Note: Descriptions are shown in the official language in which they were submitted.
~L2~02~
BACKGROUND OF THE INvEN~rIoN
Recent times have seen the develop~ent o~ dry medium
culture plates, a typical example of which is tlle one ~old
by 3M Company (Medical Products Division, St. Paul, Minnesota
55144, under the trademark PetriEilm), whose products
typlcally consist oE a dry, self-contained, ready-to-use
bacterial culture medium coated onto a ~ilm base and overlaid
with, for example, a pol~ethylene film. The base carries
Standard Method nutrients and a cold water solu~le ~elling
agent. The overlay film is also coated with the qelling
agent, and in addition, 2,3,5-triphenyltetrazolium chloride
indicator dye, in order to facilitate counting. Grids (each
square 1 cm x 1 cm) outlined on the bottom film also aid~the
counting proces~. The overall dimension of a single Petrifilm *
plate is 20 cm sq.
Such dry medium culture plates, as those previously
described, are often advantageous over the more conventional
Petri dishes and ag~r plates, commonly used for inoculation
and bacterial growth. For example, the older, more conven-
tional use of Petri dishes and nutrient agar medium involves
col~iderable bulk, weight~ and space. This makes transport
o organism samples from the field to the laboratory settin~
die~icult, at best. On the other hand, ~he dry medium
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¦ cult~re film ~hi~h becores supp~rtive of bacterial growth
Il 3ilnply by the addition of an aqueous sample woul~ represent
¦ a-lnore efficient way of doing viable bacterial counts than
¦l present conventional methods.
¦ It i~ a fact, however, that the available dry medium
¦ culture films only contain a Standarcl Method culture medium
¦ coated onto the film ba~e. Put another way, the presently
use(3 culture medium is one suitable for growing all types of
organi~ms and iB not selective ~or growth of any particular
type. Thu~, a bacteriologi~t ~eeking to grow a specific
organism in the pa~ ha3 had to abandon the possibility of
using tlle very desirable culture media films and return to
use of tedious conventional agar plates, with ~pecifically
¦ tailored culture media known to enhance the growth o the
de~ired ~pecific organisms, which at the ~ame time intliblt
growth of undesirable ones. Thu~, because bacteriologist~
¦ and microbiologi~ts are often de~irous of growing only a
specific organism or further isolation, study, evaluation
and use, the Standard Medium containing dry media culture
ilms have not been useful in many lnstances becau~e of
thelr non-selective medium.
A diluent or carrier solution
was developed or making dry medla culture ~ilmq, specific
for growth of Lactobacillaceae, which includes both lactobacilli
I
and streptococci. Whlle the proces~
works ~ati~factorily for most purpose~, there are instances
wllere one may wi~h to further delineate between lactobacilli
and cert~in 9 reptococci. 0~ ~xample of auch an in~tance
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i8 in situatiorls involving quality assurance checks of
forage innoculants. For such forage, or ~ilage innoculants
to properly per~orm, it is essential they have adequate
level~ of Lactobacillaceae ~trains present. If not, the
innoculant wlll have no significant value.
It is not uncommon for silage innoculant product sample~
to have both Lactobacillu~ ~trains and Streptococcus strains
present in the ~ame ~ample. While the Streptococcu~ strain
i9 not harmful to the performance characteristics of the
eorage innoculant product, if one's objective i8 to determine
whetller a satl3factory level of the Lactobacillus strain i~
present, a positive te~t with the dry media culture plate,
using the diluent of the prior art may not be determini-
tive, ~ince it may indicate either tlie presence of LactobacilIus
strain~ or Streptococcus strains, or both. There i~ therefore
a need in certain instance3 to develop a further refinement
in my ~culture media to allow dry media culture plates to be
used to diferentiate between Lactobacillus ~trains and
_reptococcus strains. Doing ~o would allow quality assurance
I
cl~ecks of forage innoculant, with a positive test providing
certainty of the desired presence of Lactobacillus strains,
without any lingering doubt that a "false" reading exists
because of the presence of Streptococcu~ strains.
Accordingly, it i8 a primary objective o the present
invention to develop a method and means for making normally
dry, self-contained, ready-to-u~e medium culture ilm
speciic for ~elective growtll of certain lactobacilli
while at the same time inllibiting the growth of Streptococcus
strains.
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In particular, it is an objective of tlte present invention
j to make the above de~cribed culture film speciic for
! selective growth of lactobacilli under anaerobic condition~,
¦ while at the same time inllibiting the growth of other
¦ streptococcus straing.
¦ Another objective of the pre~ent invention i~ the
development of a metllod, mean~ and technique of malcing 3M
* Petrifilm medlum culture film ~pecific for tlle growth of
¦ ~actobacillaceae organism~, or in other words the lactic
I
acid bacteria.
Another ~pecific objective of the present invention i~
to develop a diluent ~olution for use in preparing ~amples
for inoculation onto ~elf-contained, ready-to-use medium
cultuee film~, which make the film ~pecific for lactobacilli,`
while at the same time inhibiting the growth of Streptococcus
~trains.
Another more specific objective i9 to develop a dilu~nt
~olution for use in teqting forage innoculant product~ to
determine the presence of de~ired Lactobacillaceae ~train~,
and to differentiate between Lactobacillus and Streptococcus
strain~ which might be pre~ent in said innoculant.
Another objectlve is to prepare a diluent ~olution for
use with dry, ~el-contained, ready-to-u~e Inedium cult~re
fllm ~uch as Petrifiln~ which eliminates the pos~ible growth
of pathogens.
The method and means for accomplishing each of the above
objectives, as well as others, will become apparent from the
detalled description of the lnvention.
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¦ SUI~N~RY OF THS INVENTION
Normally dry, self~contained, ready-to-use bacterial
medium culture films are made specific for 3elective growth
of lactic acid bacteria, under anaerobic conditions, while
at the ~ame time inhibiting Streptococcus strain3. This i3
done with a water ba~ed diluent 301ution, or diluent, which
contains a gram negative organism inhibitor, in combination
Wittl a water soluble anti-fungal agent, a water 301uble
nitrite salt, and a source of ~luoride ion. The solution
has a pH within the range o about 6.5 to about 7.5, preferably
neutralO The ~olution, when u3ed a~ a diluent or carrier
for bacterial inoculation3, functions to make the otherwi~e
Standard Method3 cwlture medium~ coated onto the base film,
Lactobacillus ~pecific, while also being inl)ibiting to
Streptococcu~ strains.
DB~rAIL~D DESCRIPTIOt1 OF TI~E INVENTION
Dry medium culture film containing Standard ~lethods
culture medium will become supportive of bacterial growth,
simply by the addition of an aqueous ~ample. In accordance
wlth tl~l3 reeinement it has been
discovered that if the aqueou~ ~ample is inoculated onto tlle
culture film b~ u3e of a specific solution which contain~ a
30urce of fluoride ion a3 hereinafter described, then, and
only then, will the film become organi3m specific for
~actobacillus, to the exclu3ion of Streptococcus strain3.
There are numerou3 rea~oll~ why bacteriologist3 and
microbiologi~t3 may desire to grow a specific organism, to
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¦ the exclu~ion of others. One such instance earlier mentioned
¦ is in doing quality assurance checking of forage inoculants.
¦ This lnvention allows convenient use of the desirable
advantages of culture medium film3 to enu~erate only the
Lactobacillus organi3m without getting a "fal3e" reading of
I
¦ Streptococcus ~train.
Milnes, et al. in J. Dent. Res., March, 1985 at pp. 401-
404 recognize generally that sodium fluoride may inhibit
growth oE Streptococcus ~train~ and not Lactobacillu~
strainq, but they do not discuss or ~uggest combination of
this information with Petrifil ~ technology and forage
ino~ulant technology to develop a quality control program
for forage inoculant~. Their inve tigation dealt with
caries lesions.
The diluent for u~e in thi~ invention is a four component
9y8tem ~ and i~ water based. The solution contains in
combination a gram negative organi~m inhibitor, an anti-
fungal agent, a water ~oluble nitrite salt, and a fluo~ide
ion source. The ~olution also mu~t have a p~ within the
range o~ from about 6.5 to about 7.5, and i~ preferably
neutral. When the~e conditions are met, it allows for a
diluent which, when placed on a dry madium culture film,
make~ the film organi~m specific for Lactobacillu9, while
inhibiting Streptococcuq ~tralns.
The gram negative organi~m inhibitor can be a Polymixln*
antiblotic and ~hould have a concentration withln the range
of 24 unit3/ml of diluent eolution to about 40 unit~/ml of
diluent 901ution with 24 unit~/ml unctionally very ~ati~-
.
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¦I factory for*Polymixin B sulfates. The preferred antibiotic is
¦l * Polymixinand * Polymixin 8 sulfate is the preferred
l * Polymixill.
I The inoculating solution also contains a water soluble
anti-fungal agent, in a concentration within the range of
10 micrograms/ml to about 250 micrograms/lnl, with about
10 microgram3/ml to about 20 microgral~s/ml being preferred.
A preferred anti-fungal agent i9 cycloheximide. ~owever, in
addition to cycloheximide, one may u~e other anti-~ungal
agents and preservative~ such as pota~ium sorbate, butylated
! hydroxyani~ole (B~A) and butylated hydroxytoluene (~IT).
l The third component of the carrier solution, or diluent,
¦ is a water solublç nitrite salt at a concentr~tion within
the range of ~00 micrograms/ml to about 1800 micrograins~ml.
The nitrite ~alt may be any water soluble metal nitrite
salt, but iq preferably a Group I ~etal nitrite salt and i9
mo3t preferably either sodium nitrite or potassium nitrite.
Potas~ium nitrite i~ mo~t preferrea. A nitrite salt, when
u~ed within thi~ range of concentrations has been found to
inhibit growth of organisms other than lactic acid bacteria.
¦ The fourth component is an aqueous solution of a source
of fluoride lon. Suitable exalnples includ~ Group t and
Group II metal fluoride salts. The mo~t preferred salt is
i ~odiuln fluoride, simply because of ease o availability.
¦ The amount of fluori~e ion needed can functionally be
¦ described as an amount ~ufficient to inh~bit Streptococcus
¦ strain growth, without inhibiting Lactobacillus strain
growth. Thi~ can furtller be defined a~ an amount suficient
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¦ t~ ~rop plate count oE Streptococcu~ ~tr~ln by 2-3 1099~
¦ i.e. from 10 for example down to at leaqt 10 during serial
dilutions. Generally sati~factory re~ults can be achieved
whell the fluoride ion concentration i~ at least 4~ mM/ml
(millimoles/milliliter), preferably at lqast 50 mM/ml. The
upper limit of range of addition is Merely practical, there
being little value in larger do~e concelltration~ than the
minimu~ needed to accomplish the intended result.
The carrier solution must have a p~l within the range of
6.5 to about 7.5, preferably from about 6.5 to about 7.0~
Ideally, the diluent ~olution i~ neutral. It has been found
critical that in ~aking the culture ~ilm LactobaclIlaceae
3pecific that the pH remains generally within the range o
neutral, i.e. from about 6.5 to about 7.5. This i~ A0
because the commercially available films, such as the
* Petrifilm' sold by 3~ Company, contain a tetrazolium dye for
dying the cultured bacteria red, to enhance counting.
However, the tetrazolium dye may become toxic to all growing
organi~ms, if the pH become~ more acid, such a~ for example
at a level of pH 5.5. Thus, the carrier solution of thl~
invention must have a p~l within the range ~pecified, which
avoid~ the adver~e reaction with tetrazoliu~ dye.
In order to maximize the inhibitory affect Oe the growth
of other organlcms it i9 al90 e~ential for thi~ invention
that the inoculated film be incubated under anaerobic
condition~. Put another way, the diluent ~olution is
organis~ ~pecific to Lactobacillaceae ~ under anaerobic
conditions. Thls anaerobic environment may be induced in
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many con~entioal way~, such a~ use of*Ga~ Pac ~ anaerobic
¦ generators, manufactured by BBL Microbiology Syste~o~ o~
Cokey~ville, Maryland.
The previou~ description ha~ been given in connection
Wittl the u~e of*Polymixin antibiotics a~ the gram negative
organism inhibitor. A pre~erred formulation llas been
developed which i9 ~uitable for many purposes, and in
instance~ o~ quality a~surance testing of forage inoculant
pecforms even hetter than the previously described formulation.
It is based upon u3e of 2-phenylethanol as the graln negative
organism inhihitor. When 2-phenylethanol i9 u~ed a preferred
compo~ition i~ one having the following de~cription. The
concentration of 2-phenylethanol should be from about 1 mg/ml
to about 4 mg/ml. The concentration of the anti-fungal
agent ~hould be from about 10 ~icrograms/ml to about 250
micrograms/ml, the concentation o the water soluble nitrite
salt should be from about 600 micrograms/ml to about 800
micrograms/ml, and the concentration of fluoride,
! 50 micrograms/ml. Tile pH will thus be within the range of
rom about 6.5 to about 7.5, preferably from about 6.5 to
~bout 7Ø
The use of the dry medium coated film with th~ carrier
.solutions of this invention i~ a conventional technique
known to microbiologists and bacteriologlsts. Basically,
the use involve~ the ollowing technique. The film is
placed on a flat surface, the top transparent film layer
lifted 90 that a 1 milliliter sample of the diluent carrying
the organism can be plac2d on the bottom film. The top film
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i~ then closed carefully down over the bottom film. A
plastic ~ample spreader i3 then lightly pre~sed down over
the top film to ~peead the carrier ~olution across the
¦ bottom me~ium containing film. Thereafter, the film i3 left
¦ undisturbed for approximately a minute to allow gelling.
¦ The Eilm 1~ then incubated in a horizontal position with the
clear ilm side up at 32~C for up to 48 hours. The organisms
will reduce the tetrazolium indicator dye in the l~ottom film
making the organi~m colonies on the film appear as red dot~,
which can be counted.
The following examples are offered to further illustrate
but not limit the proce~, technique and prod~ct composition
of tlle pre3ent invention. Those examples through Table V
illu~trate my earlier ~vén~ion.
In each of the example~ demon~trated herelnafter, the
dry medium culture film was the*Petrifilm plate developed
by the 3M Company (Medical Proauct~ Divi~ion, St. Paul,
Minnesota 5$144). This*Petrifilm con~isted Oe a dry self-
contalned, ready-to-use bacterial culture medium coated onto
a ellm base and overlaid with a polyethylene film. The ba~e
carried Standard Method nutrient~ and a cold water ~oluble
gelling agent. The overlay film, which wa~ also coated with
a gelling agent, also contained 2,3,5-trlphenyltetrazolium
chloride indicator dye to facilitate organi~m counting. A
grid of 1 cm x 1 cm squares was outlined on the bottom film
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to also aid in counting. The overall ~imen~ions o~ the
* Petrifilm plate wa~ 20 ~quare cm.
¦ The ba~ic procedure involved preparation of ~tock
~olutions of the diluent, inoculating the*PetriEilms
followed by incubation at 32C for 4~ hours under anaerobic
condition~, and thereaEter counting organi~ms tc~u/lnl). A
~oil sample from a greenhouse wa~ used as the test culture
and another sample designated S/80, known to be a Lactobacillus
culture wa~ u~ed as a control. The soil sample was known to
contain many organisms other than Lactobacillu~.
The diluent stock ~olutions were prepared at tlle f~ollowing
¦ level~ of sodiu~ nitrite, by percent weight/volume~:
1~ weight/volu~ne, 6% weight/volume, 12~ weight/volume, and
18~ weight/volume. Cycloheximide was used at percent
weight/volume levels of: 0.1~, 0.5%, 1~ and 2.S$ weight/volume.
*Polymixin B, in this first series of examples, cince they
were de~igned to study the variations in concentration of
I ¦ ~odium nitrite and cyclohexlmide, was u~ed at a constant
0.03g weight/volume.
¦ The stock solutions were filter sterilized and the final
diluent was prepared by u~ing 97 ml of sterilized distilled
~ water and addition 1 ml per 100 ml of wate~ Oe eflch of the
¦ three ccmponents, i.e. sodium nitrite, cycloheximide and
*Polymixin B all witilin tlle ranges previously .~pecified.
¦ Sterile mixture~ were then asceptlcally delivered to test
tube~ at a rate of 9 ml/tube. The following mixtures were
prepared:
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¦ Final Concentration
of In~redients
Control~utterfleld's buEEer plus
! o.l~ Tween 80*
Variation 1lnO ~/ml nitrite
10 ~g/ml cycIoheximide
24 units/ml *Polymixin
Variation 2600 ~g/ml nitrite
10 ,~g/ml cyclohexilnide
24 units/ml *Polymixin B
Variation 31200 ~/m]. nitrite
10 ~g/ml cyclolleximide
24 units/ml *Polymixin B
Variation 41800 ~g/ml nitrite
10 ~g/ml cyclohexil~ide
I 24 units/ml *Polymixin B
: I Variation 5600 ~g/ml nitrite
: ¦ 50 ~g/ml cyclolle%imide
24 units/ml *Polymixin ~
Variation 6600 ~g/ml nit~ite :
I 100 ~/ml cycloheximide
24 units/ml *Polymixin B
Variation 7600 ~g/ml nitrite
250 ~g/ml cycloheximide
24 units/ml *Polymixin
: ~
Standard*Petrifilm dilution procedures were followed in
: platin~ the S/80 control, in particular 1 ml of an overnight
culture was serially diluted usln~ the test solution and 1 ml
volume~ were delIvered to the plates. An 11 graln sample Oe
greenhouse soil was initially diluted in 100 ml of ~uttereield's
buEeer. Thi~ preparation was then seria.lly diluted using
the test solutlon.
1.0 ml volumes of 10 6, 10 7 and 10 8 dilutions of S/80
were delivered to*PetriEilm plating medium. 1.0 ml volumes
_ _ _ . _ __ _
~Butterfield's bu~fer is a conventional potassium phosphate
: . bufferin~ solution in food microbiology.
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o~ 10 4, 10 5 and 10 6 dilutions of soil were delivered to
* Petri~ilm plating mediumc Plated samples were incubated
anaerobically in jars containing Gas Pack~ generators tBsL).
They were incubated at 32C for 48 h.
Table I below sllows the resulting organism count, using
the diluent solution herein specified.
TABLE I
Colony formin~
Sam~leUnits/m.l (cujml)
Soil: Control 5 x 107
Variation 13.3 x Io6
: Variation 22.3 x 107
Variation 35 x 104
Variation 40 x 104
Variation 57.0 x 106
Variation 67.1 x 107
Variation 74.0 x 1~7
S/80: Control 2.4 x 109
Variatlon 17.5 x 108
Variation 21.3 x 109
Variation 31.3 x 109
Variation 41.3 x 109
Variation S9.6 x 108
Variation 61.2 x 109
Variation 71.0 x 109
~ can be ~een, there is a signiflcant increase ln the
inhibitory efeect o~ the diluent on soil micro-organi5ms as
the concentration of the nitrite was increased from
600 ~g/ml to 1200 ~g/ml. The S/80 was unaffected by the
variation of either the nitrite or cycloheximide concentration
over the ranges of 100 ~g/ml ~o 1800 ~g/ml and 10 ~g/ml to
250 ~g/ml, respectively. It can also be ~een that the
diluent allowed the culture film to be organism specific for
:: . the Lac obacillaceae organism, and inhibited the ~rowth o~
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otller or~anisms. Similar experiments to those shown in
variations 1-7 were done varying the*Polymixin s conc~ntration
which was shown to have no e~fect on the S/80 organism over
a range of 24-40 units/ml. For soil organisms levels of
below 24 units/ml of Polymixin B decreased the inhibitory
eEfect.
Examples Showing Replacement of Polymixin_B
Sulfate W rh 2-Phenylethanol
2-Phenylethanol (PEA) purchased from Si~ma Scienti~ic as
a liquid (1 ml = 1.02 gra~ns) was used in the diluent. the
PEA is added to obtain the concentrations specified below,
to a given volume of distilled water, and autoclaved.
Filter sterilized 6~ weight/volume sodium nitrite and 0.1~
wei~ht/volume cycloheximide solutions were used to complete
the below listed mixtures by adding 1 ml per 100 ml of
diluent prepared.
TABLE II
Mixtures
Prepared % By Wt./Vol.
8 600 ~g/ml nitrite
10 ~g/ml cycloheximicle
0.05~ PEA
9 600 ~g/ml nitrite
10 ~g/ml cycloheximide
0.10% PEA
600 ~g/ml nitrite
10 ~g/ml cyclohexi~ide
0.20~ PEA
11 600 ~g/ml nitrite
10 ug/ml cycloheximide
0.4~ PEA
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Inoculation and technique ~as a~ in the previou~ly
descrlbed example~. An S/80 solution and soil were the
tested ~ampleq. The S/80 was serially dllut~d from an
overnight culture through the diluent mlxture~, and plated
on*Petrifil ~ at 10 7 and 10 8, The 90il wa~ initially
diluted 1:10 with 99 ml modif~ed Butterfield'~ buffer and
then serlally diluted in the gi~en diluent. The 10 4,
10 5 and 10 6 dilution~ of soil were deliver~d to*Petrifilm.
Films were incubated anaeroblcally u~ing BBL Gas Pack~O for
48 hours at 32C. The re~ult~ are ~hown in Table III.
TABLE III
Results
Inoculating
Solution~ #cfu/ml
Soil: Variation 86.2 x 105
Variation 95 x 103
Variatlon 100 x 103
Variation 110 x 103
S~80: Variation 119.4 x 108
As can be 3een, the phenylethyl alcohol substitution in
the formulation or the*Polymixin B ~ulfate antibiotic
appear~ gulte effective in inhibitlng the 30il microorganisms.
The followlng example~ de~onstrate various modi~ications
of the formulation~ using phenylethyl alcohol (PEA) as the
gram negative inhibitor.
TABLE IV - Formulations
Phenylethyl
Mixture3 Sodium Nitrite Cycloheximide Alcohol
Prepased ConcentrationConcentration Concentration
Control (~utterfield's
Buffer~
12 800 ~g~ml10 ~g/ml 0.1~
13 800 ~g/ml10 ~g/ml 0.2%
14 600 ~g/ml10 yg/ml 0.1
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Phenylethyl
Mixture~ Sodium Nitrite Cycloheximide Alcoho~
PreparedConcentration Concentration Concentration
I
600 ~g/ml 10 ~g/ml 0.2%
16* 800 ,ug/ml 10 ,ug/ml 0.1
17* 600 yg/ml 10 jug/ml 0.1
S/80 and 90il were the test ~amples. S/80 lilutions
were plated at 10 7 and 10 8, Soil wa~ diluted initially in
¦ 99 ml ~1:10) of modified ~utterfield's buffer and plated at
10 3 or through 10 6.**petr1film ~ were incu~ated anaerobically
using BBL Ga~ Packs~ at 32C for 48 hours, a~ similarly
described in earlier examples. The re~ult~ are inclicated below.
TABLE V - Re~ults
Sample Mixture cfu/ml
; Control~Control 9.8 x 108
S/80: 12 8.7 x 108
13 8.5 x 108
14 8.7 x 10
1.0 x 109
16 7.4 x 10
17 1.1 x 10
Control: -- 2.7 x 107
Soil: 12 1.8 x 104
13 3.0 x 102
14 3.4 x 106
3.3 x 104
16 4.2 x 104
17 9.0 x 104
I
l As can be ~een, solution ~13 gave the best inhibitory
¦ re.sults with ~oil, without adver~ely affecting the growth of
S/80.
* Include~ Polymixin B at a level of 24 unit~
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i Exampleq of Differentiation Between
Lactobacillus Strains and Streptococcu~ Strain~
The following examples demonstrate tlle use of sodium
fluoride when selectively applied to dry medium culture
film, to select against the lactic acid cocci an~ permit
growth of Lactobacillus strain~.
I'he ~elective diluent used
in this instance
comprised: 5 ml of 8~ sodium nitrite, 5 ml of 0.1~ cyclo-
heximide, 1 mi of phenylethyl alcohol, and 489 ml of sterile
distilled water. In the table below, the solution is
referred to in shorthand abbreviation a~ PECAN.
I A 1~ sodium fluoride solution was prepared an~ the
appropriate volume used in obtaining solutions with the
following concentration~ o ~odium fluoride: O, 20, 30, 40
and SO 0M. The ~peci'fic solutions are ~hown in Table VI.
I TABLE VI
Grams Grams mls 1% NaF/
mM NaF ~ NaF ~00 500mls PECAN
840 mg 420 mg 42
1.26 g 0.~3 g 63
I40 1.6~ g 0.84 9 84
2.1 g 1.05 9 105
100 4.2 g 2.10 g 210
'rhe diluent solutions containing sodium fluoride were
Eilter sterillzed and dispensad into 9 ml blanks. ~our
i901ate9 were screened, 2 Streptococci and 2 Lactobacillus~
One ml o~ an overnight culture ~uspension (standard broth) was
pipeted into a 99 ml of Butterfield's Buffer. The five
sodium fluoride 801ution8 were used to serially dilute the
i901ate9 onto*Petrifilm from 10 to 10 . Standard
¦ plating procedure a~ earlier described wa~ u~ed. The
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¦~* PetriEllm~ ~!re inc~lbated at 32 araerobic~lly for 2 day~. ¦
The colony forming unit~ were counted and the following
data obtained.
TAsLE VII
Isolate Tested mM NaF
ID Specie~ 0 20 30 40 50 100
PC202 Streptococcu~ 9.1xlO9 7.8X10~ 1.5x108 3.0x105 N6 0
l PC301 Steeptococcu~ 7.6x108 9.0xlOB 2.5x105 1.4x104 2x103 o
1 286 Lactobacillu~ 1.8xlO9 1.7xlO9 1.6X109 1.7xlO9 1.4X109 1.5xlO9
287 Lactobacillus 4.0xlO9 3.8X109 3.7X109 4.6X109 3.8X10g 3.4X109
The ID number~ refer to identification ~umber~ for the
samples. A~ Table VII demon~trates, the two ~treptococci te~ted
we~e inhibited by the ~odium fluoride. Inhibition wa~ demon-
~trated at 30 mM NaF for 1 lso1ate, and at 50 mM NaF inhibition
was almo~t complete. The lactobacilli tested were not
inhibited by the sodium fluoride at these concentrations.
Other Lactobacillaceae ~rgani~ms were tested in order to
I ,
determine whether or not the soaium fluoride selectivity wa~
wide-~pread. It was ~o found as indica~ed in Table VIII
below. Other fluoride ion ~ources such a~ other water
~oluble Group I and Group II metal~ w~ll work equally well
in that suitable lactic acid cocci inhibition i9 achieved.
TABLE VIII
:
Organi~m Organism
Sample Count At Count At
Species Origin I.D. Tolerance O.O~M NaF 5~ NaF
1 L. Acidophilu~ Portland 04 - 1.7E9* 0
2 L. Acidophilu~ Product S/Chris ~n~en - 5.0E8 O
_
~ 1.7E9 refer3 to 1.7 x 109.
: -19-
~:
**Tra~e Mark
~ ~,
.' - :
.~ , .
272
Organis~ Organism
¦ Sample Count At Count At
Species Origin I.D. Tolerance O.OmM NaF 50n~ NaF
I
3 L. Acidophilus Portland l9/RL421 - 5.6E8 0
4 L. Acidophilu~ Porcine 296/PIG75 - 9/2E8 0
L. Acidophilu~ Portland 6/KL321 - 2.4E9 O
6 L. Amylovorus Gift 41 - 3.1E9
7 L. Brevis Bovine 289/D15B ~ 2.0E9 l.9E9
8 L. Brevi~ Bovirle 289/A14A ~ 1.28E9 1.34E9
9 L. Brevis Porcine 288/37 ~ 2.4Eg 2.4E9
L. 8uchlleri Bovine 289/D27B -~ 1.06E9 6.8E8
11 L. Ca~ei Portland 02/KL322 + 2.9E9 2.2E9
12 L. Casei (Psuedo) Corn 285/129A -~ 1.6E9 2.1E9
13 L. Cellobiosus Bovine 2~9/D24A -~ 1.36E9 1.24E9
14 L. Cellobio~us Bovine 289/D24B + 1.75E9 1.51B9
L. Cellobiosus Bovine 289/A44B -~ 1.07E9 3.5E8
16 L. Cellobio~us Bovine 289/A22A + 1.26E9 9.4E8
17 L. Cellobiosu~ Bovine 289/A13B +/- 1.5E9 l.OE6
18 L. Cellobiosus Bovine 289/C26A - l.OE6 0
19 L. Coryniformis Corn 285/66A + l.OOE9 7.2E8
L. Corynifonmi~ Bovine 289/B9B +/- l.lE9 5.5E6
21 L. Coryniformis Corn 285/81A - 1.16E9 5.7E4
22 L. Curvatis Bovine 289/B3~ 4.4~8 2.4E8
23 L. Delbrueckii Alfalfa285/148A + 3.4E8 2.7E8
24 L. Fermentum Porcine 305/PIG38 + 6.3E8 5.4E8
L. ~lelveticus Portland 129 - 1.5E7 0
26 L. I~omohiochii Wheat 285/151C + 1.18E9 1.04E9
27 L. Jensenii Bovine 289/D2A + l.OlE8 1.13E9
28 L. Jen~enii Bovine 2a9/D2B + 1.07E9 8.1E8
29 L. Jensenii Bovine 289/A6A - 9.0E8 0
L. Jensenii Bovine 289/C3B - 4.6E8 0
31 L. Lactis Corn 377/504C + 1.08E9 1.20E9
32 L. Lacti~ Portland 124/L. Lac 15808 - 2.2E8 O
33 L. Oenu~ Hay 285/17A - 2.0E9 0
34 L. Plantarum Portland 29/C7 -~ 1.03E9 9.8E8
L. Plantarum Portland 28/C54 + 8E8 1.3E9
3G L. Plantarum Portland 26/S80 + 7.9E8 6.5E8
37 L. Plantarum Alfalfa 31/Alpha5 + 2.8~9 1.9E9
38 rJ~ Plant.~rwn Gra~ 286 + 3.2E9 2.0E9
39 L. Planta~um Corn 287 -~ 5.1~.9 4.1E9
L. Plantar~n Gras~ 318 ~ 2.8E9 2.7E9
41 L. P~antarum Gras~ 319 ~ 1.8E9 2.5E9
42 L. Plantarum Al~alfa 346 -~ 2.13E9 2.18Eg
43 L. Plantarum Alfalfa 347 + 7.6E8 4.8E8
44 L. Plantarum Alfalfa 345 + 2.0E9 1.7E9
Leu.~lesenteroides Corn 377/618B -~ 2.1E8 l.lE8
46 L~u.Mesenteroides Bovine 289/A13A +/- 7.3R8 9E6
47 Lsu.Mesenteroides Corn 285/12A -~/~ 3.7E8 2.00E8
48 Pediococci Corn 247/P-2 + 4.7E9 4.8E9
49 S. ~nginosus Grass 377/629A - l.OE8 0
S. Anginosus 80vine 289/D23A - 9.3E7 l.OE4
51 S. Avium Grass 377/622A - 4.8E8 0
52 S. Bovi~ Bovine 289/A368 - 1.4E8 0
53 S. Equi 80vine 289/C15A - 1.35E8 l.OE4
-20-
~Z9027
1.
I
I
Organism Organisln
Sample Courlt At Count l~t
Species Origin IoD Tolerance 0.0mM t~aF 50m~ NaF
1 54 S. E~ui~imiles Corn 377/615A - 7. lE~ O
i 55 S. Faecalis ? 97/Jl~2-2 ~ 9.5E8 5.0E4
¦ 56 S. Faecali~ ? 98/DS16-C3 - 1.35E9 O
¦ 57 S. Faecium Portland ~2/PC201 + 1.29E9 1.3~9
¦ 5~ S . Faecium Portland 18/PClOl - 1.2~E9 1.07E5
¦ 59 S. Faecium Portland PC102 - 6.0E8 0
S. I;aeciwn Portland 8/PC202 - 9.1~9 0
61 S. Faeciurn Portland 3/PC301 - 7.6e8 2.0E3
62 S. Faeciurn Portland 13/PC401 - 9.3E8 9.0E4
63 S. Faecium Portland 9/PC402 - 8. SE~ E4
1 64 S. Faecium Portland UR~ll - 1.6~9 3.6E4
~65 S. r.actis Gift 101 -- 1.00E8 0
i66 S. Mitis Porcine 288/C51 - 2.9~ 1.6E4
¦67 S. Pneumoniae Alfalfa 285/122B + 2.0E9 3.0E9
6~ S. Pneumoniae Porcine 288/A6 + 1.9E9 1.4E9
169 S. Salivarus Porcine PIG99 - 2. OE8 0
170 S. S3nguis Grass 377/622B - 4.7E8 O
¦71 S Ubens Bovine 289/B5A - 2.23~ 0
~s can be seen, nearly all of the 44 isolates of
Lactobacillus strain~ tested were tolerant to fluoride ion.
ll
Moreover, those few that indicated some intolerance are
species which are not likely to be found in a forage inoculant.
Likewise, nearly all Streptococcus strains likely to be
found in a forage inoculant was successfully inhibited.
It can be seen from each of the above examples as
presented in the varlous tables that effective diluent
composition~ can make media eilms lactic acid bacteria
speclflc e.nd therefore effectively accomplish their intended
pur~)oses .
I
I
