CA1115652A - Pectin culture media and method - Google Patents
Pectin culture media and methodInfo
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- CA1115652A CA1115652A CA342,300A CA342300A CA1115652A CA 1115652 A CA1115652 A CA 1115652A CA 342300 A CA342300 A CA 342300A CA 1115652 A CA1115652 A CA 1115652A
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Abstract
ABSTRACT
A gelled biological growth medium is prepared by adding a liquid growth medium and low methoxyl pectin material to a culture growth container to produce a gelled growth medium having the low methoxyl pectin material as essentially the sole functional gelling agent. The culture growth contain-er has a growth compatible gel therein which contains a multi-valent metal cation material which is suitable to produce gelling of the low methoxyl pectin material.
A gelled biological growth medium is prepared by adding a liquid growth medium and low methoxyl pectin material to a culture growth container to produce a gelled growth medium having the low methoxyl pectin material as essentially the sole functional gelling agent. The culture growth contain-er has a growth compatible gel therein which contains a multi-valent metal cation material which is suitable to produce gelling of the low methoxyl pectin material.
Description
l~S65;~
PECTIN CULTURE MEDIA AND METHOD
The present invention relates to the field of culture media and methods for producing the same, and more particularly to culture media including pectin as the gelling agent.
A considerable variety and number of culture media and methods for their production are disclosed in the prior art. In general, media used for the growth of living cells, tissues or organisms contain certain ingredients. These ingredients include water nutrients (generally a carbon source, a nitrogen source, and smaller amounts of other essential elements), buffers, and often a gelling or solifying agent.
The majority of the biological media present in the prior art utilize agar, gelatin or silica gel as solidifying agents, also referred to herein as gelling agents. Disadvantages are associated with each of these materials as solidifying agents.
Agar is obtained from marine algae which must be harvested from naturally occurring populations. The supply of agar correspond-ingly fluctuates from year to year, while the demand for solidi-fying agent continues to grow. The price for agar has steadily increased as a result, and the present price is relatively high.
Another problem associated with the use of agar is the need to dispense the agar into its container while quite warm, since the agar solution may solidify at about 40-45C. A temperature of 45C is too high for some cells to withstand without adverse effects.
Gelatin is easily obtained at a relatively reasonable cost, but it is easily hydrolyzed by many micro-organisms, which causes the gel to become a liquid. This is undesirable except in those cases where the hydrolysis is being used as a diagnostic biochemical test. Further, gelatin is generally available as a nutrient source for the organisms in contact with it, and as a ~S65Z
result may interfere with the testing of specific nutrient sources. Gelatin also has the undesirable property of liquifying at quite low temperatures, so that media incorpora-ting it as a gelling agent cannot be incubated above 2~C
with assurance that the medium will retain its solid consis-tency. Disadvantages associated with silica gel include the relatively high cost of silica gel, and the complicated procedure required to prepare a medium using silica gel.
Pectins are routinely used as the thickening or gelling agent in the production of jams and jellies. However, the process generally used involves high sugar concentrations and low pH, neither of which is suitable for general microbial or tissue culture work. In fact, the high sugar and low pH characteristics are useful factors in preventing the establishment of growing, contaminating organisms in the jelly products.
The present invention in one aspect involves the preparation of a gelled biological growth medium having pectin as the gelling agent. The method involves the combination of a liquid growth medium and a low methoxyl pectin material with a growth-compatible gel containing a suitable amount and type of metal cation material to produce gelling of the llquid growth medium. In a preferred embodiment, an agar gel containing calcium chloride is provided in a container, such as a Petri dish, and an aqueous mixture of the liquid growth medium and pectin is poured into the container, whereby gelling of the medium subsequently occurs. The present invention in another aspect further comprises the combination of a culture-growth container and a growth-compatible gel containing a multivalent metal cation material.
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Thus, various aspects of the invention are asfollows:
A method for preparing a gelled biological growth medium which comprises the steps of:
a. providing a predetermined amount of a liquid growth medium;
b. providing a predetermined amount of a low - methoxyl pectin material;
c. providing a culture-growth container having a growth-compatible gel therein, the growth-compatible gel containing a multivalent metal cation material suitable to produce gelling of the low methoxyl pectin material;
and d. adding the liquid growth medium and low methoxyl pectin material to the culture growth container to produce a gelled growth medium having the low methoxyl pectin material as essentially the sole functional gelling agent.
The combination useful as a biological growth medium which comprises:
a culture growth container;
a growth-compatible gel forming a first layer in said container, said growth-compatible gel including a material other than a pectin material as the substantial functional gelling agent, said growth-compatible metal cation material suitable to produce gelling of a low methoxyl pectin material; and a growth-medium forming a second layer adjacent said growth-compatible gel in said container, said growth-medium gel including a growth medium and as essentiallythe sole functional gelling agent a low methoxyl pectin material.
- 2a -1~56S2 The combination useful in preparing a gelled biological growth medium comprising:
a culture-growth container; and a growth-compatible gel located in said container said growth-compatible gel including a material other than a pectin material as the substantial functional gelling agent, said growth-compatible gel further including a multi-valent metal cation material in an amount of at least about 4 grams/100 milliters.
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Referring to the drawings:
FIG. 1 is a perspective view of one embodiment of the present invention.
FIG. 2 is a side elevational view of the embodiment of FIG. 1.
FIG. 3 is a perspective view of an alternate embodiment of the present invention.
FIG. 4 is a side elevational view of the embodiment of FIG. 3.
In accordance with the present invention, a liquid culture growth medium is prepared which includes a pectin material as the gelling agent. The present invention utilizes a low methoxyl pectin which is defined for the purposes herein as having from about one percent to about eight percent methoxyl content. Stated in other terms, the low methoxyl pectin has a degree of methoxylation of about seven to about fifty percent, the degree of methoxylation referring to the extent of esterification of the carboxyl groups with methoxyl groups. In a preferred embodiment of the present invention the low methoxyl pectin has between about three and ahout seven, and most preferably approximately a five percent methoxyl content, or a degree of methoxylation of from about twenty-five to about fourty percent.
The pectin should be present in the growth medium in an amount effective to provide sufficient gelling of the growth medium upon combination of the growth medium with a suitable metal cation material. The amount of pectin will vary with the degree of methoxylation, and also upon other factors such as the extent of gelling desired. However, the amount of pectin desired may be readily determined by simple and ~565Z
direct experimentation. It has been determined that ~ost preferably the pectin, particularly with an approximately five percent degree of methoxylation, is present in an amount of from about ten to about thirty grams of pectin per liter of growth medium.
The liquid growth medium containing the low methoxyl pectin may include a variety of other constituents. In general, the medium may correspond to the wide variety of growth media used in the prior art for microbial and/or tissue cultures, except to the extent that components which would break down or interfere with the pectin should generally not be included. Typically, the culture medium would include several other constituents including 2-10 grams/liter of a carbon source, such as glucose or other sugars, 2-10 grams/
liter of nitrogen, and other micronutrients in the form of natural products (e.g. tryptone, peptone, beef extract, yeast extract, etc.) or synthetic materials (potassium nitrate and various other microelements). The exact nutrients and concentrations which are useful in the liquid growth medium employed in the present invention are innumerable, and as alway8 in the preparation of a growth medium would be Belected according to the particular situation.
The limitations for the other constituents of the medium prepared in accordance with the present invention are generally the same as exist for any other culture media.
Typical ranges for certain media components have already been stated. In another aspect, the sugar concentration of the growth medium of the present invention would generally be less than about ten percent, and the pH would preferably range from 3.5 to 8, primarily from about 6 to about 7. In contrast, food products such as jellies or jams typically include more than fifty percent and perhaps eighty percent sugar as the percent of total solids in the product.
1~15~5Z
The biological growth medium produced in accordance with the present invention would also preferably include one or more buffers to control the pH of the media. The buffers must be non-toxic to micro-organisms and must not degrade the pectin to a point of uselessness. Generally, buffers containing the elements potassium or sodium in combination with phosphate or carbonate groups are non-toxic in minor amounts. Any buffers may cause breakdown or complexing with pectins, and therefore the best results are obtained if the growth medium and the buffers are separately sterilized and then combined after cooling. The variety of buffers which would be useful wiht the pectin-containing medium include g 2P4~ Na2HPO4' Na3Po4, NaHCO3 and Na CO
with the tribasic sodium phosphate (Na3P~4) being found to be particularly suitable. Other useful buffers include citric acid and sodium citrate; acetic acid and sodium acetate, citric acid and dibasic sodium phosphate, succinic acid and sodium hydroxide; monobasic sodium or potassium phosphate and dibasic sodium or potassium phosphate; and tris-maleate. The suitability of other buf~ers or buffer systems are readily predictable and/or determinable by direct and simple experimentation.
In accordance with this invention, an acceptable, solid gel is obtained at a pH of as high as about 9. The prior art literature suggests that pectin as a gelling agent requires an acid pH, typically below about 4. It was there-fore an unexpected result that a culture medium useful at pH
as high as about 9 could be obtained using pectin as the gelling agent.
The pectin-containing growth medium is combined with a multivalent metal cation material suitable to produce gelling of the growth medium. It is known that low methoxyl pectin ~5t~5Z
is sensitive to the presence of various multivalent cations such as calcium, and will form gels when combined with such cations. As is well known, the various multivalent metal cations may be provided most readily as the metal salts, most preferably those which are water soluble. As in the case of the pectin included in the growth medium, a sufficient amount of metal cations must be provided to produce the desired gelling of the growth media.
In general, the relative amounts of pectin and metal cations to produce adequate gelling are known and understood in the art, and additionally the amounts desired for use in the present invention may be readily determined by direct experimentation. Sufficient amount of cation is reguired to produce a good, firm gel formation, but not so much that the gel is hard, brittle or tends to syneresis (weeping). The a~ounts of the pectin-containing growth medium and/or metal cation material are typically and preferably predetermined to provide the proper gelling, particularly in preparing the materials in a kit form. In a preferred embodiment of the present invention, the cation concentration is from about 50 to about 125 milligrams of calcium cations per gram of pectin. Equivalent amounts of other multivalent metal cations could equally be used. In any event, the amount of cation most preferred will depend on the degree of methoxy-lation and the amount of the pectin.
Referring to Figures 1 and 2, the combination of the growth ~edium and metal cations within a culture-growth container is generally disignated 10. A suitable culture growth container 11, such as a test tube or Petri dish is provided, such container being defined as one which is appropriate and used for containing the medium during culture growth. Typically, the culture growth container is one which permits ox facilitates observation or evaluation of the culture growth. A growth-compatible gel 13 containing the metal cation material is caused to Porm in the culture-growth container typically on the bottom 12 thereof, and the liquid growth medium and pectin material are added thereto.
Contact of the medium with the growth-compatible gel will result in a diffusion of the cations through the pectin solution and the consequent formation of a growth-medium gel 14. As indicated, the concentration of the cations in the growth-compatible gel and/or the amount of the growth-compatible gel in the culture-growth container is determined to provide a suitable amount of the metal cations for gelling of the growth medium.
The growth-compatible gel may comprise a variety of gels presently known for use in connection with media for biological growth. As recited and described int he prior art, gels of this type may include, for example, gels utili~-~ng agar, gelatin, silica gel, or carageenan as the solidify-ing or gelling agents. Other materials could also suitably be used as the carrier for the multivalent metal cations.
Such carriers should be inert and nontoxic to living cells, and should not be hydrolized during the process. The growth-compatible gel caused to be formed in the culture-growth container is prepared and handled in the usual fashion.
Although these growth-compatible gels may include growth nutrients and other non-toxic or non-interfering components, such as not required or preferred. The growth-compatible gel may simply and preferably does include only the constitu-ents necessary to provide the gel, as well as the multivalent metal cation material as previously described.
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It has been noted that the amount of multivalent cation material necessary will vary with a number of factors which need not be fully described since the factors are readily apparent and the determination may be readily accomplished by simple and direct experimental techniques. As indicated, a sufficient amount and type of multivalent cation material must be included to produce the desired gelling of the growth medium as a result of the presence of the low methoxyl pectin material. Understandably, the characteristics of the growth-compatible gel will to some extent control the amount of multivalent metal cation material necessary to provide sufficient interaction with the low methoxyl pectin material to produce the desired gel. Further, the configuration of the culture-growth container and of the growth-compatible gel contained therein will to some extent control the amount of multivalent ~etal cation material required to be present in the growth-compatible gel. For example, the ratio of the surface area of the growth-compatible gel to the volume of the growth-compatible gel will affect the amount of metal cation material required.
In a preferred embodiment of the present invention, the growth-compatible gel is located within a Petri dish as a thin, generally uniform film layer coating the bottom of the dish. In this embodiment, a concentration of calcium chloride of about 2-5 grams per 100 milliliters of a 2~ agar solution has been found to be most preferable, particularly when combined at a ratio of about one to ten with a liquid growth medium solution containing from about ten to about thirty grams of pectin per liter of medium.
The growth-compatible gel preferably would not include a pectin material as a substantial functional gelling agent.
Thus, although minor amounts of a pectin material may be ~ 56S2 present, it is preferred that the substantial extent of the gelling affect is the result of other gelling agents.
reason for utilizing gelling agents other than the pectin in the growth-compatible gel is the relationship of the pectin material with the multivalent metal cation material for which the growth-compatible gel is a support and carrier.
As previously indicated, an excess of the multivalent metal cation material can produce syneresis or weeping. The amount of the multivalent metal cation material which may suitably be provided in the growth-compatible gel is therefore limited in the instance of using pectin as the solidifying or gelling agent.
A net or mesh of natural or synthetic material may be used with the medium of the present invention as with prior art media. ~ net with a uniform mesh size, such as five millimeters, provides the function of allowing the observer to have measured fields outlined on the Petri dish or other container. A net or mesh also permits the observer to see completely through the solidified medium rather than limiting viewing from one side as would occur with an opaque, abosrbent pad or paper.
It is a particular aspect of one embodiment of the present invention that the low methoxyl pectin material is utilized as the sole or essentially the sole, functional gelling agent. The term sole functional gelling agent is used herein as meaning the only constituent of the composition which has a significant function as a gelling agent. This owuld, for example, exclude agar or gelatin as significant functional gelling agents. However, just as minor traces of impurities would not necessarily interfere with or contribute to the functioning of the pectin material, minor amounts of agar or other gelling agents could be present and are contem-plated in this particular embodiment, if not present in such _ g _ " 11156S;~ -quantities and forms as to significantly contribute to the gelling of the culture medium.
The term low methoxyl pectin refers to pectin having from about one to about eight percent methoxyle content, or from about seven to about fifty percent degree of methoxyla-tion. As previously indicated, the amount of metal cation required will depend upon the degree of methoxylation of the pectin material, as well as other factors. It has been determined that the lower the degree of methoxylation of the pectin material, the more sensitive the pectin becomes to contact with the metal cation. As a result, pectin having a ~ary low degree of methoxylation, such as pectic acid which essentially has a zero percent methoxyl content, is too sensitive to be readily useful in the preparation of a gelled culture medium. The high sensitivity of metal cation causes a gel to immediately for in the vicinity of the cations upon contact, and lumps and uneven surfaces in the medium will typically result. The opposite effect resulting from a reduced sensitivity to metal cations occurs with pectin having a degree of methoxylation substantially above the range recited for the present invention.
In a preferred method of the present invention, a gelled biological growth medium is preapred by combining predetermined amounts of a liquid growth medium and a low methoxyl pectin material with a predetermined amount of growth-compatible gel containing a multivalent metal cation material. These amounts are selected in accordance with the prior descriptions to pro~ide a suitable gel formation. In this aspect of the invention, the low methoxyl pectin material is essentially the sole functional gelling agent, and suitably is the sole functional gelling agent.
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As previously described, one or more of these components is preferably presterilized, and may conveniently be provided in a pre-packaged kit form. In such a kit form, the three components may be separately packaged and sterilized, or the liquid growth medium and low methoxyl pectin material may be mixed in predetermined proportions to later be combined with the growth-compatible gel. The liquid growth medium and low methoxyl pectin material, either separately or as a pre-mix, are then added to the culture growth container and gelling results upon contact of the low methoxyl pectin material with the multivalent metal cation material.
Other methods for providing the multivalent metal cation material in the culture-growth container are contem-plated. For example, the metal cation material may be conveniently provided by impregnating a support material, such as a filter pad or paper, with a solution of the material, or by spraying the material directly onto thesurface of the liquid growth medium/pectin material, or simply by mixing the components together prior to delivery to the culture-growth container. Alternatively, a solution of the metal cation material may be directly applied to the culture-growth container and allowed or caused to dry to deposit the metal cation material upon the surface of the culture-growth container. For example, a solution of themultivalent metal cation material, with water or other solvents, may be sprayed, painted, or otherwise aplied to the surface of the culture-growth container and permitted to dry thereon. Most desir-ably, the method of applying tthe solution would provide a uniform depositing of the metal cation material upon the surface.
11~5652 Referring again to the drawings, there is shown in Figures 3 and 4 and alternative embodiment of the present invention generally designated 10' in which a culture-growth contain 11' is utilized. The multivalent metal cation material forms a thin film layer 13' on a surface of the container, such as the bottom 12' of container 11'. The growth-medium gel 14' solidifies thereover.
The present invention further provides a biological growth medium gel as produced in accordance with the methods of the present invention. The growth medium gel preferably comprises growth nutrients and as the essentially sole functional gelling agent a low methoxyl pectin material.
The pectin material is suitably the sole functional gelling agent in the growth medium gel of the present invention. In this regard, the term gelling agent is understood as refèrring to the functioning of the pectin to form the gel and in the existence of the pectin material as the gel network or structure. The gel of the present invention may comprise various constituents in accordance with the earlier descrip-tions relating to methods of forming such a gel.
The biological growth medium gel suitably consists essentially of growth nutrients, buffers, water, and a low methoxyl pectin material as the essentially sole or sole functional gelling agent. The low methoxyl pectin material is preferably present in an amount of from about ten to about fifty grams of pectin per liter of medium.
In one aspect, the present invention provides the combination useful in the preparation of a gelled biological growth medium which comprises a culture-growth container and a growth-compatible gel located in the container. The growth-compatible gel is constituted as previously described, l~S65Z
and in this particular embodiment includes a multi~alent metal cation material in an amount of at least about 4 grams per 100 milliliters of the water/agar mix. Preferably the growth-compatible gel consists essentially of the multivalent metal cation meterial, agar and water. As previously indi-cated, the growth-compatible gel in this embodiment of the invention is prepared and caused to form within the culture-growth container in accordance with usual techniques. The combination of the culture-growth container and the growth-compatible gel containing a multivalent metal cation material may then be subsequently used to provide a gelled biological growth medium in accordance with the methods previously described. Briefly, a liquid growth medium and a low methoxyl pectin material are delivered to the culture-growth container, and upon contact with the growth-compatible gel will solidify.
An alternate embodiment of the present invention comprises the combination useful as a biological growth medium which includes a culture-growth container, a growth-compatible gel and a growth-medium gel. The growth-compatible gel is constituted as previously described, and forms a first layer in the container. The growth-medium gel forms a second layer adjacent to the growth-compatible gel in the container, and also i6 consituted as previously described. The growth-medium gel may comprise the components previously described with respect to the liguid growth medium, and further comprises a low methoxyl pectin material as essentially the sole functional gelling agent. Suitably the low methoxyl pectin material is the sole functional gelling agent for the growth-medium gel.
With respect to the growth-medium gel, the low methoxyl pectin material is preferably present in an amount from about ten to about thirty grams of pectin per liter of ~l~S6S2 growth-medium. The growth-medium gel may further comprise one or more buffers selected from the group consisting of ~a2HPo4~ NaH2P04, Na3P04, NaHC03 and Na2C03. It is furhter preferred that the low methoxyl pectin material have approxi-mately a 5% methoxyl content, and that the multivalent metal cation material comprise calcium cations. Most preferably the calcium cations are present in an amount from about 5 to about 125 milligrams of calcium cations per gram of pectin. It is preferred that the growth-compatible gel comprise an agar gel although the other gels as previously indicated bay be utilized. Preferably, the volume of the growth-medium gel is from about three to about twenty times the volume of the gro~th-compatible gel, and in a particularly preferred embodiment there is combined about ten milliliters of growth-medium gel to about one milliliter of gxowth-compatible gel which may suitably be performed ina sixty millimeter diameter Petri dish.
The present invention introduces unique concepts and methodology into the area of preparing biological growth media, and specifically incorporates pectin into such media as the sole gelling or solidifying agent. The present invention provides a simple, straightforward method of utilizing pectin as the gelling agent. Particularly in the preferred method of the present invention, preparation of a biological growth medium utilizing pectin as the gelling agent is accomplished by the use of presterilized components not requiring the use of special sterilizing equipment such as an autoclave or oven~ For example, the liquid growth medium including pectin may be and preferably is presterilized and packaged, and the culture-growth container having the growth-compatible gel including the metal cation material - 14 ~
~ilS652 th~rein is corresp~ndingly presterilized and packaqed. Als~
any or all of the materials (liquid growth medium, low methoxyl pectin material, growth-compatible gel, growth-medium gel and solution of ~etal cation material) may be and preferably are presterilized and packaged for later use.
The preparation of a gelled growth medium in a culture growth container may therefore be easily accomplished without substantial time and without the use of other equipment.
The present invention is therefore particularly suited for use in teaching laboratories or other environments where equipment is limited and the time and/or expertise of persons preparing a growth medium are also limited.
Assitional advantages are also associated with the method of the present invention. In one aspect, the liquid growth medium including the low methoxyl pectin may be dispen8ed either hot, warm or chilled, with solidification occurring in any case upon combination in the culture-growth container. Temperature independence is a particularly notable advantage over the classical agar medium in techniques such as dilution plating for population de~ermination or separation of a mixture of various microbial types. In the dilution technique, an agueous mixture of the microbes is added to the ungelled medium, mixed for homogeneity, and then poured into Petri dishes and allowed to gel. If agar i~ used as the gelling agent for the growth-medium gel, the medium must be at a temperature of about 45C or higher when the microbes are added due to the ~act that solidification will occur below that temperature. Such a high temperature will be harmful to many delicate microbes, and may actually kill or inactivate many, or cause undesirable changes such as mutation. This would be expected to result in an inaccurate picture of the original microbial mixture. Such problems are avoided by the present invention since the microbes can be mixed with the liquid ~rowth medium at as cool a tempera-ture as desired.
The usefulness of the biological growth medium produced in accordance with the present invention is evidenced by the lack of temperature dependence previously described, as well as the fact that most micro-organisms are incapable of hydrolyzing the gelled pectin. The medium may also be used with Procaryotic organisms of the kingdom Protista, with Eucaryotic micro-organisms, or in cell or tissue culture techniques. The medium may also be used in demonstrating which microbes produce pectolytic enzymes, since such organ-isms may effect the hydrolysis of the media. In addition, the media produc~d by the procedures of the present invention are easily and accurately reproducible such that a continuing series of experiments or a duplication of an experiment can be performed with accuracy.
The procedures utilized in the present invention are very straightforward and well known to those skilled in the art. As a particular example of the method of the present invention, the folllowing procedure is recited in detail.
Pirst, the pectin is blended with the nutrient broth solution at an amount of 20-30 grams per liter and in a manner to avoid the formation of insoluble lumps. ~he nutrient-pectin broth is then buffered with Na3PO4 to provide a pH in the range of 6-7. The nutrient-pectin broth is then sterilized in an autoclave at 10-15 pounds per 10-15 minutes. Alterna-tively the nutrient-pectin broth and the buffers may be sterilized separately and combined following sterilization.
Or alternatively, the pectin may be dissolved in water at an amount of 20-30 grams/liter and in a manner to avoid the formation of insoluble lumps, while the nutrient and buffer _ 16 -11~565Z
may be combined separately and the two solutions unified following separate sterilization.
A solution containing 2% (2 grams per 100 milliliters deionized or distilled water) agar-agar and calcium chloride is prepared. Alternatively, other compounds including multivalent metal cations may be employed as is well known in the art relating to agents for use with pectin to provide a gel. Typically, the calcium compounds including chloride, nitrate or phosphate are particularly desirable, and the ideal agent would be water soluble. As previously indicated, the concentration of the calcium chloride or other multivalent metal cation material is determined to provide the proper metal cation concentration to cause solidification of the nutrient-pectin composition when poured over the solidified agar gel.
The solution of the 2% agar and metal cation material i~ prepared by suitable means, such as by dissolving the materials in water heated at 15 pound pressure and about 120C in an autoclave. The sterile agar mixture is then dispensed while hot into a Petri dish sufficiently to cover the bottom of the sterile dish. The mixture solidifies quickly forming a gel film adhering to the inside of the Petri dish base. Alternatively, the agar mixture may be dispensed into non-sterile Petri dishes or containers and, after ~olidification, they may be sterilized in customary a~hions such as by ethylene oxide gas or radiation.
The nutrient-pectin broth, or liquid growth medium, is preferably presterilized, and then is dispensed into the dishes or other culture-growth container on top of the agar, growth-compatible gel fil~. The presence of the multivalent metal cations causes the liquid growth medium to solidify generally in about 2-4 hours. The solidified growth-medium is then inoculated with micro-organisms, and is incubated either right side up or upside down.
The present invention is useful with a variety of culture-growth containers. The invention is particularly well-suited to use with disposable containers. Preferably the volume of the growth-medium gel is about three to about twenty times the volume of the growth-compatible gel. In the instance of use with Petri dishes and other containers, a suitable combination has been found to be about two milliliters of growth-compatible gel to about ten milliliters of growth medium gel in a sixty milliliter Petri dish.
The following examples further exemplify biological media prepared in accordance with the present invention.
A general microbiological medium for the growth of bacteria, molds and yeasts was formulated as follows:
Tryptone 2 gm Peptone 2 gm Yeast extract 2 gm Glucose 2 gm LM Pectin 25gm Deionized Water 1 liter This formulation is sterilized by autoclaving and following the autoclaving and cooling of the medium, a combination of Na3P04 and ~a2C03 (presterilized) is added to adjust the p~ of the medium.
Numerous bacteria, yeast, and molds have been grown successfully on this formulation.
EXAMPLE 2 ~
A specific differential medium known as Eosin Methylene blue agar is used to identify the presence of Escherichia 1~1S6SZ
coli fro~ other similar bacteria. E. coli grows with a green sheen on this medium in comparison to Enterobacter aeroqenes which grows as a gummy pink culture.
The following medium was prepared which, in preliminary tests, worked very well in defferentiating these 2 organisms.
Peptone 5 gm Lactose 5 gm Eosin y 0.4 gm Methylene blue 0.065 gm LM Pectin 25 gm Deionized water 1 liter The above formulation was sterilized and then adjusted to a pH of 7.1 with Na3PO4 and ~a2CO3 (presteril).
As previously indicated, the above media of Examples 1 and 2 were utilized in preparing a gelled biological growth media in accordance with the present invention. Initially, an agar mixture prepared as previously described was delivered to a Petri dish and allowed to cool and therefore solidify.
The formulations of Examples 1 and 2 were then added to diferent Petri dishes containing the solidified agar mixture.
This procedure is followed using the different porportions of the formulations with the agar gel ranging from about three to about twenty times the volume of each formulation to the volume of the agar gel. Excellent results are obtained.
The procedures of Example 3 are followed except that instead of the agar mixture, other standard gel formulations were utilized to provide the growth-compatible gel. The other growth-compatible gels included gelatin, carageenan and silica gel as the solidifying or gelling agents. The addition of the formulations of Examples 1 and 2 produced excellent growth-medium gels.
EXA~IPLE 5 The procedures of Example 3 are repeated except that instead of utilizing an agar gel as a carrier for the calcium chloride, a solution of the calcium chloride was aplied directly to Petri dishes. The solutions were variously applied by spraying with an atomizer and by painting the solution directly onto the dishes. Upon combination with the formulations of Examples 1 and 2, suitable gels were produced. T~ese procedures are performed utilizing an aqueous solution of calcium chloride, and also a mixture comprising 5-10 grams of methyl celulose and about 4 grams of calcium ~hloride to lO0 milliliters of water, and similar results were obtained.
As further examples of the methods and medium gel of the present invention, variations of the above examples are conducted. Performing the methods of the above examples utilizing alternatively a low methoxyl pectin material having about one, three, five and eight percent methoxyl content provides in each instance a suitable culture medium gel. Practicing the methods of the prior examples similarly produces a suitable gel when the liquid growth medium and low methoxyl pectin material are either separate or in combination, and the method also suitably comprises the addition of the li~uid growth medium and the multivalent metal cation prior to addition of the low methoxyl pectin material thereto. Varying the amount of pectin present in the culture medium gel in the range of from about ten to about thrity grams of pectin per liter of medium also produces suitable gels under the prior examples. Most preferably the multivalent metal cation material comprises a calcium salt, and variation of the amount of calcium cations from about 50 to about 125 milligrams of cations per gram of pectin provides a suitable gel in accordance with the prior examples.
Although the most preferred ranges for amount of pectin and amount of multivalent metal cation materials have been recited herein, suitable gels may be provided outside of these ranges and applicant does not intend to be limited to these ranges which from the most preferred embodiments of the invention.
PECTIN CULTURE MEDIA AND METHOD
The present invention relates to the field of culture media and methods for producing the same, and more particularly to culture media including pectin as the gelling agent.
A considerable variety and number of culture media and methods for their production are disclosed in the prior art. In general, media used for the growth of living cells, tissues or organisms contain certain ingredients. These ingredients include water nutrients (generally a carbon source, a nitrogen source, and smaller amounts of other essential elements), buffers, and often a gelling or solifying agent.
The majority of the biological media present in the prior art utilize agar, gelatin or silica gel as solidifying agents, also referred to herein as gelling agents. Disadvantages are associated with each of these materials as solidifying agents.
Agar is obtained from marine algae which must be harvested from naturally occurring populations. The supply of agar correspond-ingly fluctuates from year to year, while the demand for solidi-fying agent continues to grow. The price for agar has steadily increased as a result, and the present price is relatively high.
Another problem associated with the use of agar is the need to dispense the agar into its container while quite warm, since the agar solution may solidify at about 40-45C. A temperature of 45C is too high for some cells to withstand without adverse effects.
Gelatin is easily obtained at a relatively reasonable cost, but it is easily hydrolyzed by many micro-organisms, which causes the gel to become a liquid. This is undesirable except in those cases where the hydrolysis is being used as a diagnostic biochemical test. Further, gelatin is generally available as a nutrient source for the organisms in contact with it, and as a ~S65Z
result may interfere with the testing of specific nutrient sources. Gelatin also has the undesirable property of liquifying at quite low temperatures, so that media incorpora-ting it as a gelling agent cannot be incubated above 2~C
with assurance that the medium will retain its solid consis-tency. Disadvantages associated with silica gel include the relatively high cost of silica gel, and the complicated procedure required to prepare a medium using silica gel.
Pectins are routinely used as the thickening or gelling agent in the production of jams and jellies. However, the process generally used involves high sugar concentrations and low pH, neither of which is suitable for general microbial or tissue culture work. In fact, the high sugar and low pH characteristics are useful factors in preventing the establishment of growing, contaminating organisms in the jelly products.
The present invention in one aspect involves the preparation of a gelled biological growth medium having pectin as the gelling agent. The method involves the combination of a liquid growth medium and a low methoxyl pectin material with a growth-compatible gel containing a suitable amount and type of metal cation material to produce gelling of the llquid growth medium. In a preferred embodiment, an agar gel containing calcium chloride is provided in a container, such as a Petri dish, and an aqueous mixture of the liquid growth medium and pectin is poured into the container, whereby gelling of the medium subsequently occurs. The present invention in another aspect further comprises the combination of a culture-growth container and a growth-compatible gel containing a multivalent metal cation material.
, .
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Thus, various aspects of the invention are asfollows:
A method for preparing a gelled biological growth medium which comprises the steps of:
a. providing a predetermined amount of a liquid growth medium;
b. providing a predetermined amount of a low - methoxyl pectin material;
c. providing a culture-growth container having a growth-compatible gel therein, the growth-compatible gel containing a multivalent metal cation material suitable to produce gelling of the low methoxyl pectin material;
and d. adding the liquid growth medium and low methoxyl pectin material to the culture growth container to produce a gelled growth medium having the low methoxyl pectin material as essentially the sole functional gelling agent.
The combination useful as a biological growth medium which comprises:
a culture growth container;
a growth-compatible gel forming a first layer in said container, said growth-compatible gel including a material other than a pectin material as the substantial functional gelling agent, said growth-compatible metal cation material suitable to produce gelling of a low methoxyl pectin material; and a growth-medium forming a second layer adjacent said growth-compatible gel in said container, said growth-medium gel including a growth medium and as essentiallythe sole functional gelling agent a low methoxyl pectin material.
- 2a -1~56S2 The combination useful in preparing a gelled biological growth medium comprising:
a culture-growth container; and a growth-compatible gel located in said container said growth-compatible gel including a material other than a pectin material as the substantial functional gelling agent, said growth-compatible gel further including a multi-valent metal cation material in an amount of at least about 4 grams/100 milliters.
~.
~L5~5Z
Referring to the drawings:
FIG. 1 is a perspective view of one embodiment of the present invention.
FIG. 2 is a side elevational view of the embodiment of FIG. 1.
FIG. 3 is a perspective view of an alternate embodiment of the present invention.
FIG. 4 is a side elevational view of the embodiment of FIG. 3.
In accordance with the present invention, a liquid culture growth medium is prepared which includes a pectin material as the gelling agent. The present invention utilizes a low methoxyl pectin which is defined for the purposes herein as having from about one percent to about eight percent methoxyl content. Stated in other terms, the low methoxyl pectin has a degree of methoxylation of about seven to about fifty percent, the degree of methoxylation referring to the extent of esterification of the carboxyl groups with methoxyl groups. In a preferred embodiment of the present invention the low methoxyl pectin has between about three and ahout seven, and most preferably approximately a five percent methoxyl content, or a degree of methoxylation of from about twenty-five to about fourty percent.
The pectin should be present in the growth medium in an amount effective to provide sufficient gelling of the growth medium upon combination of the growth medium with a suitable metal cation material. The amount of pectin will vary with the degree of methoxylation, and also upon other factors such as the extent of gelling desired. However, the amount of pectin desired may be readily determined by simple and ~565Z
direct experimentation. It has been determined that ~ost preferably the pectin, particularly with an approximately five percent degree of methoxylation, is present in an amount of from about ten to about thirty grams of pectin per liter of growth medium.
The liquid growth medium containing the low methoxyl pectin may include a variety of other constituents. In general, the medium may correspond to the wide variety of growth media used in the prior art for microbial and/or tissue cultures, except to the extent that components which would break down or interfere with the pectin should generally not be included. Typically, the culture medium would include several other constituents including 2-10 grams/liter of a carbon source, such as glucose or other sugars, 2-10 grams/
liter of nitrogen, and other micronutrients in the form of natural products (e.g. tryptone, peptone, beef extract, yeast extract, etc.) or synthetic materials (potassium nitrate and various other microelements). The exact nutrients and concentrations which are useful in the liquid growth medium employed in the present invention are innumerable, and as alway8 in the preparation of a growth medium would be Belected according to the particular situation.
The limitations for the other constituents of the medium prepared in accordance with the present invention are generally the same as exist for any other culture media.
Typical ranges for certain media components have already been stated. In another aspect, the sugar concentration of the growth medium of the present invention would generally be less than about ten percent, and the pH would preferably range from 3.5 to 8, primarily from about 6 to about 7. In contrast, food products such as jellies or jams typically include more than fifty percent and perhaps eighty percent sugar as the percent of total solids in the product.
1~15~5Z
The biological growth medium produced in accordance with the present invention would also preferably include one or more buffers to control the pH of the media. The buffers must be non-toxic to micro-organisms and must not degrade the pectin to a point of uselessness. Generally, buffers containing the elements potassium or sodium in combination with phosphate or carbonate groups are non-toxic in minor amounts. Any buffers may cause breakdown or complexing with pectins, and therefore the best results are obtained if the growth medium and the buffers are separately sterilized and then combined after cooling. The variety of buffers which would be useful wiht the pectin-containing medium include g 2P4~ Na2HPO4' Na3Po4, NaHCO3 and Na CO
with the tribasic sodium phosphate (Na3P~4) being found to be particularly suitable. Other useful buffers include citric acid and sodium citrate; acetic acid and sodium acetate, citric acid and dibasic sodium phosphate, succinic acid and sodium hydroxide; monobasic sodium or potassium phosphate and dibasic sodium or potassium phosphate; and tris-maleate. The suitability of other buf~ers or buffer systems are readily predictable and/or determinable by direct and simple experimentation.
In accordance with this invention, an acceptable, solid gel is obtained at a pH of as high as about 9. The prior art literature suggests that pectin as a gelling agent requires an acid pH, typically below about 4. It was there-fore an unexpected result that a culture medium useful at pH
as high as about 9 could be obtained using pectin as the gelling agent.
The pectin-containing growth medium is combined with a multivalent metal cation material suitable to produce gelling of the growth medium. It is known that low methoxyl pectin ~5t~5Z
is sensitive to the presence of various multivalent cations such as calcium, and will form gels when combined with such cations. As is well known, the various multivalent metal cations may be provided most readily as the metal salts, most preferably those which are water soluble. As in the case of the pectin included in the growth medium, a sufficient amount of metal cations must be provided to produce the desired gelling of the growth media.
In general, the relative amounts of pectin and metal cations to produce adequate gelling are known and understood in the art, and additionally the amounts desired for use in the present invention may be readily determined by direct experimentation. Sufficient amount of cation is reguired to produce a good, firm gel formation, but not so much that the gel is hard, brittle or tends to syneresis (weeping). The a~ounts of the pectin-containing growth medium and/or metal cation material are typically and preferably predetermined to provide the proper gelling, particularly in preparing the materials in a kit form. In a preferred embodiment of the present invention, the cation concentration is from about 50 to about 125 milligrams of calcium cations per gram of pectin. Equivalent amounts of other multivalent metal cations could equally be used. In any event, the amount of cation most preferred will depend on the degree of methoxy-lation and the amount of the pectin.
Referring to Figures 1 and 2, the combination of the growth ~edium and metal cations within a culture-growth container is generally disignated 10. A suitable culture growth container 11, such as a test tube or Petri dish is provided, such container being defined as one which is appropriate and used for containing the medium during culture growth. Typically, the culture growth container is one which permits ox facilitates observation or evaluation of the culture growth. A growth-compatible gel 13 containing the metal cation material is caused to Porm in the culture-growth container typically on the bottom 12 thereof, and the liquid growth medium and pectin material are added thereto.
Contact of the medium with the growth-compatible gel will result in a diffusion of the cations through the pectin solution and the consequent formation of a growth-medium gel 14. As indicated, the concentration of the cations in the growth-compatible gel and/or the amount of the growth-compatible gel in the culture-growth container is determined to provide a suitable amount of the metal cations for gelling of the growth medium.
The growth-compatible gel may comprise a variety of gels presently known for use in connection with media for biological growth. As recited and described int he prior art, gels of this type may include, for example, gels utili~-~ng agar, gelatin, silica gel, or carageenan as the solidify-ing or gelling agents. Other materials could also suitably be used as the carrier for the multivalent metal cations.
Such carriers should be inert and nontoxic to living cells, and should not be hydrolized during the process. The growth-compatible gel caused to be formed in the culture-growth container is prepared and handled in the usual fashion.
Although these growth-compatible gels may include growth nutrients and other non-toxic or non-interfering components, such as not required or preferred. The growth-compatible gel may simply and preferably does include only the constitu-ents necessary to provide the gel, as well as the multivalent metal cation material as previously described.
~565Z
It has been noted that the amount of multivalent cation material necessary will vary with a number of factors which need not be fully described since the factors are readily apparent and the determination may be readily accomplished by simple and direct experimental techniques. As indicated, a sufficient amount and type of multivalent cation material must be included to produce the desired gelling of the growth medium as a result of the presence of the low methoxyl pectin material. Understandably, the characteristics of the growth-compatible gel will to some extent control the amount of multivalent metal cation material necessary to provide sufficient interaction with the low methoxyl pectin material to produce the desired gel. Further, the configuration of the culture-growth container and of the growth-compatible gel contained therein will to some extent control the amount of multivalent ~etal cation material required to be present in the growth-compatible gel. For example, the ratio of the surface area of the growth-compatible gel to the volume of the growth-compatible gel will affect the amount of metal cation material required.
In a preferred embodiment of the present invention, the growth-compatible gel is located within a Petri dish as a thin, generally uniform film layer coating the bottom of the dish. In this embodiment, a concentration of calcium chloride of about 2-5 grams per 100 milliliters of a 2~ agar solution has been found to be most preferable, particularly when combined at a ratio of about one to ten with a liquid growth medium solution containing from about ten to about thirty grams of pectin per liter of medium.
The growth-compatible gel preferably would not include a pectin material as a substantial functional gelling agent.
Thus, although minor amounts of a pectin material may be ~ 56S2 present, it is preferred that the substantial extent of the gelling affect is the result of other gelling agents.
reason for utilizing gelling agents other than the pectin in the growth-compatible gel is the relationship of the pectin material with the multivalent metal cation material for which the growth-compatible gel is a support and carrier.
As previously indicated, an excess of the multivalent metal cation material can produce syneresis or weeping. The amount of the multivalent metal cation material which may suitably be provided in the growth-compatible gel is therefore limited in the instance of using pectin as the solidifying or gelling agent.
A net or mesh of natural or synthetic material may be used with the medium of the present invention as with prior art media. ~ net with a uniform mesh size, such as five millimeters, provides the function of allowing the observer to have measured fields outlined on the Petri dish or other container. A net or mesh also permits the observer to see completely through the solidified medium rather than limiting viewing from one side as would occur with an opaque, abosrbent pad or paper.
It is a particular aspect of one embodiment of the present invention that the low methoxyl pectin material is utilized as the sole or essentially the sole, functional gelling agent. The term sole functional gelling agent is used herein as meaning the only constituent of the composition which has a significant function as a gelling agent. This owuld, for example, exclude agar or gelatin as significant functional gelling agents. However, just as minor traces of impurities would not necessarily interfere with or contribute to the functioning of the pectin material, minor amounts of agar or other gelling agents could be present and are contem-plated in this particular embodiment, if not present in such _ g _ " 11156S;~ -quantities and forms as to significantly contribute to the gelling of the culture medium.
The term low methoxyl pectin refers to pectin having from about one to about eight percent methoxyle content, or from about seven to about fifty percent degree of methoxyla-tion. As previously indicated, the amount of metal cation required will depend upon the degree of methoxylation of the pectin material, as well as other factors. It has been determined that the lower the degree of methoxylation of the pectin material, the more sensitive the pectin becomes to contact with the metal cation. As a result, pectin having a ~ary low degree of methoxylation, such as pectic acid which essentially has a zero percent methoxyl content, is too sensitive to be readily useful in the preparation of a gelled culture medium. The high sensitivity of metal cation causes a gel to immediately for in the vicinity of the cations upon contact, and lumps and uneven surfaces in the medium will typically result. The opposite effect resulting from a reduced sensitivity to metal cations occurs with pectin having a degree of methoxylation substantially above the range recited for the present invention.
In a preferred method of the present invention, a gelled biological growth medium is preapred by combining predetermined amounts of a liquid growth medium and a low methoxyl pectin material with a predetermined amount of growth-compatible gel containing a multivalent metal cation material. These amounts are selected in accordance with the prior descriptions to pro~ide a suitable gel formation. In this aspect of the invention, the low methoxyl pectin material is essentially the sole functional gelling agent, and suitably is the sole functional gelling agent.
--` 111565Z
As previously described, one or more of these components is preferably presterilized, and may conveniently be provided in a pre-packaged kit form. In such a kit form, the three components may be separately packaged and sterilized, or the liquid growth medium and low methoxyl pectin material may be mixed in predetermined proportions to later be combined with the growth-compatible gel. The liquid growth medium and low methoxyl pectin material, either separately or as a pre-mix, are then added to the culture growth container and gelling results upon contact of the low methoxyl pectin material with the multivalent metal cation material.
Other methods for providing the multivalent metal cation material in the culture-growth container are contem-plated. For example, the metal cation material may be conveniently provided by impregnating a support material, such as a filter pad or paper, with a solution of the material, or by spraying the material directly onto thesurface of the liquid growth medium/pectin material, or simply by mixing the components together prior to delivery to the culture-growth container. Alternatively, a solution of the metal cation material may be directly applied to the culture-growth container and allowed or caused to dry to deposit the metal cation material upon the surface of the culture-growth container. For example, a solution of themultivalent metal cation material, with water or other solvents, may be sprayed, painted, or otherwise aplied to the surface of the culture-growth container and permitted to dry thereon. Most desir-ably, the method of applying tthe solution would provide a uniform depositing of the metal cation material upon the surface.
11~5652 Referring again to the drawings, there is shown in Figures 3 and 4 and alternative embodiment of the present invention generally designated 10' in which a culture-growth contain 11' is utilized. The multivalent metal cation material forms a thin film layer 13' on a surface of the container, such as the bottom 12' of container 11'. The growth-medium gel 14' solidifies thereover.
The present invention further provides a biological growth medium gel as produced in accordance with the methods of the present invention. The growth medium gel preferably comprises growth nutrients and as the essentially sole functional gelling agent a low methoxyl pectin material.
The pectin material is suitably the sole functional gelling agent in the growth medium gel of the present invention. In this regard, the term gelling agent is understood as refèrring to the functioning of the pectin to form the gel and in the existence of the pectin material as the gel network or structure. The gel of the present invention may comprise various constituents in accordance with the earlier descrip-tions relating to methods of forming such a gel.
The biological growth medium gel suitably consists essentially of growth nutrients, buffers, water, and a low methoxyl pectin material as the essentially sole or sole functional gelling agent. The low methoxyl pectin material is preferably present in an amount of from about ten to about fifty grams of pectin per liter of medium.
In one aspect, the present invention provides the combination useful in the preparation of a gelled biological growth medium which comprises a culture-growth container and a growth-compatible gel located in the container. The growth-compatible gel is constituted as previously described, l~S65Z
and in this particular embodiment includes a multi~alent metal cation material in an amount of at least about 4 grams per 100 milliliters of the water/agar mix. Preferably the growth-compatible gel consists essentially of the multivalent metal cation meterial, agar and water. As previously indi-cated, the growth-compatible gel in this embodiment of the invention is prepared and caused to form within the culture-growth container in accordance with usual techniques. The combination of the culture-growth container and the growth-compatible gel containing a multivalent metal cation material may then be subsequently used to provide a gelled biological growth medium in accordance with the methods previously described. Briefly, a liquid growth medium and a low methoxyl pectin material are delivered to the culture-growth container, and upon contact with the growth-compatible gel will solidify.
An alternate embodiment of the present invention comprises the combination useful as a biological growth medium which includes a culture-growth container, a growth-compatible gel and a growth-medium gel. The growth-compatible gel is constituted as previously described, and forms a first layer in the container. The growth-medium gel forms a second layer adjacent to the growth-compatible gel in the container, and also i6 consituted as previously described. The growth-medium gel may comprise the components previously described with respect to the liguid growth medium, and further comprises a low methoxyl pectin material as essentially the sole functional gelling agent. Suitably the low methoxyl pectin material is the sole functional gelling agent for the growth-medium gel.
With respect to the growth-medium gel, the low methoxyl pectin material is preferably present in an amount from about ten to about thirty grams of pectin per liter of ~l~S6S2 growth-medium. The growth-medium gel may further comprise one or more buffers selected from the group consisting of ~a2HPo4~ NaH2P04, Na3P04, NaHC03 and Na2C03. It is furhter preferred that the low methoxyl pectin material have approxi-mately a 5% methoxyl content, and that the multivalent metal cation material comprise calcium cations. Most preferably the calcium cations are present in an amount from about 5 to about 125 milligrams of calcium cations per gram of pectin. It is preferred that the growth-compatible gel comprise an agar gel although the other gels as previously indicated bay be utilized. Preferably, the volume of the growth-medium gel is from about three to about twenty times the volume of the gro~th-compatible gel, and in a particularly preferred embodiment there is combined about ten milliliters of growth-medium gel to about one milliliter of gxowth-compatible gel which may suitably be performed ina sixty millimeter diameter Petri dish.
The present invention introduces unique concepts and methodology into the area of preparing biological growth media, and specifically incorporates pectin into such media as the sole gelling or solidifying agent. The present invention provides a simple, straightforward method of utilizing pectin as the gelling agent. Particularly in the preferred method of the present invention, preparation of a biological growth medium utilizing pectin as the gelling agent is accomplished by the use of presterilized components not requiring the use of special sterilizing equipment such as an autoclave or oven~ For example, the liquid growth medium including pectin may be and preferably is presterilized and packaged, and the culture-growth container having the growth-compatible gel including the metal cation material - 14 ~
~ilS652 th~rein is corresp~ndingly presterilized and packaqed. Als~
any or all of the materials (liquid growth medium, low methoxyl pectin material, growth-compatible gel, growth-medium gel and solution of ~etal cation material) may be and preferably are presterilized and packaged for later use.
The preparation of a gelled growth medium in a culture growth container may therefore be easily accomplished without substantial time and without the use of other equipment.
The present invention is therefore particularly suited for use in teaching laboratories or other environments where equipment is limited and the time and/or expertise of persons preparing a growth medium are also limited.
Assitional advantages are also associated with the method of the present invention. In one aspect, the liquid growth medium including the low methoxyl pectin may be dispen8ed either hot, warm or chilled, with solidification occurring in any case upon combination in the culture-growth container. Temperature independence is a particularly notable advantage over the classical agar medium in techniques such as dilution plating for population de~ermination or separation of a mixture of various microbial types. In the dilution technique, an agueous mixture of the microbes is added to the ungelled medium, mixed for homogeneity, and then poured into Petri dishes and allowed to gel. If agar i~ used as the gelling agent for the growth-medium gel, the medium must be at a temperature of about 45C or higher when the microbes are added due to the ~act that solidification will occur below that temperature. Such a high temperature will be harmful to many delicate microbes, and may actually kill or inactivate many, or cause undesirable changes such as mutation. This would be expected to result in an inaccurate picture of the original microbial mixture. Such problems are avoided by the present invention since the microbes can be mixed with the liquid ~rowth medium at as cool a tempera-ture as desired.
The usefulness of the biological growth medium produced in accordance with the present invention is evidenced by the lack of temperature dependence previously described, as well as the fact that most micro-organisms are incapable of hydrolyzing the gelled pectin. The medium may also be used with Procaryotic organisms of the kingdom Protista, with Eucaryotic micro-organisms, or in cell or tissue culture techniques. The medium may also be used in demonstrating which microbes produce pectolytic enzymes, since such organ-isms may effect the hydrolysis of the media. In addition, the media produc~d by the procedures of the present invention are easily and accurately reproducible such that a continuing series of experiments or a duplication of an experiment can be performed with accuracy.
The procedures utilized in the present invention are very straightforward and well known to those skilled in the art. As a particular example of the method of the present invention, the folllowing procedure is recited in detail.
Pirst, the pectin is blended with the nutrient broth solution at an amount of 20-30 grams per liter and in a manner to avoid the formation of insoluble lumps. ~he nutrient-pectin broth is then buffered with Na3PO4 to provide a pH in the range of 6-7. The nutrient-pectin broth is then sterilized in an autoclave at 10-15 pounds per 10-15 minutes. Alterna-tively the nutrient-pectin broth and the buffers may be sterilized separately and combined following sterilization.
Or alternatively, the pectin may be dissolved in water at an amount of 20-30 grams/liter and in a manner to avoid the formation of insoluble lumps, while the nutrient and buffer _ 16 -11~565Z
may be combined separately and the two solutions unified following separate sterilization.
A solution containing 2% (2 grams per 100 milliliters deionized or distilled water) agar-agar and calcium chloride is prepared. Alternatively, other compounds including multivalent metal cations may be employed as is well known in the art relating to agents for use with pectin to provide a gel. Typically, the calcium compounds including chloride, nitrate or phosphate are particularly desirable, and the ideal agent would be water soluble. As previously indicated, the concentration of the calcium chloride or other multivalent metal cation material is determined to provide the proper metal cation concentration to cause solidification of the nutrient-pectin composition when poured over the solidified agar gel.
The solution of the 2% agar and metal cation material i~ prepared by suitable means, such as by dissolving the materials in water heated at 15 pound pressure and about 120C in an autoclave. The sterile agar mixture is then dispensed while hot into a Petri dish sufficiently to cover the bottom of the sterile dish. The mixture solidifies quickly forming a gel film adhering to the inside of the Petri dish base. Alternatively, the agar mixture may be dispensed into non-sterile Petri dishes or containers and, after ~olidification, they may be sterilized in customary a~hions such as by ethylene oxide gas or radiation.
The nutrient-pectin broth, or liquid growth medium, is preferably presterilized, and then is dispensed into the dishes or other culture-growth container on top of the agar, growth-compatible gel fil~. The presence of the multivalent metal cations causes the liquid growth medium to solidify generally in about 2-4 hours. The solidified growth-medium is then inoculated with micro-organisms, and is incubated either right side up or upside down.
The present invention is useful with a variety of culture-growth containers. The invention is particularly well-suited to use with disposable containers. Preferably the volume of the growth-medium gel is about three to about twenty times the volume of the growth-compatible gel. In the instance of use with Petri dishes and other containers, a suitable combination has been found to be about two milliliters of growth-compatible gel to about ten milliliters of growth medium gel in a sixty milliliter Petri dish.
The following examples further exemplify biological media prepared in accordance with the present invention.
A general microbiological medium for the growth of bacteria, molds and yeasts was formulated as follows:
Tryptone 2 gm Peptone 2 gm Yeast extract 2 gm Glucose 2 gm LM Pectin 25gm Deionized Water 1 liter This formulation is sterilized by autoclaving and following the autoclaving and cooling of the medium, a combination of Na3P04 and ~a2C03 (presterilized) is added to adjust the p~ of the medium.
Numerous bacteria, yeast, and molds have been grown successfully on this formulation.
EXAMPLE 2 ~
A specific differential medium known as Eosin Methylene blue agar is used to identify the presence of Escherichia 1~1S6SZ
coli fro~ other similar bacteria. E. coli grows with a green sheen on this medium in comparison to Enterobacter aeroqenes which grows as a gummy pink culture.
The following medium was prepared which, in preliminary tests, worked very well in defferentiating these 2 organisms.
Peptone 5 gm Lactose 5 gm Eosin y 0.4 gm Methylene blue 0.065 gm LM Pectin 25 gm Deionized water 1 liter The above formulation was sterilized and then adjusted to a pH of 7.1 with Na3PO4 and ~a2CO3 (presteril).
As previously indicated, the above media of Examples 1 and 2 were utilized in preparing a gelled biological growth media in accordance with the present invention. Initially, an agar mixture prepared as previously described was delivered to a Petri dish and allowed to cool and therefore solidify.
The formulations of Examples 1 and 2 were then added to diferent Petri dishes containing the solidified agar mixture.
This procedure is followed using the different porportions of the formulations with the agar gel ranging from about three to about twenty times the volume of each formulation to the volume of the agar gel. Excellent results are obtained.
The procedures of Example 3 are followed except that instead of the agar mixture, other standard gel formulations were utilized to provide the growth-compatible gel. The other growth-compatible gels included gelatin, carageenan and silica gel as the solidifying or gelling agents. The addition of the formulations of Examples 1 and 2 produced excellent growth-medium gels.
EXA~IPLE 5 The procedures of Example 3 are repeated except that instead of utilizing an agar gel as a carrier for the calcium chloride, a solution of the calcium chloride was aplied directly to Petri dishes. The solutions were variously applied by spraying with an atomizer and by painting the solution directly onto the dishes. Upon combination with the formulations of Examples 1 and 2, suitable gels were produced. T~ese procedures are performed utilizing an aqueous solution of calcium chloride, and also a mixture comprising 5-10 grams of methyl celulose and about 4 grams of calcium ~hloride to lO0 milliliters of water, and similar results were obtained.
As further examples of the methods and medium gel of the present invention, variations of the above examples are conducted. Performing the methods of the above examples utilizing alternatively a low methoxyl pectin material having about one, three, five and eight percent methoxyl content provides in each instance a suitable culture medium gel. Practicing the methods of the prior examples similarly produces a suitable gel when the liquid growth medium and low methoxyl pectin material are either separate or in combination, and the method also suitably comprises the addition of the li~uid growth medium and the multivalent metal cation prior to addition of the low methoxyl pectin material thereto. Varying the amount of pectin present in the culture medium gel in the range of from about ten to about thrity grams of pectin per liter of medium also produces suitable gels under the prior examples. Most preferably the multivalent metal cation material comprises a calcium salt, and variation of the amount of calcium cations from about 50 to about 125 milligrams of cations per gram of pectin provides a suitable gel in accordance with the prior examples.
Although the most preferred ranges for amount of pectin and amount of multivalent metal cation materials have been recited herein, suitable gels may be provided outside of these ranges and applicant does not intend to be limited to these ranges which from the most preferred embodiments of the invention.
Claims (15)
1. A method for preparing a gelled biological growth medium which comprises the steps of:
a. providing a predetermined amount of a liquid growth medium;
b. providing a predetermined amount of a low methoxyl pectin material;
c. providing a culture-growth container having a growth-compatible gel therein, the growth-compatible gel containing a multivalent metal cation material suitable to produce gelling of the low methoxyl pectin material; and d. adding the liquid growth medium and low methoxyl pectin material to the culture growth container to produce a gelled growth medium having the low methoxyl pectin material as essentially the sole functional gelling agent.
a. providing a predetermined amount of a liquid growth medium;
b. providing a predetermined amount of a low methoxyl pectin material;
c. providing a culture-growth container having a growth-compatible gel therein, the growth-compatible gel containing a multivalent metal cation material suitable to produce gelling of the low methoxyl pectin material; and d. adding the liquid growth medium and low methoxyl pectin material to the culture growth container to produce a gelled growth medium having the low methoxyl pectin material as essentially the sole functional gelling agent.
2. The method of claim 1 in which steps a. and b.
comprise providing a mixture of predetermined amounts of a liquid growth medium and a low methoxyl pectin material.
comprise providing a mixture of predetermined amounts of a liquid growth medium and a low methoxyl pectin material.
3. The method of claim 2 in which the low methoxyl pectin material has approximately a five percent methoxyl content.
4. The method of claim 2 in which the liquid growth medium includes between about ten and about fifty grams of pectin per liter of growth mediium.
S. The method of claim 1 in which the method volume of the liquid growth medium and low methoxyl pectin material if from about three to about twenty times the volume of the growth-compatible gel.
6. The method of claim 5 in which the culture-growth container is a Petri dish having a growth-compatible gel present as a film layer therin.
7. The combination useful as a biological growth medium which comprises:
a culture growth container;
a growth-compatible gel forming a first layer in said container, said growth-compatible gel including a material other than a pectin material as the substantial functional gelling agent, said growth-compatible metal cation material suitable to produce gelling of a low methoxyl pectin material;
and a growth-medium forming a second layer adjacent said growth-compatible gel in said container said growth-medium gel including a growth medium and as essentially the sole functional gelling agent a low methoxyl pectin material.
a culture growth container;
a growth-compatible gel forming a first layer in said container, said growth-compatible gel including a material other than a pectin material as the substantial functional gelling agent, said growth-compatible metal cation material suitable to produce gelling of a low methoxyl pectin material;
and a growth-medium forming a second layer adjacent said growth-compatible gel in said container said growth-medium gel including a growth medium and as essentially the sole functional gelling agent a low methoxyl pectin material.
8. The combination of claim 7 in which the low methoxyl pectin material is present in an amount of from about ten to about thirty grams of pectin per liter of growth-medium.
9. The combination of claim 7 and which includes at least one buffer selected from the group consisting of Na 2 HPO 4, HaH2P04, Na3P04 NaHC03 and Na2CO3.
10. The combination of claim 7 in which the low methoxyl pectin material has approximately a five percent methoxyl content.
11. The combination of claim 10 in which the multivalent metal cation material comprises calcium cations.
12. The combination of claim 11 in which the calcium cations are present in an amount of from about 50 to about 125 milligrams of calcium cations per gram of pectin.
13. The combination useful in preparing a gelled biological growth medium comprising:
a culture-growth container; and a a growth-compatible gel located in said container said growth-compatible gel including a material other than a pectin material as the substantial functional gelling agent, said growth-compatible gel further including a multivalent metal cation material in an amount of at least about 4 grams 100 milliters.
a culture-growth container; and a a growth-compatible gel located in said container said growth-compatible gel including a material other than a pectin material as the substantial functional gelling agent, said growth-compatible gel further including a multivalent metal cation material in an amount of at least about 4 grams 100 milliters.
14. The combination of claim 13 in which the growth-compatible gel consists essentially of the multivalent metal cation material, agar and water.
15. The combination of claim 14 in which the culture-growth container is a Petri dish having a base including an interior bottom surface, the multivalent metal cation material forming a thin film upon the interior bottom surface.
24 ~
24 ~
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/970,348 US4284554A (en) | 1977-12-23 | 1978-12-18 | Monoazo compounds having a 6-(2'-N-alkylanilino--4-chloro-1',3',5'-triazinyl-6'-amino or alkylamino)-1-hydroxy-3-sulfonaphthalene coupling component radical |
| US325879A | 1979-01-15 | 1979-01-15 | |
| US3,258 | 1979-01-15 | ||
| US6,253 | 1979-02-21 | ||
| US06/006,253 US4282317A (en) | 1979-01-15 | 1979-02-21 | Pectin culture media and method |
| US970,348 | 1992-11-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1115652A true CA1115652A (en) | 1982-01-05 |
Family
ID=27357360
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA342,300A Expired CA1115652A (en) | 1978-12-18 | 1979-12-18 | Pectin culture media and method |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1115652A (en) |
-
1979
- 1979-12-18 CA CA342,300A patent/CA1115652A/en not_active Expired
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