Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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.
IMPROVED METHOD FOR PRODUCING SINGLE AND/OR
MIXED STRAIN CONCENTRATES OF BACTERIA
BACKGROUND OF THE INVENTION
1. Field of the Invention
.. . . _ _
The present invention relates to an impro~ed me-thod
device for differentiating or separating heterogeneous
populations of fast and slow acid producing strains of bacteria
to produce single strains or clones. In particular, the
present invention relates to a method wherein special growth
media and conditions are utilized to achieve the
differentiatiQn an~ wherein the differentiated and selected
strains are preferably provided as cultures to producers of
~ermented products.
2. Prior Art
.
The principal prior art is described in:
McKay et al., Applied Microbiology, Vol. 23, pages
1090-10~6 (1972);
Limsowtin, G. K. and _e~ B. E., New Zealand
Journal of Dairy Science & Technology, Volume 11, pages 65 and
66 (1976);
Limsowtin, G~ K., et al., New Zealand Journal of Dairy
.
Science Technology 13, pages 1 to 8 (1978);
R J Marshall et al., Dairy Research, Vol. 43, pages
-
449 to 458 (1976); and
Rull, R. R., rrhe Australian Journal of Dairy
Technology, pages 65 and 77 (June 1977)~
McKa~ et al. describe the problem of the 105s oE
lactose fermPnting ability in lactic acid producing cultures in
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a broth medium. A non-milk agar containing bromocresol purple
as an indicator is described for separating colonies which
product acid (yellow) from non-acid producing strains (white~
under aerobic conditionsO There is no attempt at selection of
phage insensitive mutants or recognition of ~he problem. Thus,
McKay was studying loss of lactose fermentation and used the
non-rnilk agar medium containing an acid-base indicator to
detect non-lactose fermenting (lac ) cells. 1~he selection of
lac cells on the McKay medium would not be a worthwhile
approach to isolating cells which would yield fast acid
producing cultures in milk. Another important determinant for
fast acid production in milk is proteolysis (prt)O The McKay
medium only distinguishes between lac and lac and lac
prt and lac prt cells appear the same on his rnedium,
yet the former would be slow in milk while the latter fast.
The problem not solved by McKay et al. is -to distinguish
between lac~ prt and lac+ prt~ cells, particularly
since the large majority of slow acid-producing ~ariant cells
in milk cultures are lac+ prt .
Limsowtin et al. (1976) describe a glycerophosphate
buffered, nonfat milk-basedl agar medium (GMA) for the
differentiation of fast and slow milk coagulating lactic
streptococci. Aerobic (air) growth conditions were used for
the growth of ~he bacteria. The medium has been found to be
impractical to use since it produced uncertain diEferentiation
of fast and slow acid producing cultures of certain
Streptococcus crernoris or Streptococcus lactis and i~ was
dificult to see white or translucent streptococcal colonies on
the white bac;~ground of this medium. I~hus, two strains known
to be fast acid producing strains produced colonies which had
RJP/paa 1/18/83 1255E 24767
only a 0~5 mm colony diameter thereby erroneously indicating
that they were all slow acid producersO Oblique illumination
was used to obtain the published photographs, however visual
selection is difficulto Marshall et al. describe other
~ .
phosphate buffered media for the selection process under
aerobic conditions where selection is difficult. In selecting
star~er strains for commercial fermenkations, par~icularly for
making fermented dairy products, the method for differentiating
and selecting the strains must be completely reliable because
of the large volumes of milk or other food being fermented.
Hull describes a method wherein the method of
Limsowtin et al. can be used by producers of cultures to
provide phage resistant, fast acid producing bacterial cultures
to producers of fermented dairy products. By this process a
portion of the fermented product or a by-product (whey)
provides a source for phage which are mixed on the plating
medium with the bacteria for differentiation and selection of
phage resistant strains. New phaye resistant strains are
provided to the producers of fermented dairy products and older
strains are dropped as phage appear before they have a chance
to propagate and to vitally infect the older strains~ The
problem is that the Limsowtin et al. method is not reliable or
certain enough to make the method suggested by Hull
commercially feasible for the culture producers.
References which discuss the use of buffered plating
media include:
Barach, J. T~, I'Improved Enumeration of Lactic Acid
Streptococci on Elliker Agar Containing Phosphate," _p~
~nviron. Microbiol. 38:173-174 (1979);
RJP/paa 1/18/83 1255E 24767
Douglas, JO~ "A Critical Review of the Use of
Glycerophosphates in Microbiological Media," Lab. Pract.
20:414-416, 42~ (1971);
Huggins, A~ R~, and W. E. Sandine, I'Selection and
Characterization of Phage-Insensitive Lactic Streptococci," J.
Dairy Sci. 62:70 (1979);
Hunter, Go J. E., "A Simple Agar Medium for the Growth
of Lactic Streptococci: The Role of Phosphate in the Medium,"
J~ Dairy Res. 14:283-290 (1946);
Keogh, B. P., "Appraisal of Media and Methods for
Assay of Bacteriophages of Lactic Streptococci, Ap~l. Environ~
Microbiol. 40:798-802 (1980),
Lee, S. Y. et al., "An Agar Medium for Differential
Enumeration of Yogurt Starter Bacteria," J. Milk Food Techno1.
37:272-276 (1974); the Limsowtin (1976) article;
Mullan, W. M. A. "Lactic Streptococcal Bacteriophage
Enumeration; A Review of Factors A~fecting Plaque Formation,"
Dairy Ind. Int. 44(7):11-15 (1979);
Reddv, M. S., et al., "Agar Medium for Differential
Enumeration of Lac~ic Streptococci," Appl. Microbiol.
2~:947-952 (1973);
ReiterJ B., "Lysogenic Strains of Lactîc
Streptococci," Nat~re 1640667-668 (1949~;
Shankar 7 Po A. ~ et al., 'IA Note on the Suppression of
Lactobacillus bulgaricus in Media Containing B-glycerophosphate
and Application of Such Media to SelectiYe Isolation of
Streptococcus thermophilus from Yoghurt," _ Soc. Dair~
Technol. 30:28-30 (1977);
She~, D. I., "Effect of Calcium on the Developmenk of
Streptococcal Bacteriophages," Nature 164:492-493 ~1949);
____
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Stainer, R. Y. "The Microbiol World, p. 37-40, 68
(1976); and
~ erzaghi, B. E., and W. E. Sandine, "Improved Medium
for Lactic Streptococci and their Bacteriophages," Ap~l.
Microbiol. 29:807-813 (1975).
Hunter demonstrated that with addition of sodium
phosphate (3307mM) to a simple plating medium, more growth and
larger colonies resulted. Buffering provided by the phosphate
allowed more cell divisions before the hydrogen ion
concentration became inhibitory to further growth.
Improved growth of lactic streptococci results when
disodium-B-glycerophosphate (62mM) is incorporated into a
complex medium by Terzaghi and Sandine. This medium is also
lactose-limiting which enables the final pH to remain well
above pH 500 and avoid any acid injury to the cells~ Douglas
claimed one of the advantages of glycerophosphates over
inorganic phosphates was that microbiological media could be
formulated with high phosphate concentrations without
precipitating out many essential metals. This feature i5
especi~lly useful in pla~uing lactic bacteriophages where
Ca~2 is required for optimum formation of plaques (articles
by Reiter; Shew).
Sodium-B-glycerophosphate is also utilized in two
different plating media designed to diEferentiate "fast" and
"slow" colonies of lactic streptococci (articles by ~uggins and
Sandine; Limsowtin). These buffered media enable fast
acid producing colonies to grow to a larger si~e so that they
can be distinguished from smaller slow coloniesO
Shankar and Davies found B-glycerophosphate inhibited
some strains of L~ bul~aricus and more recently there have been
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reports of inhibition of a few strains of lactic streptococci
(articles by Keogh; Mullan).
Barach improved the enumeration of lactic streptococci
by as much as 7. 75 times by incorporating 3UmM diammonium
phosphate into unbuffered Elliker agar.
Insoluble CaC03 has been added to agar media to help
preserve neutral conditions for isolation and cultivation of
acid-producing bacteria. Production of acid is localized
around colonies and can be detected by clear halos resulting
from acid-solubilization of CaC03 immediately surrounding the
colonies as described by Stanier. Such a buffering system is
used in a plating medium for differential enumeration of lactic
strep~ococci by Reddy, et al.~ and in another for
differentiating colonies of S _ hermophilus from I. bulgaricus
by Leer et al.
OBJECTS
_, ~ . .
It is therefore an object of the present in~Jention to
provide a method wherein heterogeneous populations of fast and
slow acid producing strains of bacteria are readily and
reproducibly differentiated or separated as to acid producing
ability~ Further it i5 an object of the present invention to
provide a method wherein phage insensitive strains can be
reliably selected. Further still it is an object of the
present invention to provide the selected strains as c~ltures
or concentrates of phage insensiti~Je strains of bacteria as a
single strain or as a mixture of several such single strains.
These and other objects will become increasingly apparent by
reference to the following description and to the drawingO
-- 6 ~
In _e Drawing
The drawing is a photograph showing clearly differen-
tiated fast (f) and slow (s) acid producing single strain
bacterial colonies produced by the method and plating device of
the present invention using an indicator and anaerobic fermen-
tation conditions.
General Descri.ption
The present invention relates to an improved method
for the differentiation of heterogeneous populations of fast
acid producing strains of a species of bacteria from slow acid
producing strains of the same bacteria which comprises
providing a gelled, solid bacterial growth medium containing
milk protein, a milk protein derivative or a milk protein
substitute and containing a p~l sensitive indicator which
changes color upon contact with acid in the pH range between
about 4 and 7 and growing the heterogeneous populations of a
strain or strains of acid producing bacteria on ~he medium to
produce single strain acid producing bacterial colonies,
wherein the colonies are of varying si~es and have a
contrasting color from the growth medium around and within the
colonies because of the reaction of the acid in the colonies
with the indicator
A bacterial plating device is used for differen-
tiating and selecting fast acid producing strains of a species
of bacteria in a heterogeneous population with slow acid
produ~ing strains or variants of the same bac-teria. A closed
container is used containing a gelled, solid growth medium for
the bacteria in admixture with milk protein, a milk protein
derivative or a milk protein substitute and a pH sensitive
indicator which changes color upon contact with acid in the pH
range between about 4 and 7. Preferably anaerobic or near
anaerobic conditions are provided in the space.
The present invention also relates to an
improved method for the differentiation of heterogeneous popu-
lations of fast acid producing strains of a species of bacteria
from slow acid producing strains by visual observation of color
differences amony colonies grown on a gelled, solid bac-terial
growth medium by providing a medium that includes milk protein,
a milk protein derivative or a milk protein substitute, a pH
sensitive indicator which changes color upon contact with acid
in the p~ range between about 4 and 7 even in the absence of
milk proteolysis, and a substantial non~oxic, insoluble
buffering agent.
The method of the present inventi.on also relates
to the production of a concentrate o:E a single strain or a
mixture of single strains of a species of bacteria of the gen-
era Streptococcus or Lactobacillus of the type used :Eor produc-
... . . . ~
ing lactic acid in foods by -fermentation. The selected bacter
ium is characterized by being a fast acid producer and by hav-
ing been grown anaerobically on a gelled solid growth medium,
wherein the medium includes a pH sensitive indicator which
changes color upon contact with acid in the pH range between
about 4 and 7, a bufferiny agent and a milk protein, a milk
protein derivative or a rnilk protein substitute, and by being
able to grow in the presence of a phage which kills or impairs
the growth of parent strains of the same species to produce the
single strains which are then selected and regrown in a second
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fluid growth medium to produce the concentrates which contains
at least about 1 x 106 cells per ml.
The present invention also relates to the method for
selecting phage resistant strains of acid producing bacteria
and concentrating the cells for use in producing fermented food
products which comprises: growing heterogeneous or homogeneous
populations of a strain or strains of the acid producing
bacteria anaerobically or nearly anaerobically in the presence
of phage on a solid growth medium so as to produce colonies
wherein fast acid producing strains produce relatively larger
colonies than slow acid producing strains; selecting a large
colony; and growing the cells in a fluid growth medium to at
least about 106 cells per ml to provide a concentrate of
cells.
The present invention particularly relates to the
method for providing phage resistant strains of acid producing
bacteria to producers of fermented food produc~s which
comprises growing heterogeneous or homogeneous populations of a
strain or strains of an acid producing bacteria anaerobically
or nearly anaerobically in the presence of phage from a sample
of the food product or a by-product of the food product
obtained from a producer of the fermented food product on a
growth medium which differentiates slow acid producing strains
from fast acid producing strains of the bacteria by producing
relatively larger colonies of cells of fast acid producing
strains in the presence of phage; selecting cells in a large
colony having a diameter of at least about 1 mm and growing the
cells in the larger colony to at least about 106 cells per ml
to provide a culture of ce:Lls; and providing the producer which
supplied the sample with the culture for use in prsducing
fermented food productsO
The method for differentiation and selection produces
a eoneentrate of improved homologous cells of a single strain
which is from a clone of a single member of the species and
which has the most desirable acid producing properties as well
as preferably phage insensitivity. The term "strain" or
"variant" as used herein means a member of a single species of
bacteria which has a common source or parent with other members
anc~ whieh generally has almost all of the same fermen-tation
characteristics with other members, but which can have a weak-
ness in the ability to produce acid or in the case of mi]k
fermentations have a poor proteolytic ability or have phage
sensitivity. A single species of bacteria may have many
strains of the same bacterium which differ by one or more fer-
mentation characteristics and thus form a "heterogeneous popu-
lation'l. The general fermentation eharacteristics are deter-
mined in relation to sugars and other assimilable carbon
sources for the species as listed in Bergey's Manual, Eighth
Edition (1974). There are commercially available devices for
determining -fermentation characteristies on sugars and other
substrates using microassay techniques. The API series Erom
Analytab Products, Inc., in Carle Place, N.Y., is particularly
suitable~
The ~erms "slow acid producing" and "fast acid pro-
ducing" are used in the milk fermentation industry in relation
to lactie acid produeing bacteria. Slow lactic acid producing
strains are those whieh fail to eoagulate milk in 18 hours at
21C using a 1~ by volume inoculum (Citti, J.E., et al~
"Cornparison of slow and fast acid producing streptococcus lac-
tis," J. Dairy Sci~ 48.14-18 (1965~). Fast lactic acid produc-
ing strains develop 'che relatively larger colonies.
The baeteria are grown so that there are between about
30 and 300 colonies per petri plate or between about 0~5 and 5
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per square centimeter of gelled growth medium. PreEerably
there are less than 100 colonies per plate (about 1.6 per
square cm of medium). The reason for these preferred colony
densities is that there can be too few or too many colonies on
the plate outside of the broad ranges for reliable results.
The fast acid producing colonies are at least about 1 mm in
diameter.
Many acid indicators can be used in the present
invention. One such indicator is litmus which changes from
blue to red upon contact with acid. Litmus makes proteolysis
of the milk protein in the medium by the bacteria readily
visible in a ring (p) around the fringes of the fast acid
producing colonies (f) as shown in Figure 1. Milk proteolysis
is necessary in most fermentations. Other indicators can be
used, such as bromcresol purple (pH range 5.2 to 6.8) and
bromthymol blue (pH range 6~0 to 7.6) which do not rely on milk
proteolysis.
Anaerobic growth conditions provide unexpectedly
superior results in terms of the diferentiation of fast and
slow acid producing colonies. The anaerobic conditions can be
provided in the confined space by a vacuum or by providing a
reducing or rare gas in the confined space. The preferred
gases are nitrogen, hydrogen or other nontoxic gases alone or
mixed with carbon dioxide which is assimilated by the lactic
acid producing bacteria. Preferably between about 5~ and 50
by volume carbon dioxide is used with the balance being
hydrogen or nitrogen. Other useful non-oxidizing ~ases include
the rare gases, such as neon and krypton and particularly argon.
An important improvement of the present invention is
the use of the acid indicator combined with anaerobic growth
RJP/paa 1/18/83 1255E 24767
conditions which provides a synergistic resul~ ih the
differentia~ion and selection and in the homogeneity of ~he
single strain of cells produced. Unexpectedly it has been
found that significantly i.mproved differentiation and selection
of fast acid producing strains can be achieved with this
combinakion.
The growth medium includes milk protein, a milk
protein derivative, preferably nonfa~ milk or a casein digest,
or a milk protein substitute in an amount between about 5 and
15 percent by weight of the gelled solid medium (W/V).
Another preferred ingredient is a buffering agent,
particularly a compound that is substantially nontoxic to the
bacteria in the medium and that is an alkali metal carbonate,
phosphate, hydroxidel oxide/ or an organic sulfonate D The
organic sulfonates include alkali metal salts of piperazine-N~
N'-bis~2-ethane-sulfonic acid (PIPES), morpholinopropane
sulfonic acid (~IOPS) and 2(n-morpholino)ethane sulEonic acid
(MES) as described in the abstract of the American Dairy
Society Association Meetings June 24 to 27 (1979). Disodium
glycerophosphate is a suitable buffering agent when present in
an amount between about 0.5 and 5 percent by weight based upon
the volume of the gelled solid growth medium (W/V). But,
substantially nontoxic! insoluble buffering agen~s enable even
better visual differentiation, by color, of "fast" and "slow"
colonies, when the buffering agent is present :in an amoun~ of
between 0.1 and 1 percent, preferably about 0~25 percent.
Specific insoluble buffering agents are phosphatesJ including
ammonium phosphates, carbonates and hydroxides of magnesium and
carbonates and hydroxides of calcium and zinc oxide.
Outstanding results are obtained using phosphates oE magnesium,
particularly trimagnesium phosphate.
- 12 -
The agars are preferably Davist.m. agar which is
produced by the Davis Gelatine Company, Christ Church, New
Zealand or Bacto-Agart m from Difco, Detroit, Michigan.
Agar substitutes and other gelled solids such as gelatin are
also available. Preferably the plating device has a trans-
parent window on the confined space so that the growth of the
bacteria can be observed. A conventional petri dish containing
the gelled solid growth medium sealed with a confined space
around the medium is generally used.
After grow~h of the bacteria 7 the relatively large
colony is picked from the gelled solid medium and transferred
to a second fluid growth medium including assimilable carbohy-
drate and nitrogen sources and is grown to at least abou~
1 x 106 cells per ml and preferably to 108 to 109 cells
per ml. In this manner, large numbers of bacterial cells can
be produced as a concentrate wherein virtually every individual
cell has the same fast acid producing capability. This can be
demonstrated by replating the cells on the gelled solid growth
medium.
The cells can be held as a concentrated, refrigerated
milk culture where milk is the fluid growth medium containing
about 1 x 106 to 1 x 109 cells per ml or can be concen-
trated further to above about 1 x 109 to 1 x 1012 cells
per ml by removing some of the growth medium~ The single
strains can be mixed with other single strains produced by -the
rnethod. The thus concentrated bacteria can be frozen for
storage and/or shipment preferably with a freezing stabilizing
agent such as glycerol in an amount up to 20 percent by volume
or they can be lyophilized. The bacteria can also be
stabilized as described in U.S~ Paten-t No. 4~282/255.
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RJP/paa 1/18/~3 1255E 24767
The bacteria which can be differentiated are
preferably species selected from the genera Streptococcus and
Lactobacillus and are used for lactic acid production in food
products by fermentation. Included are ~lS,
Stre~tococcus lactis, ~ cus lactis subspecies
-
diacetylactis, Streptococcus_~ ermophilus~ Lactobacillus
bul~aricus, Lactobacillus acidophilus~ Lactobacillus caseii,
Lactobacillus lactis, and Lactobacillus helveticusO These are
. _ _
bacteria which are sensitive to phages.
The homologous or heterogeneous phages for the
particular strain of bacteria are preferably present when they
are grown by the plating method and thus the bacteria produced
by the method are thus also phage insensitive. The method can
also be repeated using different races of phages when the host
bacterium is sensitive to more than one phage. In these ways,
cells resistant to more than one phage can be generated.
Phayes occur in whey from cheese making. Whey can be
used to continuously produce phage insensitive strains by
repeated periodic exposure to these phages. Other sources of
phage from the producers oE fermented products can also be
used. This method prevents failures resulting in the loss of
hundreds or thousands of gallons of milk in making cultured
dairy products where the bacteria are phage sensitive.
SPECIFIC DEscRIprrIoN
In the following Examples 1-3, the bufering agent was
disodium glycerophosphate; the indicator was litmus; and
anaerobic growth conditions were used~
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Example 1
The composition of the growth med,ium, referred to as
fast-slow differential agar (FSDA), was as shown in Table 1.
Table 1
Percent
Grams/liter ~Y_~ gh~
Nonfat milk powder (NFM) 100.0 g 76.9%
Davist m agar (New Zealand) 10.~ g 7.7%
Disodium glyce.rophosphate 19.0 g 14.6%
Bactolitmust m (litmus) 1.0 g 0.8%
100 . 0%
The medium was prepared by dispersing lOg of Davis
agar in 550 ml of double distilled wa~er in a 2 liter flask by
steaming for 30 minutes. To the melted agar, 1.0 g of
Bactolitmust m and 19 g of disodium glycerophosphate were
added and mixed until dissolved. In a separate 1 liter flask,
100 g of the NF~ powder was dissolved in 450 ml of double
distilled water. The two mixtures were then autoclaved
separately for 17 minutes and rapidly cooled to 55C in a water
bath, combined, and poured into petri plates and flamed to
eliminate bubbles as needed. The p~ates were dried by
inverting overnight at room temperature~
Single colony strains were obtained by spreading or
streaking a mixture of a heterogeneous population of a specific
species o~' bact~ria on the agar and growing the mixtureO An
atmosphere of hydrogen or hydrogen and carbon dioxide was
generated using Gas Pakt m gas generating envelopes
(Bio-Quest, Cockeysville, MD) in the confined space over the
growth medium in petri plates which were seal.ed in an air
RJP/paa 1/18/83 1255E 24767
evacuated Gas Pakt m jar. Typical colonies produced within
24 to 36 hours at 30C were as shown in Figure 1, wherein the
large fast acid producing colonies (f) were readily
distinguishable from the smaller slow acid producing colonies
(s). The large colonies (f) measured 1 to 3 mm in diameter and
the small colonies (s) between 0.2 to ~5 mm in diameter or
less. The large colonies were selected and had a clear
proteolysis ring (p).
It is speculated that the problems encountered by the
prior art with aerobic growth of the bacteria on GMA were due
to the higher oxygen tension relative to liquid NFM. The
effect of anaerobic incubation on colony appearance was
remarkable. Not only was excellent colony differentiation
achieved, but the incubation period required at 30C was about
one-half of the time re~uired when incubated under aerobic
conditions in air.
Subsequently, 20 differen~ active strains were
differentiated and fast acid producing strains selected in the
manner of Example 1 including: Sc A2, Sl C2, Sc H2, Sc
HP, Sc 205, Sl c10, Sl ML8, Sc 104, Sc 286, Sc 287, Sc 288/
Sc 289A, Sc 289C, Sc 290, Sc 290A, Sc 291, Sc 292~ Sc 134~ Sc
108 and Sl E where "Sc" represents Streptococcus cremoris and
"S1" represents Strep~ococcus lactis. These strains were from
the culture collection of Oregon State University, Corvallis,
Oregon and samples are freely available to the public without
charge~
All of these species produced fast acid strain
colonies (f) within 24 to 36 hours at 30C using the improved
plating methodO Slow acid producing colonies (s) were apparent
in different proportions in many strains~
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When fast and slow acid producing strains were
isolated, cultured separately in NFM for 16 hr, and again
plated separa~el~ on FSDA using the anaerobic method of the
invention, both types yielded all of the same single or
homologous colonies produced by the method. Concentrated
cultures of fast acid producing strains (f) produced from the
selected strains usually were significantly more active in acid
production than the parent cultures that consisted of a
heterogeneous population of fast and slow strains, which was
unexpected. The differentiation and selection of phage
resistant strains by the method was also very unexpected.
The following Example 2 describes the differentiation
and selection of phage resistant strains.
Example 2
Using strains Sc A2, Sc H2, S1 C2~ and Sc 104,
active phage-insensitive strains were isolated on FSDA when
incubated aerobically. These strains were isolated by directly
plating the host with an excess of phage by spreading on FSDA
as in Example 1~ E`ollowing 2 to 4 days incubation at 30C,
fast and slow acid producing colonies were apparent. Ten fast
acid producing colony strains were picked from each plate and
subcultured in NFM as a second fluid growth medium to about 1 x
cells per ml at 21C and at 30C, with and without added
phage, and the phage insensitivity ~as confirmed. Preliminary
characterization of the fast acid producing strains lndicated
that they adsorbed phage but without subsequent DNA
penetration. Similar variants that adsorb phage withou~
subsequent plaque formation were produced as had been reported
by Limsowtin et al., N~Z. Jr. Dairy Sci. Technol., 13:1~ 1978.
-
RJP/paa 1/18/83 1255~ 24767
The first three strains grew aerobically on GL~A; however thiswas not true of Sc 104 which had to be grown anaerobically.
This demonstrates the reported difficulty that some
investigators have had in selecting phage-insensitive mutants
that have adequate acid production as described by Limsowtin et
al., N.Z. J. Dairy Sci. Technol., 13-1297 1978.
As phag~s appeared for fast acid producing strains,
isolates of phage-insensitive strains were obtained and used in
place of the original phage-insensitive parent strains in
Cheddar cheese making. Whey was used as the phage source.
Strain Sl ML8 produced by the method was a classic
phage-resistant mutant that did not absorb phages, as
determined by conventional phage absorption experiments.
The finding of improved colony growth and
differentia ion using the method of Example 2 was the same for
other phage-host combinakions. Another alternative is a
modification of that reported by Xull, Aust. J~ Dairy TechnolO
32, 65 ~1977) called "Whey Adaptation". This involved
culturing the host strain with a whey sample containing phages
and then streaking this infected culture and selecting
resistant colonies on FSDA. The method of selection and
diferentiation appeared to be universal for acid producing
bacteria,
Example 3 shows the growth of the bacteria under
aerobic conditions~
~ ple 3
Example 1 was repeated under aerobic conditions using
the FSDA medium. Recognition of typical fast acid producing
colonies was elther poor or inapparent for 12 (43%~ out of 28
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RJP/paa 1/18/83 1255E 24767
single strains incubated aerobically. In general, colony
growth was markedly retarded under aerobic incubation for all
strains. Consequently, differentiation of fast and slow acid
producing colonies was either unr~liable or inapparen~ as all
colonies were small (0.5 mm in diameter) and appeared as slow
acid producers.
This explains why the method in the original report by
Limsowtin and Terzaghi (1976) was unsuccessful in the
differentiation of fast and slow colonies. They reported that
some fast acid-producing strains gave rise to only slow
appearing colonies on glycerophosphate milk agar incubated
aerobically. They did not investiga~e the use of anaerobic
incubation to overcome this problem. The problem was that
milk-based media such as glycerophosphate milk agar and FSDA
would not effectively differentiate the fast and slow colonies
because of growth inhibition under aerobic conditions~
In the following Example 4, the b~ffering agent was an
insoluble alkali metal compound, trimagnesium phosphate7 the
indicator was bromcresol purple~ and anaerobic growth
conditions were used. Tests were also conducted using FSDA
medium for comparison.
_x~
Trimagnesium phosphate was substitu~ed for disodium
glycerophosphate in a plating medium Eor differentiation of
"fast" and "slowi' acid-producing colonies oE lactic
streptococci developed by Limsowtin and Terzaghi and later
improved by Huggins and Sandine, "Selection and
Characterization of Phage-Insensitive Lactic Streptococci," JO
Dairy Sci. 62:70 (1979). This medium, entitled FSDA-I L, was
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RJP/paa l/18/83 1255E 24767
prepared in three separate components. Component A was made by
adding 20.0 ml of a 0.25% (w/v) bromcresol purple solution and
10.0 g Davis agar to 400 ml of distilled water. Three drops of
an anti-foaming agent, Pourite (Scientific Products) were also
added to this component to reduce entrapped air bubbles in the
medium when pouring plakes. Incorporation of anti Eoaming
agent obviated the need to flame plates during pouring.
Component B was prepared by dissolving lO0 g NDM in 500 ml of
distilled waterO Component C was made by adding 5.0 g
trimagnesium phosphate (Stauffer Chemical Co.) to lO0 ml of
distilled water~ Component C was generally prepared in
multiple units and stored at room temperature. Components A
and C were autolaved for 15 min~ a~ 121C. Component ~ was
autoclaved for only lO min. at 121C7 The three components
were tempered to approximately 55C in a water bath at which
time components B and C were aseptically added to A. The
complete medium was then mixed on a magnetic stirrer and
~urther cooled to 45C in a water bath. Plates were poured and
allowed to air dry for 48 h at xoom temperature. Occasional
swirling of the flask was necessary to maintain Mg3(po~)2
in suspension while pouring plates~ Streaked or spread plates
of FSDA-II were incubated at 30C for 48 h under arlaerobic
conditions. After incubation, there was excellent visual
contrast of yellow colonies on a blue background.
A comparison between E~SDA-II and FSDA was made by
spread plating S. lactis C2 (lac~, prt+) and the three
plasmid-charac~erized slow variants of S. lactis C2, LM0210
(lac ~ prt ), LM0220 (lac t prt ) and LM0231 (lac ,
prt ) (provided by L, L~ McKay) on the two media. The C2
parent and the three variants were grown in litmus milk (1L%
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w/w3 at 21C for 24 h~ The following mixtures of freshly grown
cultures were made: 0.1 ml (lac+, prt~) + 0.25 ml (lac~,
prt ) -~ 0.5 ml (lac , prt+) + 0O5 ml (lac , prt );
0.25 ml (lac+, prt+) + 0O5 ml (lac+, prt ); 0.~5 ml
(lac+, prt ) + 0.5 ml (lac , prt )O Each of the three
mixtures was serially diluted and spread-plated on FSDA and
FSDA-II.
Streptococcus thermophilus strains 404, 410, 440,
19987, L12l S122 and C3; ~ strains 404
and 448; and Lactobacillus helveticus strain6 L112, 15807 and
450 were all streaked from freshly coagulated litmus milk
cultures onto FSDA and FSDA-II. The pla~es were incuba-ted at
37C for 30 h under anaerobic conditions. Qualitative
comparisons of growth by these thermophilic strains were made
on FSDA and FSDA- I I .
Halos resulting from solubilized Mg3(po4)2 were
not detectable on FSDA-II because of the opaqueness contributed
by the NDM solidsu However, as with FSDA, differentiation
between fast and slow acid-producers was possible on FSDA-IIo
Differentiation between lac , prt+ and lac , prt was
not possible on FSDA-II as with FSDA. Three distinct colony
types were evident on FSDA-II. The S. lactls C2 parent
appeared as a full-colored yellow colony against an opaque
baby-blue background. The lac~ prt colonies were less
brightly colored and resembled a yellow doughnut~ Colonies of
+
lac / prt and lac , prt were indistinguishable from
each other but clearly differentiated from the other two colony
types. They appeared as colorless, translucent colonies; best
visualized by observing their projection above the surface oE
the plating medium.
RJP/paa 1/18/83 1255E 24767
In a comparative platiny of lac , prt and lac ,
prt on both FSDA and F5DA-II media, ~isual observation of
the FSDA plate revealed only a single colony type. A precise
measurement of colony size enabled differentiation between the
two colony types (lac~, prt being slightly larger than
lac , prt ). Howe~ler, with FSDA-II it was visually
apparent that two distinct colony types were present.
A comparative plating of all four phenotypes mixed
toyether was conducted. On FSDA, two distinctly different
colony types were evident, and again precise measurement would
have allowed differentiation of an intermediate-sized colony
representing the lac , prt variant. On FSDA-II, three
distinctly different colony types were clearly di~tinguishable
without any measurementO The lac , prt+ was a bright,
fully-colored yellow colony with a darkened halo; the lac~
prt was not as brightly colored and was without a halo. The
lac , prt+ and lac , prt appeared as colorless
colonies.
When lac~, prt and lac+, prt were plated on
both media, two colony types were discernible on both FSDA and
FSDA-II.
Fast-slow differentiation on F5DA was dependent on
size differences. Crowded plates or dehydrated plates (surface
concentration of agar) make SiZQ differentia~ion somewhat
difficult. Injured cells also appear initially as slow
colonies on FSDA but with additional incubation time they
slo~ly transform into typical fast colony typesO
Differentiation on F5DA-II is based primarily on color
differences and not size. This enables ~Jisual differentiation
without the need for colony size measurement~ Be~ause of the
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lower phosphate concentration in FSDA-II (0.5% w/v) compared to
nearly 2~ (w/v) in FSDA, injured cells are more likely to
reco~rer and develop their true colony types on FSDA-II within
the 48 h incubation period.
All the S. thermophilus st~ains examined grew equally
well on ~SDA or FSDA-II4 However, ~ aricus 404 and 448
and L. helveticus 450 did not grow nearly as well on FSDA as on
FSDA-II and MRS agar (control mediumr Difco). And L~
helveticus L112 and 15807, while growing poorly on ~SDA-II,
were completely inhi~ited on FSDA.
There are numerous applications for the improved
method and plating device~ The most important is the direct
selection of phage-insensitive fast acid producing strains.
~he improved method and plating device can also be used as a
tool to directly study various chemicals or conditions
influencing the appearance of variants in bacterial strains.
Commercially the method is advantageous in the isolation,
selection, and screening of fast lactic acid producing starter
strains, particularly Cottage and Cheddar cheese and buttermilk
producing strainsO Thus a Cheddar cheese plant in Olympia/
Washington, has been on the same culture developed for them for
about 2 years, producing about 20,000 pounds of cheese a day or
over 5 mil]ion pounds. A plant in Tillamook, Oregon, now has
over 1200 consecutive 38,000 pound vats (over 4 million pounds
of cheese) using a culture developed for their use. The FSDA
agar is used to isolate phage-resistant fast acid-producing
mutants whenever viruses appear for any of the 6 strains in the
multiple strain starter cultures~ We are convinced that all
cheese plants can operate this well when this method is used to
keep the cultures active in plants~ The cheese is of excellent
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quality and these cultures eliminate public health problems
because vf slow vatsO The FSDA agar facilitates selective
manipulation oE starter strains which has not been possible
previously.
Tests indicate that the addition of optimum amounts
(1000 units per petri plate and 0.5~ by weight) of catalase and
pyruvate, respectively; are substitutes for the anaerobic
incubation and addition of ferrous sulfate appears to be
effective. These additives degrade the hydrogen peroxide that
colonies of laçtic streptococci produce when grown aerobically
on milk-based media. This minimizes auto-inhibition of cell
growth due to hydrogen peroxide accumulation. ~owever, these
additives are not ~uite as good as anaerobic incubation. Other
additives which simulate anaerobic conditions by stimulating
the growth of the bacteria under aerobic or anaerobic
conditions can be used.
