as a preservation or stabilization agent by adding a compound of thc above formula to the sample in an amount o~, e.g. .5 to about .8% boric acid in the form of the above complex.
In the above-described method of producing the boric acid complexes, the preferred aspects of a method according to the present invention as outlined comprise heating the -eactants within predetermined temperature and time limitations, so as to evolve a predetermined amount of water from the reaction products produced -i.e. between 1 and 2 moles whereby the resulting products are water-soluble - i.e. they have a predetermined wa~er concentration in the reaction product. The concentration of the boric acid in the complexes can be regulated by an additional amount of the complexes o- by the water thai is included in the complex.
Preferred embodiments of the reaction comprise reac'ing --30 the rcactants at a temperature in the range o~ lrom 80 to 160C, -6~
pre~erably in the range of fr~m 110-129C. The re~ction is carried out over a relatively short period of time, within about 1 to 12 minutes and preferably within 3 to 4 minutes. The boric acid em-ployed in the reaction is well known per se; preferred reactants comprise the compounds ethylene glycol, propanediol-1,2, glycerol, fructose, mannitol and sorbitol or other like di- or polyols which give -the aforementioned wa~er-soluble complexes and the corres-ponding residues of this groups in those complexes. The parti-cular choice o~ reactant which is utilized with the boric acid can be chosento provide a reaction product which, in addition to being ~ater-soluble, has wax-li]ce properties. Pre~erably, the react~n-ts are chosen so as to obtain a reaction product havins between 10 to 20Y of boric acid and desirably 11 to 13% oE boric acid concentration in the complex~
If desired, various additives may be incl~lded with the complexes for preparing compositions and for use in -the method of the present invention; such additives may include, -or example, coloring agents to identify the inclusion of an additiv~ in a urine sample - e.gO coloring agents such as methylene blue or other like and equivalent additives may be employed. It will be under~
stood by those skilled in the art that any such additives con-ventionally used for this purpose may be employed provided that they do not interfere with subsequent laboratory examination pro-cedures or do not adversely in*luence the bacteria in the samples.
T~en a coloring agent is employed, such as methylene blue, the concentration may be in the order of from 5 to 50 par-ts per million (ppm).
In addition, in the method o* the present invention, the comple~es are preferably added to the sample in an amount of .2 ~ %, desirably .~ - .8% boric acid concentration. I.i~tures ~v~s~
OI the boric acid comple.Yes may be employecl in the method for stabilizing the samples.
Having thus ~enerally described the invention, reference will now be made to the accompanying Examples, illustrating pre-ferred embodiments and preferred techniques.
In this example, 100 parts by weight of ethylene glycol was employed together with 17 par~s by weight of boric acid (BP).
This mixture was heated to 9~C, during which reaction water was lQ formed. Thereafter, the temperature was raised to 112C for a period o four minutes. It was found that 2 moles of water were evolved. The product was found to contain 17.7% boric acid; it had a pH in the concentrated reaction product of 2.5, while a 10%
aqueous solution had a pH of a . a.
EXAMP~E 2 In this example, 124 parts by-weight of glycerol DAB d-1.23 and 17 parts by weight of boric acid (BP) were mixed and heated to 110C, whereupon the reaction was initiated and 2 mol2s o~ water was evolved. The amount of water increased in the reaction mixtu-e f-om 11.3% to 22.3%. The temperature was raised ~or a period of 3 minutes to 129C whereafter the heating was stopped and the reaction product was allowed to cool down. The reaction product contained 15.5% water; 12.4% boric acid concentration and a pH, in the concentrated product, o 1.3. A 10% aqueous sol~ltion of the reaction product had a pH o 3.9.
Methylene blue, a coloring agent, was added in a con-centxation of 5 to 50 parts per million. ~ater is present in the above Ex~ample, as in all of theother Examples, as it is created during the reaction or is included withthe reactants, -e.g. in accompanying Example 4- propanediol~ contains 5% wa~er.
~ 8 ~ QOS~L
EX~LE 3 100 parts by weight of fructose, 150 parts by weight of water, and 36 parts by weight of boric acid were-heated to 80C, whereafter the temperature was increased in 6 minutes to 190C, resulting in 2 moles of water being driven of~.
The boric acid content was determined to be 12.1%; the pH of ~he concentrated reaction product was 1.1; and the pH of a 10% aqueous solution of the product was 4.2. The reaction product was pressed into tablets.
~XAMPLE 4 100 parts by weight of 1,2-propanediol and 25 parts by weight of boric acid were heated to 98& , wherea~ter the tem-perature was increased to 160C during a period of 10 minutes.
Analysis of the reaction product revealed the boric acid content of 20,'; a pH of the concentrated reaction product was 1.7 and a pH of a 10% aqueous solution of the same was 4.6. The reac-tion product was dyed with methylene blue to color the same.
A glycerol boric acid complex, produced according to Example 2 has been studied to determine if individual sensitivity exists between different bacteria types, at different bacteria counts and in samples that contain many different types of bacteria.
This has been carried out by testing samples both before and after normal conventionally used transport types (less than 24 hours), as well as in samples that have been stored for longer periods of time before initiation of the bacteria growth. It ~as also the intention to individually investigate the different components of the products with regard to anti-bacterial effects and also to try to investigate how large the discrepancies from the instructions that can be tolerated as regards to added amounts of the boric acid containing a preservation agent relative to the amount of urine.
_9_ ~.~40~115~
To this end, two tests have been carried out.
1. Samples have been taken ~rom patients with a urine containing bacte~ia. Samples have been taken by laboratory personnel and by the personnel of a urology department and have been transported by conventional transport means to a laboratory.
In the cases where samples and transport have been carried out by the laboratory personnel, complete control of the sampling technique and the transport time was obtained. Sanlples have been taken after washing and drying - i.e. this so-called "mid-stream"
samples and the bacteria growth was initiated within 1 hour. The bacteria gro~th has been carried out on blood agar plates, Drigalski agar and sorbitol acid agar plates. The quantification has been carried out after bacteria growth with series dilution of urine samples in sodium chloride solutionO The type definition has been carried out by conventional methods after primary incubation under 18 hours at 37C.
2. Laboratory tests with experimentally produced bacteria containing urine to make possible special type tests. For this purpose, eight bacteria types have been employed which are common urinary-tract infection types, namely, Escherichia coli, Klebsiella, Pseudomonas aeruginosa, Pseudomonas mirabilis, Enterokockerr Staphylococcus aureus, Staphylococcus epidermidis, and ~taphylococcus saprophyticus.
.
-,, ' 05~
In trials that demand particularly sensitive test types, gonococci were used.
The research has mainly been carried out as comparisons between samples stored under refrigeration, and at room -tempera-ture, respectively, with boron-glycerol additives.
As a reference value, the growth result that was used was determined after gro~th within one hour after the sampling.
For the tests, hoth pure boric acid in powder form and different concentrations of boric acid complexes according to the example 2 were used~
Experimental bacteriuri-Urine , From bacteria growth on agar plates,a sus~ension was made in a physiolo~ical sodium chloride solution. A c~antifying was made to receive a bacteria amount of between about 100,000 and 10,000,000/ml urine after adding the suspension to uninfected urine.
RESULTS
Tests were carried out with boric acid in powder form;
to 9 ml of urine there was added .1 g of boric acid. The following test types of bacteria were employed accorcling to the following table.
h ~D ~D u~ ~ ~9 u~
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s~ l In Table 1, the results given are the 10th log of the number of bacteria.
The bacteria-containing urine produced according to the method description was quantified and divided into two portions. One portion was placed under temperature conditions of +4C; to tlle other was added boric acid and stored at room temperature. Quantification was carried out after ~4 hours and after 48 hours. From the Table, it will be seen that there are no remarkable difierences regardless of whether urine is stored under refrigeration or at room temperature with the boric acid addition.
Patient samples: Samples were taken from six geriatric patients in a geriatric ward. The samples were grown within one hour whereafter the samples were treated in an analogous method according to the above~ From accompanying Table 2, it will be seen that the differences for refrigerated and boric-acid-containing samples stored at room temperature were less than 11.
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To determine whether glycerol has a lowering or depressing effect on bacteria, the following tests were carried out.
Fifteen freshly isolated bacteria from patients having bacteria in the urine were added to urine which was quantified and then divided into three portions, for storage at +~ C., for storage at room temperature with boric acid being added to it, and for storage at +22C. with additional glycerol. From Table III
it will be seen that the tested bacteria types and the method changes are given to log 10. In none of the cases has there been a lowering through the addition of glycerol. The changes in the refrigerated samples and the room temperature stored samples with boric acid additive, respectively, were in all cases less than 1l. To find out if a negative effect could be ascertained through the combination of boric acid complex according to Example 2 and refrigeration storage samples, the following tests were carried OU L
Flve different bacteria types were added to urine, and the samples were quantified and divided into portions. An untreated urine portion was stored at -20 C. Another portion of the sample had added to it the boric acid complex of ~xample 2 and was stored both at +4C. and at room temperature ~or a further sample. (The further sample contained the complex of Example 2).
~o~
!
Table 3 Growth After*
1 hr. 24 hrs 24 hrs. Boric 24 hrs. Glycerol ~4C Acid +22C +22 C.
Proteus 1 5.8 5.85 6.0 > 9 Proteus 2 5.7 5.8 5.9 > 9 Proteus 3 6.5 6.5 7.1 8.15 Proteus 4 8.05 8.05 8.25 8.20 Proteus 5 8.2 8.0 8.25 8.7 ProLeus 6 6.05 6.3 6.15 > 9 Klebsiella 1 4.75 4.4 4.55 >9 Klebsiella 2 6.7 6.7 6.5 > 9 Klebsiella 3 7.3 8.3 7.5 8.2 Klebsiella 4 6.05 5.7 5.45 ~ 9 Klebsiella S 6.8 6.75 6.9 7.5 Pseudomonas 1 7.5 7.0 7.3 7.8 Pseudomonas 2 7.8 8.0 7.8 8.2 Enterococci 5.35 4.4 5.5 > 9 E coli 7.7 7.7 7.8 8.05 Table 4 ..
Bacteria lnoculation Boric Acid Glycerol Complex 18 hrs.
of Example 2 -20C.
18 hrs -~4C 18 hrs -~22C
E. Coli 5.7 SOS 5.8 3.5 Prot. mira-bilis 6.5 6.0 6.2 2.4 Pseudomonas 6.9 6.1 6.3 3.9 Enterococci 5OS 6O0 6.0 4~2 Staph~ aureus 6~0 6.0 6.0 5.7 *Denoted the amount of time it was stored as indicated and then growth was permitted after that time.
-16_ ~a~sl From the above table, it can be seen that the com-bination of refrigerated storage and the boric acid glycerol complex according to Example 2 does not give a deviating result after storage for 18 hours. Freezing of the urine ~ives com-pletely unreliable quantitative results. The deviations are greater for gram nega~ive bacteria rods than for gram positive cocci.
To determine the glycerol effects on-bacteria, tests were made with a particularly sensitive type. Urine, with and without ~lycerol additives were prepared, to 9 ml of urine, there was added .65 ml of glycerol. Gonococci were added to this urine as well as to the urine which did not contain the glyce!ol additive~
Quantifying was carried out after 1, 2, 4 and 6 hours. From the following Table 5, it will be seen the percentage of the originally added gonococci that could be re~isolated after ths respective time. From the table it w111 be seen that the gonococci bacteria are quickly ~illed in the urine and cannot be found 4 hours after the addition, while addition of glycerol still permits the gonococci bacteria to be isolated after 6 hours.
Table 5 Test Organism- Hours Urine with .65 ml Urine glycerol/~ml Gonococci 1 100% 80~
2 100% 60%
4 40% c1%
6 10% _ c1%
~17-The experiments were carried out using additives of di ferent dyestuffs. Loefflers methylene blue (.5 gram/1000 ml~
was diluted and added to urine. The color and the concentration of .01 ml/9 ml urine was clearly visible to the untrained eye.
To determine the effect of me~hylene blue on bacteria, tests were made with a 10 times stronger solution. Boric acid glycerol complex with and withcut methylene blue, was added to samples.
From Table 6, it is seen that the addition of methylene blue did not detrimentally affect the quantification of the tested bacteria types.
Table 6 . . .
Bacteria type Inoculation After 24 hrs with Without methylene blue Methylene blue ~22C ~22C.
E. coli 5.6 5.5 5.3 Proteus mir. 6.0 5.9 6.0 Pseudomonas aeruginosa 6.0 5.9 5.9 Enterococci 6.2 6.0 5.9 20 Staph. aureus 6.0 6.0 6.2 MARGI~ OF ERROR
To determine the variance permitted from the described method (to determine how much it can deviate from the prescribed method), regarding the amount of urine and the amount of boxic acid complex respectively, the following tests were made with different concentrations.
-18_ ::
.~ .
" ~40~5~L
For the tests, urine with the addition of E-coli bacteria were used. Starting from earlier reports re~arding suitable concentrations of boric acid, after adjustments both on e,~periences, a boric acid with glycerol comple~ was made with 12.4% boric acid. To a norrnal amount of urine sample, 9 ml, was added 9 different amounts varying from .4 to .8 ml (.65 ml = .82%) boric acid. The samples were stoîed at ~22QC
for 24 hours. ~uantification was made both ~rom a 5 month old solution as well as from a freshly prepared solution ~i.e. two different types of solution were used). Changes within a dilution area were less than 11. The boric acid co~plex was pac~;aged in a portion bag by employing .65 ml per package or bag.
The result would probably not be affected if one, by carelessness, did not empty 1/3 o~ the bag contents into the urine sample, which would thereby have a lower concentration of boric acid than was intended. The method of this invention also permits a certain concentration increase by utilizing a smaller amount of urine in the tube. According to estimates, the method is~still effective even though one utilizes 3 to 4 ml less than the recommended 9 ml o~ urine.
Final Tests Final tests were made on bacteria containing urineO
All sampling was carried out using the portioned packages, and transport o~ the samples were made by conventional routines.
One portion of the sample was added with the boro glycerol com-plex and stored at room temperature; the other portion was refri-gerated and refri~eration-transported. Both the test types were stored for 24 hours,thereaf-.er quantification was carried out.
The results are as shown in Table 7.
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0 ~51 In the above table, the 10th log of the number of bacteria after preservation of 9 ml of urine sample with .~5ml boro glycerol complex equivalent to .82~ boric acid at room temperature compared with urine preserved at ~4& .
Significant problems are present using the transportation of samples for bacteriological growth. Unwanted changes take place in growth if not made within very short time after sampling.
Changes can be prevented by refrigeration transport. Refrigeration of samples can sometimes cause freezing of samples which is deleterious for many bacteria types. The refrigeration method is clumsy and uneconomical. The use of boric acid as a preservative in urine samples has been shown earlier and verified by new trials.
The disadvanta~es of boric acid in powder form have initiated attempts to get boric acid in solution. ~he boric acid glycerol complex that has been produced according to Example 2, and methylene blue has been added to color the sample in this example.
Laboratory or clinical test research has shown that the composi-tion according to the invention has had the desired effect.
Changes have not arisen under 48 hours. A large number of differ-ent bacteria types normally causing urine tract infections havebeen tested. No anti-bacterial effects have been found for any of the ingredients at the disage concentration~ Titration of the security margins for wrong usage of the product has shown that one can probably tolerate a 30~ deviation, plus or minus, from the recommended concentration without deleteri~us effects , -22-_ _ Through the concentration of the product, one is not tied to any special tube type, but can flexibly adapt to sample volumes of the tube types that are used by dif~erent laboratoriesr It is evident that applicant's system is con$iderably easier than the conventional refrigeration transport and it eliminates fro~en damage to samples~ From the transportation point of view, the method offers greater conformability and considerable economic savings. A properly carried out refrigera-tion transport has, in practice, shown to be very difficult to achieve. The boric acid complexes of the present invention are not to be confused with similar products produced in the prior art.
Thus, or example, British Patent 511,641 teaches a reaction pro-duct which does nGt have the characteristics of the compounds of the present invention and for ex~ample, such compounds of the afo3~ementioned British Patent are not hydrolyzable in water. More-over, the complexes of the present invention contain between 10 to 20% boric acid which is again different from the prior art compositions.
The products of the present invention, when employed, could be compounded o tabletted according to conventional pro-cedures and as such conventional expedients may be employed.
Still further, mixtu^es of the boric acid complexes could be used according to the invention in a method of preserving and stabilizing urine samples or for the compositions.
It will be understood that various modifications can be made to the ab~ve-described embodiments without departing from the spirit and sco~e of the invention.
r ~
~~3~
~ . -
From the above, it will be seen that in practice, one cannot work with a solution higher than a 3% concentration o~ boric acid to avoid crystallization during temperature changes. Addition-ally, for economic reasons, it is also desirable to use a dosage unit which is as small as possible for transportation purposes, and to to this end, standardized transport containers with a volume of 10 ml are the most common ones in usage.
h~hen using a 3% boric acid solution, 13 rnilliliters must be added to 9 ml of urine to achieve a concentration of 1.8% boric acid which is necessary for the boric acid to act as a preservative.
This means that a large amount of water is added to the sample and ~0 that the resulting volume increases to more than double.
. _.
s~
With this invention, applicant has developed a novel series of compounds which have preservation properties for urine samples, and in addition, a method of preparing such compounds~
Still ~urther, there is also provided a method of using such compounds to stabilize urine samples during transpor-tation to laboratories and as well, a composition containing a functional preservation agent for urine samples which contain the novel com~
pounds.
More particularly, in accordance with the present inven-tion, there are provided novel boric acid complexes of the formula [ \ / \ ~ [ ~ ~ \ / R ¦ H
wherein the group R represents a member selected from ethyleneglycol; propanediol-1,2; glycerol fructose; mannitol; sorbitol;
and as well, other water-soluble dioxy or polyoxy complexes of the above formula. In accordance with a still fur-ther aspect o~ the invention, there is pxovided a method of preparing the above bor.ic acid complexes which comprises heating boric acid with the afo.re-mentioned dioxy or polyoxy compounds including ethylene glycol,propanediol-1,2; glycerol, fructose; mannitol; or sorbitol for a relatively short period of time and until 1 to 2 moles of reaction water have been driven off, whereby water-soluble complexes of the above formu~a are obtained.
The above compounds have been Found to achîeve a more efficient and effective preservation of the bacteria in urine samples and may be employed in relatively low amounts compared to boric acid per se. More particularly, in accordance with a further aspect of this invention, it has been found that the above ~ .~
compounds function as an effective preservative for the bacteria ` _, !
in urine samples employing between about .5 to about .8% boric acid utilized in the form of the aforementioned boric acid com-plexesO In the compounds of the present invention, the concentra-tion of boric acid is relatively high and the addition of between about .4 to about 1.4 ml to 9 ml of urine has been -~ound to be sufficient. Thus, in accordance with the present invention, aompared to samples which contain no preservation or stabili~ing agent, the volume of the urine samples utilizing the complexes of this invention are only increased in a very limited manner so that standardized transport tubes or containers may be utilized for this purpose. Thus, in brief summary of the latter aspect of the invention, there is provided a method o~ preserving samples, par-ticularly urine samples, on which bacteriological laboratory tests are to be subsequently carried out, characterized in that a boric acid complex of the above formula is employed as a preservation or stabilization agent by adding a compound of thc above formula to the sample in an amount o~, e.g. .5 to about .8% boric acid in the form of the above complex.
In the above-described method of producing the boric acid complexes, the preferred aspects of a method according to the present invention as outlined comprise heating the -eactants within predetermined temperature and time limitations, so as to evolve a predetermined amount of water from the reaction products produced -i.e. between 1 and 2 moles whereby the resulting products are water-soluble - i.e. they have a predetermined wa~er concentration in the reaction product. The concentration of the boric acid in the complexes can be regulated by an additional amount of the complexes o- by the water thai is included in the complex.
Preferred embodiments of the reaction comprise reac'ing --30 the rcactants at a temperature in the range o~ lrom 80 to 160C, -6~
pre~erably in the range of fr~m 110-129C. The re~ction is carried out over a relatively short period of time, within about 1 to 12 minutes and preferably within 3 to 4 minutes. The boric acid em-ployed in the reaction is well known per se; preferred reactants comprise the compounds ethylene glycol, propanediol-1,2, glycerol, fructose, mannitol and sorbitol or other like di- or polyols which give -the aforementioned wa~er-soluble complexes and the corres-ponding residues of this groups in those complexes. The parti-cular choice o~ reactant which is utilized with the boric acid can be chosento provide a reaction product which, in addition to being ~ater-soluble, has wax-li]ce properties. Pre~erably, the react~n-ts are chosen so as to obtain a reaction product havins between 10 to 20Y of boric acid and desirably 11 to 13% oE boric acid concentration in the complex~
If desired, various additives may be incl~lded with the complexes for preparing compositions and for use in -the method of the present invention; such additives may include, -or example, coloring agents to identify the inclusion of an additiv~ in a urine sample - e.gO coloring agents such as methylene blue or other like and equivalent additives may be employed. It will be under~
stood by those skilled in the art that any such additives con-ventionally used for this purpose may be employed provided that they do not interfere with subsequent laboratory examination pro-cedures or do not adversely in*luence the bacteria in the samples.
T~en a coloring agent is employed, such as methylene blue, the concentration may be in the order of from 5 to 50 par-ts per million (ppm).
In addition, in the method o* the present invention, the comple~es are preferably added to the sample in an amount of .2 ~ %, desirably .~ - .8% boric acid concentration. I.i~tures ~v~s~
OI the boric acid comple.Yes may be employecl in the method for stabilizing the samples.
Having thus ~enerally described the invention, reference will now be made to the accompanying Examples, illustrating pre-ferred embodiments and preferred techniques.
In this example, 100 parts by weight of ethylene glycol was employed together with 17 par~s by weight of boric acid (BP).
This mixture was heated to 9~C, during which reaction water was lQ formed. Thereafter, the temperature was raised to 112C for a period o four minutes. It was found that 2 moles of water were evolved. The product was found to contain 17.7% boric acid; it had a pH in the concentrated reaction product of 2.5, while a 10%
aqueous solution had a pH of a . a.
EXAMP~E 2 In this example, 124 parts by-weight of glycerol DAB d-1.23 and 17 parts by weight of boric acid (BP) were mixed and heated to 110C, whereupon the reaction was initiated and 2 mol2s o~ water was evolved. The amount of water increased in the reaction mixtu-e f-om 11.3% to 22.3%. The temperature was raised ~or a period of 3 minutes to 129C whereafter the heating was stopped and the reaction product was allowed to cool down. The reaction product contained 15.5% water; 12.4% boric acid concentration and a pH, in the concentrated product, o 1.3. A 10% aqueous sol~ltion of the reaction product had a pH o 3.9.
Methylene blue, a coloring agent, was added in a con-centxation of 5 to 50 parts per million. ~ater is present in the above Ex~ample, as in all of theother Examples, as it is created during the reaction or is included withthe reactants, -e.g. in accompanying Example 4- propanediol~ contains 5% wa~er.
~ 8 ~ QOS~L
EX~LE 3 100 parts by weight of fructose, 150 parts by weight of water, and 36 parts by weight of boric acid were-heated to 80C, whereafter the temperature was increased in 6 minutes to 190C, resulting in 2 moles of water being driven of~.
The boric acid content was determined to be 12.1%; the pH of ~he concentrated reaction product was 1.1; and the pH of a 10% aqueous solution of the product was 4.2. The reaction product was pressed into tablets.
~XAMPLE 4 100 parts by weight of 1,2-propanediol and 25 parts by weight of boric acid were heated to 98& , wherea~ter the tem-perature was increased to 160C during a period of 10 minutes.
Analysis of the reaction product revealed the boric acid content of 20,'; a pH of the concentrated reaction product was 1.7 and a pH of a 10% aqueous solution of the same was 4.6. The reac-tion product was dyed with methylene blue to color the same.
A glycerol boric acid complex, produced according to Example 2 has been studied to determine if individual sensitivity exists between different bacteria types, at different bacteria counts and in samples that contain many different types of bacteria.
This has been carried out by testing samples both before and after normal conventionally used transport types (less than 24 hours), as well as in samples that have been stored for longer periods of time before initiation of the bacteria growth. It ~as also the intention to individually investigate the different components of the products with regard to anti-bacterial effects and also to try to investigate how large the discrepancies from the instructions that can be tolerated as regards to added amounts of the boric acid containing a preservation agent relative to the amount of urine.
_9_ ~.~40~115~
To this end, two tests have been carried out.
1. Samples have been taken ~rom patients with a urine containing bacte~ia. Samples have been taken by laboratory personnel and by the personnel of a urology department and have been transported by conventional transport means to a laboratory.
In the cases where samples and transport have been carried out by the laboratory personnel, complete control of the sampling technique and the transport time was obtained. Sanlples have been taken after washing and drying - i.e. this so-called "mid-stream"
samples and the bacteria growth was initiated within 1 hour. The bacteria gro~th has been carried out on blood agar plates, Drigalski agar and sorbitol acid agar plates. The quantification has been carried out after bacteria growth with series dilution of urine samples in sodium chloride solutionO The type definition has been carried out by conventional methods after primary incubation under 18 hours at 37C.
2. Laboratory tests with experimentally produced bacteria containing urine to make possible special type tests. For this purpose, eight bacteria types have been employed which are common urinary-tract infection types, namely, Escherichia coli, Klebsiella, Pseudomonas aeruginosa, Pseudomonas mirabilis, Enterokockerr Staphylococcus aureus, Staphylococcus epidermidis, and ~taphylococcus saprophyticus.
.
-,, ' 05~
In trials that demand particularly sensitive test types, gonococci were used.
The research has mainly been carried out as comparisons between samples stored under refrigeration, and at room -tempera-ture, respectively, with boron-glycerol additives.
As a reference value, the growth result that was used was determined after gro~th within one hour after the sampling.
For the tests, hoth pure boric acid in powder form and different concentrations of boric acid complexes according to the example 2 were used~
Experimental bacteriuri-Urine , From bacteria growth on agar plates,a sus~ension was made in a physiolo~ical sodium chloride solution. A c~antifying was made to receive a bacteria amount of between about 100,000 and 10,000,000/ml urine after adding the suspension to uninfected urine.
RESULTS
Tests were carried out with boric acid in powder form;
to 9 ml of urine there was added .1 g of boric acid. The following test types of bacteria were employed accorcling to the following table.
h ~D ~D u~ ~ ~9 u~
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'`
s~ l In Table 1, the results given are the 10th log of the number of bacteria.
The bacteria-containing urine produced according to the method description was quantified and divided into two portions. One portion was placed under temperature conditions of +4C; to tlle other was added boric acid and stored at room temperature. Quantification was carried out after ~4 hours and after 48 hours. From the Table, it will be seen that there are no remarkable difierences regardless of whether urine is stored under refrigeration or at room temperature with the boric acid addition.
Patient samples: Samples were taken from six geriatric patients in a geriatric ward. The samples were grown within one hour whereafter the samples were treated in an analogous method according to the above~ From accompanying Table 2, it will be seen that the differences for refrigerated and boric-acid-containing samples stored at room temperature were less than 11.
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s~
To determine whether glycerol has a lowering or depressing effect on bacteria, the following tests were carried out.
Fifteen freshly isolated bacteria from patients having bacteria in the urine were added to urine which was quantified and then divided into three portions, for storage at +~ C., for storage at room temperature with boric acid being added to it, and for storage at +22C. with additional glycerol. From Table III
it will be seen that the tested bacteria types and the method changes are given to log 10. In none of the cases has there been a lowering through the addition of glycerol. The changes in the refrigerated samples and the room temperature stored samples with boric acid additive, respectively, were in all cases less than 1l. To find out if a negative effect could be ascertained through the combination of boric acid complex according to Example 2 and refrigeration storage samples, the following tests were carried OU L
Flve different bacteria types were added to urine, and the samples were quantified and divided into portions. An untreated urine portion was stored at -20 C. Another portion of the sample had added to it the boric acid complex of ~xample 2 and was stored both at +4C. and at room temperature ~or a further sample. (The further sample contained the complex of Example 2).
~o~
!
Table 3 Growth After*
1 hr. 24 hrs 24 hrs. Boric 24 hrs. Glycerol ~4C Acid +22C +22 C.
Proteus 1 5.8 5.85 6.0 > 9 Proteus 2 5.7 5.8 5.9 > 9 Proteus 3 6.5 6.5 7.1 8.15 Proteus 4 8.05 8.05 8.25 8.20 Proteus 5 8.2 8.0 8.25 8.7 ProLeus 6 6.05 6.3 6.15 > 9 Klebsiella 1 4.75 4.4 4.55 >9 Klebsiella 2 6.7 6.7 6.5 > 9 Klebsiella 3 7.3 8.3 7.5 8.2 Klebsiella 4 6.05 5.7 5.45 ~ 9 Klebsiella S 6.8 6.75 6.9 7.5 Pseudomonas 1 7.5 7.0 7.3 7.8 Pseudomonas 2 7.8 8.0 7.8 8.2 Enterococci 5.35 4.4 5.5 > 9 E coli 7.7 7.7 7.8 8.05 Table 4 ..
Bacteria lnoculation Boric Acid Glycerol Complex 18 hrs.
of Example 2 -20C.
18 hrs -~4C 18 hrs -~22C
E. Coli 5.7 SOS 5.8 3.5 Prot. mira-bilis 6.5 6.0 6.2 2.4 Pseudomonas 6.9 6.1 6.3 3.9 Enterococci 5OS 6O0 6.0 4~2 Staph~ aureus 6~0 6.0 6.0 5.7 *Denoted the amount of time it was stored as indicated and then growth was permitted after that time.
-16_ ~a~sl From the above table, it can be seen that the com-bination of refrigerated storage and the boric acid glycerol complex according to Example 2 does not give a deviating result after storage for 18 hours. Freezing of the urine ~ives com-pletely unreliable quantitative results. The deviations are greater for gram nega~ive bacteria rods than for gram positive cocci.
To determine the glycerol effects on-bacteria, tests were made with a particularly sensitive type. Urine, with and without ~lycerol additives were prepared, to 9 ml of urine, there was added .65 ml of glycerol. Gonococci were added to this urine as well as to the urine which did not contain the glyce!ol additive~
Quantifying was carried out after 1, 2, 4 and 6 hours. From the following Table 5, it will be seen the percentage of the originally added gonococci that could be re~isolated after ths respective time. From the table it w111 be seen that the gonococci bacteria are quickly ~illed in the urine and cannot be found 4 hours after the addition, while addition of glycerol still permits the gonococci bacteria to be isolated after 6 hours.
Table 5 Test Organism- Hours Urine with .65 ml Urine glycerol/~ml Gonococci 1 100% 80~
2 100% 60%
4 40% c1%
6 10% _ c1%
~17-The experiments were carried out using additives of di ferent dyestuffs. Loefflers methylene blue (.5 gram/1000 ml~
was diluted and added to urine. The color and the concentration of .01 ml/9 ml urine was clearly visible to the untrained eye.
To determine the effect of me~hylene blue on bacteria, tests were made with a 10 times stronger solution. Boric acid glycerol complex with and withcut methylene blue, was added to samples.
From Table 6, it is seen that the addition of methylene blue did not detrimentally affect the quantification of the tested bacteria types.
Table 6 . . .
Bacteria type Inoculation After 24 hrs with Without methylene blue Methylene blue ~22C ~22C.
E. coli 5.6 5.5 5.3 Proteus mir. 6.0 5.9 6.0 Pseudomonas aeruginosa 6.0 5.9 5.9 Enterococci 6.2 6.0 5.9 20 Staph. aureus 6.0 6.0 6.2 MARGI~ OF ERROR
To determine the variance permitted from the described method (to determine how much it can deviate from the prescribed method), regarding the amount of urine and the amount of boxic acid complex respectively, the following tests were made with different concentrations.
-18_ ::
.~ .
" ~40~5~L
For the tests, urine with the addition of E-coli bacteria were used. Starting from earlier reports re~arding suitable concentrations of boric acid, after adjustments both on e,~periences, a boric acid with glycerol comple~ was made with 12.4% boric acid. To a norrnal amount of urine sample, 9 ml, was added 9 different amounts varying from .4 to .8 ml (.65 ml = .82%) boric acid. The samples were stoîed at ~22QC
for 24 hours. ~uantification was made both ~rom a 5 month old solution as well as from a freshly prepared solution ~i.e. two different types of solution were used). Changes within a dilution area were less than 11. The boric acid co~plex was pac~;aged in a portion bag by employing .65 ml per package or bag.
The result would probably not be affected if one, by carelessness, did not empty 1/3 o~ the bag contents into the urine sample, which would thereby have a lower concentration of boric acid than was intended. The method of this invention also permits a certain concentration increase by utilizing a smaller amount of urine in the tube. According to estimates, the method is~still effective even though one utilizes 3 to 4 ml less than the recommended 9 ml o~ urine.
Final Tests Final tests were made on bacteria containing urineO
All sampling was carried out using the portioned packages, and transport o~ the samples were made by conventional routines.
One portion of the sample was added with the boro glycerol com-plex and stored at room temperature; the other portion was refri-gerated and refri~eration-transported. Both the test types were stored for 24 hours,thereaf-.er quantification was carried out.
The results are as shown in Table 7.
.. .
~ Si ~ ~ *
r~ X O ~ O O ~ ~D ~ O 11~ o ~-1 Q
5-1 C) O ~d~ N O ~9 N 1~ 0 ~D ~D r-l O
~r-l ~ ~
r l ~/ ~D1~tlO t~ ~9 1~ ~9 N 1~ ~ D CO 0 E~
O S~
r~ ~
'a)~ *'' *' ~) O O O 1~ N O ~r) o O O O O 1~ Q ~ Il) U !~1 N C ) ~ D N (O ~ 1~11-1C~ rl N U) ~1 ~ O ~ ~
m m + ~
..
~ ~ s~ 'u UI ~
rl r l ~ U U~1 ~ ~r~ ~~rl U~ ~rl rl ~ 1 U U ~q r~ rl rlr-l ~5 r-l rlrl r l O ~ rl r-l u o u~ o ~ ~ o o oo s~ o ro ~ ,D U Q, ~ U U U U (~) ~ ~R C) m ~ ,a o ~ o ~ ~ ~ h ~ r~ -~` .
Q
E~
X
u~ h :~: h 1~ h h h h ~ h ~D ~ O ~ 0~
~1 ~ r) rl N~ N N r-lr-l O ~1~) O~ ~I N O O N N ~:P
~1 0 0 0 0 ~I rl r~ O ~ O
.,1 ~D O ~S) ~ I~ ~~:) CD ~I N~) m a~ o ~o ,a) u r~ N ~ d' U') ~0 1~0:) ~ O ,~1 ~1 r1r-l --20_ 5~
a~ . ~
~ ~ lD ~ O O a) ~ I` ~ ~ I` o ,1 X O ~ ~ o r~ I~ o h O ~
~ In d' ~) 1- ul 1~ OD 1` ~9 1` U~
~ O
O h o U
ra :~
,1 ~
*
O ot~. U~ U) ~ CO O 0 1~ 1~ ~ ~` O
U h ~ 0~ ~1 0 ~r ~ r-l o ~ o ~ ~ O
m m ~ ~
.
h ~ tJ~
Ill U ~ U ~ U rl ~1 Ou n~O ~J O E~
QlQ U ~ ~1 U
~ U ~ O~ O -~ ~1 0 _ a ~ h ~ t~ 0 5~
u a) ~ ~ ~:4 ~ O ~ ~HO ~ C~ ~ O O ~ , O h ~ rl h ~ h 5 J ~ h t~ P~ h 1` ~ U
Q~ X rQ
~ . a) ~ ~ .
. h 1~
. _ . ,~ ~
h $ U ~
. td U7 In ~
, Q o o o Q ~o h O '~ O Q :~
, m o ~ 0 ~ Q
, u ~
/ , ~ ~ h ~ ~Z; ~C *
:~' ~
: / ~: , :, .~
. ~ (~ N ~) ~ 111 : / ~ ~
0 ~51 In the above table, the 10th log of the number of bacteria after preservation of 9 ml of urine sample with .~5ml boro glycerol complex equivalent to .82~ boric acid at room temperature compared with urine preserved at ~4& .
Significant problems are present using the transportation of samples for bacteriological growth. Unwanted changes take place in growth if not made within very short time after sampling.
Changes can be prevented by refrigeration transport. Refrigeration of samples can sometimes cause freezing of samples which is deleterious for many bacteria types. The refrigeration method is clumsy and uneconomical. The use of boric acid as a preservative in urine samples has been shown earlier and verified by new trials.
The disadvanta~es of boric acid in powder form have initiated attempts to get boric acid in solution. ~he boric acid glycerol complex that has been produced according to Example 2, and methylene blue has been added to color the sample in this example.
Laboratory or clinical test research has shown that the composi-tion according to the invention has had the desired effect.
Changes have not arisen under 48 hours. A large number of differ-ent bacteria types normally causing urine tract infections havebeen tested. No anti-bacterial effects have been found for any of the ingredients at the disage concentration~ Titration of the security margins for wrong usage of the product has shown that one can probably tolerate a 30~ deviation, plus or minus, from the recommended concentration without deleteri~us effects , -22-_ _ Through the concentration of the product, one is not tied to any special tube type, but can flexibly adapt to sample volumes of the tube types that are used by dif~erent laboratoriesr It is evident that applicant's system is con$iderably easier than the conventional refrigeration transport and it eliminates fro~en damage to samples~ From the transportation point of view, the method offers greater conformability and considerable economic savings. A properly carried out refrigera-tion transport has, in practice, shown to be very difficult to achieve. The boric acid complexes of the present invention are not to be confused with similar products produced in the prior art.
Thus, or example, British Patent 511,641 teaches a reaction pro-duct which does nGt have the characteristics of the compounds of the present invention and for ex~ample, such compounds of the afo3~ementioned British Patent are not hydrolyzable in water. More-over, the complexes of the present invention contain between 10 to 20% boric acid which is again different from the prior art compositions.
The products of the present invention, when employed, could be compounded o tabletted according to conventional pro-cedures and as such conventional expedients may be employed.
Still further, mixtu^es of the boric acid complexes could be used according to the invention in a method of preserving and stabilizing urine samples or for the compositions.
It will be understood that various modifications can be made to the ab~ve-described embodiments without departing from the spirit and sco~e of the invention.
r ~
~~3~
~ . -
Claims (14)
1. A method of stabilizing urine samples, for preser-vation during transportation comprising adding to the sample an effective amount of a boric acid complex of the formulae or wherein R represents the residue of a group derived from a member selected from the group consisting of ethylene glycol, 1,2-propanediol, glycerol, fructose, mannitol, sorbitol and the corresponding water-soluble di- or polyoxy derivatives thereof after reaction with boric acid, said boric acid complex con-taining between about 10% to about 20% boric acid and having between 1 to 2 moles of water evolved from the reaction product.
2. A method according to claim 1, wherein a mixture of boric acid complexes are employed.
3. A method as defined in claim 1, wherein said complex is added to the sample in an amount of from .2 - 1.4% boric acid concentration.
4. A method as defined in claim 1, wherein said complex is added to the sample in an amount of from about .4 - .8% boric acid concentration.
5. A method as defined in claim 1, wherein the boric acid complex contains a colouring additive.
6. A method as defined in claim 5, wherein methylene blue is employed as an additive in an amount of from about 5 to about 50 ppm.
7. A method as defined in claim 1 wherein said boric acid complex is added in the form of a tablet derived from the reaction of boric acid with a polyoxy complex including mannitol and fructose yielding a wax-like substance.
8. A method as defined in claim 1, wherein said boric acid complex contains between 11 to 13% boric acid.
9. The composition suitable for use as a stabilizing agent for urine samples comprising a boric acid complex of the formulae wherein R represents the residue of a group derived from a member selected from the group consisting of ethylene glycol, 1,2-propanediol, glycerol, fructose, mannitol, sorbitol and the corresponding water-soluble di- or polyoxy derivatives thereof after reaction with boric acid, said boric acid complex containing between about 10% to about 20% boric acid and having between 1 to 2 moles of water evolved from the reaction product.
10. A composition as defined in claim 9 containing a mixture of said boric acid complexes.
11. The composition of claims 9 or 10 wherein the boric acid complex contains a colouring additive.
12. The composition of claim 9 or 10 wherein methylene blue is employed as a colouring additive in an amount of from about 5 to 50 ppm.
13. The composition of claim 9 or 10 wherein said composition is in the form of a tablet derived from the reaction of boric acid with a polyoxy complex including mannitol and fructose yielding a wax-like substance.
14. The method of claim 9 or 10 wherein said boric acid complex contains between 11 to 13% boric acid.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE7802147-4 | 1978-02-24 | ||
SE7802147A SE419346B (en) | 1978-02-24 | 1978-02-24 | PROCEDURE FOR STABILIZING BACTERIOLOGICAL SAMPLES |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1140051A true CA1140051A (en) | 1983-01-25 |
Family
ID=20334100
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000321844A Expired CA1140051A (en) | 1978-02-24 | 1979-02-16 | Boric acid complexes and their use as functional preservation agents |
Country Status (7)
Country | Link |
---|---|
CA (1) | CA1140051A (en) |
DE (1) | DE2906730C2 (en) |
DK (1) | DK80479A (en) |
FI (1) | FI63595C (en) |
GB (1) | GB2016446B (en) |
NO (1) | NO147054C (en) |
SE (1) | SE419346B (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8800502D0 (en) * | 1988-01-11 | 1988-02-10 | Cerestar Holding Bv | Method of adding borax/boric acid to mixing/reaction-zone |
US5505953A (en) | 1992-05-06 | 1996-04-09 | Alcon Laboratories, Inc. | Use of borate-polyol complexes in ophthalmic compositions |
FR2731008B1 (en) * | 1995-02-24 | 1999-05-07 | Dev Activites Chimiques Distri | AQUEOUS BOREAL SOLUTION, PARTICULARLY FOR THE ADJUVANTATION OF AMYLACEOUS ADHESIVE |
WO2008131499A1 (en) * | 2007-04-26 | 2008-11-06 | Realco Sa | Method for treating banana leafspot disease and treatment composition |
US9102700B1 (en) * | 2014-08-29 | 2015-08-11 | Vdf Futureceuticals, Inc. | Compositions and methods for borocarbohydrate complexes |
-
1978
- 1978-02-24 SE SE7802147A patent/SE419346B/en unknown
-
1979
- 1979-02-16 NO NO790518A patent/NO147054C/en unknown
- 1979-02-16 CA CA000321844A patent/CA1140051A/en not_active Expired
- 1979-02-19 FI FI790541A patent/FI63595C/en not_active IP Right Cessation
- 1979-02-21 GB GB7906062A patent/GB2016446B/en not_active Expired
- 1979-02-21 DE DE2906730A patent/DE2906730C2/en not_active Expired
- 1979-02-23 DK DK80479A patent/DK80479A/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
DE2906730C2 (en) | 1982-04-22 |
NO147054C (en) | 1983-01-26 |
FI63595C (en) | 1983-07-11 |
NO147054B (en) | 1982-10-18 |
DK80479A (en) | 1979-08-25 |
GB2016446A (en) | 1979-09-26 |
NO790518L (en) | 1979-08-27 |
SE419346B (en) | 1981-07-27 |
DE2906730A1 (en) | 1979-09-06 |
FI790541A (en) | 1979-08-25 |
GB2016446B (en) | 1982-12-01 |
SE7802147L (en) | 1979-08-25 |
FI63595B (en) | 1983-03-31 |
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