Note: Descriptions are shown in the official language in which they were submitted.
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.
, .
,~,.
~Sf~5~
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.
-- 4 --
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
65Z
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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~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.
-- 10 --
--` 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 --
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,
- 12 -
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
156SZ
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
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111565Z
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.
EXAMPLE 1
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
- 18 -
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).
EXAMPLE 3
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.
EXAMPLE 4
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.
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111S6SZ
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
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111565Z
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.
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