CA1290272C - Method for differentiation with film medium of lactobacillus organisms from streptococcus organisms - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A carrier solution for making normally dry, self-contained ready-to-use bacteria culture medium film specific for selective growth of Lactobacillus strains by applying as a diluent a solution containing a gram negative organism inhibitor, water soluble anti-fungal agent, a water soluble nitrite salt, and a fluoride ion, with the solution having a pH within the range of from about 6.5 to about 7.5.

Description

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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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~ 90'~7~: 1 ¦ 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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12902~ 1 ¦ 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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go2~2 ¦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 ¦ ~ Trade Mar ~L290~72 ¦ 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 *Trade Mark . ~9~

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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 I
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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 *Trade Mark I!

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!l 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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~L29~)272 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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9~2~2 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 * Trade Mar]c .:..,~, I
-j , ,~ , 1290;~72 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~

**Trade Mark `, .~ ~l ~29~7 Il 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 ~Trade Mar ;~ ;J

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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
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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.
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Organis~ Organism ¦ Sample Count At Count At Species Origin I.D. Tolerance O.OmM NaF 50n~ NaF
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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 ~Z9027 1.
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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 .
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Claims (10)

1.
An aqueous diluent solution for making normally dry, self-contained, ready-to-use bacterial culture films specific for selective growth of Lactobacillus strains under anaerobic conditions, comprising a diluent solution having:
(a) a concentration of from about 1.0 mg/ml to about 4.0 mg/ml of diluent of 2-phenylethanol;
(b) from about 10 micrograms/ml to about 250 micrograms/ml of a water soluble anti-fungal agent;
(c) from about 600 micrograms/ml to about 800 micrograms/ml of a water soluble nitrite salt:
(d) at least 40 mM/ml concentration of fluoride ion; and (e) said solution having a pH within the range of from about 6.5 to about 7.5.

2.
The diluent solution of claim 1 wherein the source of fluoride ion is selected from Group I and Group II fluoride salts.

3.
The diluent solution of claim 2 wherein the source of fluoride ion is sodium fluoride.

4.
The diluent solution of claim 1 wherein the concentration of fluoride ion is at least 40 mM/ml.

5.

The diluent solution of claim 1 wherein said anti-fungal agent is selected from the group consisting of cycloheximide, potassium sorbate, butylated hydroxytoleuene (BHT) and butylated hydroxyanisole (BHA).

6.
The diluent solution of claim 5 wherein the anti-fungal agent is cycloheximide.

7.
The diluent solution of claim 1 wherein the pH is from about 6.5 to about 7Ø

8.
The diluent solution of claim 1 wherein the nitrite is a Group I metal nitrite.

9.
The diluent solution of claim 1 wherein the nitrite is sodium nitrite.

10.
The method of quality assurance checking of a forage inoculant to determine the presence of Lactobacillus straine without also counting cocci, comprising:
obtaining a biological plate containing standard methods culture medium;
preparing an aqueous diluent solution which contains in combination from about 24 units/ml of diluent solution to about 40 units/ml of diluent solution of a gram negative organism inhibitor, from about 10 micrograms/ml of solution to about 250 micrograms/ml of solution of a water soluble anti-fungal agent, frim about 600 micrograms/ml to about 1800 micrograms/ml of a water soluble nitrite salt, and a fluoride ion concentration of at least 40 mM/ml, said solution having a pH within the range of from about 6.5 to about 7.5; and applying a small but effective amount of said solution to said plate in conjuction with a forage inoculant to be checked; and incubating said inoculated plate under anaerobic conditions to provide culture growth of only Lactobacillus strains while simultaneously inhibiting growth of lactic acid cocci.