All lactobacilli and Pediococcus were grown on APT broth or agar (Merck). The medium for acetic acid bacteria contained 1% yeast extract (Oxoid), 3% CaCO3,.
J. hist. Brew., September-October, 1983, Vol. 89, pp. 361-363
361
GROWTH OF LACTOBAC1LLUS SPECIES ON DIFFERENT MEDIA By C. Van Keer, L. Van Melkebeke, W. Vertriest, G. Hoozee and E. Van Schoonenberghe
(Katholieke Industriele Hogeschool van Oost- Vlaanderen, Gent, Belgium) Received 7 February 1983
Growth and recovery of thirty strains of Lactobacillus from different origin and four other bacteria
were tested on various media commonly recommended for rapid detection and enumera tion of lactobacilli. Racka Ray No. 3 yielded the highest colony-counts and allowed enumeration of most strains within 48 h of semi-anaerobic incubation. A new medium has been proposed which is comparable to Racka Ray No. 3 in recovery results, however, enumeration can easily be done 24 h after inoculation.
Key words: Lactobacillus, media, contamination. Introduction Members of the genus Lactobacillus are widely distributed throughout our food chain; they play an important role in the manufacturing of different dairy products, but they also might be detrimental infectants in brewery processes. In both cases it is of particular interest to isolate them selectively and enumerate them accurately as fast as possible. These lactic acid bacteria possess a wide diversity of physiological properties. Apparently the use of one single, universal medium for the isolation and cultivation of all lactobacilli is impossible. Many media have already been described and many of them are commercialised. Some media are recommended by official agencies, e.g. the Sub-committee ofthe International Committee on Nomenclature of Bacteria recommended the use of MRS4-7 for the determination of physiological proper ties of lactobacilli, the Microbiology Sub-committee of the Analysis Committee of the Institute of Brewing recom mended sucrose agar for detection of lactobacilli in brewery samples3 and the ASBC Sub-committee on Microbiologi cal Control prefers Brewer's Tomato Juice Medium and Universal Beer Agar for the same purpose.1-210 Reviews on media for lactobacilli were published by Sharpe18 and Sharpe & Fryer.19 An extensive study on media for beer-infecting lactobacilli was performed by Wackerbauer & Emeis;21 they tested forty-two media, but they neither characterised their strains, nor mentioned the numbers of strains. They recommended their own medium VLB-L41-agar.
A less extensive study was accomplished by Meyling, who proposes the Racka Ray No. 3 medium (RR3) for routinecontrol in breweries.1517 Hsu & Taparowsky compared nine media for growth of Lactobacillus gayonii and five other common brewery bacteria.8 From their results HRM and RR3 can be pointed out as most suitable for rapid detection.9 Another comparative investigation was carried out by Matsuzawa el al;14 they used seven uncharacterised
Lactobacillus-strains on eleven media. RR3, MRS and LS-
agar (Mieth, Mannheim) were recommended for semianaerobic cultivation of these organisms. To evaluate the value of different media for recovery and colony growth, the use of a large number of well characterised strains is necessary. Growth must be com pared on a significant number of different media in well defined conditions. For our investigation we used thirty Lactobacillus-slrams and four strains belonging to other genera, but known as common brewery infectants. Eleven media have been investigated and a new one is proposed.
Materials and methods
Organisms used.—Cultures were obtained from the American Type Culture Collection (ATCC), the National Collection of Type Cultures (NCTC), the National Collec
tion of Industrial Bacteria (NCIB), the Laboratorium voor Microbiologie en microbiele Genetica, R. U. G. Gent (LM), the Laboratorium voor Industriele Microbiologie, G.R.L.H. Gent (LIM) and J. C. Meyling (Skol Brewery, Breda). The following strains have been used: Lactobacillus acidophilus LIM La 115, LIM D 10, LIM La 357,79/4, LIM La 168; L. bulgaricus 79/1; L. casei LIM L 151,80/1, ATCC 7469, 79/2; L. delbruckii NCTC 4033; L. helveiicus casei 8010; L. leichmanii ATCC 7830; L. plantarum ATCC 8014, LIM T9, LIM T108, 79/3, 81/4; L. salivarius LIM JD 166, LIM 276; L. brevis NCIB 8847, 81/1; L. brevis pastorianus 81/3; L. fermentum ATCC 9338, LIM La 415, LIM JD 184a; L. fructivorans 81/2; Lactobacillus sp DR, 80/2; Pediococcus cerevisiae NCTC 8066; Escherichia coli NCTC 9001; Hafnia protea 520 Shinwell 10/71; Gluconobacter oxydans oxydans NCIB 9013; Actobacter aceti aceti NCIB 8621.
Growth media.—All lactobacilli and Pediococcus were grown on APT broth or agar (Merck). The medium for acetic acid bacteria contained 1% yeast extract (Oxoid), 3% CaCO3, 5% D-glucose and 2-5% agar in distilled water. Other bac teria were grown on Nutrient Agar (Oxoid). MYGT-medium consisted of malt extract 3%, yeast extract 0-4%, D-glucose 0-2% and trypticase 0-4% in distilled water. Ringer-solution was prepared with Oxoid-tablets (BR 52). Media under investigation.—Sucrose Agar (SA),3 Lactobacillus-Streptococcus Differential Medium (LSDM, Oxoid CM 495),s Man-Rogosa-Sharpe Agar (MRS)," Racka Ray No. 3 agar (RR3),17 Lee's Multi Differential Agar (LMDA),'2 VLB-L41 agar,21 Micro Assay Culture Agar (MACA, Difco), Tomato Juice agar (TJ, Difco), All purpose medium containing Tween 80 (APT, Merck) and Sorbic Acid Agar (SAA, Merck). To all media 7 mg actidione per litre was added.
The Lactobacillus-GTOWth-Medium (LGM) consisted of (in g/litre medium): yeast extract 5; lab Icmco 8; liver extract 1; pepton 5; trypticase 5; D-glucose 20; di-ammoniumcitrate 20; CaCoj 5; K2HPO4 2; K-aspartate 0-75; K-glutamate 0-75; MgSO4.2aq. 0-5; MnSO4.7aq. 2; FeClj0-0l; mevalonic acid 0-005; betaine.HCl 0-6; biotin 10"5; Ca-panthotenate 0-5; folic acid 10"6; nicotinic acid 10"4; pyridoxal 2.10~4; riboflavin 10"4; thiamine 10~4; L-cysSH.HCl 0-3; adenine 5.IO"3; N-acetyl glucosamine 0-5; Tween 80 10; actidione 7-IO-3; bromocresolgreen 22-10"3; and agar 20. Final pH after sterilisation 6-25.
Cultures.—All strains were grown semi-anaerobically6 on
APT agar-slants at 30°C, unless stated otherwise. For evalu ation of the investigated media, cells from a young culture agar slant were suspended in 3 ml of Ringer-solution and diluted to an appropriate number of cfu/ml (colony forming units per ml). When the experiment was started with a liquid pre-seed culture medium 01 ml of this medium was diluted
with Ringer-solution before inoculation of the solid media
under investigation. Petri-dishes filled with 15 ml of the investigated media were inoculated by spreading 0-1 ml of
362
[J. Inst. Brew.
VAN KEKR HTAL: GROWTH OF LACTOBACIUMSSPECfES
TABLE I. Number of cfu and colony-Diameter of Lactobacillus spp and Pediococcus spp on Different Solid Culture Media (incubation: semi-anaerobic. 30°C, 3 days) Number of cfu in%* RR3
LSDM
LMDA
SA
MRS
MACA
TJ
too 100 100 100 100 100
139 151 125 103 109 130
61 88
126
19 102
125
152 98 109
88 93 98
9 107 112 42 92 107
L. acidophilus 79/4 L. bulgaricus 79/1 L. casei 79/2
L. planwrum 79/3 L brevis NCIB 8847 P. cerevisiae NCTC 8066
69
75 82
99
79 96 112
87 78
58
59 67
SAA 1 12
67
30 86 100
APT
88 91 66 45 19
62
Average colony-diameter in mm
L. acidophilus 79/4 L. bulgaricus 79/1 L. casei 79/2
L. plantarum 79/3 L. brevis NCIB 8847 P. cerevisiae NCTC 8066
RR3
LSDM
LMDA
SA
MRS
MACA
TJ
SAA
APT
1-5 1-5 20 20 20 30
05 0 1 10 0-2 0-5 0-5
10
0-5 10 10 10 0-5 1-5
1-5 0-5 1-5 0-5 10 1-5
10 10
10 0-5 1-5 10 01
0-5 0-5
0-5 0-5 1-5
1-5 10
1-5 1-5 1-5
10
10 01 1-5
10
0-5 10 10 10
1-5 1-5 20
'Results on RR3 are taken arbitrarily as 100%.
the diluted cell suspension and incubated scmi-anacrobically at 30°C. During 5 days, the cultures were inspected daily, the numbers and the diameters of the colonies were recorded. Results
Five strains of Lactobacillus and one of Pediococcus were grown in MYGT or MRS broth; 01 ml of the culture was inoculated into S ml of nine different liquid culture media. After 3 days at 30°C a sample was taken from each culture, diluted 104 times and 01 ml of this dilution was transferred to the respective solid medium. The results (average values of 5 experiments) are presented in Table I. Our results with LSDM, RR3 and LMDA were superior to those with SA and MRS. In these conditions the results
with MACA, TJ, SAA and APT were not satisfactory. Best recovery was obtained with LSDM, followed by RR3 and LMDA. While after three days of incubation all colonies stay small (less than I mm) on LSDM, good growth was obtained on RR3, allowing enumeration 24 to 48 h after inoculation. Based on these observations and a study of literature con cerning vitamin requirements of lactobacilli"1316-20 the composition of LGM was formulated. In a series of seven media 18 strains were tested. From a S ml seed culture in APT broth, 0-1 ml was diluted in Ringer solution to obtain a suspension of maximum 3000 cells/ml; 01 ml of the suspension was used to inoculate the different solid media. Incubation was performed at 30°C during 48 h. The average number of cfu and the colony-diameter were recorded. Results are given in Table II.
TABLE II. Growth of 15 Lactobacillus spp and 4 Other Strains on Different Solid Culture Media (incubation: semi-anaerobic, 30°C, 48 h) Number of cfu in %*/colony-diameter in mm
L. acidophilus L1M La 115
79/4 LIM D 10 LIMU357 LIM La 168 L. bulgaricus 19/i L. plantarum LIM T9 LIM T108 79/3
L. salivarius LIM JD 166
LIM 276 L.fermentum LIM La 145 LIMJDI84a Lactobacillus sp DR iHafnia protea 520 tE.coli NCTC 9001 P. cerevisiae NCTC 8066 t/1. aceti aceti NCIB 8621
RR3
LSDM
LMDA
SA
100/1 100/01 100/3 100/2 100/1 100/0-5 100/2 100/2 100/1 100/3 100/0-5 100/3 100/1 100/2 100/1 100/1 100/1 100/01
0 40/0-5 224/0-1 d d 137/0-1 160/01 d d 0 d 62/3 89/1 113/2 d d d d
62/2 80/0-5 97/2 125/2 500/0-1
79/2 60/1 141/2 d d
'Results on RR3 arc taken arbitrarily as 100%. tGrowth in aerobic conditions at 25°C. ^Growth in aerobic conditions at 37°C. d=not tested.
88/3 33/1
40/2 0 15/2 109/0-1
56/4 100/0-5 97/1 68/0-5 100/4 11/1-5 40/0-1
82/3
33/0-1 d d 31/2 d 51/3 73/0-5 38/2 d d d d
MRS 38/3 4/0-1 0
d d 0 80/1 d d 33/0-1 d 3/1 73/0-1 13/0-5 d d d d
VLB-L41 0 0 0 d d 0
20/0-1 d d 0 d 0 0 0
d d d d
LGM 100/1 60/1 96/3 50/4 200/1 122/1 75/1 40/2 125/1 100/3 273/0-5 100/5 88/1 108/4 42/1-5 83/3 101/2 40/0-5
Vol. 89, 1983]
363
VAN KKI-R XT.\L: GROWTH OF LACTOB.-ICILLUSSPi-CIES
TABLE HI. Influence or Adaptation to the Culture Medium (incubation: semianaerobic, 30"C, 48 h) Number ofcfu in %•/colony-diameter in mm
Seed culture medium Enumeration medium L. casei L1ML 151 80/1
ATCC 7469 L. delbruckii NCTC 4033 L. helveticus casei NC1B 8010 L leichmaniiATCC 7830 Lplantarum ATCC 8014 81/4 Lbrevis 8[/i L brevis/pastorianus 81 /3 Lfermemum ATCC 9338 L. fruciivorans
Laciobacillus spp DR
80/2
P. cerevisiae NCTC 8066
Seed culture medium RR3
RR3 100/2 100/1 100/1
100/1-5 100/0-1 100/0-5 100/2 100/0-5 100/1 100/0-5 100/0-5 100/0-5 100/0-5 100/1-5 100/1
LGM
148/2 61/5 126/4 449/2-5 116/0-5 82/0-5 151/1-5 57/0-1 40/1 24/0-1 1/0-1 0 34/4 190/4 90/2
Seed culture medium LGM RR3
LGM
100/2-5
156/2-5 28/5
100/1-5 100/01 100/0-1 100/2 100/01 100/0-5 100/0-5 100/0-5
542/2-5 131/0-5 11/1 101/2 27/0-1 68/1 96/0-1
100/1 100/1-5 100/1
30/5 336/4 119/2-5
100/2 100/1
117/5
0
'Results on RR3 are taken arbitrarily as 100%.
The results of this experiment confirmed previous data. For several strains LSDM yielded high cfu numbers, but very small colonies. Growth results on RR3 and LMDA were comparable, but the recovery on RR3 was better. The results on SA and MRS did not match those on RR3. VLB-L41 could not be compared. The recovery on the new medium, LGM, was equal to the one on RR3, but LGM offered one important advantage: Tor all strains enumeration was possible 24 h after inoculation. This characteristic of the medium permits very fast detection and enumeration of contaminating lactobacilli. To determine the influence of adaptation to the medium, several strains were grown semi-anaerobically on RR3 and LGM agar-slants. These cells were suspended in Ringersolution at an appropriate dilution and used to inoculate solid RR3 and LGM culture media. Results are presented in Table III. Adaptation on LGM favours only those strains which showed already high numbers of cfu before. Exception has to be made for L. brevis 81/1 and L. brevis pastoriamis. which are clearly stimulated. L. casei 80/1, L. leichmanii and L plantarum 81/4 are inhibited. No growth was ob tained with L. fermenmm ATCC 9338 and L. fructivorans. This latter fact can easily be explained, since LGM lacks D-fructose as the essential source of carbohydrate for L. fruciivorans. The replacement of D-glucose as a single carbon source in LGM by 10 g/litrc D-glucose and 10 g/litre D-fructose raises this inhibition and supports growth com parably to that on RR3. The same effect was observed with L. plantarum 81/4, but in that condition growth of Lactobacillus sp 80/2 (isolated from saliva) was completely inhibited.
Discussion
Out of eleven media tested three could be selected that gave good recovery (a high cfu number) and fast growth of colonies. In general a sufficiently high recovery was obtained with RR3, allowing enumeration after 48 h of incubation. On LMDA cells grew faster, but with a significant loss of recovery. LSDM supported a high recovery for a limited number of strains, but gave poor growth. From these results RR3 is recommended as the most convenient medium for routine-analysis of lactobacilli.
A new medium, LGM, was formulated forculturing lacto bacilli. On this medium, recovery generally equalised that on RR3 and allowed abundant growth. Almost all strains tested could be easily enumerated 24 h after inoculation. The medium also allows growth of other kinds of bacteria, e.g. Escherichia. Hafitia and acetic acid bacteria.
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