Note: Descriptions are shown in the official language in which they were submitted.
913,'ll')
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GRO~TII LIMITIli i~lEDI~
This invention relates to a medium for ~rowing
bacteria from an initial population to another hi~her
po~ulation, which medium then causes the bacteria to
substantially cease growin~ due to the lack of nutrient.
In a number of procedures utilized to identlfy
bacteria or to determine the susceptlbility of the bacteria
to certain antibiotics, it is necessary to have the bacteria
at the be~innin~ of the test procedure in a certain concen-
tration ran~e (colony formin~ units per ml (CPU/ml)) or
the rinal test result wlll not be accurate. ~or example,
in an article entitled "~ntibiotics Susceptibility Testing
by Standardized Single-Disc Method", Thc h .r~a~ n-l
of Clinical Patholo~y, Vol. 45, i~o. 4~ April, 1966, pages
.
493-496, and in the 'iPerformance Standards for ~ntimicrobial
Disc Susceptibility Tests", ~S~1-2, promul~ated b~ the
National Committee for Clinical Laboratory Standards of the
Unlted States, the Klrby-~auer procedure for determirllng
the suSceptibility of rapidly growin~ bacteria to anti-
biotics and chemotherapy a~ents is described.
The Kirby-Bauer procedure involves ~rowing on an
~- a~ar plate colonies of bacterla ob~ained from a patlent.
A wire loop is used to plcl from ll to 5 colonies of the
bacteria and introduce them lnto a test tube containing
Il to 5 milliliters of soybean casein di~est broth. The
. .
~: 25 tubes are then incubated for 2 to ~ hours to produce a r
bacterial suspension of moderate cloudiness. The sus-
, .
pension is then diluted~ if necessary, with saline ~olution
.
or lil-~e broth to a density visually equivalent to that Or
a standard prepared by acldin~ 0.5 milliliter of l~a BaC12 to
i~K`b :
~ ~ , ~ , - , ; - ,
.5 milliliters of l~ 2S0l~ (0.3~ 1~) (0.5 ~i1cFarland
standard hereina~ter rei`erred to as the McFarland standard).
A plate containin~, Mueller-~linton a~ar is then streaked wlth
the bacterial broth suspension using a cotton swab. A~ter
the inoculum has dried, a paper disc containing an anti-
biotic or chemotherapeutic agent is applied to the a~ar,
The ~lates are incubated. After overni~,ht incubatlon the
area around each disc wherein there is an absence Or
bacteria growth is measured. This is known as the zone of
- lO inhibition and is used to determine which antibiotic will
be useful in combating the particular bacteria.
In or~er ~or the Kirby-Bauer technique to be
accurate there must be approximately l x 108 C~U/ml included
in the medium which is streaked onto the a~ar plate.
~`; 15 Usually the level of F,rowth is determined by using the
visual cbmparison with the licFarland standard descrlbed
above. The ti~e period to reach this concentration of
bacteria rnay vary from 2 to ~ hours depending on the
bacteria. If the ~acteria are allo~Jed to ~,row in excess
Or l x lO~ CFU/ml and becomc more turbi~ than the McFarland
standarc~ t~le medium must be diluted in order to be e~uivalent
to the standard.
; Another method of determinin~ the susceptibility
of bacteria to various antibiotics is called the MIC or
Minimum Inhibitory Concentration test. This test is
discussed in Current 1'echniques for Antibiotics Suscept-
ibilit~ Testin~ Albert Balows, ~ 19741 pa~es 77-87. This r
method involves preparation of a series of concentrations on
an antibiotic, either in a li~1uid or solid medlum which will
; 30 support the ~rowth o~ a bacteria to be tested. Liquid media
~ - 2
are conveniently dispensed in test tubes and solid media are
usually poured into petri dishes. It is common practice to
prepare a ran~e of antibiotic concentrations as a series of
two-fold dilutions in order to carry out the test. Each
tube or petrl dlsh i5 inoculated with the bacteria in
question. After a period Or incubation the bacterial growth
or absence of ~rowth of each antibiotic concentration is
observed. In this way, the minimum inhibitory concentration
the antibiotic is determined to the nearest dilution when
used in a serles. This is the most accurate method of
determinin~ the inhibitory concentration. However~ this
metllod di~ not gain popularity until recently when the
laborious effort of makin~ the dilutions was simplified.
The diluted antibiotic i8 inoculated in the MIC test with
bacteria at a certain concentration, i.e., normally 105
to 106 CFU/rnl. Broth containing bacteria equivalent to
the rlcFarland standard, i.e., approximately 1 x 10
CFU/ml is diluted to obtain this concentration
The aforesaid susceptibility tests a~ well as
other tests for determinin~ the types of bacteria or
,'` susceptibility ti~ereof to antibiotics require that a
certain ~redeterrnined amount of bacteria be utilized in
the test to inoculate the plates u~on which the paper disc
will be lilaced ln the case of the Kirby-~auer test or to
; 25 inoculate the diluted antibiotics in the case of the MIC
test. This is required in order for the test to be
accurate. Ir 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
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bacteria are present in a hi~her concentration, the test
results would indicate that a hi~iler concentration of
the antibiotic would be required in order to inhi~it the
growth of the bacteria. ~oth 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 or 5 colonies of bacteria from the agar plate upon which
the bacteria have been growing and place lt in a ~roth
growth 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 examin-
ation of the medium, one will find that some rnedium culturesOr bacteria have grown to be equivalent to the McFarland
standard. Also, one may find that some cultures have not
rown to the proper concentration while others have ~rown
:`
~eyond the appropriate concentration. The former requires
tilat the technician allow the bacteria to grow longer,
. .
whereas the latter requires a dilution to brin~ the con-
centration back to that of the standard. All of these
; measures are teclious and time consuming. ~ ;
Applicants have discovered a medium upon which
bacteria can ~row but which limits the concentration level
to which the bacteria will ~row. ~pecifically, applicants
have discovered an aqueous medium capable o~ growin~ at
~ least one species from two dlfferent genera of aerobic,
; patho~enic, rapidly growing bacteria from a beginning
population to a determined endin~ population at which
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growth of the bacteria substantially subsides due to the lack of nu~rient in
the medium and wherein the bacteria remain viable for at least 18 hours.
The medium comprises an aqueous solution comprising a carbon source, a nitro-
gen source, vitamins and minerals o-f sufficient quantity to provide growth
and in a form usable by the bacteria for said growth.
Accordingly, the present invention provides an aqueous medium
capable of growing at least one species from two different genera of aerobic,
pathogenic, rapidly growing bacteria from a beginning population to a deter-
mined ending population of about 6 x 10 to 3 x 108 CFU/ml at which said
growth of said bacteria substantially subsides due to the lack of nutrient
in said medium and wherein said bacteria remain viable for a sufficient
period to allow antibiotic susceptibility testing, said medium comprising
an aqueous solution comprising about 0.~2 to 0.70 milligram of carbon per
milliliter of medium and 0.09 to O.lS milligram of nitrogen per milliliter
of medium in the form in which said carbon and nitrogen are present in pep-
tone, or about 0.16 to 0.27 milligram of carbon per milliliter of medium and
about 0.035 to 0.056 milligram of nitrogen per milliliter of medium in the
form in which said carbon and nitrogen are present in proteose peptone.
The bacteria upon which applicants' medium is useful are aerobic ~i-
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 broth gram-negative as well as gram-
positive aerobic, pathogenic, rapidly growing 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 re-
quired to obtain the requisite concentration for gram-positive tends to be
longer than that for gram-negative bacteria.
.~ _ 5 _
,
2'7~t
Applicants' 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 susceptibil-
ity testing are performed. These are Staphylococcus and Streptococcus. If
the medium will grow species from each of
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these two ~enera, therl it allows one to merely test the
bacteria to determine whether they are gram-positive or
ne~ative USillg a gram stain test. If the bacteria are
granl-positive, mediurn which grow 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 ~ram stain test, the bacterla could be from a
much larger number Or ~enera. Sixteen genera represent
the bacteria found to be the cause of 9~% of illnesses
caused by ~ram-ne~ative, aerobic bacteria. T~lese gram-
negative genera include: Escherichia, Shi~ella, Edward-
siella, Salmonella, Arizona, Citrobacter, Klebsiella~
Enterobacter, Serratia, Proteus, Providencia, Yerslnia,
Pseudomonas, hcinetobacter, Moraxella and Pasteurella.
The medium ~rows the bacteria from a certain
po~ulation which will normally be described ln terms of
C~IJ as above defined and will normally be referenced to
the population in a certain volume of medium, i.e.,
CFU/ml. The be~inning concentration can ran~e as low as
~; 1 CFIJ but wlll normally be, and i5 preferable, at least
5 x 10~ C~U/ml.
Starting with a lower initial population or con-
centration will not cause the medium to ~row the bacteria
to a significantly different final population or concen-
tration than wlth a hiher starting concentration, but will
affect the time it takes for the final concentration to be
reac~ed The final concentration to which the bacterla
~row is referred to as the stationary phase.
As above discussed the time to reach a concen-
7~
tration equivalent to the McFar~and standard varles
accordln~ to the test procedure from 2 to 8 hour~ with
most bacteria taking at least 5 to 6 hours to reach that
concentration. Applicantst media preferably reaches the
final concentration or skatlonary phase within 5 hours.
However, a feature of appllcants' medium is that if the
bacterla reaches the stationary phase in 2 hours it will
remain there even if the techniclan 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 concentratlon
ls reached with applicants' medium, the growth of the
bacterla substantially subsides. This is due to exhaust-
- ion of at least one nutrient critical to the continued
growth Or the bacteria and not to the formation of toxic
byproducts by the bacteria which stop growth and can cause
the population to decrease substantially, With the
standard media used prior to the present lnventlon and
described in the Kirby-Bauer procedure, the population of
bact,eria which would be reached in order for growth to
subside would be determined by toxic byproducts of the
bacteria. This population of bacteria i8 above the
McFarland standard which is described in the Kirby-Bauer
procedure. '~
The final concentration or maximum stationary
phase is obtained because of exhaustiorl of one or more
nutrients. At that point the viable CFU/ml count levels
off and rernains substantially unchanged ror at least about
18 ~lours. rhe bacteria remain viable for a period Qf time
useful for carrying out the various tests to be per~ormed
'. ~
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thereon, ~or example, those descrlbed above. Normally
the time period for such viability is at least 18 hours.
The final concentration which i8 desired to be
reached with most bacteria will be between 6 x 107 to
3.0 to 10~ CFU/ml. This is equivalent to the 0.5 Ma-
Farland standard. The medium can be modified to reach
different desired concentratlon 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 bacterla belng 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 3 vitamins
and minerals are also present. However, as noted, the amount
of one or more of-these in~redients is limited to cause the
;~ bacteria to reach a final predetermined concentration and
substantially cease growin~,.
For the gram~negative bacteria, a preferred
medium comprlses about 0.42 to about 0.70 milligram of
carbon per milliliter Or medium. The carbon is in a form
useful by the bacteria for growth. This form has been
found to be the form which carbon is pre3ent in peptone
or a similar form. The preferred medium al~o comprises
0.09 to 0.15 milligram of nitrogen per milliliter of
medium in a form similar to the nltrogen present in
peptone. The preferred medium has a pH from about 7 to 8
With proteose peptone, the ran~e for carbon i5 from
about 0.16 to a~)out 0.27 milligram of carbon per mllllliter
of medium, the nitrogen is 0.035 to o.os6 milligram
nitrogen per milliliter of medium, and the carbon and
~ . .
nitrogen are as found in proteose peptone or a form
fimilar thereto. A typical analysis of the peptone and
proteose peptone is set forth below:
Proteose
Percent ~ one Peptone
Total Nitrogen 16.16 14.37
Primary Proteose N 0. o6 o.60
Secondary Proteose N 0.~8 l~.03
Peptone N 15.38 9.74
Ammonia N 0.04 0.00
Free Amino 3.20 2.66
Amide N - 49 0-94
Mono-amino ~ 9.42 7.61
Di-amino ~ 4.07 4.51
Tryptophane 0. 29 0.51
Tyrosine 0.98 2.51
Crystlne 0. 22 0.56
Org,anic sulrur o . 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. 5~ 0.137
Ma~nesium 0. 056 0~ll8
Manganese nil 0.0002
Iron ~33 o.oo56
Ash 3~53 9.61
Ether Soluble Extract 0. 37 0.32
Reaction, pH 7- 6.8
p~ solution in distilled water after autoclaving
15 minutes at 121C.
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 growlng gram ne~ative bacterla to a
7 8
final concentration of from ~ x 10 to 3 x 10 CFU/ml in
less than 5 hou:rs comprises a mixture of 1000 ml Or water
; containing o.8 gram peptone or 0.3 gram proteose peptone,
0.03 gram dextrose, 2.5 grams dipotassium phosphate3 1.25
grams monopotassium phosphate and 5.0 grams sodium
chlor~de. The phosphates are added as a buffer material
.
.
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to maintain the composition at a p~l of approximately 7Ø
rfering is necessary with certaln of the bacteria. The
aforesaid two formulations provide a medlum upon which ~p~-
cies from most o~ the genera o~ the~ gram-negative, aerobic,
pathoFenic bacteria can grow to the~ above described CFU/ml
ran~es within 5 hours.
As noted, the preferred carbon and nitrogen
sources are peptone and proteose peptone for gram-nega~ive
bacteria. Neopeptone, tryptone and polypeptone can also
be used, but it has been ~ound that these do not produce
growth to the levels of the McFarland standard wlthin the
same time fraMe, and with some of the bacteria withln this
group, they do not provide the approprlate nutrlenks to
~row the bacterla to any significant degree. Therefore,
! 15 these materlals are useful for more limited numbers of
bacteria. I~owever, combinations of such materials with
peptone or proteose peptone can be used to provide a
medlum useful with a larger number Or bacteria.
A preferred medium for use with gram-positive
bacteria comprises a solution o~ 1000 ml of water con-
taining 1.7 ~rams tryptlcase, 0 3 gram phytonel 0.25 gram
dextrose, 0.5 gram sodium chloride and 0.25 ~ram di-
potassium phosphate.
All the various media are used by inoculating
the medium with the bacteria and incubatin~ the medlum ~or
2 to ~ hours. A device for such inoculation is described
below with reference to the Figure. ~his device is the
subJect o~ a separate patent application ~iled con-
currently herewlth.
In the following examples, re~erence will be
i .
made to the use of a growin~ device containing medium
which is made for use in plcking up baoteria from growing
colonles of bacteria and ~or providing medlum for growing
the bacteria. The concentratlon of the bacterla, a8 re- ~ ,
moved from 4 to 5 colonies rrom whi.ch the bacterla are ''
obtalned, is from 5 x 1o6 to 1 x 108 CFU's per mllllliter.
The devioe comprl~es a sleeve 1 whlch i8 made
of a deformable materlal such as poIypropylene, polyamlde, ~i!
cellulose acetate butyrate or various polyesters. The -:
sleeve is transparent, cloæed on one end and contains with-
in it a franglble ampoule 2 contalnlng the growth medium
3 of the present inventlon. In use, the deformable sleeve
1 is squeezed by means of a crushing device (not shown) ;~
and ~lass ampoule 2 breaks allowing the medium 3 to mlx
: 15 with and grow the bacteria which are lntroduced into the
sleeve 1 by means of wand 4. Wand 4 is a~flxed to cap 5
and contalns wlthin lt a tapered groove 6 which allows ror
pick-up Or bacteria by means o~ capillary action, l.e., ;~-
~; when the wand i9 pushed into a colony o~ bacteria the
bacteria move up the groove 63 and a majorlty o~ the air
prevlously occupying the groove is pushed out o~ the top
of the ~roove 6. Cap 5 contains within it two ridges 7
and 8 which provide a tlght seal a~alnst sleeve 1 but does .-~
allow for remova]. of cap 5 from sleeve 1. Cap 5 also con-
tains hole 9 which allows the bacteria to be expresaed
from the sleeve ]. after growth by deformatlon o~ sleeve 1.
~fole 9 ls covered by adhesive tape 10 whloh covers the hol~e ~;i
9 until it is desirea to exude ~he grown bacteria and ~ j;
; medium 3 from the sleeve 1. Cap 5 also contalns pro- ;
trusions 11 which are three in number and prevent~the glas~
. ~ ..
7~
rrom ~lass ampoule 2 from pluggin~ hole 9 durin~ the
exudation process.
In the examples below the growlng devlces were
filled by placing 0.6 ml of medium 3 in the glass ampoule
2 and heat sealing the ampoule 2. The ampoules were
steam sterlllzed for 10 minutes at 121~C. The sleeve 1,
cap 5 and tape 10 were ~as sterilized with ethylene oxide
for 3 hours and 100C and then aerated for 8 hours The
sealed and sterilized glass ampoules were asceptically add-
ed to tile sleeves; the caps were attached and the tapes
were pressed into place. The ~,rowin~, devices were then
ready for use.
In the followin~ examples the followin~ mater-
ials and bacteria are re~erences. The source is set forth
belo~:
Tryptone, an enzymatic hydrolysate o~ casein,
Dirco, Inc , Detroit, Michi~an, U.S.A.
Peptone, an enzymatic hydrolysate of casein,
Dirco, InG, Detroit, Michigan, IJ.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.
i~eopeptone, an enzymatic hydrolysate of ~rotein,
~irco, Inc., ~etroit, Michigan, U.S.A.
Propteose peptone, an enzymatic hydrolysate of
protein, Difco, Inc., Detroit, Mlchi~a~, U.S.A.
Phytone, an en%ymatic hydrolysate of soybeans, I
Bio~uest, Inc., Baltimore~ Maryland, IJ.S.A.
Salmonella typhimurium, Amerlcan Type Culture
Collection, (ATCC) No. 19028
Shigella Sonel, (ATCC 25331)
Enterobacter cloacae (ATCC 23355) and St. Paul
Ramsey l~ospltal, St. Paul, Minnesota, U.S.A.
I Klebsiella pneumoniae, ~ATCC 23357) and St. Paul
Ramsey ~Iospltal, St, Paul, Minnesota, U.S.A.
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Proteus vulgaris (ATCC 6380)
Proteus mirabills, St. John's Hospital, St. Paul,
Minnesota and St. Paul Ramsey HospLtal, St. Paul,
Minnesota, U.S.A.
; 5 Serrakia marcescens (ATCC 8100) and St. Paul
Ramsey Hospital, St. Paul, Minnesota, U.S.A.
Providencia species, University o~ MlnneRota,
Minneapolis, Mlnnesota, U.~.A.
Citrobacter species3 University of Minnesota,
Minneapolis, Minnesota, U.S.A.
Edwardsiella, Unlversity of Minnesota3
Minneapolis, Minnesota, U.S.A.
Arizona, University of Minnesota, Mlnneapolis,
Minn~sota, U.S.A.
Yersinia, University of Minnesota, Minneapolis,
Minnesota, U.S.A.
Pseudomonas aeruginosa (ATCC 27853) St. Paul
Ramsey ~1ospital, St. Paul, Minnesota, U.S.A.
Escherichia coli (ATCC 25922j and St. Paul
; 20 Ramsey ~1ospltal, St. Paul, Minnesota, U.S.A.
Acinetoba¢ter calcoaceticus, St. Paul Ramsey ~`
Hospital, St. Paul, Minnesota, U.S.A.
Proteus morganii9 S~. Paul Ramsey ~1ospital,
St. Paul, Mlnnesota, U.S.A.
Enterobacter aerogenes, St Paul Ramsey
Hospital, St. Paul, Minnesota, U.S.A.
Pasteurella (species), St. Paul Ramsey ~1ospital~
St. Paul, Minnesota, U.S.A.
CDC C,roup II F, St. Paul Ramsey ~1ospital, St.
Paul, Mirmesota, U.S.A.
r;10raxella, St. Paul Ramsey Hospital, St. Paul,
Minnesota, U.S.A.
Citrobacter freundii, St. Paul Ramsey ~10spital,
St. Paul, M1nnesota, U.S.A.
Trypticase, an enzymatic hydrolysate of aasein~
Bio~luest, Inc., Baltimore, ~aryland) U.S.A.
,~
~"
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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 containin~ 0~2 eram peptone
and 1.6 ~rams peptone were also prepared. Growing devices
were then prepared using each of the media. Utilizin~ the
wand 4 of the growlng devlce bacteria were picked ~rom 4
to 5, 18 to 24 hour old bacterial colonies of the varlou~
bacteria set forth in the table below. Five growing de-
vices were used ~or each bacteria to obtain a mean of 5
samples for each bacteria. Fourteen different bacterla
were tested; thus, there were 70 growing devices utilized
in the test for each of the 3 media. Each growlng devlce
was vor~exed, 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 rorth in the table
below:
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Table 1
Count (x 107 C~U/ml)
Bacteria Time ~ ~ 1. K
Escherichia coli 0 hours 1.6 1.44 2.4
4 hours o'l~ 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.o 12.2 21.0
Klebsiella pneumoniae 0 hours2.6 2.86 2.6
4 hours 5,3 13.4 11.6
5 hours 4.9 13.6 13.6
6 hours l~,g 12.0 16.0
Enterobacter cloacae 0 hours 5,3 I~.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
,
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.
: Proteus mirabilis 0 hours 4.2 4.66 5.l~
4 hours~ 8.9 21.4 21.4
5 hours 8.6 19.8 33.2
6 hours 9.7 24.0 37.2
Salmonella typhimurium 0 hours2.4 2.32 3.8
4 hours 6.6 16.4 27.8
3 5 hours 7.1 16.4 31.0
: 6 hours 6.8 19.0 33.2
Pseudomonas aeruginosa 0 hours1.0 0,7 0.8
4 hours 5.6 14.6 11.4
5 hours 3.6 16.6 18.6
~:35 - 6 hours 5.3 26,4 29.2
Citrobacter specles 0 hours 3.8 31.0 6.6
4 hours 9.2 ! 20.8 21.0
5 hours 9 3 20.8 30.6
6 hours12 6 31.4 32.6
Arizona 0 hours 1.1 1.46 2.0
4 hours 4.1 15.2 16.0
5 hours 4.9 16.0 21.8
6 hours 4.9 17.6 26.2
Edwardsiella 0 hours 2.2 2.98 1.9
l~ hours2~3 6.04 8.3
5 houra 2.5 ~.02 8.8
6 hours 2.4 5.9 10.5
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Table l (Cont.)
Count (x 107 CFU/ml~
Bacteria Time
Yersinia 0 hours 3.3 3,02 3-7
'j 4 hours 5.0 9.916~0
5 hours 5.4 11.519.2
6 hours 6.7 13.021.2
Serrati marcescens0 hours 1.1 1.621.1
1~ hours 5.2 10.014.0
:~ 10 5 hours 6.3 16.817.8
6 hours 6.8 26.323.4
Proteus vulgaris 0 hours 1.7 1.360.5
4 hours 6.5 19.3619.2
5 hours 6.3 23.231.4
`~: 15 6 hours 7,3 22.034.5
,
~: Example 2
Example 1 was repeated except that polypeptone
was substit~ted for pepton. The results are set ~orth
:
:~ in the table below: :
Table 2
: . Count (x 107 CFU~ml~
acteria Time 0.2g0. g1. g
Escherichia coli 0 hours 1.l1 o.81.1
~: 4 hours 7.8 9O86.8
5 hours 6.0 5.55,5
6 hours 6.7 7.97.9
~ Shigella sonnei 0 hours 1.71.682.8
:` , - 4 hours 7.19.9810.6
: 5 hours 6.49.8811.4
5 hours 8.0 11.4 11.0
Klebsiella pneumoniae0 hours 3.o3.643.3
4 hours 6.94 787.1
5 hours 7.0 7.38.0
; 6 hours 7.812.69,5
`:. 35 Enterobacter cloacae 0 hours 4.2 3.2 1.8
4 hours 16.219.08.1
. 5 hours 15.812.813~2
: 6 hours 11.613.616.8
,
. Providencia species 0 hours
4 4 hours - - _
5 hours
~: ~ 6 ~lours
16 -
~$~4;~7~ .
Table 2 (Cont.)
-
_unt (x lO7 CFU/ml)
~acteria . Tlme
Proteus mirabilis O hours0.46 0.46 0,34
4 hours2.7 3.86 3~6
; 5 hours7.6 14.6 73.0
6 hours7.5 10.6 11.4
Salmonella typhimurium O hours1~3 ~ 0.91
4 hours6.8 6.9 7,1,
5 hours10.0 11.8 10,7
6 hours9.0 14.1 11.0
Pseudomonas aeruginosa O hours5.8 7~7 5.6
l~ hours 8.o ~.3 11.0
5 hours - 22.2 2ll.8
6 hours7,5 20.3 23.6
Citrobacter species O hours15.4 21.6 34.2
4 hours16.6 22.4 22.2
5 hours14.8 31.l~ 25.6
6 hours15.2 27.2 30.4
- 20 Arizona O hours3.7 4~0 3.2
4 hours5.3 6.6 5.6
5 hours6.6 13.2 12.5
6 hours7.0 20.8 17.2
Edwardsiella O hours No ~rowth
ll hours No growth
~ 5 hours No growth
: 6 hours No ~rowth
~: Yersinia O hours4.ll 2.85 5.7
4 hours l~,4 5,o 10.7
5 hours 8.0 9.73 17.2
6 hours 8.6 11.0 18.6
:
Serratia marcescens O hours 4.1l l~.6 4.1
- Il hours 13.6 11.1 8.8
5 hours 15.2 14.4 10.1
6 hours 15,4 15.0 . 13.6
Proteus vulgaris O hours 4.5 2.64 1.4
4 hours 6.9 7.92 4.2
5 hours 15.5 15.4 11.3
~: - 6 hours 16.5 18.0 15.3
Example 3
Examp].e 1 was repea~ed except that neopeptone
was substituted for the peptone. The result~ are set
~; forth in the table below:
. ~ .
: . .
~ 17
Z7~)
Table 3
Count (x 107 CFU/ml~
Bacteria Time ~ ~ 1 6
Escherichia coli 0 hours o.84 l.0 0.55
4 hours o.54 o.6 0.3
5 hours 1.8 1.96 l.l
6 hours 1.3 4.0 3~0
Shigella sonnei 0 hours l. 8 0.8 0.79
4 hours 3.3 2.3 1.0
5 hours 4.6 6.2 4.2
6 hours 6.3 6.1 4.0
Klebslella pneumoniae 0 hours 0.2 0.13 0.1
4 hours 3,7 2.5 4.0
5 hours 7.2 2. 5 8.o
6 hours 5.3 5. 8 8.2
Enterobacter cloacae 0 hours No growth
4 hours No growth
5 hours No ~row~h
6 hours No ~rowth
Providencia species 0 hours0.4 -- 0.2
4 hours 0.7 - o.6 - .
5 hours 1.4 - 0. 7
6 hours 1.9 - 1.8
Proteus lairabilis 0 hours2.0 l.0 4.2
: 25 4 hours 6.3 8. 5 6.8
5 hours 10.0 17.6 16.0
6 hours 10.5 21.8 22.2
Salmonella typhimurium 0 hours 3.3 2.48 2.6
4 hours 7.2 8.92 8.0
5 hours 13.2 17.2 19.0
- G hours12.6 16.6 18.0
Pseudomonas aeruginosa 0 hours 0. 2 0.16 0.24
4 hours 1.8 5.9 4.9
: :~ - 5 hours 5.3 9.5 9. 6
35~ 6 hours 7.2 18.0 16,0
:~ Citrobacter spec:les 0 hours5.4 7.6? 13.0
. 4 hours 7.4 7.44
5 hours 12.2 24.6 27.2
~ 6 hours 15.6 30. 2 31.4 ~ :
: 40 Arizona 0 hours 3.4 3.0 2.6
~ 4 hours 6.3 6.6 7.6
- ~ 5 hours 7,~ 13.0 11.8
6 hours 9.8 16.4 17.4
Yersinia 0 hours 4. 9 6.24 6.0
4 hours 6.0 10.7 10.8
~l:- 5 hours 9.7 15.5 16.Q
6 hours 10.4 20.6 21.0
~ .
; ~ - 18 -
~Ll 4 Z'70
Table 3 (Cont.)
~ Count (x 107 CFU/ml?
; Bacteria Time ~ ~E 1~
.
. .Edwardsiella 0 hours No data
4 hours No data
5 hours No data
6 hours No data
Serratia marcescens0 hour~ 3.53.16 5.1
1 4 hours 8.710.1 9.8
5 hours 11.311.6 12.3
6 hours 9~712.7 12.8
Proteus vul~aris 0 hours 2.43.56 2.0
4 hours 5.512.5 8.1
5 hours 8.623.8 16.4
15 1 6 hours 10.728.4 23.2
; Example 4
I
Example 1 was repeated except that tryptone was
substltuted ~or peptone. The results are set forth in the
table below:
Table 4
Count (x 107 CFUJml)
: Bacteria : Time ~ 1.6g
Escherichia coli 0 hours 3.5 3.9 2.
:~ 4 hours 12.2. 17.8 13.5
5 hours 13.222 A 8 18.4
- 6 hours 13.025.2 23.2
Shigella sonnel 0 hours 1.0 o.4 0.33
: 4 hours 6.311. o lo .0
~; 5 hours 7.116.0 16.0
~:~ 30 6 hours 8.221.3 22.3
~:~: Klebsiella pneumoniae 0 hours5.9 5.32 4.7
. : i 4 hours 13.214. 2 16.2
~ 5 hours 14.015.8 15. 4
:~ . 6 hours 13.019.6 19.4
Enterobacter cloacae 0 hours8.7 7.08 10.6
4 hours 15.42~.0 24.2
5 hours 15. 234.2 33.4
~:: . 6 hours 17.440 . 6 39,4
Providencia species0 hours 3.23.62 3.9
4 hours 8.216.4 17.0
5 hours a . 222. 4 22.2
6 hours 5.824.6 27.4
,,~
~: .
19 ~
... .
~427~.
Table 4 (Cont )
Coun~ (X 1~ C~
Bacteria T _ O.2~
Proteus mlrabilis0 hours 3,3 2.083.3
4 hours 11.8 16.219.8
5 hours 11.1 21.619.0
6 hours 13.0 26.224.0
Salmonella typhimurlum 0 hours 1.11.56 1.0
4 hours 7,~ 15.611.6
5 hours 8.0 21,413.8
6 hours 10.6 27.619.2
Pseudomonas aeruginosa 0 hours 3.42.52 2.9
4 hours 10.0 11.011.6
5 hours 11.0 10,413.0
6 hours
Citrobacter specie~0 hours 3,3 2.662.4
4 hours 16.6 15.817.2
5 hours 16.8 25.420.4
. 6 hours 17.0 34.l~27.7
Arizona 0 hours 1.4 1.00.66
l~ hours 5-5 6.26.3
5 hours 5.7 12.010.2
6 hours 6.4 17.314.2
Edwardsiella 0 hours o.78 o.851.6
4 hours 2.3 1.93.4
5 hours 3.1 2.33.9
~: 6 hours 2.9 2.84.8
. Yersinia 0 hours 2.0 1.582.7
4 hours 5.1 5.810.0
~: 30 5 hours 6.1 8.4213.6
6 hours 7.1 12.818,3
Serratia marcescens0 hours 3.2 4.983.0
4 hours 16.6 20.815.6
5 hours 19.2 25.421.4
6 hours 23.2 29.825.8
Proteus vul~aris 0 hours 1.46 1.11.0
4 hours 8.0 16.213.3
5 hours 10.0 16.518.0
6 hours 10.0 25.722.3
ExampIe 5
Examp.le l was repeated except that proteose
.
peptone was substituted for the peptone. The results are
: set forth in the table below:
::
1~ .
!: ~
I
` - 20 -
, ~
Table 5
___._
Count (x 107_CFU/ml)
Bacteria Time0.2
Escherlchia col~ 0 hours2.2 1.78 1.9
4 hours10.015.4 14.5
5 hours7,1,22.0 22.0
6 hours7.620. 4 29.0
Shi~ella sonnei 0 hours6.9 6038 I~.6
4 hours14.023. 2 20.6
5 hours13.420.8 28.8
6 hours13.825.4 . 33.4
Klebsiella pneumoniae 0 hours 4.1 3.52 3.5
4 hours12.615.2 15.4
5 hours12.826.0 23.4
. 6 hours13. 421,2 17.8
Entero~acter cloacae 0 hoursIl. 20.8 4.3
4 hours12.224.2 17.6
5 hours13.028.3 27.0
6 hours - 27.0 35.2
Providencia species O hours6.4 6.8 7.7
4 hoursl9o 23302 32.0
5 hours22. 8I~4.~ 41.8
6 hours21l.6l~g. 644.6
Proteus mirabilis 0 hours5.6 5.98 5.3
4 hours18. 838.2 32. 4
~ 5 hours18.838.2 38.0
:~ 6 hours18.439.6 48.6
-: Salmonella typhimurium 0 hours 3.0 3. 86 5.0
4 hours15.028.0 25.2
: 3 . 5 hours15.034.2 30.6
6 hours17.835.2 38.4
: Pseudomonas 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 hours8 7 15 8 l9.Q
Citrobacter species 0 hours4.4 3.82 5 ~ 2
4 h~urs 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 Z.7
4 hours 10.8 14.2 15.0
: 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
i . 6 hour~ 3.9 10.0 15.8
' '
`i. - 21 -
Z7~
Table 5 (Cont.)
e~
Bacteria Time 0~
Yersinia 0 hours6.4 3.0 5.9
4 hours11.4 12,2 13.2
5 hour~3 13.4 18.0 19.6
6 hour~3 13.4 22.4 26.4
Serratia marcescens 0 hourl3 9.~ 6.14 6,5
4 hours28.0 26~2 25.8
5 hour~27.6 30.2 27.6
6 hours33.8 37,8 35.8
Proteus vulgaris 0 hours5.2 7.6 7,4
4 hours12.6 19.6 18.4
5 hours11,2 29.4 27.2
6 hours13.6 33.2 33.6
Example 6
In order to compare the medium o~ the present
invention with the results obtalned utilizlng a standard
broth ~rowth technlque, i.e., tryptic soy broth prlor to
placing the bacteria onto discs for use ln the Kirby-Bauer
~ procedure, 10Q growlng devices were prepared which con-
; tained the same medium as set ~orth in Example 1. One
hundred clinical isolates of bacteria that were received
from patients were run using both the growing device and
the standar~ growin~, technique Or the standard set ~orth
, . .
; ~or the Kirby-Bauer test. The bacteria tested included:
Escherichla coli
Klebsiella pneumoniae
Pseudomonas aeruglnosa
Acinetobacter calocoaoeticus
Proteus mirabilis
Proteus morganii
Enterobacter aerogenes
nterobacter cloacae
Serratia marcescen~
::
22
:, . ;, . ~. , ,
27
Pasteurella (species)
CDC Group II F
Moraxella
Cikrobacter freundli
Specl~ically, 4 to 5 isolated colonies were touched with
the wand from the growing devlce and the wand was u~ed to
inoculate the growing medi~m in the growing devlce. The
units were incubated a~ 35C in a 3M brand incuba~or Model
107 for 4 hours. The top kape seal was removed from the
cap of the ~rowing unit and 6 to ~ drops of bacterial
suspension were dispensed on.to a cotton swab. The swab
was streaked ln three directione over a Mueller-Hinton agar
plate and the Kirby-Bauer test was completed according to
the NationaI Cllnical Commlttee for Laboratory Standards
(NCCLS) Ankibiotlcs Susceptibility Standard set forth above.
A comparision was made between khe results obtalned ln
respect to the susceptiblllty of the~`organism tested in
using the growkh medla of the present inventin versus the
.standard technique for growlng bacteria. The results were` :~
~; 20 comparable.
xamp}e 7 o
The following materials were dlssolYed in 1000
milliliters of deionized water and steam s~erill~ed at
: 121C for 15 minutes:
: 25 1.7 gram Trypkicase
` 0.3 gram Phytone
~: . 0.25 gram Dextrose
: 0.5 gram NaCl
2 4
The growing devices utilized in thls exampl~
:
:
~ .
~ 23
: . . . . . ` ,;, . - . ` ` , . .
;. . ..
~L9LZ7~
were polypropylene ~leeves as above described with the
medium placed directly therein. The sleeves were capped
with a plastic oap containlng a "Tyvec" filter. ~he
medium was inoculated using a wire loop with Staphy-
loccocus aureus bacteria obtained from 4 to 5 coloniesof said bacteria on an agar plate. The ~rowing devices
were vortexed, i.e., mixed ~or 10 seconds and then in-
cubated at 35C. Vlable bacterla counts were per~ormed
at 0, 1, 2-1/2, 4-1/2~ 5-1/2, 6-1~2, 11-1/2, 13-1/2,
23-1/2 and 31 hours. The result3 show that the count
initiàlly was 1 x 104 and at 11-1/2 hours the count had
lncreased to about 1.7 to 1.9 x 108 CFU/ml and did not
substantially decrease therefrom through the remainder
of the aforesald tlme/count intervals.
; ~
.
~ '
~ 24 -
.. . . . : . . , ~ .
,: ~ : . .. . ~ : : -
