CA1322733C - Differential agars medium for separating streptococcus lactis and streptococcus on the basis of butyrate esterase activity - Google Patents
Differential agars medium for separating streptococcus lactis and streptococcus on the basis of butyrate esterase activityInfo
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- CA1322733C CA1322733C CA000600756A CA600756A CA1322733C CA 1322733 C CA1322733 C CA 1322733C CA 000600756 A CA000600756 A CA 000600756A CA 600756 A CA600756 A CA 600756A CA 1322733 C CA1322733 C CA 1322733C
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- agar
- lactis
- butyrate
- faecalis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
ABSTRACT OF THE DISCLOSURE
The characteristic ability of Streptococcus faecalis and inability of Streptococcus lactis to produce the butyrate esterase formed the basis for the development of a differential agar medium to separate these species in pure and mixed cultures. The differential agar medium contained 0.5% bacto-peptone, 0.3% bacto-yeast extract, 1.0% 2-naphthyl butyrate or 1.0% indoxyl butyrate, 1.5% agar and the remaining 96.7% water (pH 6.5). S. faecalis colonies grown on naphthyl butyrate agar (24 h;
30°C) produced an intense violet color on immediately after the addition of detector reagents, while S. lactis did not. A solid medium with indoxyl butyrate as indicator was found suitable for both qualitative and quantitative differentiation by direct observation of indigo blue formation around the colonies of S. faecalis. This is a first rapid new medium developed for separating S. faecalis and S. lactis colonies without inhibition.
The characteristic ability of Streptococcus faecalis and inability of Streptococcus lactis to produce the butyrate esterase formed the basis for the development of a differential agar medium to separate these species in pure and mixed cultures. The differential agar medium contained 0.5% bacto-peptone, 0.3% bacto-yeast extract, 1.0% 2-naphthyl butyrate or 1.0% indoxyl butyrate, 1.5% agar and the remaining 96.7% water (pH 6.5). S. faecalis colonies grown on naphthyl butyrate agar (24 h;
30°C) produced an intense violet color on immediately after the addition of detector reagents, while S. lactis did not. A solid medium with indoxyl butyrate as indicator was found suitable for both qualitative and quantitative differentiation by direct observation of indigo blue formation around the colonies of S. faecalis. This is a first rapid new medium developed for separating S. faecalis and S. lactis colonies without inhibition.
Description
A DIFFERENTIAL AGAR MEDIUM FOR SEPARATING STREPTOCOCCUS LACTIS AND
STREPTOCOCCUS FAECALIS ON THE BASIS OF BUTYRATE ESTERASE ACTIVITY
Cheese and Eermented milk products contain a deEined starter flora, associated flora and an undefined highly variable adventitious flora.
Mesophyllic starters (Streptococcus _actis, Streptococcus cremoris) and thermophilic starters (Streptococcus thermophilus, Lactobacillus bulgarius, Lactobacillus helveticus or Lactobacillus lactis) are normally added to provide a deslred texture and flavors in fermented dairy products. Unlike starter and associated flora, adventious flora are accidentally acquired through the survival of thermoduric microorganisms from pasteurized milk or postpasteurization contamination in the plant.
The separation of the group N-streptococci (S. lactis, S.
dlacetylacti.s, S. cremoris) from Leuconostoc species and S. thermophilus,`
and further differentiation between S. lactis, S. diacetylactis from the group D streptococci (S. faecalis) is, difficult and requires many tests (1, 2, 3, 4). The fecal Streptocoecus, S. faeealis oeeurs in eheese as high as 104 - 107 eells per gram (5) due to its high metabolic activities.
There is no simple and rapid proceclure for separating S. lactis from S. fae.calis, though the separation of these mixtures was recently tried by affinity chromatography on an ion exchange resin (6) without success.
A differential agar medium for separating Streptococcus lactis and Streptococcus cremoris was developed by Reddy et al. (7). However, there is no available differential medium for S. faeeal~.s and S. lactis in the market.
.' ~
STREPTOCOCCUS FAECALIS ON THE BASIS OF BUTYRATE ESTERASE ACTIVITY
Cheese and Eermented milk products contain a deEined starter flora, associated flora and an undefined highly variable adventitious flora.
Mesophyllic starters (Streptococcus _actis, Streptococcus cremoris) and thermophilic starters (Streptococcus thermophilus, Lactobacillus bulgarius, Lactobacillus helveticus or Lactobacillus lactis) are normally added to provide a deslred texture and flavors in fermented dairy products. Unlike starter and associated flora, adventious flora are accidentally acquired through the survival of thermoduric microorganisms from pasteurized milk or postpasteurization contamination in the plant.
The separation of the group N-streptococci (S. lactis, S.
dlacetylacti.s, S. cremoris) from Leuconostoc species and S. thermophilus,`
and further differentiation between S. lactis, S. diacetylactis from the group D streptococci (S. faecalis) is, difficult and requires many tests (1, 2, 3, 4). The fecal Streptocoecus, S. faeealis oeeurs in eheese as high as 104 - 107 eells per gram (5) due to its high metabolic activities.
There is no simple and rapid proceclure for separating S. lactis from S. fae.calis, though the separation of these mixtures was recently tried by affinity chromatography on an ion exchange resin (6) without success.
A differential agar medium for separating Streptococcus lactis and Streptococcus cremoris was developed by Reddy et al. (7). However, there is no available differential medium for S. faeeal~.s and S. lactis in the market.
.' ~
DESCRIPTION OF THE INVENTION
Six strains of Streptococcus lactis and Streptococcus faecalis were used in this study (Table 1).
Lyophilized cultures were reconstituted at 30C for 24 h. All cultures were activated in the same broth through two transfers before each experiment For APIZYM procedure, designed for the detection of 19 constitutive enzymes (API Laboratory Products Ltd., St. Laurent, Quebec), bacterial growth (24 h) from the surface of an MRS agar plate was removed with a loop and suspended in 5 ml of Lactobacilli MRS medium (Difco) broth with a turbidity of a McFarland ~o. 6 standard. Two drops of the resulting suspensions were inoculated with a pasteur pipet into each microcupule of the gallery containing 19 dehydrated chromogenic enzyme substrates. Each suspension was assayed in duplicate. AEter incubation of the strips of 4 h at 37C, reactions were visualized after the addition of the detector reagents supplied with the system.
Arginine dihydrolase and citratase activities were detected by minitek arginine disk (BBL) and citrate disk prepared by the procedure of Gilliland and Speck (8). Growth at 15, 39 or 45C was determined by streaking the culture on MRS agar and incubating it in an anaerobic jar (BBL) for 48 h.
The results of the APIZYM technique, showed that S. faecalis differed from S. lactis in producing a unique bu-tyrate esterase (Table 2).
S. faecalis was also characterized by a stronger activity of caprylate esterase-lipase than S. lactis. However, simple and fast butyrate ~, . . !` "~ .,;
~- `
~l 3 ~. ,.J .~ .3 esterase test for differentiating S. faecalis and S. lactis is very useful and offer a reasonable substitute :Eor the more cucumbersome and time -consuming conventional methods.
Arginine and citrate tests were not able to differentiate these two species but S. faecalis differed from S. lactis by their thermophilic nature (45C) which is in agreement with the results for known species in the literature.
BRIEF DESCRIPTION OF THE PHOTOGRAPHY
In the appended figure: Figure 1 shows the differentiation of S. lactis (4 strains) from S. faecalis (4 strains) on the differential agar medium. S. lactis, butyrate esterase-negative strains are white (no color on actual medium), whereas S. faecalis, butyrate esterase positive strains produce violet color on this medium.
The differential medium developed was designated as SFNB and contains 0.5% bacto-peptone, 0.3% bacto-yeast extract, 1.0% 2-naphthyl butyrate (sigma), 1.5% agar and the rest 96.7% distilled water. The pH of the medium was adjusted to pH 6.5 and the medium was sterilized by autoclaving it at 121C for 15 min. A spread - or pour - plating method was used for growth of S. lactis and S. faecalis. Decimal dilutions of the cultures for the platings were made according to standard methods (9).
Excellent results were obtained when the culture dilutions were spread on the agar with a bent glass rod and incubated in anaerobic jar (BBL) at 30C
for 24 or 48 h. The plates were flooded with drops of detector reagents (A and B) supplied with the APIZYM system. Reagent A contains tris amino methane (25%), HCl (37%, 11 ml), laurylsulfate (10%), while reagent B
contains fast blue BB (0.35%) in 2-methoxyethanol.
Figure 1 shows the differential agar medium containing 2-naphthyl butyrate as a rapid means for separating four strains of S.
faecalis and S. lactis; S. faecalis produced a violet color on this medium because of their ability to produce B-naphthol from naphthyl butyrate.
Mixed cultures of S. faecalis and S. lactis were also tested but the differentiation was not as distinct as when pure cultures were tested because of smearing with color reagents. There was no difference in numbers of counts between 24 h and 48 h incubation.
The differential agar medium was further modified for both qualitative and quantitative differentiation by direct observation of indigo blue (bright) formation around the colonies of Stree~coecus faeealis. The differential agar medium, designated SFBL, eontained 0.5%
baeto-peptone, 0.3'~ baeto-yeast extraet, 1.0~ indoxyl butyrate and 1.5%
agar (pH 6.5). When mixed cultures were made 1.0 ml of eaeh of the respeetive dilutions of the strains was mixed together in a sterile serew-cap test tube, and 0.1 ml of-the resul-tant mixture was spread on the agar surfaee. After ineubation of the agar plate prepared by spread-plating at 30C for 24 - 48 h in anaerobie jar, only S. faeealis produced bright blue eolonies while S. laetis did not.
As indoxyl butyrate is not eommereially available, it was prepared aceording to the proeedure of Barrnett and Seligman (10). A
portion (64 g.) of sodium indoxyl obtained from Allied chemieal (Produit Chimique Général, Montréal) was plaeed in a liter of water and iee in a ..~`~ :
2-liter Erlenmeyer flask into which i:Lluminating gas was constantly passed to prevent extensive oxidation. As soon as it dissolved, 40 ml 50% ~v/v) H2S04 was rapidly added and stirred. This was followed by the addition oE
50 ml butyric anhydride. Illuminating gas was bubbled through the mixture vigorously for lO min. The reaction mixture was allowed to stand for several days (25C) until all the anhydride had decomposed and indigo-stained crystals had formed. After the crystals were washed free oE butyric acid, treated with charcoal, and diluted, the product was recrystallized from dilute alcohol in white needles.
Four strains of Strep-tococcus faecalis and Streptococcus lactis were tested on this medium in both surface - and pour - plating techniques. S. faecalis formed bright blue colonies on the agar plate, while S. lactis produced white colonies and the differential agar medium allowed the differentiation of two species directly on the same plate.
Both techniques of surface - and pour - plating provided better recoveries than naphthyl butyrate agar (Fig. l). These results indicate that the proposed mediwn and techniques could be used both for qualitative and quantitative differentiation of mixtures of S. faecalis and S. lactis.
This differential agar will offer many advantages to the dairy industry and starter culture manufacturers for rapid and economical differentiation or selection of potential starter strains, and for investigations of compatibility and dominance of mixed strain starters (7).
~. , ;. . ~.
`d~ "i~
TABLE 1 Strains and sources of Streptococcus lactis and Streptococcus faecalis.
StrainsIdentification No. Obtained (fro~) 1. Streptococcus lactis ML3 Agriculture Canadaa 2. lactis 14871 ATCCb 3. lactis 19435 ATCC
4. lactis 11454 ATCC
Six strains of Streptococcus lactis and Streptococcus faecalis were used in this study (Table 1).
Lyophilized cultures were reconstituted at 30C for 24 h. All cultures were activated in the same broth through two transfers before each experiment For APIZYM procedure, designed for the detection of 19 constitutive enzymes (API Laboratory Products Ltd., St. Laurent, Quebec), bacterial growth (24 h) from the surface of an MRS agar plate was removed with a loop and suspended in 5 ml of Lactobacilli MRS medium (Difco) broth with a turbidity of a McFarland ~o. 6 standard. Two drops of the resulting suspensions were inoculated with a pasteur pipet into each microcupule of the gallery containing 19 dehydrated chromogenic enzyme substrates. Each suspension was assayed in duplicate. AEter incubation of the strips of 4 h at 37C, reactions were visualized after the addition of the detector reagents supplied with the system.
Arginine dihydrolase and citratase activities were detected by minitek arginine disk (BBL) and citrate disk prepared by the procedure of Gilliland and Speck (8). Growth at 15, 39 or 45C was determined by streaking the culture on MRS agar and incubating it in an anaerobic jar (BBL) for 48 h.
The results of the APIZYM technique, showed that S. faecalis differed from S. lactis in producing a unique bu-tyrate esterase (Table 2).
S. faecalis was also characterized by a stronger activity of caprylate esterase-lipase than S. lactis. However, simple and fast butyrate ~, . . !` "~ .,;
~- `
~l 3 ~. ,.J .~ .3 esterase test for differentiating S. faecalis and S. lactis is very useful and offer a reasonable substitute :Eor the more cucumbersome and time -consuming conventional methods.
Arginine and citrate tests were not able to differentiate these two species but S. faecalis differed from S. lactis by their thermophilic nature (45C) which is in agreement with the results for known species in the literature.
BRIEF DESCRIPTION OF THE PHOTOGRAPHY
In the appended figure: Figure 1 shows the differentiation of S. lactis (4 strains) from S. faecalis (4 strains) on the differential agar medium. S. lactis, butyrate esterase-negative strains are white (no color on actual medium), whereas S. faecalis, butyrate esterase positive strains produce violet color on this medium.
The differential medium developed was designated as SFNB and contains 0.5% bacto-peptone, 0.3% bacto-yeast extract, 1.0% 2-naphthyl butyrate (sigma), 1.5% agar and the rest 96.7% distilled water. The pH of the medium was adjusted to pH 6.5 and the medium was sterilized by autoclaving it at 121C for 15 min. A spread - or pour - plating method was used for growth of S. lactis and S. faecalis. Decimal dilutions of the cultures for the platings were made according to standard methods (9).
Excellent results were obtained when the culture dilutions were spread on the agar with a bent glass rod and incubated in anaerobic jar (BBL) at 30C
for 24 or 48 h. The plates were flooded with drops of detector reagents (A and B) supplied with the APIZYM system. Reagent A contains tris amino methane (25%), HCl (37%, 11 ml), laurylsulfate (10%), while reagent B
contains fast blue BB (0.35%) in 2-methoxyethanol.
Figure 1 shows the differential agar medium containing 2-naphthyl butyrate as a rapid means for separating four strains of S.
faecalis and S. lactis; S. faecalis produced a violet color on this medium because of their ability to produce B-naphthol from naphthyl butyrate.
Mixed cultures of S. faecalis and S. lactis were also tested but the differentiation was not as distinct as when pure cultures were tested because of smearing with color reagents. There was no difference in numbers of counts between 24 h and 48 h incubation.
The differential agar medium was further modified for both qualitative and quantitative differentiation by direct observation of indigo blue (bright) formation around the colonies of Stree~coecus faeealis. The differential agar medium, designated SFBL, eontained 0.5%
baeto-peptone, 0.3'~ baeto-yeast extraet, 1.0~ indoxyl butyrate and 1.5%
agar (pH 6.5). When mixed cultures were made 1.0 ml of eaeh of the respeetive dilutions of the strains was mixed together in a sterile serew-cap test tube, and 0.1 ml of-the resul-tant mixture was spread on the agar surfaee. After ineubation of the agar plate prepared by spread-plating at 30C for 24 - 48 h in anaerobie jar, only S. faeealis produced bright blue eolonies while S. laetis did not.
As indoxyl butyrate is not eommereially available, it was prepared aceording to the proeedure of Barrnett and Seligman (10). A
portion (64 g.) of sodium indoxyl obtained from Allied chemieal (Produit Chimique Général, Montréal) was plaeed in a liter of water and iee in a ..~`~ :
2-liter Erlenmeyer flask into which i:Lluminating gas was constantly passed to prevent extensive oxidation. As soon as it dissolved, 40 ml 50% ~v/v) H2S04 was rapidly added and stirred. This was followed by the addition oE
50 ml butyric anhydride. Illuminating gas was bubbled through the mixture vigorously for lO min. The reaction mixture was allowed to stand for several days (25C) until all the anhydride had decomposed and indigo-stained crystals had formed. After the crystals were washed free oE butyric acid, treated with charcoal, and diluted, the product was recrystallized from dilute alcohol in white needles.
Four strains of Strep-tococcus faecalis and Streptococcus lactis were tested on this medium in both surface - and pour - plating techniques. S. faecalis formed bright blue colonies on the agar plate, while S. lactis produced white colonies and the differential agar medium allowed the differentiation of two species directly on the same plate.
Both techniques of surface - and pour - plating provided better recoveries than naphthyl butyrate agar (Fig. l). These results indicate that the proposed mediwn and techniques could be used both for qualitative and quantitative differentiation of mixtures of S. faecalis and S. lactis.
This differential agar will offer many advantages to the dairy industry and starter culture manufacturers for rapid and economical differentiation or selection of potential starter strains, and for investigations of compatibility and dominance of mixed strain starters (7).
~. , ;. . ~.
`d~ "i~
TABLE 1 Strains and sources of Streptococcus lactis and Streptococcus faecalis.
StrainsIdentification No. Obtained (fro~) 1. Streptococcus lactis ML3 Agriculture Canadaa 2. lactis 14871 ATCCb 3. lactis 19435 ATCC
4. lactis 11454 ATCC
5. lactis 14872 ATCC
6. lactis 14873 ATCC
7. Streptococcus faecalis 7080 ATCC
8 faecalis A69 ATCC
9 faecalls19433 ATCC
10. faecalis8043 ATCC
11. faecalis 882 ATCC
12. faecalis6057 ATCC
.
` aAgriculture Canada Food Research Centre, Ottawa, Ontario.
bThe Amerlcan type culture collection, Rockville, Md.
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4~o o ~ o 0~ U~ U~ U~ U! D~ ~ ql ~
~ ~ u~ u~ ,~ u~ u~ ~n ~ 1~~ ~d ~1 l ~1 o bO~3 C~l ~ ~ J- ~ ~ ~ ~ ~ ~ t~ ~ ~ ~ .,~
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. .,~, .
~ 3 ! J; i `,, REFERENCES CITED
1. Diebel, R.H. and H.W. Seeley. Family, Streptococcaceae. In: Bergey's Manual o~ Determinative Bacteriology, (Buchanan, R.E. and N.E.
Gibbons, eds.), 8th ed., Williams and Wilkins Co., Baltimore, 1978.
2. Huggins, A.R Food Technol. 1984. 38(6): 41-50.
3. Law, B.A. and M.E. Sharpe. Streptococci in the dairy industry Streptococci, (Skinner, F.A. and L.B. Quesnel, eds.), pp. 263-278, Academic Press, London, 1978.
4. Lee, G.H., S. Haché and R.E. Simard. J. Ind. Microbiol. 1986.
1: 209-217.
5. Clark, W.S. and G.W. Reinbold. J. Dairy Sci. 1966. 49: 1214-1218.
6. Paquin, C. and J. Goulet. Can. Inst. Food Sci. Technol. J. 1984.
17(3): 33(abstract).
7. Reddy, M.S., E.R. Vedamuthu, C.J. Washam and G.W. Reinbold. Appl.
Microbiol. 1969. 18: 755-759.
8. Gilliland, S.E. and M.L. Speck. Appl. Environ. Microbiol. 1977. 33:
1289-1292.
9. APHA. Standard Methods for the E~amination o~ Dairy Products, 14th ed.
(Ld.) E.H. Marth, Amer. Pub. Health Assoc., Washington, DC. 1978.
10. Barrnett, R.J. and A.M. Seligman. Science. 1951. 11~: 579-582.
. j ~. ~
.
` aAgriculture Canada Food Research Centre, Ottawa, Ontario.
bThe Amerlcan type culture collection, Rockville, Md.
.s ,? i, ~ ) ~:
6 t~ ,~ ++++++
~ ,1 O ~0~ ql~ + ~ ++ ~ ~ ++++++
~ 4 o ++++++++++++ o X
ql~ ql u~ ~ , ., ~ + I ~ + I I ~
. I ~ O~ ql U~ a) R+, + + o + + ~ ~
q ¢ ~ a) ,~ u a ~ a ~ ~ O u~ ~ ~ ,1 a I ~ ~
. ~ o u~ a) ~ 1 S~ o I ~ ~ a) ~4 .C o u~ o ~ ~ .~ ~ O
'~ ~ ~ ~ a) O
1~ ~1 ~ ql E!~ O a) o P' O _ . q r C~
P~ O ~,~ qlV ql ~ ~ c~ .
Q ~ o u~ q ~ q u~ a) ~ 1 ~ 1 ~ ) 3 ~ ~ ¢ ~ ~ ql ,~ ~ ~,~
4~o o ~ o 0~ U~ U~ U~ U! D~ ~ ql ~
~ ~ u~ u~ ,~ u~ u~ ~n ~ 1~~ ~d ~1 l ~1 o bO~3 C~l ~ ~ J- ~ ~ ~ ~ ~ ~ t~ ~ ~ ~ .,~
r~ ~ ql ql ql q~ Cq) ~q, , I q, $ ~ ql O ~ ~ ~1 ~1 ~1 ~1 4~ 4~ 4~ 4~ ~ 4~ ~ + I ~ ~0 ¢ U~ U~ ~ ~ C17 If~ ~ R o ~ ~1 ` . ~
. .,~, .
~ 3 ! J; i `,, REFERENCES CITED
1. Diebel, R.H. and H.W. Seeley. Family, Streptococcaceae. In: Bergey's Manual o~ Determinative Bacteriology, (Buchanan, R.E. and N.E.
Gibbons, eds.), 8th ed., Williams and Wilkins Co., Baltimore, 1978.
2. Huggins, A.R Food Technol. 1984. 38(6): 41-50.
3. Law, B.A. and M.E. Sharpe. Streptococci in the dairy industry Streptococci, (Skinner, F.A. and L.B. Quesnel, eds.), pp. 263-278, Academic Press, London, 1978.
4. Lee, G.H., S. Haché and R.E. Simard. J. Ind. Microbiol. 1986.
1: 209-217.
5. Clark, W.S. and G.W. Reinbold. J. Dairy Sci. 1966. 49: 1214-1218.
6. Paquin, C. and J. Goulet. Can. Inst. Food Sci. Technol. J. 1984.
17(3): 33(abstract).
7. Reddy, M.S., E.R. Vedamuthu, C.J. Washam and G.W. Reinbold. Appl.
Microbiol. 1969. 18: 755-759.
8. Gilliland, S.E. and M.L. Speck. Appl. Environ. Microbiol. 1977. 33:
1289-1292.
9. APHA. Standard Methods for the E~amination o~ Dairy Products, 14th ed.
(Ld.) E.H. Marth, Amer. Pub. Health Assoc., Washington, DC. 1978.
10. Barrnett, R.J. and A.M. Seligman. Science. 1951. 11~: 579-582.
. j ~. ~
Claims (6)
1. A differential agar medium, designated SFNB for enumeration of colored S. faecalis and non-colored S. lactis which is composed of 0.5% bacto-peptone, 0.3% bacto-yeast extract, 1.0% 2-naphthyl butyrate, 1.5% agar and 96.7% distilled water with the final pH of 6.5 ? 0.1.
2. A differential agar medium, designated SFBL for enumeration of colored S. faecalis and non-colored S. lactis which is composed of 0.5% bacto-peptone, 0.3% bacto-yeast extract, 1.0% indoxyl butyrate, 1.5% agar and 96.7% distilled water with the final pH of 6.5 ? 0.1.
3. A process for making a differential agar medium, designated SFNB as defined in claim 1 in which bacto-peptone, bacto-yeast extract, 2-naphthyl butyrate and agar are combined, boiled to dissolve agar, sterilized by autoclaving at 121°C (15 lbs) for 15 min, cooled to 45°C
to 50°C before making agar plates where the final pH after combining ingredients is 6.5 ? 0.1.
to 50°C before making agar plates where the final pH after combining ingredients is 6.5 ? 0.1.
4. A process for making a differential agar medium, designated SFBL as defined in claim 2 in which bacto-peptone, bacto-yeast extract, indoxyl butyrate and agar are combined, boiled to dissolve agar, sterilized by autoclaving at 121°C (15 lbs) for 15 min, cooled to 45°C
to 50°C before making agar plates where the final pH after combining ingredients is 6.5 ? 0.1.
to 50°C before making agar plates where the final pH after combining ingredients is 6.5 ? 0.1.
5. A process for making a differential broth medium which comprises carrying out the process of claim 3 wherein agar is omitted.
6. A process for making a differential broth medium which comprises carrying out the process of claim 4 where agar is omitted.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000600756A CA1322733C (en) | 1989-05-25 | 1989-05-25 | Differential agars medium for separating streptococcus lactis and streptococcus on the basis of butyrate esterase activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000600756A CA1322733C (en) | 1989-05-25 | 1989-05-25 | Differential agars medium for separating streptococcus lactis and streptococcus on the basis of butyrate esterase activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1322733C true CA1322733C (en) | 1993-10-05 |
Family
ID=4140104
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000600756A Expired - Fee Related CA1322733C (en) | 1989-05-25 | 1989-05-25 | Differential agars medium for separating streptococcus lactis and streptococcus on the basis of butyrate esterase activity |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1322733C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2875241A1 (en) * | 2004-09-16 | 2006-03-17 | Biomerieux Sa | METHOD OF DETECTING STREPTOCOCCUS AGALACTIAE USING ESTERASE ACTIVITY |
| US7626018B2 (en) | 2004-09-10 | 2009-12-01 | bioMérieux | Enzymatic substrates derived from phenoxazinone and their use as developer in detection of microorganisms with peptidase activity |
-
1989
- 1989-05-25 CA CA000600756A patent/CA1322733C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7626018B2 (en) | 2004-09-10 | 2009-12-01 | bioMérieux | Enzymatic substrates derived from phenoxazinone and their use as developer in detection of microorganisms with peptidase activity |
| US7932050B2 (en) | 2004-09-10 | 2011-04-26 | Biomerieux | Enzymatic substrates derived from phenoxazinone and their use as developers in detection of microorganisms with peptidase activity |
| FR2875241A1 (en) * | 2004-09-16 | 2006-03-17 | Biomerieux Sa | METHOD OF DETECTING STREPTOCOCCUS AGALACTIAE USING ESTERASE ACTIVITY |
| WO2006032809A2 (en) * | 2004-09-16 | 2006-03-30 | Biomerieux | Method for detecting streptococcus agalactiae using esterase activity |
| WO2006032809A3 (en) * | 2004-09-16 | 2006-05-11 | Biomerieux Sa | Method for detecting streptococcus agalactiae using esterase activity |
| EP2487235A1 (en) * | 2004-09-16 | 2012-08-15 | bioMérieux | Medium for detecting streptococcus agalactiae using esterase activity |
| US9309552B2 (en) | 2004-09-16 | 2016-04-12 | Biomerieux | Method for detecting Streptococcus agalactiae using esterase activity |
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