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(1961),26, 185-197 Printed in Great Britain
The Cultural and Physiological Characters of the Pediococci
BY HELGE L. GUNTHER AND HELEN R. WHITE Bacteriology Department, Queen Elizabeth College (University of London), London, W. 8
(Received 17 November 1960)
The cultural and physiological characters of 89 strains of pediococci have been studied. Proposals are made for extension of the genus and its subdivision into two and possibly three groups. The reactions of eleven strains of Aerococcus viridans were also investigated.
Organisms described as pediococci have been studied for some considerable time, mainly in relation to problems in the brewing industry (Balcke, 1884; Mees, 1934; Shimwell & Kirkpatrick, 1939). More recently these organisms have been found in appreciable numbers in fermenting vegetable material (Pederson, 1929 ; Pederson & Albury, 1950; Pederson, Albury & Breed, 1954), in ‘caecal faeces’ of turkeys (Harrison & Hansen, 1950), in the rumen of cows (Bauman & Foster, 1956), in summer sausage (Deibel & Niven, 1957), and in cheese (Naylor & Sharpe, 1958; Dacre, 1958a, b). Systematic studies of the physiological characters (Pederson, 1949; Felton & Niven, 1953; Jensen & Seeley, 1954; Pederson et al. 1954) and of the nutritional requirements (Jensen & Seeley, 1954) have led to recognition of the pediococci as belonging to a separate genus and to its classificationwithin the family Lactobacillaceae in the tribe Strephcocceae. This view has been incorporated in the seventh edition of Bergey’s Manual (1957), where Pediococcus is described as a genus of Gram-positive cocci occurring singly and in tetrads, pairs and short chains, microaerophilic, generally catalase-negative, homofermentative, producing optically inactive lactic acid from carbohydrates, producing acidity and cloudiness in beer, and found as saprophytes in fermenting vegetable juices. Within the genus thus described two species only are recognized, P . cermisiae Balcke, 1884, and P . acidilactici Lindner, 1887, distinguished by their optimum growth temperatures and ability to grow in beer. In the course of a study of bacterial changes occurring during the ensilage process (Hoffman, Wolf & Barker, 1957) a large number of Gram-positivecocci was isolated which resembled the pediococci in certain characters. However, some of the silage isolates differed sufficiently from the descriptions of pediococci to suggest that the genus might be wider than previously thought. We have therefore made a survey of the cultural, physiological and serological characters of strains from a wide range of sources. Such an investigation seems to be particularly appropriate at the present time since the possibility of utilizing pediococci in fermentation processes has recently been proposed (Pederson & Albury, 1950; Dacre, 1958a; Dr C. F. Niven
0. except for strains M-1. 0. Most of the experimental work was carried out with all isolates. 8520 and Tc. 1 which required 72 hr. The Gram reaction (Jensen's modification. 1948) with 24 hr. vigorously growing cultures were obtained by making a t least three successive subcultures. 1953) and Conklin's modification of Wirtz's method (Conklin.8519 and 8520. as yo. and the form of growth in agar stab cultures observed after 48 or 96 hr. . and for the aerococci glucose Lemco broth (Shattock & Hirsch. In addition to the pediococcus cultures. glucose. Eighty-nine isolates (including 39 new isolates from si1age)l were collected. For maintenance of stock cultures. 1953) was suggested by Jensen & Seeley (1954) and by Dr Ellen I. L. diameter) of such a vigorously growing culture/5 ml. 1947) or glucose yeast extract (GY) agar (containing. 1934). A possible relationship between the pediococci and members of the genus Aerococcus (Williams. For use as inocula.6. 1953) of 24 and 48 hr. agar cultures was used to search for capsules. Maintenance o f stock cultures and methods o f cultivation. 1. 0-25. A 'standard' inoculum consisted of one loopful (about 4 mm.w/v. together with the references for the named species. 11 isolates of Aerococcus viridans were studied. 31 of these were representatives of named species.. 1.3. personal communication). arrangement and size of individual organisms were determined in Gram-stained smears prepared from 24 hr. cultures fixed by heat in the usual way. where necessary. Ability to form mucoid colonies on media containing sucrose was tested on the appropriate agar medium to which Seitz-filtered sucrose had been added to a final concentration of 5 yo(w/v).186 H. New transfers were made at 3-monthly intervals. test medium. Muir's technique (Tanner. liquid cultures was determined. were used unless otherwise stated. peptone. 1sodium chloride ( 5 yo. Growth characters in liquid cultures were observed after incubation for 48 or 72 hr. The following were exceptions to this rule: for strain Tc. The incubation period was 24 hr. Cultural characters. 8519. NaC1. Garvie (personal communication). adjusted to pH 6. Yeastrel. Cultures were incubated aerobically except where otherwise stated. Hanging drop preparations of 24 hr. Smears from 72 hr. Morphology and staining reactions. agar. METHODS Sources of cultures. We have therefore included in the survey some Aerococcus strains. WHITE Jun. The shape.0.0. ' Oxoid ' tomato juice (TJ) broth or tomato juice (TJ) agar. a t pH 7. R. agar cultures were examined for the presence of spores according to Fleming's method (Mackie & McCartney. broth cultures were used to determine motility. Stock cultures were maintained as stab cultures and stored a t 4". and was used except where otherwise stated. where this was not practicable isolates considered to be representative were selected for investigation. A list of the isolates is given in Table 1. preparation of inocula and in all experimental work.4) was used. Hirch & Cowan. 1.w/v) was added to the medium. The normal incubation temperature was 30' except for isolates M-1. Mackie & McCartney. for which i t was 22". GUNTHER AND H. The kind of surface colony was noted after incubation for 72 hr.
C-1 2-170. U S A . Dacre. 4-60. Shinfield. 7597. Niven Jun. 1934) - Aerococcus viridans (Williams. 53-524. 1934) P. L-352. S-188. C-2. (3) Dr C. W. Torry Research Station. Shave. L-351. Edinburgh.S. 1953) Air of occupied rooms Milking machines 1 NCTC (1) Professor C. Visual estimation of growth was made after incubation for 24 or 72 hr. C-6. S-533. for slow growing strains. L-24. Hirch RS Cowan. L-92. NCIB = National Collection of Industrial Bacteria. FP-6 P-60 M-1 8519. S-532. S-447. K-64. NCTC = National Collection of Type Cultures. Ithaca. Pederson.. To find the optimum growth temperature the amount of + . SS-101. B-168 A-1. A-181. 3-129. S-182. 7598. (5) Dr M.C. S-101. SS-61 SS-69. N. 1958a) C-1. L-354 7592. University of Chicago. and 7 days of incubation. U S A . N-91 FP-1. L-223. 7602 7764. L-95. L-347. near Reading. 5-51. The Edinburgh and East of Scotland College of Agriculture. (2) Professor H. 1 PUE EJ-1 M-31 A-140. NCDO = National Collection of Dairy Organisms. 7765. Geneva. 6-163. Aberdeen. 7767 P. Gibson. 7601. Shinfield. species name. Cornell University. Holland. salicinaceus (Mees. F. cerevisiae (Garvie.. pentosaceus (PvIees. S-336. D-95. 4-89. S-290. cerewisiae (Pederson. habitat and sources of cultures Code numbers F-166. B-190 SS-50. BP-2 559 (strain A2. near Reading. 1949) P. Growth temperatures. Oxygen requirement. cerewisiae (Pederson. halophilus (Mees. Species name Habitat Fermenting vegetable Spaghetti sauce Fermenting vegetable Summer sausage Unknown Unknown Beer Beer Unknown Horse urine Fermenting cabbage Milk Cheese Air of dairy Air of cowshed Hay Saliva Cheese Silage Silage P. 1050) P. S-333. New York. L-16. S-338.A. 8520 Tc. London. 1934) P. State Agriculture Experiment Station. N. S-190. 7595. L-20. L-171. L-148. all results of experiments to determine the conditions which affected growth were recorded after 24. S-340. C-14 P-45. cerevisiae (Pederson. S-526. S-525. Code number. U. Illinois. L-22. B-137. 1949) P. National Institute for Research in Dairying. darnnosus (Claussen. 48 hr. (4) Technische Hoogeschool. damnosus var. 1949) P. L-345. 6-107. cerevisiae (Pederson. S-18. SS-128 HY -22s BP-1. S-180. E-68. D-32. 7766. S-339. K-106. Table 1. E. S-334. S-344. 5-61. N-82.The pediococci 187 Conditions aflecting growth Except where otherwise stated. 7599.Y. Delft. 1903) P. Duplicate broth cultures were incubated aerobically. 3-124. and anaerobically in an atmosphere of 95 yo (v/v) hydrogen 5 yo (v/v) carbon dioxide.Y. (6) Dr T. 1934) P. 6-159. S-527. Seeley. S-342. P-128. D-118. S. 1949) P. urinae equi (Mees.
The nutrient gelatin medium had the same formula as the nutrient broth. Delwiche & Seeley (1957) recommended that cultures to be used for catalase tests should be neutralized after incubation.2. Haemolysis.) hydrogen peroxide were added and the cultures examined up to 30 min. In the present work. 40" and 45" in water baths controlled to within & 1". a t pH 7. of incubation.1M-glycine buffer recommended by Shattock & Hirsch (1947) were added. cultures were incubated at lo". 1-0. preliminary tests were carried out with 12 isolates of pediococci to compare nutrient broth (containing (yo. Liquefaction of gelatin. medium and incubation was carried out for 24 hr.0 suitable quantities of the 0. Growth at pH 9. Horse blood ( 5 yo. These media were sterilized by Seitz filtration. In view of this and of the recommendations of the above workers. hopped wort and beer. Gutekunst. peptone. 0 4 ~ was selected.6. Pour plates were also made and incubated similarly. a t 22". London) and the beer was commercially available bottled Carlsberg Lager. Growth in wort. . With slow growing strains the results were read after 72 hr. Results were read after incubation for 48 hr. and again after overnight storage at 4".w/v): Yeastrel. The wort and hopped wort (about 6 % hops) were obtained through the courtesy of Mr C.0) with T J broth and GY broth as media for catalase tests. for the medium a t pH 4. The following modifications were made: tomato juice (TJ) broth was substituted for glucose Lemco broth.01.188 H. or 72 hr.05 or 0. when necessary. No qualitative differences were found but the reactions in nutrient broth were sometimes stronger. sodium acetate+acetic acid buffer (Clark. 0. R. 14 and 28 days. Felton. Everitt (Watney Mann Breweries. Known positive and negative control cultures were included in each test series. Biochemical tests Media used for biochemical tests were based on those commonly used for testing lactobacilli since optimal media for pediococci have not yet been devised. NaC1. In these experiments the technique was based on that described by Shattock & Hirsch (1947) for testing growth of streptococci a t pH 9. nutrient broth was retained as the experimental medium.0 and pH 4. 30" and 37" was estimated visually.v/v) agar streak plates were prepared and incubated both aerobically and anaerobically. Stab cultures were incubated at optimum temperature and examined for liquefaction after chilling at 7. with the addition of 14% (w/v) gelatin. Two ml.WHITE growth after incubation for 24 hr. Tolerance to sodium chloride and Teepol. Evans & Niven (1953) found that a medium of low carbohydrate content (YTG) gave a greater number of positive reactions than a medium of high carbohydrate content (APT). since some inhibitory effects were noted at higher concentrations. 0.5 yo(w/v) sodium chloride and in 0. To indicate the range of growth temperatures.3. S. 0. L. GUNTHER AND H. for visible gas bubbles. Ability to grow in 4 and 6.2. Twice the 'standard inoculum' (above) was used for 5 ml. to obtain the medium a t pH 9. hopped wort and beer. of freshly prepared 3 yo (10 vol. 1928) a t 0 . Where consistent with satisfactory results the lactobacillus media were simplified.1 yoTeepol was tested in T J broth cultures. The amount of growth was observed visually in wort.5. Catalase activity..
05. glycerol. Reaction in litmus milk. Hydrolysis of aesculin. The method used was a modification of that outlined in the Difco Manual (1953).05 %. NaCl.The pediococci Reduction of nitrate. w/v) and magnesium sulphate (0. Production of ammonia f r o m arginine. change in pH value or coagulation during 28 days of incubation. and Seitz-filtered carbohydrate added to give 1 % (w/v) final concentration. Cultures were incubated for 6 days and tested for acetylmethylcarbinol by Barritt’s (1936) modification of the Voges-Proskauer test. lactose. Glucose (1 yo.04 % (w/v) bromcresol purple. except that Tween 80 (which according to Jensen & Seeley. The method of Gibson & Abd-el-Malek (1945) was used. Acid and gas production were determined after 7 days of incubation (indicator. Yeast-extract peptone broth (containing. w/v) were added. xylose. Litmus milk cultures were examined for reduction of indicator. Production of carbon dioxide f r o m glucose. Incubation was carried out for 7 days in the medium of Davis (1955) from which salt solutions ‘A’ and ‘B ’ had been omitted. The medium and method described by Hucker (1924) were used. 0.5. 1. The method described by Niven. 5 . glucose. 0 . extracting the sample for 48 hr. sucrose. The carbohydrates tested were : arabinose. The cultures were examined daily for 7 days. Acid once produced was not masked by subsequent production of alkaline substances.w/v) yeast-extract liquid cultures were incubated for 18 days and the final pH values measured electrometrically. raffinose. sorbitol. Production of acetylmethylcarbinol f r o m glucose and f r o m lactose. mannitol.05 %. trehalose. yeast extract.2 %. 0. Utilization of ammonium salts as sole source of nitrogen.. The method of Pederson. The method of Davis (1955) was used. salicin. 1954 is not required by pediococci) and salt solutions ‘A’ and ‘B’ were omitted from the medium. MgSO. T y p e of lactic acid produced. since preliminary results had shown that many isolates. inulin. 0. maltose. especially fresh ones. Tests were carried out in triplicate in 4 ml. Folinic acid requirement. and sodium chloride (0. using the anhydrous salt in 1 yo (w/v) aqueous solution.05. Incubation continued for 14 days. were slow in producing acid. a t pH 7. Six pediococcus strains were examined. MnSO. Peterson & Fred (1926) was followed except that a continuous ether extraction apparatus was used. cultures were examined daily for gas production during a 2-week incubation period. manganese sulphate (0. Final hydrogen ion concentration. fructose. The zinc content of the isolated zinc lactate was determined by the titrimetric method of Kolthoff & Sandell (1950) and the optical rotation determined polarimetrically. Carbohydrate reactions. added after incubation).0) was used as a basal medium for fermentation tests. The medium was tested for the presence of nitrite before incubation and for residual nitrate after incubation. Some isolates grew poorly in this medium but the use of tomato juice broth was considered inadvisable because of its natural content of reducing sugar which might have resulted in the production of acids from compounds other than glucose. w/v). Tests were carried out in the medium of Swartling (1951). dextrin.. Cultures were then tested for the presence of nitrite and of nitrogen gas as described in the Manual for Pure Culture Study (1954). 0. amounts in . modified in one series of experiments by the substitution of glucose for lactose. Smiley & Sherman (1942) was used.0. Yo w/v : peptone.
acidified and coagulated only rarely. P-128. A number of additional features was possessed by some but not all isolates examined. RESULTS A comparison of named pediococcus cultures and unnamed strains showed certain features to be shared by all. pentosaceus. The lactic acid produced by the six isolates tested was optically inactive. and was used in concentrations of 0. 4 3 . Characters common to all pediococci Morphology and staining reaction. of which 27 were cultures received as Pediococcus cerevisiae. L. the results are listed in Table 2. nor did they produce detectable amounts of carbon dioxide from glucose.36 to 1 . BP-1. Surface colonies were greyish-white. Biochemical tests. abundant growth . two distinct physiological groups could be differentiated and there was some indication of a less well-defined third group. Small amounts of steam-volatile acids were produced in addition to lactic acid. were arranged in clusters. On the basis of these additional features. This group includes 38 isolates. A small zone of p haemolysis was produced on blood agar by a few isolates only. BP-2.6 mpg. Growth after 18 hr. NO growth was observable in media in which ammonium salts constituted the sole source of nitrogen. In the media described. these are regarded as characteristic of the genus.190 H. in diameter. SS-128. ~They . No strains were able to grow a t p H 9.0. EEL colorimeter tubes. GMTHER AND H. Mucoid colonies were not formed on agar media containing sucrose. The criteria on which the subdivisions have been based are summarized in Table 3. Wort provided a suitable substrate but the addition of hops exerted some inhibitory effect. Growth was initiated at 10". non-sporeforming and not encapsulated.2 mm. growth was similar under aerobic and anaerobic conditions. SS-50. C-2. With the majority of isolates a zone of 'bleaching' similar to that described by Davis & Rogers (1939) for lactobacilli was noted.WHITE 12 x 80 mm. with a small amount of surface growth. The organisms did not liquefy gelatin.0-15. and the following were unnamed: C-1. for these it was 22'.3and 0. smooth. The reactions given by the aerococci are included for comparison. were non-motile. The results are presented in two sections : (i) characters common to all ' pediococci ' . Litmus milk was reduced. no growth took place in the particular beer used. varying between 0. EJ-1. ranging in diameter from 0. pairs or singly. Growth in stab culture was beaded throughout the entire length of the stab. circular. Characters on which was based differentiation into groups (Tables 2 and 3) Group I .0. Twenty isolates were examined for this requirement. P-45. The organisms were spherical. did not reduce nitrate to nitrite or nitrogen gas. R./ml. The folinic acid used in these experiments was supplied as ' leucovorin ' by Lederle Laboratory Division Ltd. one was received as P. Members of this group were distinguished readily by: size of surface colonies on tomato juice agar. tetrads. Growth conditions. occasionally ovoid. (ii) characters used to differentiate the groups. C-6. low convex with entire margins. The optimum temperature was 30' for all but three isolates. strongly Gram-positive.5 and 1. Cultural characters. of incubation wasmeasured turbidimetrically with an EEL colorimeter.
30" Growth a t 40" +++ + +++ 20/22 .9-6. When only a proportion of strains was tested the number positive/the number tested are given as a fraction TJB = tomato juice broth. of strains 24 P . temp.7-3-9 8/13 + .2 - - Growth in NaCl4 yo (w/v) Growth in NaCl6.4 +++ profuse growth.618 - + + . temp.Table 2 .(7) - + (late) 14/15 + (late) + (late) + (late) 5 + + + (late) Y ++ + - 5 r - - + + + + (15) + (16) + + + 5.(20/20) + - - Catalase NHBfrom arginine Acid from Arabinose Xylose Glucose Fructose Maltose Lactose Sucrose Trehalose Raffinose Inulin Dextrin Glycerol Mannitol Sorbitol Salicin Litmus milk Acid Dye reduction Coagulation Hydrolysis of aesculin AMC from glucose AMC from raactose Final pH in GYB after 18 days Leucovorin requirement 3.(21) ++ + ++ + + + (late) + (late) + (5) 10/14 Growth at 45" Growth a t pH 4. urinae equi PUE 1 IIb Growth on TJB (pH 6.01 yo Growth in Teepol 0-05yo Growth in Teepol 0-1yo +++ + (late) .8 .(7) + . Physiological characters of pediococci and aerococci Pediococci A Aerococci r ~~ I > w H Not grouped L \ Group \ I1a 8 No.(6) - .(22) .6) Opt.012 3.(20120) . 22" Opt. AMC = acetylmethylcarbinol. Figures in brackets refer to number of strains giving indicated reaction when all strains were teste? and not all gave identical reactions.5 yo Growth in Teepol 0.(5) 3-9-5.(7) . + moderate growth.014 .(20/20) + (late) . GYB = glucose yeast-extract broth. ++ good growth.(19) . .(7) . .(23) + (12) .0 - + + + + + + + .
Table 3. absence . S-338. and 33 produced acetylmethylcarbinol from glucose and lactose.. Main characters on which subdivision of pediococci may be based Diameter Opti.05 yo(v/v) did not affect growth but a t 0. Growth abundant . absence of catalase activity. SS-61.cerevisiae P. S-527. TJB = tomato juice broth. L-20. L-345. B-137.dex24 h r .) 1 Species name Growth in TJB P . Group I I . AMC Acid NaCl from from from . S-180. S-335. A-181. S-182.glu. S-339. S-190. They were unable to grow at 45' or in 4 % (w/v) NaCl within 24 hr. nine cose trin +++ +++ + + 11 111 30" 0-3-0-4 0. Catalase activity was demonstrated in 26 isolates.. 25 of these isolates were capable of growth a t 45'.r argi. AMC = acetylmethylcarbinol.+ + - - TJA = tomato juice agar. HY-ZZS. and grew a t pH 4. The 24 isolates listed (S-18. to some degree. SS-101.-h.0 0-3-0. L. L-24. B-190. moderate growth.6-1. S-290. 48 hr. vigorous growth a t pH4. these isolates form a well-defined and easily recognizable group. failed to grow a t 40" within 24 hr. S-340. S-447. production of ammonia from arginine. GUNTHERAND H. This group was less well defined but is tentatively suggested for the following 14 unnamed isolates: L-16. diameter. R. S-344. 'parvulus' P. +++ + Possible group I I I . halophilus? 30" 30' 0. They could be separated from group I1 isolates by: colony size. WHITE in tomato juice broth. consistently low final pH value ( 3 .01 and 0. L-223. Eight isolates in this group exhibited a specific requirement for leucovorin.. for 3 isolates. L-171. L-148. C-14.1 % initiation of growth was delayed by 24 hr.5 % delayed the growth of 27 isolates. Thus. S-188. . showing less vigorous growth in tomato juice broth.6-1-2 +++ Growth pH4. S-191. L-22. A-140. and generally did not produce acid from trehalose. absence of catalase activity. Two subdivisions were recognized within this group. S-334.192 H. All isolates were tolerant of 4 % (w/v) sodium chloride but a concentration of 6. These isolates resembled group I in size of surface colonies on tomato juice agar and abundant growth in tomato juice broth but could be differentiated by their inability to grow a t pH 4.of surface mum colonies growth onTJA Group temp. no acetylmethylcarbinol from glucose or lactose. S-526.2 even when incubated for 7 days and by their failure to produce ammonia from arginine. In addition. SS-69).. S-524. S-532. L-354.2 only after prolonged incubation (up to 7 days). This group consisted of the cultures received as Pediococcus halophilus (Tc. failure to produce acetylmethylcarbinol from glucose or lactose. L-347. 1 ) and the following 32 unnamed isolates: M-31. L-351. 7 3 . L-95. damnosus? P. S-342. S-333. failure to produce ammonia from arginine . L-92. grew poorly in tomato juice broth. (mm. They differed also from the majority of group I isolates in: failure to grow at 45'.. ss-69. S-18.2 +++ - Growth in 4 % (w/v) NH. Its members differed from group I in : producing surface colonies on tomato juice agar of only 0-3-0. L-352. S-525.. The addition of Teepol a t 0. + + good growth..6 0-6-0-8 22' 30" Late Late + ++ Late - - ++ essential 5% ++ - - - almost always + - - . 9 ) in GY broth. unlike the others.4 mm.2. S-533.
They resembled the pediococci. however. 1 was exceptional in its requirement for 5 yo (w/v) sodium chloride. mode of division and high lactic acid-producing capacity. Nine of the 11 isolates were catalase-positive. 1887. Pediococcus urinae equi was indistinguishable in cultural characteristics from members of group I but differed from the majority of those in failure to grow at pH 4.. Lindner. salicinaceus strains 8519 and 8520 were readily distinguishable from the other pediococci by their lower optimum growth temperature of 22'.2. in most of its other reactions it resembled members of group 1 1 1 . higher final pH value in glucose broth (pH 5. These organisms were fairly uniform in character. All members of this genus are easily recognizable by their morphology. (1954) and by Jensen & Seeley (1954). Pediococcus cerevisiae Balcke 1884. that the genus should include a rather wider range of organisms than suggested by Pederson et al. one was negative and one gave a variable reaction. Some isolates did not fall readily into any of the above three groups. Pederson. DISCUSSION Relationship of pediococci to other genera The results of the present work provide additional evidence in support of the recognition of a separate genus Pediococcus as suggested by Balcke (1884). Unlike the pediococci. However. is regarded as capable of growth in wort. 1934. in fact. Pediococcus damnosus strain M-1 and P . The present results show that the leucovorin (folinic acid) requirement. 1903) was the ability to multiply vigorously in beer. in morphology. They did not produce ammonia from arginine and P. suggested by Felton & Niven (1953) and by Jensen & Seeley (1954) to be typical of all pediococci.8 exerted an inhibitory effect).2.T h e pediococci 193 of growth at pH 4. 1949). grew vigorously at pH 9. Pederson (1957) in Bergey's Manual (7th ed. although more recently strains have been described which failed to multiply in this medium (Mees. Another property considered by some investigators to be an outstanding character of pediococci (Balcke.0) and lower resistance to Teepol. 1949. Felton & Niven (1953) and Jensen & Seeley (1954). acidilactici Lindner 1887 will grow in unhopped 13-2 . Claussen. however. Other isolates. darnnosus strain M-I produced acetylmethylcarbinol from glucose but not from lactose. while P . damnosus var. Aerococci. the type species. ability to grow in media containing 4 yo (w/v) NaCl within 24 hr. hopped wort and beer. is. the aerococci grew well in nutrient broth. Shimwell.) describes the genus as producing acidification and some degree of clouding in beer. It may be regarded as a member of a possible subgroup of group I. 1884 . Mees (1934) and more recently by Pederson (1949). restricted only to some isolates within our group I.0 and were highly sensitive to acidity (even pH 6. as defined above. and the two species listed are separated according to their optimum growth temperature and ability to grow in beer and hopped wort. failure to hydrolyse gelatin. Pediococcus halophilus strain Tc. acid production from dextrin. inability to produce ammonia from arginine and acetylmethylcarbinol from glucose or lactose. in the media used all these aerococci were strictly aerobic. to reduce nitrate to nitrite or to utilize ammonia salts as sole source of nitrogen. We think. and in absence of gas formation from glucose.
as indicated in the section on results. Although there is some variability within each proposed group. However. Such differences provide sufficient evidence for separating the two groups but further experimental data are yet required before establishing their separation a t generic or specific level. (1954) suggested two possible additional species of which the first ( a )produced slime. R. GUNTHER AND H.194 H. reduce nitrate to nitrite or nitrogen gas. none showed growth in the particular beer used for the test. Morphology. grew only under aerobic conditions. Subdivision of the pediococci A subdivision of the pediococci into three groups. all of those investigated in the present study have been shown to be stable over a period of at least 18 months. as beers may show wide variation in acidity. and the second ( b ) possessed a higher optimum growth temperature. the aerococci grew well on simpler media. It resembles the lactic acid streptococci in requiring complex media for growth as shown by Jensen & Seeley (1954). experience in the laboratory handling of these organisms has enabled us to recognize easily the three groups by their cultural characters and we feel justified in suggesting the subdivision of the pediococci in this way. (1954)and confirmed in this study. in ethanol and carbon dioxide concentration. is suggested. Pederson et al. acidiluctici . the type species. The differentiation between pediococci and aerococci is less satisfactory on the basis of present results. 1959). The use of the criterion ‘growth in beer’. hydrolysing gelatin or utilizing ammonium salts as sources of nitrogen. the results were in general the same as those previously described. L. is however of little value. and for organism ( b )either P. in contrast to the pediococci. Although biochemical characters are often found to be variable and therefore unreliable as diagnostic criteria. For organism ( a ) the name P. in hop content. viscosus Lindner was suggested. as reported previously (Giinther. liquefy gelatin. and in the degree of ‘attenuation’ which influences the quantity of nutrients available in the medium. but grew profusely in alkaline media (pH 9. without further qualifications as to the kind in which growth is tested. Therefore it seems justifiable to use such characters as differential criteria. utilize ammonium salts as sole source of nitrogen. Thus pediococci can be differentiated from micrococci on the basis of their failure to : grow on simple media. WHITE wort but not in beer. Of the isolates studied in the present work. The present investigation confirms the separation of the genus Pediococcus from the other closely related genera. (1954) serve to differentiate the pediococci from the genus Leuconostoc. Garvie (personal communication) have suggested that the two groups may be related sufficiently to be included in the same genus.0). However. homofermentative character and production of optically inactive lactic acid. were highly sensitive to acid. the pediococci are also clearly distinguishable from streptococci on the basis of morphology and mode of division. as found in the present work and previously by Pederson et al. I n a later paper. The pediococci closely resemble micrococci in morphology and mode of division but may be separated from them by consideration of their biochemical characters. Where isolates obtained by other authors were investigated. Their morphology and mode of division is similar and neither group is capable of reducing nitrate. Jensen & Seeley (1954) and Dr Ellen I.and in being homofermentative as demonstrated by Pederson et al. Pediococcus cerevisiae Balcke. Pederson (1949)and Jensen & Seeley (1954)recognized only one species.
Group I11 is much less well defined. a fifth group might be recognized. cerevisiae Balcke. However. Pederson et al. salicinaceus strains 8519 and 8520 failed to fit into any of the three groups described. Jensen & Seeley (1954) and Dacre ( 1 9 5 8 ~ ) . strain A 2 (NCDO 559) of Dacre (1958a). In the present survey no isolates were observed which produced slime (in presence of sucrose) or had a high optimum temperature. halophilus of Mees (1934) are the same. urinae equi to produce a low final pH value but showed that it was indistinguishable from P. which includes the isolates received as P. No description could be found in the literature of such a species and the name Pediococcus parvulus is suggested. damnosus var.comb. Many authors have found the fermentation of pentoses and salicin to be variable and Pederson (1949) classified such strains as P. Deibel & Niven (1960) described strains of pediococci isolated from meat-curing brines which were salt tolerant and produced dextrorotatory lactic acid from glucose. They suggested that all these organisms should be placed in the genus Pediococcus with the species name Pediococcus homari nov. Pederson (1957) lists two species :the type species P. A culture of the latter organism was not available for comparative study. It is felt that insufficient evidence is at present available on which to base the establishment of this group at specific rank. I n the seventh edition of Bergey's Manual. should additional strains be isolated. to apply the species name P. P. cerevisiae. Further investigation is needed before it can be decided whether this species and the P. Deibel & Niven considered that their strains may be closely related to the marine micrococcus Gaflkya homari (Sniesko & Taylor. W/V. therefore. 1) was quite distinct in charwter (especially in its requirement for 5 %. and strain P-SO previously known as Leuconostoc mesenteroides P-SO but classified recently with the pediococci by Garvie (1959). NaCI) and. 1 and P. hennebergi Sollied. Three isolates received as Pediococcus damnosus strain M. acidilactici Lindner characterized by an optimum growth temperature of 40" and failure to grow in beer.The pediococci 195 Lindner or P. Felton & Niven (1953). pentosaceus (Mees). cerevisiae in most of its morphological and cultural characters. The present study confirmed the inability of P. damnosus. The reactions characteristic of the group I organisms of this study are in general the same as those described for the strains of Pediococcus cerevisiae Balcke studied by Pederson (1949). It would appear justifiable. cerevisiue (group I) in a number of characters (see Table 2. cerevisiae Balcke to this group. The features of the group I1 isolates are sufficiently distinct to warrant the recognition of a separate species. (1954) compared strain Pediococcus urinae equi with their culture of P. They might form the nucleus of a fourth group should other isolates with such characters be noted in future and should be regarded as members of a species P. It . The characters of this group resemble those described by Andrews & Gilliland (1952) for a dextrin-fermenting organism which they named Streptococcus damnosus var. None of them fermented salicin. it may be noted that they all have a low optimum temperature). diastaticus. 1947) and also to Aerococcus viridans. the three isolates we received resembled each other and differed from P. cerevisiae and concluded that this organism should not be included in the pediococci because of lower acid-production properties. The culture received as Pediococcus halophilus (Tc. It can therefore only be regarded as a variant of that species.
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