Note: Descriptions are shown in the official language in which they were submitted.
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¦ DRIED MEDIUM CULTURE FILM SOLUTION ~ND MET~OD
¦I FOR M~KING MEDIUM LACTOBACILLACEA SELECTIVE
i Recent times have seen the development of dry mediuln
¦ culture plates, a typical example of wtlich is the one sold
¦ by 3M Company (Medical Products Division, St. Paul, Minnesota
551~4, under the trademark Petrifilm), whose products
l! typically COIlsist of a dry, self-contained, ready-to-use
¦ bacterial culture medium coated onto a film base and overlaid
itll, for example, a polyethylene film. The base carries
talldard Me~hod nutrients and a cold water soluble gelling
0 1¦ ayent. The overlay film is also coated with the gelliny
ayent, 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 process. The overall dimension of a single Petrifilm~
plate is 20 cln sq.
Sucll dry medium culture plates, as those previously
described, are often advantageous over the rnore conventional
¦ Petri dishes and agar plates, commonly used for inoculation
and bacterial growth. Eor example, the older, more conven-
¦ tional use of Petri dishes and nutrient agar media involves
¦ considerable bulk, weight, and space. This makes transportof organism samples from the field to the laboratory setting
¦ difficult, at best. On the other hand, the dry medium
¦ culture film whicll becomes supportive oE bacterial growth
¦ simply by the addition of an aqueous sample would represent
¦ a more efflcient way of doing viable bacterlal counts than
present conventional Inethods.
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It is a fact, llowever, that the available dry medium
culture films only contain a Standard Metl~o~s culture medium
coated onto the film base. Put another way, tlle presently
used culture medium is one suitable for growiny all types oE
oryal~isms al-d is not selective for growth of ally particular
type. Tl~us, a bacteriologist seeking to grow a specific
orgarlism, in tlle past has had to abandon the possibility oE
usiny tl~e very desirable culture media films and return to
use of tedious conventional agar plates, with specifically
tailored culture media knowrl to enhance the yrowt)) of the
desired speciEic organisms, which at the same time inllibit
yrowth of undesirable ones. Thus, because bacteriologists
and microbiologists are often desirous of growirlg only a
s~ecific organism for further isolation, study, evaluation
and use, the Standard Medium containing dry media culture
films have not been useful in many instances because of
tl~eir noll-selective medium~
~ ccordingly, it is a primary objective of the presellt
invelltion to develop a method and means for makilly normally
dry, selE-contained, ready-to-use medium culture film
specific for selective yrowtll oE certain bacteria, wllile at
the same time inhibiting tlle growtl- oE others.
In particular, it is an objective oE the present invel~tio
to make the above described culture Eilm specific for
selective yrowth of lactic acid bacteria, un~er anaerobic
collditions, while at t~le same time inhibiting the growtll of
otller ulldesired oryallisms.
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`I F~llother objective oE t~le present inventior- is the
~¦ dev~lopmellt of a metho(l, means and techl-ique of making 3
Petrif ilm'~ medium culture ~ilm specif ic for tlle growtll of
~I Lactobacteriaceae orgallislll5, or ill other words the lactic
¦ aci~l bacteria.
Anotller specif ic objective of the presel-t invention is
to (3evelop a diluellt solution for use in prepariny samples
or inoculation onto self-contained, read~y-to-use medium
culture Eilms, wllicll make the film specific for lactic acid
bacteria, while at the same time inhibiting the growtil of
un~esired bacterial oryanisms, yeasts and molds.
¦ 'l'he method and means for accomplishing each of the above
objectives, as well as others, will become apparent from tlle
¦ detailed description of tl~e inventioll.
SU~ ARY OF l'~;E INVENTION
¦ Normally dry, self-contained, ready-to-use bacterial
me~lium culture f ilms are made specif ic or selective growtt
oE lactic acid bacteria, under anaerol>ic conditions. Tllis
is ~lone witll a water based diluent solution, or diluent,
whicl~ contains a gram neyative organism inhibitor, in
combina~ion with a water soluble anti-fungal agent, and a
water soluble nitrite salt. Esyecially preferred is sodium
Il nitrite. l'he solution has a pll within tt~e range o~ about
'I 6.5 to about 7.5, preferably neutral. 1'he solution, w~en
¦ use(l as a diluent or carrier for bacterial inoculation,
¦ f unctiol)s to make tl~e: otllerwise Standard Metl~ods culture
¦ me~lium, coated onto t~le base film, Lactobacteriac~e specific-
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¦¦ ~I,l'AILE~ ~ESCRI~'rlON OF ~'~IE INVENTION
~ ry medium culture film containing Standard Metllods
culture medium will become supportive of bacterial growth,
l sim~ly by the addition of an aqueous sample. In accordance
jl witll tllis invention, it has been discovered tllat if the
¦¦ a~ueous sample is inoculated onto the culture film by use o~
¦¦ a s~ecific solution, hereinafter described, then, and only
il tllen, will tlle film become organism specific for Lacto-
¦! bacteriaceae.i
¦ Tllere are numerous reasons why bacterioloyists and
I microbiologists may desire to grow a specific oryanism, to
¦ tlle exclusion of otllers. Carrier solution or diluent
solution developed in accordance Wltll tllis invention is
es~ecially designed ~or use with culture films to make tllem
¦ Lactobacteriaceae specific. Thus, it allows convellient use
of the desirable advantayes of culture mediuln films such as
field collection, transport and study, even when specifically
culturiny only the Lactobacterla,Ce3,e organism.
l Typical lactic acid organisms wl~icll can be selectively
¦ yrown on the culture films previously described, when one
uses the diluent of tlle present invention, are streptococci,
pediococci, lactobacilli and leuconostocs.
lhe diluent for use in this invention is a tllree componerlt
system, and is water based. ~`he solution contains in
combination a gram negative organism inhibitor, all anti-
fungal ayent, and a water soluble nitrite salt. lhe solution
also must llave a pll Witllisl the range of from about 6.5 to
about 7.5, and is pre~erably neutral. Wllen these conditions
are met, it allows for a diluellt wllicll, wllen placed on a dry
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il me~iuln culture film, makes tlle ilm oryanism specific or
I Lactobacteriaceae.
llle gram negative or~anism inllibitor call ~e a Polymixin
antibiotic and sllould have a concentration within tlle ranye
o 24 units/ml of diluent solution to about 40 units/lnl of
¦ diluent solution with 24 units/ml functionally very satis-
¦ factory Eor Polymixin s sulfate. The preerred antibiotic
is Polymixin and Polymixin B sulfate is the preferred
i Polymixin.
llle inoculating solution also contains a water soluble
anti-fun~al agent, in a concentration witllin the range of 1
microyrams/ml to about 250 micrograms/ml, with about 10
I microyrams/ml to about 20 micrograms/ml beiny preferred. A
I preferred anti-ungal agent is cycloheximide. Ilowever, in
ad~ition to cycloheximide, one may use other anti-fungal
ayerlts and preservatives such as potassium sorbate, butylated
ydroxyanisole ~B~IA~ and butylated hydroxytoluene (B~T).
Il lhe third component o tl-e carrier solution, or diluent,
il is a water soluble nitrite salt at a concentratioll witl~in
20 ¦I tlle range of 60~ micrograms/ml to about 18~ micrograms/ml.
Il Tlle nitrite salt may be any water soluble metal nitrite
i salt, but is preferably a Group I metal nitrite salt and is
l most yreferably eitller sodium nitrite or potassium nitrite.
i Potassium nitrite is most preerred. A nitrite salt, wllen
. ¦ used witllin tllis range of concentrations llas been found to
inlli~it yrowtll of oryanisms otller tllan lactic acid bacteria.
I llle carrier solution Inust llave a p~l witllin tlle ranye of
j 6.5 to about 7.5, preferably from about 6.5 to about 7Ø
¦ Ideally, tlle ~iluent solution is lleutral. lt llas been ound
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¦¦ critical tllat in makiny ths culture film Lactobacteriaceae
specific that tlle pl~ relnains generally witlli~ tlle ranye of
neutral, i.e from about 6.5 to about 7.5. This is 50
! because the commercially available films, sucll as tlle Pe~rifilm
i ~tr~demar~),sold by 3M Company, contain a tetra~olium dye for
I¦ dying tlle cultured bacteria red, to enllance color counting.
¦¦ llowever, tlle tetrazolium dye becomes toxic to all yrowing
!i oryal~isms, if the p~l becomes more acid, such as for example
¦ at a level of pH 5.5. rllus, the carrier solution of tllis
invention must have a p~l within ttle ranye speciEied, whict
avoids the adverse reaction with tetrazolium dye.
In order to maximi~e the inhibitory affect of the yrowtl
of other oryanisms it is also essential for tl-is invelltion
tllat the inoculated film be incubated under anaerobic
¦ conditiolls. Put anotller way, the diluent solu'ion is
¦¦ oryanism specific to Lactobacteriaceae ~ unaer anaerobic
¦ conditions. This anaerobic environment may be induced in
¦ mally conventional ways, such as use of Gas Pack'~ anaerobic
yenerators, manufactured by BBL Microbioloyy Systems of
Cokeysville, Maryland.
The previous description has been given in connection
witl~ the use of Polymixin anti-biotics as the yram negative
oryanism intlibitor. An additional formulation has been
developed whict~ is suitable for many purposes, and in some
instances performs even better than the previously described
formulation. It is based upon use of 2-phenyletllanol as
tlle gram negative oryanisln inllibitor. Wllen 2-phellyletllanol
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! is used a preferre~ coln~osition is one llaving tlle followiny
~ description. Tlle concentration of 2-pllellylethanol sllould be
¦ from about 1 mg/ml to about 4 mg/ml-. T~le concelltration of
tlle anti-Eungal ayent sllould be from about 10 microyrams/ml
¦ to al~out 25~ microyrams/ml and the concelltration oE tlle
i water soluble nitrite salt sl~ould be from about G~
¦ microyrams~ml to about ~O ~nicrograms/ml. Tlle pll wi'll thus
be witllin ti~e range of from about 6.5 to about 7.5, preferably
j from about 6.5 to about 7.~.
'l'lle use of tlle dry medium coated film with tlle earrier
solutions of this invention is a conventional tecllnique
Il knowll to mierobiologists and bacteriologists. ~asieally,
¦~ tlle use involves tlle following teellnique. Tlle film is
jl plaeed on a flat surface, tlle top transparent film layer
! lifted so that a 1 milliliter sample of the diluent carrying
1, tlle oryanism ean be placed on tlle bottom film. The top film
¦l is tllen closed carefully down over t~le bottom film. A
¦¦ plastic sample spreader is tllen lightly pressed down over
¦I tlle top film to spread tlle carrier solution across the
1¦ bottom medium containillg film. Tllereafter, tlle film is left
undisturbed for approximately a mirlute to allow yelliny.
Tlle film is tllen incubated in a llorizontal position with tlle
elear film side'up at 32C for up to 4~ llours. 'l'lle organisms
l will reduce tlle tetrazolium indicator dye in tlle bottom film
¦ makiny t~le organism colonies on tlle film appear as red dots,
I wllicll can be counted.
Tlle followirly examples are ofEered to Eurtller illustrate
but not limit tlle process, teellnique and product eompositio
of tlle present invelltion.
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'i Examples
In each of the examples demorlstrated llereir-aEter, t~le
I, dry medium culture film was the Petrifilm~ ulate developed
l! by tlie 3M Company (Medical Products Division, St. Paul,
I Minllesota 55144). r~ is Petrifilm~ consisted of a dry self-
¦ contained, ready-to-use bacterial culture medium coated onto
¦ a film base and overlaid witll a polyethylene film. Tlle base
carried Standard Method nutrients and a cold water soluble
1I yelling agent. ~lle overlay film, whicll was also coated with
¦ a yelling agent, also contailled 2,3,5-tripl-enyltetrazolium
cllloride indicator dye to facilitate organism counting. ~ ¦
yrid of 1 cm x 1 cm squares was outlined on tl~e bottom film
!¦ to also aid in counting. Tlle overall dimensions of tlle
¦I Petrifilm~ plate was 20 square cm.
i The basic procedure involved preparation of stock solutions
of tlle diluent, inoculating the Petrifilms~, followed by
I incubatioll at 32C Eor 48 llours under anaerobic conditions,
I and thereafter counting oryanisms (cfu/ml). ~ soil sample
from a greenllouse was used as the test culture and another
~ sample designated S/80, known to be a Lactobacillus culture
¦ was used as a control. The soil sample was knowll to contain
¦ many organisms otl~er tllan Lactobacillus.
lle diluent stock solutions were prepared at tlle following
levels of sodium nitrite, by percent weight/volullle: 1~
weiyllt/volume, 6~ weigllt/volullle, 12~ weight/volullle, and
. I 18~ weight/volume. Cyclolleximide was usecl at percent
weigllt/volume levels of: 0.1~, 0.5~, 1% and 2.5~ weight/volume~
Polymyx n D, in tl~is flrst se:~es oE examples, since tl~ey
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were desiyned to study tlle variations irI conce!Itratiol- of
soc,lium nitrite and cycloIIeximide, was used at a constant
i U.U3~ weiyI~t/volume.
'l'ile stock solutions were filter sterilized alId tlIe ~inal
diluent was prepared by usiny 97 ml of sterilized distilled
¦ water and addiny 1 ml per 100 ml of water of each of the
tllree components, i.e. sodium nitrite, cycloIIeximide and
Polymixin B all witllin the ranges previously specified.
¦ Sterile mixtures were tllen aseptically delivered to test
~ tubes at a rate of 9 ml/tube. The following mixtures were
¦ prepared:
¦ Final ConceIltration
of lnyredierIts
ControlButterfield's* buffer plus
U.l~ 'l'ween 80 (T.M.)
VariatiorI 1100 ~ci/ml nitrite
10 ,ug/ml cycloheY.imide
i 24 units/ml Polymixin B~T.M.)
j Variation 2600 ,uy/ml nitrite
20; I ln yq/llll cycloheximide
24 units/ml Polymixin B (T.M.
Variation 31200 ,uy/ml nitrite
10 ,uy/ml cycloheximide
24 units/ml Polymixin B (T.
l Variation 41800 ~g/ml nitrite
; 10 )~y/ml cycloheximlde
24 uIlits/ml Polymixin B.(T.M.
Variation 5600 ~y/ml nitrite
50 ~y/ml cyclohexiInide
24 units/ml Polymixin B (T.M.
I VariatioIl 6600 jucl/ml nitrite
1 100 ,uy/ml cyclolleximide
24 units/ml Polymixin B (T.M.
Variation 7600 ~y/Illl nitrite
250 )~y/ml cycloheximide
¦ 24 uIlits/ml Polymixin B (T.M
Uutter~ield's buffer is a conventloIl~l ~otassium
phos~IIate buf~ering solution ln ~ood microbiolocJy.
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¦ Stal)~ard Petri~ilm'~ dilution procedures were followed in
¦ platiny tlle S/U0 control, in particular 1 ml of an overnight
culture was serially diluted using tlle test solution and 1
ml volumes were delivere~ to tlle plate. An 11 graln sample
l of greenllouse soil was initially diluted itl 100 111l o
i Uutterfield's buffer. Tllis preparation was tllen serially
¦ diluted using tlle test solution.
¦ 1.0 ml volumes of 10 6, 10 7 and 10 8 dilutions of S/80
¦ were delivered to Petrifilm~ plating medium. 1.0 ml volumes
¦ of 10 4, 10 5 and 10 6 dilutions of soil were delivered to
l Petrifilm~ plating medium. Plated samples were incubated
¦ anaerobically in jars containing Gas Pack~ yenerators (BBL).
They were incubated at 32C for 4U h.
Table I below sllows tlle resulting organisln count, using
tlle diluent solution Ilerein specifie~.
T~BLE I
Sample _olony formlrl~
Units/llll (cfu~ml)
I Soil: Control 5 x 107
l Variation 1 3.3 x 106
Variation 2 2.3 x 107
Variation 3 5 x 10
Variation 4 0 x 10~
Variation 5 7.0 x 106
Variation 6 7.1 x 107
Variation 7 4.0 x 107
S/UU: Control 2.~ x 109
Variation 1 7.5 x 10~3
l Variation 2 1.3 x ]09
¦ Variation 3 1.3 x 10'3
Variation ~ 1.3 x lU3
I Variation 5 9.6 x 10~
I Variation 6 1.2 x 109
¦ Variation 7 1.0 x 109
¦ ~s can be seen, tllere is a significarlt increase in tlle
¦ inilii~itory ef~ect of tlle diluent 011 soil micro-organisllls as
¦ tlle concentratiorl o tlle nitrite was increased froln 600
,uy/ml to 120U J~g/llll. 'l`lle S/U0 was unaffecte~ ~y tlle variatior
~7~)c~
of eitl~er tlle nitrite or cyclolleximide concentration over
tl~e ranges of lUU ~g/ml to 1800 ,ug/ml and 10 ~g/~l to 25U
~y/ml, respectively. lt can also be seen tllat tlle diluent
allowecl tlle culture film to be organism specific for tlle
Lactobacteriaceae organism, and inhibited the growth of other
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orgallisms. Similar experimerlts to tl~ose sllown in variations
1-7 were done varying tlle Polymixin s concentration whicl~
was sllown to l~ave no effect on the S/80 organism over a
~1 ranye of 2~-40 units/ml. For soil oryanisms levels of below
24 units/ml of Polymixin H decreased tl)e in~ibitory e~fect.
Examples Si~owir)q Replacement of Polymixi
Sulfate Witil 2-PIlenyletllas)ol
2-PIIenyletllanol (PEA) purcllased from Siyma Scientific as
a li~uid (1 ml = 1.02 gratns~ was used in tlle diluent. l'l-e
PEA is added to obtain the concentrations specified below,
to ~ yiven volume of distilled water, and autoclaved.
I Filter sterilized 6~ weigllt/volume sodium nitrite and 0.1%
¦ weiyl~t/volume cyclolleximide solutions were used to complete
I tlle below listed mixtures by adding 1 ml per lOU ml of
diluent pre~ared.
¦I TA~LE II
Mixtures
; Pre~ared~ ~y Wt./Vol.
l 8 60U ,uy/ml nitrite
10 ~g/ml cyclolleximide
i 0.05~ PEA
9 600 ~y/ml nitrite
10 JJg/lnl cyclollexilnide
~ U.10~ PEA
l~ 10 600 ycl/ml nitrite
j 10 ~y/ml cyclolleximide
0.20~ PEA
ll 600 ~Jcl/lnl nitrite
10 ~g/ml cyclolleximide
0.4~ PEA
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Inoculation and teclln;que was as in tlle previously
described examples. ~n S/8U solution and soil were the
tested samples. Tlle S/80 was serially diluted from an
i overnigl~t culture through tl~e diluent mixtures, and plated
on }~etrifilm~ at 107 and lO8. Tl)e soil was initially
diluted l:l~ with 99 ml modified Butterfield's buffer and
tllen serially diluted in the given diluent. 1t1e lO 4, lO
5 and lO 6 dilutions of soil were delivered to Petrifilm.
¦ Films were incubated anaerobically using BBL Gas Packs~ for
4~ llours at 32C. The results are shown in Table III.
Table III
l~esults
Inoculatin~
i Solutions # cfu/ml
Soil: Variation 8 6.2 x 1035
Variation 9 5 x 103
Variation 10 0 x 10
Variation ll 0 x 103
S/80: Variation ll 9.4 x 108
~0 1 ~s can be seen, the pllenylethyl alcollol substitution in
tl)e formulation for the Polymixin B sulfate antibiotic
a~ears quite effective in inllibiting ttle soil microorgallisms.
llle following examples demonstrate various modifications
of tlle ~ormulations using pllenyletllyl alcollol (PE~) as the
yram negative inllibitor.
¦ l~BLE IV - Formulations
Pl)enyle tllyl
~1ixtures Sodium Nitrite Cyclolleximide Alcollol
~re~ared Concentratiorl Concentratioll Concel-tration
Control (Butter~leld's
¦ Buffer)
12 800 ,ug/llll lO ~q/ml O.l~
13 800 yg/ml lO yy/ml 0.2
1~ 6~0 U~3/llll lO u~/ml O.l~
6~ ~y/llll lO ~l/ml 0.2
16* 8()0 ~g/ml lO ~y/ml 0.1
17* 600 ~/ml 1~ yy/ml 0.1
i
* Includes Polymixill B at a levcl of 2~ unlts/ml.
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¦ S/80 and soil were tlle test samples. S/80 dilutions
¦ were plated at 10-7 and 10-~. Soil was diluted initially in
99 ml (1:10) oE modified Uutterfield's buffer and plated at
10 3 or tllrougll 10 6. Petrifilms were incubated anaerobically
l usiny B~L Gas Packs'~ at 32~C for 48 hours, as silnilarly
¦ described in earlier examples. llle results are indicated
¦ below.
lAL~LE V -_Results
l Sample Mixture cfu/ml
l Control:Control 9.8 x 108
S/80: 12 8.7 x 108
13 8.5 x 108
1~ 8.7 x 108
1.0 x 109
16 7.~ x 108
17 1.1 x 109
Control: -- 2.7 x 107
Soil: 12 1.8 x 10~
l 13 3.0 x 102
1 14 3.4 x 106
3.3 x 104
16 ~.2 x 10
17 9.0 x 10~
l As can be seen, solution 1~13 gave the best inl~ibitory
¦ results witll soil, witllout adversely affecting tlle growtil of
I S/~30.
¦ It can be seen from eacll oE the above examples as
presellted in tlle various tables tllat effective diluent
compositions can make media films lactic acid bacteria
speciic and tllerefore effectively accolnplisl~ tlleir intended
l purposes.
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