Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~094433
The present invention relates to a yrowth
medium for tuberculosis organisms, and more particularly
to a medium which may be stored under ambient conditions
for extended periods of time without degradation or
other deterioration thereof.
The detection of the disease tuberculosis is
usually performed by culturing a specimen from a
suspected host in a medium referred to in the art as the
Lowenstein-Jensen medium, or modifications thereof. The
culture is incubated and the M. tuberculosis organisms
develop $o the substantial exclusion of other organisms.
Thus, a positive test for tuberculosis is provided.
While this test is quite accurate and is widely
practiced, the test has a decided disadvantage in that
the Lowenstein-Jensen medium is not storage stable at
ambient temperatures and even when refrigerated must be
used within 30 days of preparation. In view thereof, it
has been the practice in the art to use the Lowenstein-
Jensen medium within a relatively short period after
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preparation. The preparation of the Lowenstein-Jensen
medium is quite time consuming and, therefore, the
use of the Lowenstein-Jensen medium has mainly been
restricted to organizations which conduct sufficient
tuberculosis tests as to justify the repeated time-
consuming preparations of the Lowenstein-Jensen medium.
The occasional tester or the smaller institution cannot
economically test for tuberculosis, due to the diffi-
culty and expense of frequently preparing fresh medium.
A basic ingredient in the Lowenstein-Jensen
medium is fresh whole eggs, e.g., eggs no older than
three days. The medium must be prepared in a specific
manner with specific nutrients/salts/inhibitors and fresh
whole eggs, as is well known in the art. If the medium
is not used within a relatively short time after prepar-
ation (even if refrigerated), the results obtained there-
with are questionable. Attempts in the art at extending
the useful life of a prepared Lowenstein-Jensen medium
have not met with success and the most common expedient
is to premix the dry ingredients in a sterile manner and
to place the fresh eggs in the dry mixture when the
medium is to be prepared.
It has earlier been discovered that freshly
prepared Lowenstein-Jensen medium can be stablized for
~5 extended ambient storage by spray-drying that freshly
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prepared medium under such specific and critical con-
ditions as to avoid deterioration of the dried product.
The spray-drying techni~ue is considered to be unique
in drying procedures, in this regard, i.e., as opposed
to tray drying, roller drying, etc. Furthermore, the
Lowenstein-Jensen medium, spray-dried in that specific
mannerj will remain storage stable for extended periods
of time and may be conveniently reconstituted with water
for testing for tuberculosis organisms. Even after
prolonged periods of storage, the spray-dried medium can
be reconstituted and used for testing for tuberculosis
organisms. The primary basis of that conclusion was in
vitro testing where the dried medium, after prolonged
storage periods, was reconstituted and was shown to be
capable of growing M. tuberculosis organisms.
However, clinical trials of the spray-dried
Lowenstein-Jensen medium have discovered that the recon-
stituted spray-dried material is not as accurate as the
freshly prepared medium. Indeed, the accuracy occasioned
by the reconstituted spray-dried medium is decreased to
approximately 75% as accurate as the fresh medium.
; It has now been determined that the population
of tuberculosis organisms in a clinical specimen may be
cons1derably lower than the normal populations encountered
in stock cultures used in in vitro testing. Thus, while
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the spray-dried material is quite a~Cur~te in testing
specimens with relatively large num~ers of tuberculosis
organisms therein, its accuracy substantially decreases
when the population of the organisms decreases This
is often the case encountered in clinical specimens and,
hence, decreased accuracy in the clinical situation is
experienced.
It has also been discovered that in order to
simulate the problem encountered in clinical situations by
in vitro testing, the normal stock solutions of tuberculosis
organisms must be diluted in the order of at least 103.
If adequate growth takes place at these low dilutions,
then the medium is satisfactory for clinical use.
It is therefore an object of the invention to
provide storage-stable Lowenstein-Jensen medium which may
be stored at ambient conditions for prolonged periods of
time without significant deterioration thereof. It is
another object of the invention to provide such storage-
stable Lowenstein-Jensen medium in a dxied form. It is
a further object of the invention to provide such dried
Lowenstein-Jensen medium in a form which can be easily
reconstituted with water by simple mixing~ It is a further
object of the invention to provide such storage-stable
Lowenstein-Jensen medium which, additionally, upon re-
constitution will provide clinical accuracy equal to
freshly prepared Lowenstein Jensen medium. Finally, it
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is another object of the invention to provide methods for
producing such storage-stable Lowenstein-Jensen medium.
Other objects will be apparent from the following disclosure
and claims.
It has now been quite unexpectedly discovered that
Lowenstein-Jensen medium may be stabilized for sustained
preservation by freeze drying the medium in a conventional
free~e dryer and that the reconstituted freeze-dried medium
exhibits tuberculosis testing accuracy equal to freshly
prepared Lowenstein-Jensen medium.
Thus, there is provided a dry, storage-stable
Lowenstein-Jensen medium which may be reconstituted with
water to provide a medium suitable for use in tuberculosis
testing, comprising a freeze-dried form a Lowenstein-Jensen
medium, the moisture content of the freeze-dried medium
being less than ~% by weight and the reconstituted freeze-
dried medium exhibits tuberculosis testing accuracy equal
to that of freshly prepared Lowenstein-Jensen medium.
There is also provided a method for preparing
the dry, storage-stable Lowenstein-Jensen medium, which
comprising mixing fresh whole eggs and Lowenstein-Jensen
nutrients, salts and inhibitors in Lowenstein-Jensen
proportions, freezing the mixture and subjecting the mixture
to sufficientl~7 reduced pressure that sublimation of
liquid in the frozen mixture occurs and until a moisture
content of the mixture of 8~ or less is reached.
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The initial medium prepared for freeze drying
will be the same as a conventional Lowenstein-Jensen
medium. There are a number of variations on the
procedures for preparing a Lowenstein-Jensen medium or
a modification thereof, but generally, fresh whole eggs
are aseptically combined with Lowenstein-Jensen nutrients,
salts, stabilizers, inhibitors, thickeners, etc., in
Lowenstein-Jensen proportions to provide a well-blended
mixture. Thereafter, the mixture is inspissated at less
than 80-90C for about 45 minutes.
A commonly used procedure is that of cleaning
fresh eggs in 5% soda and soap solution, rinsing thoroughly
with ~ater, aseptically breaking the eggs into a sterile
flask containing glass beads, adding the nutrients, salts,
etc., shaking to form a uniform mixture or emulsion,
placing the mixture in slant tubes and autoclaving the
tubes in a slanted position at 80-90C for 45 minutes.
Sterility is checked by incubating the tubed medium slants
at 37C. The slants are stored under refrigeration.
Irrespective of the specific procedure used,
after the medium has been prepared, according to the
present invention, it is introduced into a conventional
reeze-drylng apparatus and freeze dried under conventional
conditions. The particular conventional freeze drying
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conditions may be chosen as desired and it is only
important that the dried medium have a moisture content
of 8~ by weight or less.
Ho~ever, quite conveniently, the freeze drying
S process may be conducted as follows: Firstly, the
conventional Lowenstein-Jensen salts are added to the
appropriate portion of water with or without heat (up
to the boiling point of the water) to aid mixing.
Asparagine is then added, preferably to the heated mixture,
with stirring to dissolve the same. After the salts,
etc. are fully dissolved, the solution is cooled and
potato flour is then added (preferably a~ tepid tempera~
tures to aid in mixing). After a homogeneous mixture is
obtained, the temperature is raised to heat the potato
flour, e.g. 150-190F for 1 - 30 minutes. That mixture
is then cooled to at least below 130F, and the fresh
whole eggs are added thereto. Alternatively, a potato
flour mixture and a salt/nutrient mixture may be separately
prepared and added together. In any event, the total
mixture of the nutrients/salts~inhibitors~ etc. and
potato flour is prepared before adding the fresh whole
eggs.
After the eggs are homogeneously dispersed in
the mixture, the mixture is placed on trays and subjected
to reduced temperatures for freezing thereon. ~hile these
~; temperatures are not critical, the lower the temperature
the faster the process. For this reason r temperatures at
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least below 25~F are preferred, e.g., 10~ to -20F.
The frozen slab of Lowenstein-Jensen medium is
removed from t;ne trays and broken or crushed to provide
increased sur~dce area. Ideally, the average particle
size will be between 1/16 inch and 1/2 inch, usually
between 1/8 inch and 3/8 inch. A good average size is
about 1/4 inch, since this avoids packing of the particles
and yet provides sufficient surface area for effective
freeze drying.
The crushed frozen Lowenstein-Jensen medium is
then poured into conventional freeze-drying trays (pre-
ferably covered with a polyethylene lining) to provide a
layer of from 1/4 to 1 inch thick, usually about 1/2 inch
thick. These trays are placed in a conventional freeæe
dr~er so that the trays are suspended in juxtaposition to
heated shelves. A vacuum is drawn on the trays, e.g.
100 to 600 microns, usually between 100 and 200 microns.
Oil which is recirculated through the shelves for temperature
control is adjusted such that the temperature of the surface
of the layer of the crushed Lowenstein-Jensen medium, as
usually measured by thermocouples placed thereon, does not
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exceed at any one point a temperature of 130F. While
freeze drying is conventionally conducted at temperatures
of the surface of the layer of material in excess of 150 or
even 160F, this cannot be practiced with the present
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invention. It has been found that if temperatures at the
surface of the 1ayer exceeds about 130F, then the
resulting freeze-dried product is substantially deteriora-
ted in its accuracy of turberculosis testing. Otherwise,
the freeze-drying technique is as conventionally practiced.
The moisture content of the dried product, however, must
be about 8% or less and preferably less than ~%. Usually,
the moisture co~tent of the freeze-dried product will be
between 3% and 5~.
The dried powder is recovered from the freeze
dryer in the conventional manner and may then be packaged
in any container desired so long as the container prevents
ambient contamination. Thus, the dried powder may be simply
packaged in plastic or glass jars, flexible film, blister
packages, twisted plastic bag packages, foil packages, wax
paper packages and the like. While it is not required that
the package be hermetically sealed, this is preferred (or like
provisions be made), which provides that ambient contamina-
tion be substantially eliminated by the packaging method.
The elimination of ambient contamination is not in connection
wîth the stabilized Lowenstein-Jensen medium itself, but
in connection with unknown organisms which might be intro-
duced into the reconstituted powder when used in testing
for tuberculosis.
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The dried and packaged powder need not be stored
under special temperatures, and ambient temperatures are
quite acceptable. However, extreme temperatures should
be avoided for obvious reasons. Accordingly, it is
preferred that the powder not be subjected to freezing
temperatures, nor is it preferred that the powder be
stored at temperatures above 120F, preferably less than
115F, although neither of these extreme temperatures will
adversely affect the powder unless the powder remains at
those extreme temperatures for prolonged periods. On the
other hand, the powder may be stored for prolonged periods
of time at ambient temperatures without significant deter-
ioration thereof, i.e. at least six months and up to one
year, although as a precautionary measure and as a safety
factor, it is preferred that the powder be reconstituted
and used within 8 to 10 months of the drying time.
The powder may be reconstituted simply by mixing
with water. Of course, it is preferred that the mixing be
under sterile conditions and that the water be both sterili~ed
20 and distilled. Inspissation at 80C to 90C for 1 hour
may be used if desired. The amount of powder which is
reconstituted in water can vary considerablyl but it is
preferred that the amount which is reconstituted corresponds
to that which will produce the same solids content as the
original freshly prepared Lowenstein~Jensen medium. Thus,
~C)94433
the solid content of t)~ reconstituted medium can vary
from as low as 10% ~o ~s high as 20~ by weight, but the
preferred solid content will be between about 11~ and 17%,
especially between 13~ ~Ind 15%.
Reconstitution can be accomplished without any
special technique or equipment, and simple mixing is
quite adequate. Thus, the dried powder and water may be
simply hand mixed or mixed with a conventional laboratory
stirrer until dissolution thereof takes place. The mixing
may be at ambient temperatures or slightly depressed or
elevated temperatures and no special consideration in this
regard is required. Accordingly, temperatures abo~e the
freezing point of water and as high as 120~F can be used
for preparing the reconstituted mixture. However, there
is no special advantage obtained by such variation in
the temperature of the water for reconstitution, and
ambient water is quite satisfactory.
For purposes of the present specification, the
Lowenstein-Jensen medium is defined as a medium for the
growth of tuberculosis organisms (Mycobacterium tuber--
culosi_) and consists essentially of fresh whole egg,
Lowenstein-Jensen defined nutrien~ salts and inhibitors
in Lowenstein-Jensen defined proportions, all of which is
well known to the art. I~ desired, stabilizers,thickeners,
dyes and like non-essential ingredients may be used.
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1094433
The specific nutrients/salts/inhibitors and the rat~os with
the fresh whole egg may vary, as is known in the art.
Thus, all combinations of fresh whole egg/inhibitors/nutrients/
salts suitable for growing tuberculosis organisms are
intended to be embraced by the present disclosure and
claims. A typical Lowenstein-Jensen formulation, however,
will contain from about 0.5% to about 3~ salts. The
amount of water will be from about 2% to about 10% for
dispersing and dissolving the salts, but more generally
from about 4% up to about 8%. Very often the amount of
water will be about 5 or 6%. Additional water may be
used if desired for easy dispersement and dissolution
of the salts since this only goes to diluting the con-
centration of the Lowenstein-Jensen medium. Higher
dilutions are not preferred, however, and generally speak
ing, no more than 15% by weight of the medium will be
added water. The remainder of the medium will be
essentially fresh eggs, but if desired, aside from the
usual inh~bitors, thickenersr viscosity control agents
and the like may be added. Thus, typically, the medium
will also contain up to 7% of potato flour, especially
between 1% and 6% and more generally between 4% and 5%.
Glycerol is not used in the freeze-drie~ powder since it
will interfere with freezing, but it may be added during
reconstitution of the dried powder at typical concentrations.
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An inhibitor or a dye or a material which will function
in both manners is also generally added to the medium.
Thus, 0.1~ to about 1% of an inhibitor and/or dye, and
more generally 0.5% to 1% Malachite Green is commonly
used in a Lowenstein-Jensen medium for this purpose.
The typical salts used in the medium generally contain
magnesium and phosphorus, as well as potassium and the
particular combination of these salts is not narrowly
critical. However, as a typical example, monopotassium
phosphate, magnesium sulfate, and magnesium citrate may
be used. Also, typically, asparagine is used as a very
convenient nutrient. Asparagine is an amino acid found
in the sprouts of many weeds and hàs the formula
NH2.CH.(COOH).(CH2CONH2).H2O.
It has been found that the present freeze-dried
Lowenstein-Jensen medium is equal in accuracy of tubercu-
losis testing to freshly prepared Lowenstein-Jensen
medium, while the spray-dried Lowenstein-Jensen medium
referred to earlier has a decrease of accuracy in a
clinical setting to about 75%. The reason for this
difference is not known and it is, indeed, surprising
that the freeze-dried medium functions in such a
superior manner as compared with the spray-dried medium.
Investigations into the p~ysical differences between the
spray-dried and freeze-dried medium have not provided
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reasons for these differences. While the spray-dried
medium is more nearly in the form of spherical par~icles
and the like, while the freeze-dried material is more
normally in the form of flaked particles and the like,
differences in ability to dissolve, disperse, etc.
do not account for the differences in clinical performance.
The experimental work which culminated in the
spray-dried medium was based, primarily, on in vitro
testing of the spray-dried medium. It has subsequently
been discovered that the population of tuberculosis
organisms normally established under equilibrum condit-
ions in stock solutions of the organisms is so ~reat
that no difference in accuracy of the spray-dried medium
and the freshly prepared medium can be observed when
testing with these stock solutions in ln vitro circumstances.
However, the population ~f tuberculosis organisms often
encountered in a clincial specimen is substantially lower
- than that of the stock solution. Hence, for these clinical
specimens, the spray-dried medium is not sufficient to
detect the presence of low populations of tuberculosis
organisms, while the freshly prepared Lowenstein-Jensen
medium is sufficiently accurate for those specimens. It
can therefore be seen that the use of the spray-dried
material places the clinician at a distinct disadvantage,
as opposed to the use of a freshly prepared Lowenstein-
Jensen medium.
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It is now discovered that if classical serial
dilutions are made that at dilutions between about 104
and 105 in vitro testing can essentially simulate the
difficult clinical specimens discussed above. These
dilutions are made in the traditional manner of inocu-
lating a test culture with stock solution, incuba$ing
and transferring a portion of the incubated culture to
another culture such that a ten-fold dilution occurs.
That culture is then incubated and a further transfer
to a new culture is made with a subsequent ten-fold
dilution, etc. With this _ vitro testing, the present
freeze-dried material remains accurate at 104 and 105
dilutions, the same maximum dilutions of the freshly
prepared Lowenstein-Jensen medium.
This in vitro testing has also been confirmed
for the freeze-dried material by clinical testing where
the accuracy of the freeze-dried medium is the same as
the accuracy of the freshly prepared Lowenstein-Jensen
medlum.
Accordingly, the present invention provides a
most distinct improvement over the above-described
spray-dried medium and also provides the art with the
advantage of a storage-stable easily-reconstituted
Lowenstein-Jensen medium. Thus, the objects of the
invention have been achieved.
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The invention will be illustrated by the follow-
ing examples, where all percentages and proportions are
by weight, unless othexwise indicated, but it is to be
clearly understood that the invention is not limited
thereto and is fully applicable to the foregoing disclosure.
Exam~le 1
The following salts were added to water:
monopotassium phosphate 2.06 lbs.
magnesium sulfate 90 g.
magnesium citrate 225 g.
water~de-ionized) 59.5 gal.
The solution was prepared by adding the salts to the water
in a 200 gallon stirred kettle. The temperature was
brought to 200F and 625 grams of asparagine was added
and mixed at that temperature for 20 to 30 minutes. After
mixing, the solution was cooled to below 100F.
To the cooled solution was added 25 lbs. of
potato flour in small increments with constant agitation
to avoid lumping. Thereafter, the mixture was heated to
180F, stirred at that temperature for 20 minutes and then
cooled to below 130F.
Thereafter 840 lbs. of whole homogenized eggs
(prewashed) were added to the solution and thoroughly
mixed.
150 grams of Malachite green was added and
after mixing, the solution was placed in polyethylene
lined trays and frozen in a conventional freeze-drying
apparatus at about -10F~(Stokes-~astern Freeze-Dry Corp).
After freezing, the resulting slabs were crushed to
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109~l433
an average particle size of 1/4 inch. The trays were
filled with th~ crushed material to form a layer of about
1/2 inch thick. A vacuum of about 100 to 200 microns was
pulled on the trays and the recirculated oil in the
shelves of the dryer was adjusted so that the maximum
temperature of the surface of the layers was below about
120F. Drying was continued until the product had
moisture contents of between 3 and S~.
The dry product was pulvarized to a ~ree-
flowins powder and packaged in air-tight containers.
Example 2
Product of Example 1 was reconstituted with
distilled water by mixing and slants thereof were pre-
pared in the same manner as conventionally practiced with
fresh Lowenstein-Jensen medium. The slants were inoculated
with bacteria taken from a stock culture of ~ycobacterium
tuberculosis. After incubation, a 10 fold dilution of
the resulting culture was made and that diluted culture
was incubated. This procedure was serially repeated
to determine the maximum dilution of organism which would
grow on the reconstituted freeze dried Lowenstein-Jensen
medium. A total of 12 simultaneous tests were conducted
and it was determined that between the 104 and 105 dilutions,
no growth took place. This test determines the sensitivity
~ 25 of the freeze-dried medium for detecting low population
; nun~ers of the tuberculosis organism.
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1094433
The same test was repeated with commercially
available, freshly prepared and pre-tubed Lowenstein-
Jensen medium (Difco brand). In this case growth also
ceased between the 104 and 105 dilutions.
Thus, it can be seen that from an in vitro
point of view the freeze-dried Lowenstein-Jensen medium
is equal to the freshly prepared medium.
Example 3
Clinical trials were made with
~a) Commercially available, freshly prepared and
pre-tubed Lowenstein-Jensen medium (Difco brand);
~b) reconstituted freeze-dried medium of
Example l; and
(c) reconstituted spray-dried medium prepared
as follows. Lowenstein-Jensen medium was pre-
pared by mixing until uniformly blended the
following ingredients in the weight % shown in
parentheses. To water (5.45) was added glycerol
(1.75) and Malachite Green (0.059), monopotassi-
um phosphate (0.365) r magnesium sulfate (0.035),
magnesium citrate (0.087), asparagine (0.525),
potato flour (4.38) and the balance of fresh
eggs, were dispersed therein. After a mixture
has been made, it is whipped until air bubbles
are dispersed in the mixture, i.e,, a frothy
mixture is obtained. After mixing, the ingre-
dients were spray dried in a conventional box-
type spray dryer with an inlet temperature of
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280F and an outlet temperature of 180F.
To the spray-dried powder was mixed between
1/2 and 1~ on a weight basis of finely divided
silica of particle size between submicron and
micron range (Syloid)* to improve flowability
of the spray-dried powder. The spray-dried
composition obtained therefrom was reconstitut-
ed with water to prepare a reconstituted
medium having the same proportions of solids
as the medium from which the corresponding
sample was spray dried. The reconstituted
medium shows all of the properties of the
original medium prior to spray drying.
The freshly prepared medium was considered the
standard and the freeze-dried medium and the spray-
dried medium were compared with the standard in terms
of the ability to detect the presence of tuberculosis
organisms in clinical samples. This shows an overall
accuracy of the medium, as compared with the freshly
prepared medium.
No difference between the freshly prepared
medium (Difco brand) and the freeze-dried medium was
observed, but the spray-dried medium was only 72% as
accurate as the freshly prepared medium and the
~reeze-dried medium.
* Trademark
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Example 4
In order to demonstrate the storage properties
of the dried powder, samples of the product of Example
1 were stored in a tightly capped container for the
periods of 1,2,3,4,5,6,10 and 12 months. At the end
of each storage period, the sample for that period
was reconstituted to provide a medium with the same
- solid content as was originally present in the medium
prior to freeze drying. Slants of each sample were
tested by inoculating with tuberculosis organisms.
At the end of an incubation period of 2 weeks, the
samples were examined and each showed a positive
culture of M. tuberculosis. Accordingly, this example
illustrates that the present freeze-dried medium can
be stored or prolonged periods of time and thereafter
easily reconstituted for use in tuberculosis testing.
Example 5
The procedure of Example 4 was serially re-
peated with the following organisms instead of the M.
tuberculosis Mycobacterium avium, M. bovis, M.
kansasii, M. fortutium, M. intracellulare, and M.
scrofulaceum. Each organism was provided from a stock
culture. The freeze-dried sample had been stored for
1 year and the incubation period was 3 weeks. Each
test was compared with pre-tubed Lowenstein-Jensen
medium (Difco brand). The growths on the reconstituted
medium and the pre-tubed medium were equal.
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This example shows that the stored, dry medium
is effective for a wide range of Mycobacterium, which
are also indicated in tuberculosis-type diseases, and
may be so used. It also shows that the present
medium is most satisfactory as a growth medium for
the genus Mycobacterium.
Modifications of the foregoing can easily
be aceomplished by those skilled in the art and such
modifications are intended by the specification. Thus,
the invention is limited only by the following claims.
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