MILIEU GELOSE PERMETTANT DE DISTINGUER STREPTOCOCCUS LACTIS DE STREPTOCOCCUS D'APRES LA PRODUCTION DE L'ESTERASE DE BUTYRATE

DIFFERENTIAL AGARS MEDIUM FOR SEPARATING STREPTOCOCCUS LACTIS AND STREPTOCOCCUS ON THE BASIS OF BUTYRATE ESTERASE ACTIVITY

Abrégé anglais

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.

Revendications

- 9 -
WHAT WE CLAIM IS:
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.
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.
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.

Description

- 1 -
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.


.' ~

- 2
DESCRIPTION OF THE INVENTION
EXAMPLE 1
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
- 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.
EXAMPLE 2
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

- 4 -
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.
EXAMPLE 3
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~
- 6
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.


.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 `,,
- 8
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 ~. ~