IL22494A

IL22494A - Controlling the growth of micro-organisms

Info

Publication number: IL22494A
Application number: IL22494A
Authority: IL
Original assignee: Crown Zellerbach Corp
Priority date: 1964-02-10
Filing date: 1964-11-23
Publication date: 1968-03-28
Also published as: ES306378A1; NL6414294A; CH490001A; BE644614A; FR1518576A; GB1105749A; DE1492309A1; BR6465100D0; SE331768B; ES310544A1

Description

The present invention concerns a method for con-trolling the growth of microorganisms, compositions for effecting such control and processes for the preparation of these compositions. This invention particularly concerns the con-trol of microorganisms in an improved manner with known bio-cldal and biostatic agents.

It has been found that a control of microorganisms is obtained by contacting them with an effeotive amount of a dialkyl sulfoxide, in particular dimethyl sulfoxide. The growth of various microorganisms is stopped or markedly reduced in various cultures when an effective amount of dimethyl sulfoxide is placed in such cultures alone or together with known biooides or biostats.

The present invention provides a composition for controlling the growth of microorganisms which comprises an effeotive amount of a dialkyl sulfoxide. This composition may also contain a known blooide or biostat. It may also be combined with a carrier suitable for contacting the microorganisms The present invention also provides a method for controlling the growth of microorganisms by contacting the microorga isms with an effective amount of a composition comprising a dialkyl sulfoxide.

The present invention further provides a process for the preparation of a composition for controlling the growth of microorganisms by bringing a dialkyl sulfoxide into a form suitable for contacting microorganisms.

When the phrase "control of microorganisms'1 is used it may refer to a composition where blocidal or biostatio effects of known agents are enhnaoedj it may also refer to making resistant strains of microorganisms susceptible to known blo- antifungal action of* an agent which will be disclosed; and it may also refer to enhancing growth of vegetative form fungi, yeast or "baoteria under certain conditions.

Dimethyl sulfoxide is the preferred dialkyl sulfoxide and the invention will be desoribed in terms of this compound although various other dialkyl sulfoxides such as methyl ethyl sulfoxide, diethyl sulfoxide, methyl n-propyl sulfoxide and dibutyl sulfoxide may be used. Dimethyl sulfoxide is particularly preferred because it is highly effective and has very low toxicity.

Dimethyl sulfoxide is a water clear, water mlsoible, hygroscopic neutral organic liquid melting at about 18.5°°· and boiling at about l89°C. The compound is represented by the formula? 0 CH5-S-CH5 Dimethyl sulfoxide may be prepared by various methods such as described in U.S. 2,581,050, U.S. 2,955,555 and U.S. 2,702,824.

Tke effective amount of dimethyl sulfoxide to be used to stop growth in the culture stage is in part determined by the identity of a particular strain of microorganisms. Industrial control of microorganisms is used in general slimicide control, control of growth in recycling water as in air condi-tloning units and in protection of fibers in the textile industry. It will be recognized that critical amounts cannot be pre-established because this will depend on the particular conditions of the industrial operation, suoh as exposure to elements, content, volume and amounts.

Dimethyl sulfoxide in compositions with known bio- such agents. The mechanism of action is not understood hut it is supposed that increased penetration of the microorganism may be effective.

Dimethyl sulfoxide in compositions renders previous-ly resistant strains of microorganisms susceptible to the antimicrobial action of known agents. The mechanism of aotion is also not understood, but again is believed to be somehow related to inoreased penetration of the mioroorganlsm. The microorganism to be controlled include bacteria, fungi and viruses.

Vegetative growth of microorganisms is enhnaced with effectively small amounts of dimethyl sulfoxide. It has been found that spore growth is depressed while the vegetative growth Is stimulated. Certain products of inoreased vegeta-tive growth may be highly desirable such as antibiotics, alcohol and acetone. The amounts of dimethyl sulfoxide which stimulate vegetative growth is small and is generally at least about 20 ppm to 5OO ppm. The property of stimulated growth is demonstrated with amounts of dimethyl sulfoxide of about less than J% by weight. The stimulated vegetative growth is shown with fungi such as Aspergillus nlger f Penlollllum notatum and Penicillium digi atum: with true yeasts of the Endomyceticeal family, such as Brewer's yeast or Saochromyces oerevisiaej and with bacteria such as B. amylobaoter whloh is used in the acetone-baoterial fermentation process.

The following examples are presented to illustrate various embodiments of the invention. 5EXAMPLE 1 Control with Different Conoentratione , a Dimethyl Sulfoxide Lactic Acid parts by weight Total Nitrogen 0.7 parts Amino Nitrogen O.J parte Reduoing Sugars 0.7 parts Inert Solids 2.1 parts Potassium dlhydrogen phosphat 0.1 parts Magnesium Sulfate O.OO5 parts Water Sufficient to make 100 parts.

Two batches of fortified corn steep broth were prepared according to the foregoing formulation and strains of Penicilllum notatum were cultured in one batch and strains of Aspergillus nlger were oultured in the second batch. These strains fall within the fungi class of filamentous asoomyoetes. To each culture in each group was added dimethyl sulfoxide in sequential amounts which increase incrementally. The first addition was 500 ppm and then 10,000 ppm, whloh was equivalent to 1% by weight of the culture. Thereafter, the amount was increased by 1% through a series starting at 1% and ending at $. The culture conditions otherwise remain standard.

Concentrations of the microorganisms in the broth were determined after each addition. It was shown that concentrations of dimethyl sulfoxide up to about % measurably enhanced vegetative growth with a maximum yield of 16$ compared to a control. The spore production was found to be lowered with incremental increases of dimethyl sulfoxide concentrations. Above 5$ dimethyl sulfoxide levels the growth was depressed being at the lowest level in the highest dimethyl sulfoxide culture concentration of 10$. Enhanced vegetated growth may1 therefore be obtained with lower concentrations in the culture. Higher concentrations tend to inhibit growth of vegetative form of fungi in the culture.

EXAMPLE 2 Controls of Microorganisms on Wood Pulp Substrate A mixed culture of slime forming organisms such as E- ooli. B. sub tills, and A. aerobe ns ■ which are resistant to phenylmercuricaoetate (PMA) were eulturod on a ground wood pulp substrate . This is a dilute water slurry of raw ground wood containing wood pulp, dissolved sugars, low MW lignins, proteins etc. One liter aliquots of pulp were contacted with PMA alone, PMA plus dimethyl sulfoxide, dimethyl sulfoxide alone and a final aliquot served as a control. Each aliquot had a O.J% solids pulp and contained total microorganism counts in excess of 500,000 per ml. he following table shows the results: Total Mloroorganlsm Count per ml. Treatment 1 hour 12 hours 24 hours 3 Pm ot PMA 180,000 579,000 500,000 3 ppm of PMA plus 20 ppm of dimethyl sulfoxide 2,^10 47,600 500,000 ppm of dimethyl sulfoxide 500,000 500,000 500,000 Control - no treatment 500,000 500,000 500,000 The time in hours refers to the span from addition of treatment to plating. The plating medium was Sabourauds Dextrose Agar (Difco B) . The incubation of the plates was conducted for 24 hours at 3 °C. The example clearly shows that dimethyl sulfoxide enhanced or potentiated the effect of PMA, by, apparently, reducing the subsequent activity or affinity of PMA for the pulp substrate which would deactivate EXAMPLE Control of Microorganisms with Dimethyl Sulfoxide Different groups of cultures containing Bacillus subtilis . a spore forming soil borne bacteria and Bacherichia coli. an intestinal bacteria, were prepared. Into each culture was introduced a 0% solution of dimethyl sulfoxide. The mixtures were maintained for 24 hours and then the mixtures were diluted to with sterile water. The diluted mixture was plated using a nutrient agar. The plates were incubated at 57°0. for 48 hours, examined and counts were made. The plates involving E. coll were sterile, and plates involving subtllls contained only one colony of a foreign organism B. mycoides, whioh was believed to have been an air introduced contaminant. Dimethyl sulfoxide at 5C# concentration therefore showed blocidal or biostatic activity.

EXAMPLE 4 Control of Microorganism with Dimethyl Sulfoxide The tuberole bacillus, Mycobacterium tuberculosis was cultured in a standard liquid medium. Dimethyl sulfoxide was introduced to the medium so that it was present in a concentration of 15 by volume basis. Growth of the bacillus was completely inhibited. This proved to be bacteriostosls .

EXAMPLE 5 Control of Microorganism with Dimethyl Sulfoxide Different cultures were prepared containing E. coll. Staphylococcus aureus, and Pseudomonas aeruginosa. In eaoh culture was placed 10% by volume of aqueous dimethyl sulfoxide. Complete inhibition of growth occurred in eaoh culture. Transfers from these treated cultures were made to fresh standard culture mediums and prolific growth occurred in each new culture medium.

The example Illu strates that dime thyl sulfoxide is bacteriostatic against these strains of microorganisms . The Ε · bacteria is very common and widely distributed. It is an excellent and ubuquitous scavenger, hydrolizing a wide variety of proteins , fats , carbohydrates , and other organic compounds . They are found In soil, fresh waters and ocean waters and decomposing organic matter inoluding sewage . The 8 · aureus is a pathogenic microoocus and is distinguished by its golden yellow pigment .

EXAMPLE 6 Control of Stony Pit Virus in Pear Trees Six pear trees were uniformly infected with a virus causing pear fruit malformation termed "Stony Pit" . The fruit of the se trees was graded in the season of 196l and found to bear more than 95% infected fruit . Such fruit shows at least one visible surface blemish with tissue pit . The first group of two trees was a control and re ceived 100 ml of water . The second group of two trees re ceived 50 ml of water and 0 ml of dimethyl sulfoxide , and the third group of two trees rece ived 75 ml of water and 2 ml of dime thyl sulfoxide .

The trees were treated by directly injecting 100 ml of liquid into the Xylem under a hydrostatic head of 4 feet to force in the liquid. The total amount of liquid was injected in 24 hours or le ss , once per month in the two growing seasons of April to October , 1962 and 1963.

After the 1962 season, there was considerable symptomatic relief of the second group of 50$ dimethyl sulfoxide , and only minor relief with the third group rece iving 2 $ dimethyl 4*" After the Ι9β3 'season, more than 95% of the fruit in the first or control group was infected, only 4. $ of the fruit in the second group on 0$ dimethyl sulfoxide , and 47$ in the third group of 2$% dimethyl sulfoxide .

The foregoing example illustrate s the ant iviral effect of dimethyl sulfoxide .

EXAMPLE 7 Control of Leaf Mosaic Strawberry plants free of virus signs were art if 1-cially infected with a virus causing the disease , leaf mosaic . A known antiviral agent , 6-mercaptopurlne , and dimethyl sulfoxide were administered under controlled conditions . Applications of the liquids were made to four groups of three plants per group with an air powered de Viiiie sprayer, as follows; 1. 100 ppm of 6-mer capt opurine ( in 10/90 ethanol/water) 2. 100 ppm dimethyl sulfoxide ( in water) . 100 ppm dimethyl sulfoxide + 100 ppm 6-mercaptopurlne ( in water) . 4. Control ©f 10/90 ethanol/water .

In control group No, 4 the virus signs were seen in -4 days . With groups No . 1 and No . 2 , the virus signs were controlled for 1 week, but with group number 5 , no signs were seen after six weeks .

EXAMPLE 8 Control of Virus ftrowth Tobacco mosaic virus (TMV) suspension of higher titer was treated with solutions of 1%, 2% , $ and 25 aqueous dimethyl sulfoxide (DMSO) . The control and test plants were inooulated with air propelled carborundum particles . The con-trol plant suspended in water produced a higher number of necrotic le sions, 479 per leaf when read five days after inoculation. TMV treated1 with 1% dimethyl sulfoxide produced 283 discrete lesions per leaf; with 2% DMSO 170 lesions per leafj with 5% DMSO 169 lesions per leafj and with 25$ DMSO there was sufficient phyt©toxicity to void testing. It is seen that increasing concentrations of DMSO in water reduced the discrete lesion count. Results probably indicate a virustasls, not virucidal action.

The properties of dimethyl sulfoxide effeotive in combatting the signs and symptoms of dandruff may be also realized to advantage by providing a shampoo composition. Various detergents may be combined with dimethyl sulfoxide and, if desired, various amounts of water, perfume and acceptable colorants. The detergents used may be selected from the various cationic, non-ionic and anionic types. The anionic alkyl aryl sulfonates such as Nacconal NRSF and anionic sodium lauryl sulfate are adaptable for hair shampoo compositions. The polyoxy alkyl ethers are representative non-ionic detergents. The detergents clean the scalp and remove any excess dimethyl sulfoxide. The following example Illustrates one formulation.

EXAMPLE 9 Ingredient Parts Alkyl Aryl Sulfonate as Sodium lauryl sulfate 17.0 Dimethyl sulfoxide 50.0 Perfume 0.5 Water, q.s. to 100.0 The foregoing examples illustrate the control of microorganism growth with dimethyl sulfoxide. It has been shown how different concentrations and amounts of dimethyl sulfoxide compositions are effeotive in different applica- tions. The inventioh is practiced to particular advantage when dimethyl sulfoxide is used in combination with known biooldal and biostatlc agents to oontrol microorganisms. Such biocides inolude the various antibacterial, antifungal and antiviral agents. The advantage of the combination resides in potentiating such agents or in obtaining a synergistic combination. Such combinations are further useful because resistant microorganismshave been shown to be rendered sensitive to the known blocide or biostat in the combination.

No strict range or concentrations for use may be prescribed because none is critical. All of these are readily determined by the expertise of the attending practitioner who may rely on standard criteria for evaluating the effectiveness of a particular composition for a particular industrial or other application.

The disclosed method may be used in the laboratory for different evaluations and tests and in a multitude of industrial applications. Uncontrplled microorganism growth in many industrial uses may now be controlled with dimethyl sulfoxide alone or in combination with known biocides and biostat s. Dimethyl sulfoxide is available in a convenient liquid form which may be diluted with various liquids or used at 100$ concentration. This convenient physical form provided advantages in handling, especially in view of the low toxicity of dimethyl sulfoxide. Determined amounts may be used by mere referenoe to the concentration and volume handled. Microorganism growth is also controlled by stimulating vegetative form growth such as in various antibiotic cultures. This is done by adding to such cultures minimal amounts of di-methyl sulfoxide of about less than 3 percent by weight. greater amounts. However', in concentrations greater than about 5 percent by weight, dimethyl sulfoxide retards the growth of vegetative form microorganismsrather than stimulates it.

Claims

Hairing now particularly described and ascertained the nature I of our said invention and in what manner the same is to be performed, we declare that what we claim ie: -

1. A composition for controlling the growth of microorganisms which comprises an effective amount of a dlalk l eulfoxide .

2. The composition of claim 1 wherein each of the alkyl groups contains from one to four carbon atoms.

5· The composition of claim 1 or 2 wherein the dlalkyl sulfoxide is dimethyl sulfoxide.

4. The composition of any of the claims 1 to 5 wherein it contains a vegetative form microorganism in a growth medium and an effectively small amount of a dlalkyl sulfoxide to stimulate the vegetative microorganism growth. 5. The composition of any of the claims 1 to 5 wherein It contains an agent known to retard the growth of microorganisms and an amount of dlalkyl sulfoxide sufficient to enhance the effect ©f the agent. 6. The composition of any of the claims 1 to 5 wherein it oontalns an agent known to retard the growth of microorganisms but to which the microorganisms are resistant and an amount of a dlalkyl sulfoxide sufficient to allow the 5 mioroorganisms to become susceptible to such agent. 7· The composition of claim 5 wherein it contains an antibiotic and an amount of a dlalkyl sulfoxide sufficient to enhance the effect of the antibiotic. 8. The composition of claim 6 wherein it contains an antibiotic to which the microorganism has become resistant and an amount of a dlalkyl sulfoxide sufficient to allow the microorganism to become susceptible to the antibiotic. 9. The composition of any of the claims 1 to 3 wherein it contains an antibacterial agent and an amount of dlalkyl sulfoxide sufficient to enhance the effect of the antibacterial agent. 10. The composition of any of the claims 1 to 5 wherein it oontalns an antiviral agent and an amount of dialkyl sulfoxide sufficient to enhance the effect ©f the antiviral agent. 11. The composition of any of the claims 1 to 10 wherein it is combined with a carrier suitable for oontaoting the microorganisms. 12. The composition of claim 11 wherein the carrier is an aqueous solution. 15. A composition for controlling the growth of microorganisms substantially as described herein with particular reference to any one of the specific examples. 14. A method for controlling the growth of microorganisms which comprises oontaoting the microorganisms with an effective amount of a composition as defined in any of claims 1 to 15. 15. The method of claim l4 wherein the microorganisms are contacted with an effectively small amount of dimethyl sulfoxide sufficient to stimulate the vegetative growth of the microorganisms. 16. The method of claim 15 wherein the amount of dimethyl sulfoxide used is less than about 3% by weight. 17. The method of claim l4 wherein the microorganisms are contacted with amounts of dimethyl sulfoxide sufficiently large to retard the viral growth, fungal growth or bacterial growth thereof. 18. The method of claim 14 wherein the microorganisms are contacted with an effective amount of dimethyl sulfoxide and a known agent for retarding the growth of microorganisms . 19. The method of claim l8 wherein the mioroor- ganlsme are contaoted with an agent known to retard the growth of microorganisms and an amount of dimethyl sulfoxide sufficient to enhance the effect of the agent. 20. The method of claim l4 wherein the microorganisms are oontaoted with an agent known for retarding the growth of microorganisms but to whioh the microorganism was previously resistant and an amount of dimethyl sulfoxide suf- flclent to render the resistant microorganism susceptible to the agent and thereby retard the growth of the microorganism. 21. The method of claim 14 wherein an industrial oltttfl of growth of microorganisms is contacted with an amount of dimethyl sulfoxide and an agent known for retarding the growth of microorganisms sufficient to retard the growth thereof. 22. A method for controlling the growth ©f microorganisms substantially as described herein with particular reference to any one of the specific examples. 25 . Prooess for the preparation of a composition for controlling the growth of microorganisms as defined in any of the claims 1 to 15 which comprises bringing a dlalkyl sulfoxide into a form suitable for contacting microorganisms. 24. The process of claim 25 wherein each of the alkyl groups contains from one to four carbon atoms. 25. The process of claim 25 or 24 wherein the dlalkyl sulfoxide is dimethyl sulfoxide. 26. The process of any of the claims 25 to 25 wherein an effectively small amount of a dialkyl sulfoxide is combined with a vegetative form microorganism in a growth medium to stimulate the growth thereof. 27. The process of any of the claims 25 to 25 wherein an agent known to retard the growth of microorganisms is oomblned with an amount of a dialkyl sulfoxide suff icient to enhance the effeot of the agent . 28. The prooess of any of the claims 25 to 25 where in an agent known to retard the growth of microorganisms but to which the microorganisms are resistant is combined with an amount of a dialkyl sulfoxide suff icient to allow the microorganisms to become susceptible t o such agent . 29 . The prooess of claim 27 wherein an antibiotic is combined with an amount of a dialkyl sulfoxide sufficient to enhance the effect of the antibiotic . 50. The process of claim 28 where in an antibiotic to which the microorganism has become resistant is combined with an amount of a dialkyl sulfoxide suff icient to allow the microorganisms to become susceptible to the antibiot ic . 51. The process of any of the claims 25 to 25 whe re in an ant lbaoterlal agent is combined with an amount of a dialkyl sulfoxide sufficient to enhance the effeot of the anti bacterial agent . 52. The process of any of the claims 25 to 25 where in an antiviral agent is oomblned with an amount of a dialky sulfoxide sufficient to enhance the effect of the antiviral agent . 55. The process of any of the claims 25 to 52 where In the dialkyl sulfoxide is combined with a oarrier suitable for contacting the mi or ©organisms .

5 . The process of claim 55 wherein the oarrier is an aqueous solution. Process for the preparation of a composition for ^•controlling the growth of microorganisms substantially as described herein with part icular reference to any one of the specific examples .