Note: Descriptions are shown in the official language in which they were submitted.
-~ -1- lU99654
913, 812
BACTERIA GROWING DEVICE
This invention relates to a device for growing
bacteria. Specifically, this invention relates to a
device for growing bacteria from an initial population to
a final predetermined population, said device including a
medium for such growth and a means for obtaining a
predetermined population for inoculation of said medium.
In Canadian patent application serial No.
303,737 filed concurrently with this application growth
; 10 limiting media are described and claimed. These media are
described as being useful in a number of procedures
utilized to identify the bacteria or to determine the
susceptibility of the bacteria to certain antibiotics. In
such procedures it is necessary to have the bacteria at
the beginning of the test procedure in a certain
concentration range (colony forming units per milliliter
(CFU/ml)) or the final result will not be accurate. For
example, in an artlcle entitled "Antibiotics
Susceptibility Testing by Standardized Single-Disc
Method", The American Journal of Clinical Pathology, Vol.
45, No. 4, April, 1966, Pages 293-296 and in the
"Performance Standards for Antimicrobial Disc
Susceptibility Test", ASM-2, promulgated by the National
Committee for Clinical Laboratory Standards of the United
States, the Kirby-Bauer procedure for determining the
susceptibility of rapidly growing bacteria to antibiotics
and chemotherapy agents is described.
The Kirby-Bauer procedure normally involves
~` growing on an agar plate colonies of bacteria obtained
from a patient. A wire loop is used to pick from 4 to 5
colonies of the bacteria and introduce them into test
; tubes containing 4 to 5 milliliters of soybean casein
digest broth. The tubes are then incubated for 2 to 8
hours to produ¢e a bacterial suspension of moderate
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cloudiness. The suspension is then diluted, if necessary,
with saline solution or like broth to a density visually
equivalent of that of a standard prepared by adding 0.5
milliliter of 1% BaC12 to 99.5 milliliters of 1% H2SO4
(0.36 N) (0.5 McFarland Standard hereinafter referred to
as the McFarland standard). A plate containing
Mueller-Hinton agar is then stréaked with the bacterial
broth suspension using a cotton swab. After the inculum
has dried, a paper disc containing an antibiotic or
chemotherapeutic agent is applied to the agar. The plates
are incubated. After overnight incubation the area around
each disc wherein there is an absence of bacteria growth
is measured. This is known as the zone of inhibition and
is used to determine which antibiotic will be useful in
combating the particular bacteria.
In order for the Kirby Bauer technique to be
accurate there must be approximately 1 x 108 CFU/ml
included in the medium which is streaked onto the agar
plate. Usually the level of growth is determined by using
the visual comparison with the McFarland standard
described above. The time period to reach this concentra-
tion of bacteria may vary from 2 to 8 hours depending on
the bacteria. If the bacteria are allowed to grow in
excess of 1 x 108 CFU/milliliter and become more turbid
than the McFarland standard, the medium must be diluted in
order to be equivalent to the standard.
Another method of determining the susceptibility
of bacteria to various antibiotics is called the MIC or
Minimum Inhibitory Concentration test. This test is
discussed in Current Techniques for Antibiotics
Susceptibility Testing, Albert Balows, ~ 1974, pages
77-87. This method involves preparation of a series of
concentrations of an antibiotic, either in a liquid or
solid medium which will support the growth of a bacteria
to be tested. Liquid media are conveniently dispensed in
test tubes and solid media are usually poured into petri
dishes. It is common practice to prepare a range of
_3_ 1 ~9 g6 S 4
antibiotic concentrations as a series of two-fold
dilutions in order to carry out the test. Each tube or
petri dish is inoculated with the bacteria in question.
After a period of incubation, the bacterial growth or
absence of growth of each antibiotic concentration is
observed. In this way, the minimum inhibitory concentra-
tion of the antibiotic is determined to the nearest
dilution when used in a series. This is the most accurate
method of determining the inhibitory concentration.
~owever, this method did not gain popularity until
recently when the laborious effort of making the dilutions
was simplified. The diluted antibiotic is inoculated in
the MIC test with bacteria at a certain concentration,
i.e., normally 105 to 106 CFU/ml. Broth containing
bacteria grown to the equivalent of the McFarland
standard, i.e., approximately 1 x 108 CFU/ml, is diluted
to obtain this concentration.
The aforesaid susceptibility tests as well as
other tests for determining the types of bacteria or
susceptibility thereof to antibiotics require that a
certain predetermined amount of bacteria be utilized in
the test to inoculate the plates upon which the paper disc
will be placed in the case of the Kirby-Bauer test or to
inoculate the diluted antibiotics in the case of the MIC
test~ This is required in order for the test to be
accurate. If a lesser concentration of bacteria is
utilized in the test, the result would indicate that the
bacteria is more susceptible to the antibiotic than it
would be as an actual fact. On the other hand, if the
bacteria are present in a higher concentration, the test
result would indicate that a higher concentration of the
antibiotic would be required in order to inhibit the
growth of the bacteria. Both indications would be
erroneous.
In order for the aforesaid tests or tests
similar thereto to be performed, it is necessary for the
laboratory technician to take a sample of bacteria from 4
lV99654
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or 5 colonies of bacteria from the agar plate upon which
the bacteria have been growing and place it in a broth
growing medium such as above described for 2 to 8 hours.
The medium is checked periodically to determine whether or
not a sufficient concentration of bacteria has grown to be
equivalent to the McFarland standard. From a visual
examination of the medium, one will find that some medium
cultures of bacteria have not grown to the proper concen-
tration while others have grown beyond the appropriate
concentration. The former requires that the technicianallow the bacteria to grow longer, whereas the latter
requires a dilution to bring the concentration back to
that of the standard. All of these measures are tedious
and time consuming.
The above-mentioned Canadian patent application
serial No. 303,737 filed concurrently herewith describes a
growth-limiting medium which will grow bacteria to a
certain predetermined concentration or population level.
That medium is capable of growing at least one species
from two different genera of aerobic, pathogenic, rapidly
growing bacteria from a beginning population to a
determined ending population at which growth of the
bacteria substantially subsides due to the lack of
nutrient in the medium and wherein bacteria remain viable
for at least 18 hours; the medium comprising an aqueous
solution comprising a carbon source, a nitrogen source,
vitamins and minerals of sufficient quantity to provide
growth and in a form usable by the bacteria for growth.
The general description of that medium will be
described herein for purposes of completeness.
The bacteria upon which the medium is useful are
aerobic bacteria, i.e., those which use oxygen to grow.
The bacteria are also pathogenic in that they cause
diseases and are rapidly growing in that they have a
generation time of 50 minutes or less.
The medium is useful with both gram-negative as
well as gram-positive aerobic, pathogenic, rapidly growing
.
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bacteria. However, the type and amount of the various
ingredients in the medium are normally different for
gram-positive than for gram-negative bacteria, and the
time period required to obtain the requisite concen-
tration for gram-positive tends to be longer than that for
gram-negative bacteria.
The growth medium will grow at least one species
from two different genera of aerobic, pathogenic bacteria.
Normally, the medium will grow at least one species from
two genera of gram-positive bacteria or at least one
species from at least two genera of gram-negative
bacteria. Within gram-positive, aerobic bacteria, there
are two genera which include the bacteria that cause most
diseases for which normal bacteria and susceptibility
testing are performed. These are Staphylococcus and
Streptococcus. If the medium will grow species from each
of these two genera, then it allows one to merely test the
bacteria to determine whether they are gram-positive or
negative using a gram stain test. If the bacteria are
gram-positive, medium which grows the gram-positive
bacteria is used, and the medium will grow the bacteria to
the level desired such as to the equivalent of the
McFarland standard.
If the bacteria are determined to be gram-
negative using the gram stain test, the bacteria could befrom a much larger number of genera. Sixteen genera
represent the bacteria found to be the cause of 99% of
illnesses caused by gram-negative aerobic bacteria. These
gram-negative genera include: Escherichia, Shigella
Edwardsiella, Salmonella, Arizona, Citrobacter,
Klebsiella, Enterobacter, Serratia, Proteus, Providencia,
Yersinia, Pseudomonas, Acinetobacter, Moraxella and
Pasteurella.
The medium grows the bacteria from a certain
population which will normally be described in terms of
CFU as above defined and will normally be referenced to
the population in a certain volume of medium, i.e.,
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CFU/ml. The beginning concentration can range as low as
l CFU but will normally be, and is preferably, at least
5 x 106 CFU/ml.
Starting with a lower initial population or
concentration will not cause the medium to grow the
bacteria to a significantly different final population or
concentration than with a higher starting concentration,
but will affect the time it takes for the final concentra-
tion to be reached. Thus, if one is to control the
incubation time, the beginning concentration must be
controlled. The final concentration to which the bacteria
grow is referred to as the stationary phase.
As above discussed the time to reach a concentra-
tion equivalent to the McFarland standard varies according
to the test procedure from 2 to 8 hours with most bacteria
taking at least 5 to 6 hours to reach that concentration.
With the growth-limiting medium, the bacteria preferably
reach the final concentration or stationary phase within
5 hours. With the growth-limiting medium, if the bacteria
reach the stationary phase in 2 hours, they will remain
there even if the technician does not check the medium for
5 hours, and no dilution will be required to obtain a
concentration equivalent to the McFarland standard.
When the stationary phase or final concentration
- 25 is reached, the growth of the bacteria substantially
subsides. This is due to exhaustion of at least one
nutrient critical to the continued growth of the bacteria
and not to the formation of toxic byproducts by the
bacteria which stop growth and can cause the population to
- 30 decrease substantially. With the standard media used
prior to the growth-limiting medium and described in the
Kirby-Bauer procedure, the population of bacteria which
will be reached in order for growth to subside would be
determined by toxic byproducts of the bacteria. This
population of bacteria is above the McFarland standard.
The final concentration or maximum stationary
phase is obtained because of exhaustion of one or more
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nutrients. At that point the viable CFU/ml count levels
off and remains substantially unchanged for at least about
18 hours. The bacteria remain viable for a period of time
useful for carrying out the various tests to be performed
thereon, for example, those described above. Normally the
time period for such viability is at least 18 hours.
The final concentration which is desired to be
reached with most bacteria will be between 6 x 107 to
3.0 x 108 CFU/ml. This is equivalent to the 0.5 McFarland
standard. The medium can be modified to reach different
desired concentration levels.
The medium will contain different types and
amounts of ingredients depending upon the final concen-
tration of bacteria desired and depending upon the type of
- 15 bacteria being grown. In all cases a carbon and nitrogen
source are present which provide carbon and nitrogen in a
form useful by the bacteria for growth. Normally, vita-
mins and minerals are also present. However, as noted,
the amount of one or more of these ingredients is limited
to cauge the bacteria to reach a final predetermined
concentration and substantially cease growing.
For the gram-negative bacteria, a preferred
medium comprises about 0.42 to about 0.70 milligram of
carbon per milliliter of medium. The carbon is in a form
useful by the bacteria for growth. This form has been
found to be that form in which carbon is present in
peptone or a similar form. The preferred medium also
comprises 0.09 to 0.15 milligram of nitrogen per
milliliter of medium in a form similar to the nitrogen
present in peptone. The preferred medium has a pH from
about 7 to 8. With proteose peptone, the range for carbon
is from about 0.16 to about 0.27 milligram of carbon per
milliliter of medium, the nitrogen is 0.035 to 0.056 milli-
gram of nitrogen per milliliter of medium, and the carbon
and nitrogen are as found in proteose peptone or a form
similar thereto. A typical analysis of the peptone is set
forth below.
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Percent _ Peptone Peptone
Total Nitrogen 16.16 14.37
Primary Proteose N 0.06 0.60
Secondary Proteose N 0.68 4.03
Peptone N 15.38 9.74
Ammonia N 0.04 0.00
Free Amino 3.20 2.66
Amide N 0.49 0.94
Mono-amino N 9.42 7.61
Di-amino N 4.07 4.51
Tryptophane 0.29 0.51
Tr~osine 0.98 2.51
Crystine 0.22 0.56
Organic Sulfur 0.33 0.60
Inorganic Sulfur 0.29 0.04
Phosphorus 0.22 0.47
Chlorine 0.27 3.95
Sodium 1.08 2.84
Potassium 0.22 0.70
Calcium 0.058 0.137
Magnesium 0.056 0.118
Manganese nil 0.0002
Iron 0.0033 0.0056
Ash 3.53 9.61
Ether Soluble Extract 0.37 0.32
Reaction, pH 7.0 6.8
- p~ 1~ solution is distilled water after autoclaving 15
minutes at 121C.
1a~99654
g
The preferred formulation contains the vitamins
and minerals found in peptone or proteose peptone. Two
specifically preferred formulations which have been found
to be useful in growing gram-negative bacteria to a final
concentration of from 6 x 107 to 3 x 108 CFU/ml in less
than 5 hours comprises a mixture of 1000 ml of water
containing 0.8 gram peptone or 0.3 gram proteose peptone,
0.03 gram dextrose, 2.5 grams dipotassium phosphate, 1.25
grams monopotassium phosphate and 5.0 grams sodium
chloride. The phosphates are added as a buffer material
to main~ain the composition at a pH of approximately 7Ø
Buffering is necessary with certain of the bacteria. The
aforesaid two formulations provide a medium upon which
species from most of the genera of the gram-negative,
aerobic, pathogenic bacteria can grow to the above
described CFU/ml ranges within 5 hours if the initial
concentration of bacteria is sufficient, i.e., at least
about 5 x 106 CFU/ml.
As noted the preferred carbon and nitrogen
sources are peptone and proteose peptone for the
gram-negative bacteria. Neopeptone, tryptone and
polypeptone can also be used but it has been found that
these do not produce growth to the levels of the ~cFarland
standard within the same time frame, and with some of the
bacteria within this group, they do not provide the
appropriate nutrients to grow the bacteria to any signi-
ficant degree. Therefore, these materials are useful for
more limited numbers of bacteria. However, combinations
of such materials with peptone and proteose peptone can be
used to provide a medium useful with a larger number of
bacteria.
A preferred medium for use with gram-positive
bacteria comprises a solution of 1000 ml of water
containing 1.7 grams trypticase, 0.3 gram phytone, 0.25
gram dextrose, 0.5 gram sodium chloride and 0.25 gram
dipotassium phosphate.
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0- 1C199654
Applicants have found a device which utilizes
the above described growth-limiting medium and allows one
to obtain from colonies of bacteria a certain
predetermined amount of bacteria so that the growth-
limiting media is inoculated with a predetermined numberof bacteria. This allows the time period for growth of
the bacteria to be predetermined as well. In order for
the requisite growth to occur within the preferred time of
within 5 hours for gram-negative bacteria, the beginning
population must be at least about 5 x 106 CFU/ml.
Applicants have, therefore, discovered a device
for growing bacteria from an initial population to a
determined ending population at which the growth of the
bacteria substantially subsides due to the lack of
nutrient comprising: (a) a vessel containing a supply of
growth medium capable of growing the bacteria from the
beginning population to the determined ending population,
the vessel containing an opening; (b) means within the
vessel for obtaining a known quantity of bacteria from at
least one growth colony of the bacteria external to the
vessel and for inoculating the growth medium; and (c)
removable means for covering the opening in said vessel to
preserve the sterility of the inside of the vessel, for
permitting the means for obtaining said bacteria to be
removed to obtain the known quantity of bacteria from the
colony of the bacteria external of the vessel, which
bacteria are used to inoculate the medium and for closing
the vessel during the incubation of the inoculated medium.
The device will be described in more detail with
reference to the following drawings in which Figure 1 is
an exploded sectional view of the device of the present
invention; Figure 2 is a prospective view of the device of
the present invention with parts in section; Figure 3 is
an exploded view of a portion of the wand of the device of
the present invention Figure 4 is the wand of Figure 3
showing the inclusion therein of bacteria; Figure 5 is a
sectional view of the device of the present invention just
99654
after inoculation of the growth medium with the bacteria;
Figure 6 depicts a section of the device of the present
invention as shown in Figure 5 taken along line 6-6;
Figure 7 is a section of the device of the present
invention after inoculation and incubation to the maximum
stationary phase or the predetermined growth stage; and
Figure 8 depicts a section of the device of Figure 7 taken
along line 8-8.
The device of the present invention comprises a
vessel or sleeve 1 which is made of a transparent
deformable material such as polypropylene, polyamide,
cellulose acetate butyrate or various polyesters.
Contained within the sleeve 1 is glass ampoule 2 which
contains within it the growth limiting medium 3 as above
described. The glass ampoule, as will be discussed later,
is frangible, i.e., it breaks when the deformable sleeve 1
is squeezed. In juxtaposition to the ampoule 2 of the
device is wand 4 which contains tapered groove 5 which
will be described in more detail with reference to Figures
3 and 4. Wand 4 is used to pick bacteria from the various
colonies of bacteria and is made to injest a predetermined
amount of bacteria from the colonies into the tapered
groove 5. Wand 4 is affixed to cap 6. Both cap 6 and
wand 4 are made from plastic material such as poly-
propylene. Interior to cap 6 are two ridges 7 and 8 whichallow for an aseptic seal between the cap 6 and sleeve 1.
This prevents other microorganisms from entering the
sleeve 1 prior to the time that the device is inoculated
as well as during incubation. Also contained within cap 6
are three prongs 9, two of which are shown in Figure 1.
These prongs protect hole 10 in cap 6 from being blocked
by broken glass from ampoule 2 (caused when the device is
activated) during the time when deformable sleeve 1 is
being deformed to express or exude the bacteria and medium
3 via hole 10. Hole 10 is covered with pressure sensitive
adhesive tape 11 containing tab 12 during the time prior
to use of the device and up until incubation of the device
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is complete. At that time, tape ll is removed exposing
hole 10. This allows for the medium 3 and bacteria to be
exuded or expressed from the sleeve 1.
Figure 2 shows the device in its normal form
prior to use except the pressure sensitive adhesive tape
ll is not present. As can be seen, wand 4 is adjacent to
ampoule 2 and rims 7 and 8 are in juxtaposition to the
upper portion of sleeve l. In this case hole 10 is
exposed to show it from a prospective view and pressure
sensitive adhesive tape 11 is not shown. Prongs 9 are not
visible in this view. Glass ampoule 2 is ~ormally 35.6
millimeters by 6.3 millimeters in dimensions and contains
approximately 0.6 millimeters of growth media. The sleeve
1 is normally 43.0 millimeters long and 8.6 millimeters in
diameter.
An important feature of the device of the
present invention is the wand 4 containing tapered
groove 5. This groove 5 is shown in more detail in Figure
3. As can be seen, the groove 5 is tapered. This groove
i8 designed to provide a capillary action by which the
bacteria are pushed into the groove 5. As the bacteria
are pu~hed by means of pushing the wand 4 perpendicular to
the surface of the colony so that the larger end of
tapered groove 5 first contacts the colony, the bacteria
begin to move up the groove and air is exuded from the
tapered end of the groove. At a certain predetermined
level, no further bacteria can be pushed into the groove
because bacteria which are continually forced into the
groove 5 move out of the top of the groove 5 rather than
moving up to the tapered end of the groove 5. Figure 4
shows schematically bacteria 13 in the groove 5 at the
normal filled groove level. The groove 5 is designed to
allow a certain population of bacteria to enter the
groove. This is normally equivalent to at least 5 x lO6
bacteria per milliliter when the bacteria are placed in
the medium of the device of the present invention. A
tapered groove about 0.43 millimeter deep, 0.25 millimeter
. ~
1(~99654
-13-
wide at its largest end and 3.1 millimeters long will
allow for the aforesaid pick-up. The groove can be
modified to accomplish different desired populations of
bacteria.
The use of the device will be described now with
reference to Figures 5 through 8. Figure 5 illustrates
schematically, with the device in section, the device of
the present invention just after inoculation of the device
using wand 4. Cap 6 has been removed from the sleeve 1,
and wand 4, via tapered groove 5, has picked up bacteria
from 4 to 5 colonies of bacteria, normally at least
5 x 106 bacteria. Cap 6 has been replaced, and the
bacteria, via wand 4 and groove 5, were placed adjacent to
the ampoule 2. As shown in Figure 5, the ampoule 2 has
been broken by deforming sleeve 1 and, the bacteria 13 are
now shown schematically in the growth-limiting media 3.
The device in this form will be incubated at approximately
35C for normally from 2 to 8 hours. With the preferred
growth media, approximately 5 hours will be sufficient to
obtain the predetermined preferred amount of bacteria,
i,e., from 6 x 107 to 3 x 108 CFU/ml. Figure 6 depicts in
section the device of Figure 5 at this beginning stage of
incubation and shows the sleeve 1 containing broken
ampoule 2, growth media 3 and bacteria 13.
After incubation the device is in the form shown
in Figure 7. In this case, all of the device is just as
it was before except, as can be seen, the population of
bacteria 13 has increased significantly. Figure 8 is a
section of the device of Figure 7 at this stage. After
incubation, the device of Figure 7 shows that the tape 11
has been removed and the device is ready now for exuding
of the growth bacteria via hole 10. The device is merely
held with hole 10 in a downward direction, sleeve 1 is
squeezed and deformed, and the bacteria 13 and media 3 are
exuded via hole 10. Broken glass from ampoule 2 is
precluded from plugging the hole via prongs 9.
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1~39~65~
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The tapered groove 5 in the wand 4 can be
changed to affectuate a different size inoculum of
bacteria so the population of bacteria picked up is
greater than or less than that above described. Other
configurations than a tapered groove could be used in the
present invention as long as there is an ability of the
wand or pick-up device to pick-up, on a repeatable basis,
a predetermined amount of bacteria. The device then in
combination will allow the bacteria to grow from a certain
predetermined level which is determined by the pick-up
device to a final level which is determined by the growth-
limiting media in a certain period of time, which time
period is determined by the beginning population, the
growth limiting medium formulation and type of bacteria.
For use in a Kirby-Bauer test or MIC test, it is preferred
to have the device grow the bacteria within 5 hours to a
concentration of from about 6 x 107 to 3 x 108 CFU/ml.
The device as shown contains the growth limiting
media 3 in a glass ampoule 2. Other modi~ications can be
made to affectuate the same results. For example, the
device can merely be a threaded sleeve with a screw-on cap
which has attached to it the wand In this case the
growth-limiting medium is within the sleeve and is
inoculated directly when the cap was removed. The
bacteria are picked up with the wand and the cap screwed
back on placing the wand in the medium. Other modifica-
tions will be apparent to one skilled in the art and are
included within the claims.
The device depicted in Figure 1 is made by
molding and forming the various glass and plastic
components. Approximately 0.6 milliliter of medium 3 is
placed in glass ampoule 2, and the glass ampoule 2 is heat
- sealed. The ampoule 2 is steam sterilized for 10 minutes
at 121C. The sleeve 1, cap 6 and tape 11 are gas
sterilized with ethylene oxide for 3 hours at 100F and
then aerated for 8 hours. The sealed and sterilized glass
ampoule 2 is asceptically added to the sleeve 1; the cap 6
1~99tiS4
-15-
is attached and the tap 11 is pressed into place.
In the following examples the following
materials and bacteria are referenced. Their source is
set forth below. In the examples growing device means a
device as described above with the dimensions and prepared
as described above.
Tryptone, an enzymatic hydrolysate of casein,
Difco, Inc., Detroit, Michigan, U.S.A.
Peptone, an enzymatic hydrolysate of casein,
Difco, Inc., Detroit, Michigan, U.S.A.
Dextrose, Difco, Inc., Detroit, Michigan, U.S.A.
Polypeptone, an enzymatic hydrolysate of casein
and animal tissue, Bioquest, Inc., Baltimore, Maryland,
U.S.A.
Neopeptone, an enzymatic hydrolysate of protein,
Difco, Inc., Detroit, Michigan, U.S.A.
Proteose peptone, an enzymatic hydrolysate of
protein, Difco, Inc., Detroit, Michigan, U.S.A.
Salmonella typhimurium, American Type Culture
20 Collection, (ATCC) No. 19028
Shigella Sonnei, ~ATCC 25331)
Enterobacter cloacae (ATCC 23355) and St. Paul
Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Klebsiella pneumoniae, (ATCC 23357) and St. Paul
Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Proteus vulgaris (ATCC 6380)
Proteus mirabilis, St. John's Hospital, St.
Paul, Minnesota and St. Paul Ramsey Hospital, St. Paul,
Minnesota, U.S.A.
Serratia marcescens (ATCC 8100) and St. Paul
Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Providencia species, University of Minnesota,
Minneapolis, Minnesota, U.S.A.
Citrobacter species, University of Minnesota,
Minneapolis, Minnesota, U.S.A.
Edwardsiella, University of Minnesota,
Minneapolis, Minnesota, U.S.A.
1~99654
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Arizona, University of Minnesota, Minneapolis,
Minnesota, U.S.A.
Yersinia, University of Minnesota, Minneapolis,
Minnesota, U.S.A.
Pseudomonas aeruginosa (ATCC 27853) St. Paul
Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Escherichia coli (ATCC 25922) and St. Paul
Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Acinetobacter calcoaceticus, St. Paul Ramsey
Hospital, St. Paul, Minnesota, U.S.A.
Proteus morganii, St. Paul Ramsey Hospital,
St. Paul, Minnesota, U.S.A.
Enterobacter aerogenes, St. Paul Ramsey
Hospital, St. Paul, Minnesota, U.S.A.
Pasteurella (species), St. Paul Ramsey Hospital,
St. Paul, Minnesota, U.S.A.
CDC Group II F, St. Paul Ramsey Hospital,
St. Paul, Minnesota, U.S.A.
Moraxella, St. Paul Ramsey Hospital, St. Paul,
Minnesota, U.S.A.
Citrobacter freundii, St. Paul Ramsey Hospital,
St. Paul, Minnesota, U.S.A.
Example 1
Devices having the specifications described
above were inoculated with various bacteria using the
tapered groove of the wand of the device. An initial
concentration of bacteria was noted. The growth limiting
media included within the ampoule of each device contains
the following:
0.8 gram Peptone
0.03 gram Dextrose
2.5 grams Dipotassium phosphate
1.25 grams Monopotassium phosphate
5.0 grams Sodium chloride
Solutions of media containing 0.2 gram peptone
and 1.6 grams peptone were also prepared.
~r
1(~996S~
-17-
Four other growth limiting media were used which
include proteose peptone, tryptone, neopeptone and
polypeptone. These were substituted for the peptone of
the above formulation. The resulting initial concentra-
tions were determined and are as follows in CFU/ml. Theresults given are the mean of 15 samples, i.e., 5 samples
of each of the 3 formulations of each medium.
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--19--
Example 2
The following materials were dissolved in 1000
milliliters of deionized water and steam sterilized at
121C for 15 minutes:
0.8 gram Peptone
0.03 gram Dextrose
2.5 grams Dipotassium phosphate
1.25 grams Monopotassium phosphate
5.0 grams Sodium chloride
Solutions of media containing 0.2 gram peptone
and 1.6 grams peptone were also prepared. Growing devices
were then prepared using each of the media. Utilizing the
wand 4 of the growing device bacteria were picked from 4
to 5, 18 to 24 hour old bacterial colonies of the various
bacteria set forth in the table below. Five growing
devices were used for each bacteria to obtain a mean of 5
samples for each bacteria. Fourteen different bacteria
were tested; thus, there were 70 growing devices utilized
in the test for each of the 3 media. Each growing device
was vortexed, i.e., mixed for 10 seconds and incubated at
35C. Viable bacteria counts were performed at 0, 4, 5,
and 6 hours. The results are set forth in the table
below:
.~ , .
.
1996S~
-20-
Table 2
Count (x 107 CFU/ml)
Bacteria Time 0.2 g 0.8 g 1.6 g
Escherichia coli 0 hours 1.6 1.44 2.4
4 hours 0.4 5.2 11.6
5 hours 3.7 10.2 14.8
6 hours 1.3 7.6 15.4
Shigella sonnei 0 hours 4.2 3.62 1.2
4 hours 5.7 14.4 15.8
5 hours 8.1 17.0 19.8
6 hours 6.0 12.2 21.0
Klebsiella pneumoniae0 hours 2.6 2.86 2.6
4 hours 5.3 13.4 11.6
5 hours 4.9 13.6 13.6
6 hours 4.9 12.0 16.0
Enterobacter cloacae 0 hours 5 3 4.4 3.9
4 hours 9.4 17.0 17.4
5 hours 9.2 19.0 26.0
6 hours 9.4 19.6 38.6
20 Providencia species 0 hours 7.3 6.14 9.1
4 hours 11.6 22.2 30.2
5 hours 13.2 27.4 38.6
6 hours 13.2 22.8 39.8
Proteus mirabilis 0 hours 4.2 4.66 5.4
4 hours 8.9 21.4 21.4
5 hours 8.6 19.8 33.2
6 hours 9.7 24.0 37.2
9g654
-21-
Count (x 107 CFU/ml)
Bacteria _ Time 0.2 g 0.8 g 1.6 g
Salmonella typhimurium 0 hours 2.42.32 3.8
4 hours 6.616.4 27.8
5 hours 7.116.4 31.0
6 hours 6.819.0 33.2
Pseudomonas aeruginosa 0 hours 1.0 0.7 0.8
4 hours 5.614.6 11 4
5 hours 3.616.6 18.6
6 hours 5.326.4 29.2
Citrobacter species 0 hours 3.831.0 6.6
4 hours 9.220.8 21.0
5 hours 9.320.8 30.6
6 hours 12.631.4 32.6
15 AriZona 0 hours 1.11.46 2.0
4 hours 4.115.2 16.0
5 hours 4.916.0 21.8
6 hours 4.917.6 26.22
Edwardsiella 0 hours 2.22.98 1.9
4 hours 2.36.04 8.3
5 hours 2.56.02 8.8
6 hours 2.4 5.9 10.5
Yersinia 0 hours 3.33.02 3.7
4 hours 5.0 9.9 16.0
5 hours 5~411.5 19.2
6 hours 6.713.0 21.2
Serrati marcescens 0 hours 1.11.62 1.1
4 hours 5.210.0 14.0
5 hours 6.316.8 17.8
6 hours 6.826.3 23.4
- .
-22- ~ 09 96 54
Count (x 107 CFU/ml)
Bacteria Time 0.2 g 0.8 g 1.6 g
Proteus vulgaris0 hours 1.71.36 0.5
4 hours 6.519.3619.2
5 hours 6.323.2 31.4
6 hours 7.322.0 34.5
Example 3
~xample 2 was repeated except that polypeptone
wag substituted for peptone. The results are set forth in
the table below:
Table 3
Count (x 107 CFU/ml)
BacteriaTime 0.2 g 0.8 g 1._ g
15 Escherichia coli0 hours 1.4 0.8 1.1
4 hours 7.8 9.8 6.8
5 hours 6.0 5.5 5.5
6 hours 6.7 7.9 7.9
Shigella sonnei0 hours 1.71.68 2.8
4 hours 7.19.98 10.6
5 hours 6.49.88 11.4
6 hours 8.011.4 11.0
Klebsiella pneumoniae 0 hours3.03.64 3.3
4 hours 6.94.78 7.1
5 hours 7.0 7.3 8.0
6 hours 7.812.6 9.5
Enterobacter cloacae 0 hours4.2 3.2 1.8
4 hours 16.219.0 8.1
5 hours 15.812.8 13.2
6 hours 11.613.6 16.8
1~99654
23-
Count (x 107 CFU/ml)
Bacteria Time0.2 g0.8 9 1.6
Providencia species 0 hours - - -
(inoculum error) 4 hours - - -
5 hours
6 hours - - -
Proteus mirabilis 0 hours 0.46 0.46 0.34
4 hours 2.7 3.86 3.6
5 hours 7.6 14.6 13.0
6 hours 7.5 10.6 11.4
Salmonella typhimurium 0 hours 1.3 1.4 0.91
4 hours 6.8 6.9 7.4
5 hours 10.0 11.8 10.7
6 hours 9.0 14.1 11.0
Pseudomonas aeruginosa 0 hours 5.8 7.7 5.6
4 hours 8.0 8.3 11.0
5 hours - 22.2 24.8
6 hours 7.5 20.3 23.6
Citrobacter species 0 hours 15.4 21.6 34.2
4 hours 16.6 22.4 22.2
5 hours 14.8 31.4 25.6
6 hours 15.2 27.2 30.4
Arizona 0 hours 3.7 4.0 3.2
4 hours 5.3 6.6 5.6
;~: 25 5 hours 6.6 13.2 12.5
6 hours 7.0 20.8 17.2
`~ Edwardsiella 0 hours No growth
4 hours No growth
5 hours No growth
6 hour~ No growth
~, . ~- -
.
'
-24- 1~99654
Count (x 107 CFU/ml)
_ Bacteria Time 0.2_g 0.8 g 1.6 g
Yersinia 0 hours 4.42 85 5.7
4 hours 4.45.0 10.7
5 hours 8.09.73 17.2
6 hours 8.611.0 18.6
Serratia marcescens 0 hours 4.44.6 4.1
4 hours 13.611.1 8.8
5 hours 15.214.4 10.1
6 hours 15.415.0 13.6
Proteus vulgaris 0 hours 4.52.64 1.4
4 hours 6.97.92 4.2
5 hours 15.515.4 11.3
6 hours 16.518.0 15.3
Example 4
Example 2 was repeated except that neopeptone
was substituted for the peptone. The results are set
forth in the table below:
Table 4
Count (x 107 CFU/ml)
Bacteria Time0.2 g0.8 g1.6 9
Escherichia coli 0 hours 0.841.0 0.55
4 hours 0.540.6 0.38
5 hours 1.81.96 1.1
6 hours 1.34.0 3.0
Shigella sonnei 0 hours 1.80.8 0.79
4 hours 3 32.3 1.0
5 hours 4.66.2 4.2
6 hours 6.36.1 4.0
1099654
-25-
Count (x 107 CFU/ml)
Bacteria Time 0~2 g 0.8 g 1.6 g
Klebsiella pneumoniae 0 hours 0.2 0.13 0.1
4 hours3.7 2.5 4.0
5 hours7.2 2.5 8.0
6 hours5.3 5.8 8.2
Enterobacter cloacae 0 hours No growth
4 hours No growth
5 hours No growth
6 hours No growth
Providencia species 0 hours0.4 - 0.2
4 hours0.7 - 0.6
5 hours1.4 - 0.7
6 hours1.9 - 1.8
Proteus mirabilis 0 hours2.0 1.0 4.2
4 hours6.3 8.5 6.8
5 hours10.017.6 16.0
6 hours10.521.8 22.2
Salmonella typhimurium0 hours 3.3 2.48 2.6
4 hours7.28.92 8.0
5 hours13.217.2 19.0
`~ 6 hours12.616.6 18.0
Pseudomonas aeruginosa0 hours 0.2 0.16 0.24
4 hours1.8 5.9 4.9
~: 25 5 hours5.3 9.5 9.6
6 hours7.218.0 16.0
- Citrobacter species 0 hours5.47.62 13.0
4 hours7.47.44
5 hours12.224.6 27.2
6 hours15.630.2 31.4
-26-
Count (x 107 CFU/ml)
Bacteria Time 0.2 g 0.8 ~ 1.6 g
Arizona 0 hours 3.4 3.0 2.6
4 hours 6.3 6.6 7.6
5 hours 7.913.0 11.8
6 hours 9.816.4 17.4
Yersinia 0 hours 4.96.24 6.0
4 hours 6.010.7 10.8
5 hours 9.715.5 16.0
6 hours 10.420.6 21.0
Edwardsiella 0 hours No data
4 hours No data
5 hours No data
6 hours No data
15 Serratia marcescens 0 hours 3.53.16 5.1
4 hours 8.710.1 9.8
5 hours 11.311.6 12.3
6 hours 9.712.7 12.8
Proteus vulgaris 0 hours 2.43.56 2.0
4 hours 5.512.5 8.1
5 hours 8.623.8 16.4
6 hours 10.728.4 23.2
Example 5
Example 2 was repeated except that tryptone was
substituted for peptone. The results are set forth in the
table below:
$
99654
-27-
Table 5
Count (x 107 CFU/ml)
Bacteria Time 0.2 g 0.8 g 1.6 g
.. .. _
Escherichia coli 0 hours3.5 3.9 2.1
4 hours12.2 17.8 13.5
5 hours13.2 22.8 18.4
6 hours13.0 25.2 23.2
Shigella sonnei 0 hours1.0 0.4 0.33
4 hours6.3 11.0 10.0
5 hours7.1 16.0 16.0
6 hours8.2 21.3 22.3
Klebsiella pneumoniae 0 hours5.9 5.32 4.7
4 hours13.2 14.2 16.2
5 hours14.0 15.8 15.4
6 hours13.0 19.6 19.4
Enterobacter cloacae 0 hours8.7 7.08 10.6
4 hour.s 15.4 29.0 24.2
5 hours15.2 34.2 33.4
6 hours17.4 40.6 39.4
Providencia species 0 hours3.2 3.62 3.9
4 hours8.2 16.4 17.0
5 hours8.2 22.4 22.2
6 hours5.8 24.6 27.4
: Proteus mirabilis 0 hours3.3 2.08 3.3
4 hours11.8 16.2 19.8
5 hours11.1 21.6 19.0
6 hours13.0 26.2 24.0
-28- ~99~S4
Count (x 107 CFU/ml)
Bacteria Time 0.2 ~ 0.8 g 1.6 g
Salmonella typhimurium0 hours 1.1 1.56 1.0
4 hours 7.515.6 11.6
5 hours 8.021.4 13.8
6 hours 10.627.6 19.2
Pseudomonas aeruginosa0 hours 3.4 2.52 2.9
4 hours 10.011.0 11.6
5 hours 11.010.4 13.0
6 hours
Citrobacter species0 hours 3.3 2.66 2.4
4 hours 16.615.8 17.2
5 hours 16.825.4 20.4
6 hours 17.034.4 27.7
AriZona 0 hours 1.4 1.0 0.66
4 hours 5.5 6.2 6.3
5 hours 5.712.0 10.2
6 hours 6.417.3 14.2
Edwardsiella 0 hours 0.78 0.85 1.6
4 hours 2.3 1.9 3.4
5 hours 3 1 2.3 3.9
6 hours 2.9 2.8 4.8
Yersinia 0 hours 2.0 1.58 2.7
4 hours 5.1 5.8 10.0
5 hours 6.1 8.42 13.6
` 6 hours 7.112.8 18.3
Serratia marcescens0 hours 3.2 4.98 3.0
4 hours 16.620.8 15.6
5 hours 19.225.4 21.4
6 hours 23.229.8 25.8
- 1~9965~
-29-
Count (x 107 CFU/ml)
Bacteria Time 0.2 g 0.8 g 1.6 g
Proteus vulgaris 0 hcurs 1.46 1.1 1.0
4 hours 8.0 16.2 13.3
5 hours 10.0 16.5 18.0
6 hours 10.0 25.7 22.3
Example 6
Example 2 was repeated except that proteose
peptone was substituted for the peptone. The results are
set forth in the table below:
Table 6
Count (x 107 CFU/ml)
Bacteria Time 0.2 g 0.8 ~ 1.6 g
Escherichia coli 0 hours 2.2 1.78 1.9
4 hours 10.0 15.4 14.5
5 hours 7.4 22.0 22.0
6 hours 7.6 20.4 29.0
Shigella sonnei 0 hours 6.9 6.38 4.6
4 hours 14.0 23.2 20.6
5 hours 13.4 20.8 28.8
6 hours 13.8 25.4 33.4
Klebsiella pneumoniae0 hours 4.1 3.52 3.5
4 hours 12.6 15.2 15.4
5 hours 12.8 26.0 23.4
6 hours 13.4 21.2 17.8
Enterobacter cloacae 0 hours 4.2 0.8 4.3
4 hours 12.2 24.2 17.6
5 hours 13.0 28.3 27.0
6 hours - 27.0 35.2
1(~99654~
-30-
Count (x 107 CFU/ml)
Bacteria Time 0.2 g 0.8 g 1.6 g
. .
Providencia species 0 hours 6.4 6.8 7.7
4 hours 19.2 33.2 32.0
5 5 hours 22.8 44.8 41.8
6 hours 24.6 49.6 44.6
Proteus mirabilis 0 hours 5.6 5.98 5.3
4 hours 18.8 38.2 32.4
5 hours 18.8 38.2 38.0
10 6 hours 18.4 39.6 48.6
Salmonella typhimurium 0 hours 3.0 3.86 5.0
4 hours 15.0 28.0 25.2
5 hours 15.0 34.2 30.6
6 hours 17.8 35.2 38.4
15Pseudomonas aeruginosa 0 hours 1.8 1.78 2.2
4 hours 5.4 7.98 11.3
5 hours 9.3 14.2 21.6
6 hours 8.7 15.8 19.0
Citrobacter species 0 hours 4.4 3.82 5.2
20 4 hours 15.8 23.2 26.8
5 hours 16.6 27.6 28.2
6 hours 16.0 32.2 34.8
Arizona 0 hours 2.5 1.92 2.7
4 hours 10.8 14.2 15.0
25 5 hours 11.2 22.4 17.8
6 hours 11.6 25.6 24.0
Edwardsiella 0 hours 1.6 1.3 1.3
4 hours 3.6 5.9 10.3
5 hours 3.4 8.3 12.8
30 6 hours 3.9 10.0 15.8
~(~99654
-31-
Count tx 107 CFU/ml)
Bac~eria Time 0.2 g 0.8 g 1.6 g
Yersinia 0 hours 6.4 3.0 5.9
4 hours 11.4 12.2 13.2
5 hours 13.4 18.0 19.6
6 hours 13.4 22.4 26.4
Serratia marcescens 0 hours 9.9 6.14 6.5
4 hours 28.0 26.2 25.8
5 hours 27.6 30.2 27.6
6 hours 33.8 37.8 35.8
Proteus vulgaris 0 hours 5.2 7.6 7.4
4 hours 12.6 19.6 18.4
5 hours 11.2 29.4 27.2
6 hours 13.6 33.2 33.6
Example 7
In order to compare the mediu~ of the present
invention with the results obtained utilizing a standard
broth growth technique, l.e., tryptic soy broth prior to
placing the bacteria onto discs for use in the Kirby-Bauer
procedure, 100 growing devices were prepared which
contained the ~ame medium as set forth in Example 2. One
hundred clinical isolates of bacteria that were received
from patients were run using both the growing device and
the standard growing technique of the standard set forth5 for the Kirby-Bauer test. The bacteria tested included:
Escherichia coli
Klebsiella pneumoniae
Pseudomonas aeruginosa
Acinetobacter calocoaceticus
Proteus mirabilis
Proteus morganii
Enterobacter aerogenes
Enterobacter cloacae
Serratia marcescens
- ' .
1(;199654
-32-
Pasteurella (species)
CDC Group II F
Moraxella
Citrobacter freundii
Specifically, 4 to 5 isolated colonies were touched with
the wand from the growing device and the wand was used to
inoculate the growing medium in the growing device. The
units were incubated at 35C in a 3M brand incubator
Model 107 for 4 hours. The top tape seal was removed from
the cap of the growing unit and 6 to 8 drops of bacterial
suspension were dispensed onto a cotton swab. The swab
was streaked in three directions over a Mueller-Hinton
agar plate and the Kirby-Bauer test was completed
according to the National Clinical Committee for
Laboratory Standards (NCCLS) Antibiotics Susceptibility
Standard set forth above. A comparison was made between
the results obtained in respect to the susceptibility of
the organism tested in using the growth media of the
present invention versus the standard technique for
growing bacteria. The results were comparable.
- ,
.
'
