DE

MAN, J. C., ROGOSA,

M. & SHARPE,
M. E. (1960). J . oppl. Bact. 23 ( l ) , 130-135.

A MEDIUM FOR THE CULTIVATION OF LACTOBACILLI

BY J. C. DE MAN
Nederlands Instituut voor Zuivelonderzoek, Ede, Nederland
M. ROGOSA
National Institute of Dental Research,
National Institutes of Health, Bethesda, Maryland, U.S.A.
AND M. ELISABETH SHARPE
National Institute for Research in Dairying,
Shinfield, Reading, England

SUMMARY: An improved growth medium for lactobacilli is described. It supports

good growth of lactobacilli generally and also is particularly useful for a number of
fastidious strains which grow only poorly in other general media. In addition, tomato
juice, a highly variable material, is not required. In a slightly modified form, it can
also be used as e basal medium for fermentation tests.

ALTHOUGH
several media for lactobacilli have been described, such as the widely
used tomato juice medium of Briggs (1953) and Cox & Briggs (1954),it has been
found that a number of strains of lactobacilli, belonging to several species, did not
grow very well in any of them. Therefore, a need existed for a non-selective medium
which would support good growth of ladobacilli in general. Also, it would be desirable
t o eliminate tomato juice because it is variable and inconvenient t o prepare.
Rogosa, Mitchell & Wiseman (1951a,b) described a selective medium designed
especially for the enumeration and isolation of lactobacilli of oral and faecal origin,
although it was recognized that it was not satisfactory for certain dairy organisms.
Starting from this, it was hoped t o develop a new medium in which the lactobacilli
of dairy origin would also grow well. A medium of chemically defined composition
was not the aim of this study. Rather, it was decided t o use commercially obtainable
standard ingredients which would be convenient for general use in both routine and
research laboratories.
Most of the experiments were performed in two Institutes, the National Institute
for Research in Dairying (NIRD) and the Netherlands Institute for Dairy Research
(NIZO), using the same strains of lactobacilli and virtually identical methods,
and exchanging media components. The results obtained in both Institutes were in
agreement. A few of the experiments, by which the composition of the final medium
wm developed, are reported here.

Medium for lactobacilli

13'

MATERIALSAND METHODS
Strains of Eactobacilli. The strains used, which are listed in Table 1, were selected
a8 representative strains or because they did not grow satisfactorily in Briggs'
medium.
Table 1. The origin and designation of the Lactobacillus species
and strains used
Growth temperature 37"
Species
Origin*
Designation
4357
L. acidophilus ATCC
9857
L. bulgoricus
NIZO
3501

8014

L. casei
NIRD

L. helveticwr
L. jocgurti

L. lactis

NIRD
NIZO
NIRD

Ku 2
229
F1
0 10
3524
DR 9
8000
Ku 1

Growth temperature 30"

Species
Origin*
Designation
NIZO
3903
L.bifementuns
NIZO
3905
DL 127
V7
L. brevis
NIRD
T 1
XI
L. plantarum.
NIZO
3503
Unclassified
NLRD
v 5
A 41
streptobacteria NIRD

* NIRD,

National Institute for Research in Dairying, Shinfield, Great Britain; ATCC,

American Type Culture Collection; W, Bakteriologisches Institut der Sudd. Versuchs- und
Forschungaanstalt fiir Milohwirtschaft, FreiRing-Weihenstephan, Germany; NIH, National
Institutes of Health, Bethesde, Maryland, U.S.A. ; NIZO, Nederlands Instituut voor
Zuivelonderzoek, Ede, Holland.

Total bacteria ( IO'iml)

Fig. 1. The relationship between optical density and total (microscopical)

bacterial count for lactobacillus strain V 6 .

Chlturing methods and measurements of growth. I n the growth experiments the

organisms were cultured in 150x14 mm tubes containing 10 ml of medium. For
fermentation tests, tubes with 5 ml of medium were used. Inoculations were always
made with 0.1 ml of culture.

132

J. C . de Man, M . Rogosa and M . Elisabeth Sharpe

For evaluating a growth medium the bacteria were subcultured in it twice, using
48 hr growth periods. Finally, the bacteria were grown for 40 hr in a third subculture,
after which growth was measured by determining the optical density of the undiluted
culture against a medium blank with a n Engel photometer (Kipp, Delft) using filter 66
and a 0.5 cm cell, or with a Hilger Spekker absorptiometer using filter EEL 607. The
results are expressed as optical densityx 100. Fig. 1 illustrates the relationship
between the total number of ceIls/ml (microscopical counts) and optical density for
one of the strains.
As the purpose was the development of a general non-selective growth medium,
more optimal growth conditions were achieved at the outset by diminishing the
amount of acetate and the use of a higher pH. The final medium was obtained
after testing a number of combinations of ingredients and physical conditions, such
as pH, different peptones, addition of meat extracts, salts, various carbohydrates,
tomato juice and reducing agents. After each experimental change the effect of it
on growth was compared with growth on the medium of Briggs (1953), which was
used as a standard.
RESULTSAND DISCUSSION

Final growth medium

The medium (referred to below as MRS) as finally composed contained: Oxoid
peptone, 10 g ; Lab-Lemco (Oxoid), 10 g; Yeast extract (Difco or Oxoid), 5 g ; glucose,
20 g; polyoxyethylene sorbitan mono-oleate (Tween 80), 1 ml; KzHPO,, 2 g ;
CH,COONa.3Hz0, 5 g ;triammonium citrate, 2 g ;MgS0,.7Hz0, 200 mg;MnS0,.4Hz0,
50 mg; distilled water, 1 litre. The pH lies between 6-0and 6-5after sterilizing (about
6-2 to 6.6 before). A sterilization time of 15 min a t 120" is recommended.
The composition of the new medium is primarily justified by the fact that the
selected strains of lactobacilli grew much better in i t than in the medium of Briggs
(Table 2).
The addition of corn steep liquor did not improve growth. Tomato juice, often
used in media for lactobacilli, also was without stimulatory effect, and was therefore
considered unnecessary (Table 3). It is a n advantage t o dispense with tomato juice
because it is variable and inconvenient t o prepare.
Several kinds of peptone were tried, and of these Oxoid peptone appeared superior
to the others. In a h a 1 comparison, Difco Tryptone (a casein peptone) gave nearly
as good results (Table 4). I n further experiments, six different batches of Oxoid
peptone were compared: they gave virtually similar growth. When levels of 0.0,
0.2, 0.5, 1.0 and 4% (w/v) were investigated, different strains varied considerably
in their growth response. A concentration of 1 yo (w/v) of Oxoid peptone was chosen,
since growth was generally optimal with all the strains investigated. Therefore, 1%
(w/v) of peptone was used in all later experiments.
The growth stimulating effect of meat extract in the medium is demonstrated
in Table 5.
Rogosa found that with some basal media a mixture of glucose, arctbinose and
sucrose, instead of only glucose, improved the growth of several fresh isolates of

Medium f o r lactobacilli

'33

Table 2. A comparison of the growth of various lactobacilli in

M R S medium with that in Briggs' medium (1953)
Strain
4357
3501
8014
229
F 1
0 10
3524
DR 9

Growth (OD* x 100)

MRS
77
75
120
72
110
114
76
68

Briggs'
26
32
24
19
5
27
46
44

Strain
8000
3903
DL 127
v 7
T 1

x1
v 5

Growth (OD* x 100)

91
115
86
96
37
107
132

40
4
6
8
9
24
13

* OD, optical density.

Table 3. The effect of tomato juices on the growth of lactobacilli
in M R S medium*
Strain

Growth (ODt x 100) in MRS medium with

<

>

NIRD
No
Rijno
juice
juice
juice
4357
94
105
110
3501
76
66
74
8014
145
140
140
229
24
22
23
3524
80
79
84
DR 9
89
88
88
8000
90
85
92
3903
50
57
95
v 7
112
98
97
V5
145
140
140
* The media with tomato juice were made with 5 % of Rijno
tomato juice (a commercial product of Mij De Betuwe, Tiel,
Holland), or 10% of tomato juice prepared a t NIRD by
the method described by Briggs (1953). Correspondingly
less water was used in making up the media. OD, optical
density.

heterofermentative organisms (for the resulting medium see Fitzgerald & Jordan,
1953). However, this was not found t o be the case with the present medium and
the strains studied. Neither was growth better with 4% ( w p ) of glucose instead of
the 2% (w/v) finally used.
It has been shown that the addition of Tween 80 (Briggs, 1953), citrate (Evans &
Niven, 1951) and acetate (Snell, Tatum & Peterson, 1947 ; Guirard, Snell & Williams,
1946) to media for lactobacilli often results in improved growth. Most of the inorganic
ions necessary for growth will be present in sufficient amounts in a complex medium,
aa used by us. An exception is manganese (Orla-Jensen, 1943; MacLeod & Snell.
1949; Evans & Niven, 1951). Diminishing the amount of Tween 80, or omitting
the citrate, acetate or manganese, always resulted in diminished growth for a t least
several strains. The inclusion of magnesium in the medium is a precautionary
measure; it may be unwise t o rely on the presence of a sufficient amount of magnesium

= 34

J . C . de Man, M . Rogosa and M . Elisabeth Sharpe

Table 4. A comparison of the growth of various lactobacilli in MRS medium
made with Oxoid peptone and D i j b Tryptone
Strain

4357
9857
8014
Ku 2
229
DR 9
8000
Ku 1

Growth (OD* x 100) with:

Oxoid
peptone
92
85
133
131
100
95
95
118

Growth (OD* x 100) with:

Strain

5zr-Gz

Difco
Tryptone
85
H O 268
97
H O 66
123
3903
125
3905
76
v 7
83
v 5
94
A 41
110
* OD, optical density.

peptone
111
140
120
129
110
140
145

Tryptone
113
140
114
125
98
140
135

Table 5 . lhe effect of meat extract on the growth of various

lactobacilli in MRS medium
Strain

Growth (OD* x 100) with extract

4357
3501
3503
8014
F1
3524
8000
3903
DL 127
v7
T1
V5
A 41

Present
54
65
54
120
99
79
81
107
136
95
71
135
126
OD, optical density.
~~

Absent
33
36
38
110
97
77
87
104
133
92
52
129
121

in the yeast extract or other ingredients, especially as the presence of citrate in the
medium increases the amount necessary (MaoLeod & Snell, 1947).
No significant differences in growth were encountered within a pH range of 6.1
60 6.6.

Basal medium for fermentation studies

With slight alterations, the MRS medium may also be used for fermentation tests.
I n this case, the glucose is replaced by 2% (wlv) of the test substrate, the meat
extract is omitted since i t is known to contain variable amounts of fermentable carbohydrate, and for ease of recognition of the results 0.004% (wlv) of chlorophenol red
is added. Nevertheless, in this work not only was the colour noted, but the pH in
each tube was also determined.
I n a n investigation of the influence of initial pH value on the fermentation resulta,
using strains 3531,3541, DR 9,229, 3524, F 1 , 0 10, 3602, V 7, V 5,3903, DL 127 and
T 1 and the substrates arabinose, cellobiose, glucose, lactose, maltose, mannitol, melibiose, sucrose, salicin and trehalose, identical results were obtained with initial pH

Medium for lactobacilli

'35

values of 6.80, 6.20 and 5.75. At lower pH values the results tended to be obtained
earlier. For convenience, a p H between 6.2 and 6.5 is recommended. With considerably reduced carbohydrate concentrations (0.2% w/v) a relatively low initial
pH can be critical for obtaining consistent fermentation results with some strains,
and a pH no higher than 6.5 is suggested.
With the substrates used, no difference was found if the substrates were sterilized
together with the basal medium (pH 6.8) or sterilized separately and added later
as a 10% (w/v) solution. However, in other media and under different conditions,
Rogosa et al. (1953) showed that certain substrates, particularly arabinose, xylose,
mannose, fructose, galactose and sorbose may require separate sterilization by
atration. Although with the strains tested correct fermentation results were
obtained using these heated substrates in the MRS medium, other strains might be
encountered giving false positive results from these carbohydrates. The addition
of 0.15% of agar to the medium a t pH 6.2 did not significantly influence the results.
Positive results could nearly always be recognized after 1 day, and invariably
after 4 days. Generally more simple media like those of Orla-Jensen (1919) and
Wheater (1955) are aleo satisfactory for fermentation tests, provided that a high
quality peptone is used. The advantage of using the more complex medium is that
results are often obtained somewhat sooner and that the number of doubtful reactions
is diminished.

REFERENCES
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17, 18.
EVANS,J. B. & NIVEN,C. F. (Jr.) (1951). Nutrition of the heterofermentative lactobacilli that
cause greening of cured meat products. J . Bact. 62, 599.
FITZUERALD,
R. J. & JORDAN,
N. V. (1953). The in vitro effects of antibiotics and other inhibitory
agents on representative oral lactobacilli. Antibiot. & Ghemother. 3, 231.
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B. M., SNELL,E. E. & WILLIAMS,R. J. (1946). T h e nutritional role of acetate for
lactic acid bacteria. I. The response to substances related to acetate. Arch. Biochem.
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MACLEOD,
R. A. & SNELL,E. E. (1947). Some mineral requirements of the lactic acid bacteria.
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M., MITCHELL,
J. A. & WISEMAN,
R. F. ( 1 9 5 1 ~ ) .A selective medium for the isolation
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M., MITCHELL,
J. A. & WISEMAN,
R. F. (1951b). A selective medium for the isolation
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M., WISEMAN,
R. F., MITCHELL,
J. A., DISRAELY,
M. N. & BEAMAN,
A. I. (1953). Species
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SNELL,E. E., TATUM,
E. L. & PETERSON,
W. H. (1937). Growth factors for bacteria. 111. Some
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J. gen. Mkcrobiol. 12, 123.

(Received 15 July, 1960)