INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY
October 1972, p. 228-232
Copyright 0 1972 International Association of Microbiological Societies
Vol. 22, No. 4
Printed in U.S.A.
Taxonomic Status of Micrococcus varians
Migula 1900 and Designation of the
Neotype Strain
M. KOCUR and T. MARTINEC
Czechoslovak Collection of Microorganisms, University J. E, Purkynt?, Brno, Czechoslovakia
An amended description of Micrococcus varians Migula 1900 is given on the
basis of a study of 20 strains. CCM 884 (= ATCC 15306 = NCTC 7564) is
designated as the neotype strain of M. varians. Staphylococcus Zactis Shaw et al.
195 1 is a later objective synonym and Microccus pulcher Miiller 1961 is a later
subjective synonym of M. varians Migula 1900.
Recent advances in molecular biology, chemi- Bradford, and Niven (6), and (ii) the medium
cal analyses of the cell walls, and other factors recommended by the ICSB Subcommittee o n the
contributed considerably t o the discovery of Taxonomy of Staphylococci and Micrococci (21). The
so-called natural taxonomic groups (taxa) latter was also employed for the study of other
carbohydrates. The utilization of ammonium tartrate
among the micrococci. Some of these taxa, e.g., was
observed on a medium recommended by Gordon
Micrococcus roseus, were reexamined ( 12) and and Mihm (9). Esculin hydrolysis and nitrite reduction
were found t o form stable points around which were studied by methods recommended by Sneath
a new classification of the genus Micrococcus (20). Gelatin hydrolysis was determined b y the
could be established.
method of Clarke (3). Production of indole, hydrogen
A rather confusing situation remains, how- sulfide, acetoin, phenylalanine deaminase, and arginine
ever, in the classification of yellow-pigmented dihydrolase and the methyl red test and growth on
species of the genus Micrococcus. At present, Simmons citrate agar were studied by methods
two groups can be recognized among them: one recommended by Ewing (7). Oxidase was detected by
the method of Gaby and Hadley (8). Salt tolerance
contains the cell wall peptidoglycan of the was observed on nutrient agar with 5 , 7.5, 10, and
lysine-tetrapeptide type (10, 17) and does not 15% NaCl. Sensitivity to antibiotics was determined
attack glucose; the other has the cell wall by the use of Oxoid Multodiscs.
peptidoglycan of the lysine-alanine3 -4 type (1 0,
17) and attacks glucose oxidatively. The first
RESULTS
group of organisms is considered t o be the
species Micrococcus luteus (1 3). The second
Morphology. All 20 strains examined were
group is without any designation, and the
gram-positive cocci, 0.9 t o 1.5 p m in diameter,
present paper deals with its taxonomy and
arranged in tetrads and in irregular clusters of
nomenclature.
tetrads. Two strains, CCM 1414 and 2133,
formed packets.
MATERIALS AND METHODS
Cultural characteristics. Colonies of 17
Organisms. In the present work, 20 strains of strains were circular, slightly convex, smooth,
aerobic, yellow-pigmented micrococci were investi- and glistening. Colonies of strains CCM 552,
gated (Table 1). All the strains came from the 2 139, and 25 10 were rough, wrinkled, matted,
Czechoslovak Collection of Microorganisms (CCM), and dry. All of the strains produced a light t o
Brno. The strains were maintained on nutrient agar at dark yellow, water-insoluble pigment o n most
4 C. All morphological, nutritional, and biochemical solid media tested. After 3 days of cultivation,
studies and tests were made on cultures grown at 37 C. seven strains formed a brown, water-soluble
Methods. Morphological properties, Gram staining,
pigment on nutrient agar with gelatin. In
starch hydrolysis, and nitrate reduction were determined according to methods described in the Manual nutrient broth, all of the strains produced a
of Microbiological Methods (4).Most of the cultural slight turbidity and sediment.
Biochemical characteristics. The mole % of
and biochemical methods were those described by
Mortensen and Kocur (15). Two different media were the guanine plus cytosine (GC) content of the
used for glucose utilization: (i) the medium of Evans, deoxyribonucleic acid (DNA) of the strains
228
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VOL. 22, 1972
MICROCOCCUS VARIANS MIGULA 1900
229
TABLE 1 . List of strains
Organism
CCM no
Micro coccus co nglomeratu s
547
Micrococcu s co nglomeratu s
var. CY
Micrococcu s p erflavus
Micro co ccus p u lch er
Micrococcus varians
Micro coccu s var ian s
2189
Micro co ccu s va rian s
Micrococcus varians
Micrococcus sp.
Sarcina flava
Sarcina lu tea
Staphylococcus lac t is
Sta p hvlo coccus lact is
Staphvlococcus lactis
2139
2127
418
2132
2492
825
2133
2509
268
552
2490
25 10
884
1395,1411,1414
2141
243 1
Other designations
ATCC 401; NCTC 2677; NCIB 2677;
NCDO 76 1;
G. J. Hucker no. 62
OUT 8092
OUT 8099
ATCC 15936; G. Miiller H-52 (type strain)
J. Arpai no. 173
ATCC 19099; IAM 1392; K. Komagata no. 3-1
ATCC 399, G. J. Hucker no. 80
ATCC 19 100; IAM 1404; K. Komagata no. 6-1
E. C. D. Todd no. 289
UEM Sar. 2/49
ATCC 9341, PCI 1001
ATCC 383, G. J. Hucker no. S85
ATCC 533
ATCC 15306; NCTC 7564; NCDO 777; T. Gibson
strain G 33
M. S. Pohja no. 118; 219 and 185
NCTC 8340; NCTC 7566
NCDO 790; NCTC 7567
a Abbreviations used: ATCC, American Type Culture Collection, Rockville, Md., USA; CCM, Czechoslovak
Collection of Microorganisms, Brno; IFO, Institute €or Fermentation, Osaka, Japan; N U B , National Collection
of Industrial Bacteria, Aberdeen, Scotland; NCDO, National Collection of Dairy Organisms, Reading, England;
NCTC, National Collectiqn of Type Cultures, London, England; OUT, Collection of the Faculty of Engineering,
Osaka University, Japan; UEM, Institute of Epidemiology and Microbiology, Prague, CSSR.
studied ranged from 66.2 t o 72% (average personal communication); (iii) 70% of them
69.8%). All strains produced acid aerobically failed t o transform M. Zuteus ( 1 1; Kloos,
from glucose; they differed, however, in the personal communication); (iv) they produce a
intensity of the terminal pH values, which yellow, water-insoluble pigment; (v) they proaveraged from pH 4.3 t o 5.9. The aerobic duce acid from glucose and some other carboproduction of acid from glucose in the standard hydrates.
These strains were received at the CCM under
medium of the ICSB Subcommittee (21)
developed slowly and occurred in 2 t o 3 days. various names (Table l ) , but they belong to one
Utilization of other carbohydrates as well as the species only.
results of the other tests varied from strain to
A comparison of the characteristics of the 2 0
strain. Of the strains which grew on Simmons strains studied with t h e original descriptions of
citrate agar, five utilized ammonium tartrate micrococci whose names have been validly
and two hydrolyzed arginine.
published revealed that the earliest named
Susceptibility to antibiotics. All of the strains species whose description agrees with these
studied were susceptible t o penicillin, strepto- strains is Micrococcus varians Migula 1900 (14).
mycin, chloramphenicol, tetracycline, erythro- Therefore, the name of the species which
mycin, and novobiocin. Only 10 strains were includes the 20 strains studied here is M.
varian s.
susceptible t o lysozyme.
CCM 884 (= ATCC 15306 = NCTC 7564) is
here designated as the neotype strain of M.
varians Migula 1900. The characters of this
DISCUSSION
strain agree with those recorded in the original
The strains used in this work form natural description of M. varians and are presented
groups and have the following characters. (i) below.
Because CCM 884 is also the type strain of
The moles % GC of their DNA ranges from 66
t o 72% (1,2; BohAzek, Kocur, and Martinec, in Staphylococcus lactis Shaw et al. 195 1 (17), S.
press); (ii) the peptidoglycan of their cell walls lactis is a later objective synonym of M. varians
is of the Lys-Ala3_4 type (10, 17; Schleifer, Migula 1900, for both names are based o n the
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230
KOCUR AND MARTINEC
same type. Furthermore, inasmuch as the type
strain (CCM 21 27 [= ATCC 159361) of Micrococcu s pulcher Muller 196 1 ( 16) has characters
which are in general agreement with those of
the neotype of M. varians, M. pulcher is here
considered t o be a later subjective synonym (a
name based on a different, but similar, type) of
M. varians.
The original description of M . varians is
insufficient in terms of present-day knowledge;
therefore we suggest that it be amended as
follows:
Micrococcus varians Migula 1900, 13 5.
Obj. Syn.: Staphylococcus factis Shaw, Stitt,
and Cowan 195 1, 1022.
Subj. Syn.: Micrococcus pulcher Miiller
1961, 534.
(Percentages in parentheses indicate number
of strains studied which are positive for the
character cited. )
INT, J. SYST. BACTERIOL.
Spheres, 0.9 t o 1.5 y m in diameter, occurring
singly, in tetrads, and irregular clusters of
tetrads. Nonmotile. Nonsporeforming. Gram
positive.
The cell wall contains peptidoglycan of the
lysine-alanine3-4 type ( 10, 17).
Agar colonies: Circular entire, 2 t o 3 mm in
diameter, slightly convex, smooth with glistening surface, yellow, exceptionally matted, and
granular.
Agar slant: Good growth with yellow, waterinsoluble pig men t.
Nutrient broth: Moderate turbidity and sediment are formed.
Chemoorganotroph: Metabolism respiratory.
Produce acid but no gas from glucose and
fructose under aerobic conditions.
Acid production from lactose, maltose, galactose, and sucrose varies from strain t o strain.
Some strains anaerobically produce a weak acid
reaction from glucose in the standard medium
TABLE 2. Biochemical characters of 20 strains of Micrococcus varians'I
'I All strains studied had positive catalase and benzidine tests, produced a yellow, water-insoluble pigment,
produced acid aerobically from glucose and fructose, and grew at 37 C and on a solid medium with 7.5% NaCI.
None of the strains studied produced oxidase, free or bound coagulase, acid from mannitol, arabinose, raffinose,
rhamnose, mannose, salicin, adonitol, sorbitol, glycerol, or inositol; none produced deoxyribonuclease, acetoin,
or phosphatase; none split Tween 80, hydrolyzed esculin, produced hydrogen sulfide, indole, phenylalanine
deaminase, or hemolysis; none gave a positive egg-yolk reaction or grew on a solid medium with 15% NaC1.
Reaction: +, positive; --,negative; 0 , weak
Data by Silvestri and Hill (19); the other data on the GC contents are by BohiEek et al. ( 1 , 2; in press).
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VOL. 2 2 , 1 9 7 2
23 1
MICROCOCCUS VARIANS MIGULA 1900
TABLE 3. Differentiation o,f species o f the genus Micrococcus
Character
Pigment
Growth in 5% NaCl
Aerobic acid from glucose
Nitrate reduction
Acetoin production
Hydrolysis of esculin
M. luteus
M. varians
M. roseus
M. freudenreichii
M. mucilaginosus
Yellow
Yellow
Pink
None
None
-
+
+
+
+
+
-
-
+
+
+
+
-
+
-
-
-
-
of the ICSB Subcommittee (21). Acid and gas
not produced from arabinose, raffinose, rhamnose, salicin, adonitol, mannitol, sorbitol, glycerol, o r inositol.
Final p H in aerobic glucose broth varies with
the strain from 4.3 t o 5.9.
Catalase is produced (100%).
Porphyrin respiratory enzymes are produced
( 100%)(method of Deible and Evans [ 5 3 ).
Acetylmethylcarbinol not produced.
Simmons citrate agar: Growth occurs with
some strains (60%).
Ammonium tartrate is utilized by some
strains (20%).
Methyl red test is positive in some strains
(30%).
Indole and hydrogen sulfide are not produced.
Ammonia usually not produced from arginine.
Nitrates and nitrites are reduced (80%).
Gelatin is hydrolyzed (60%) (method of
Clarke [ 31 ).
Starch is hydrolyzed (50%).
Esculin is not hydrolyzed.
Oxidase is not produced (method of Gaby
and Hadley [ 81 ).
Phenylalanine deaminase and phosphatase
not produced.
Egg-yolk reaction is negative.
Tween 80 is not split.
Urease is produced (90%).
Human and rabbit plasmas are not coagulated.
Hemolysis is not produced.
Pigment: Produce yellow, water-insoluble
pigment (probably a carotenoid).
Good growth between 22 and 37 C.
Good growth on solid media with 0 t o 7.5%
NaC1.
Susceptibility t o antibiotics: Most strains are
susceptible t o penicillin, streptomycin, chloramphenicol, tetracycline, erythromycin, oleandomycin, and novobiocin.
The GC content of the DNA is 66 t o 72% (1,
2; BohaEek, Kocur, and Martinec, in press).
Saprophytic.
Habitat: Soil and water.
Description of ATCC 15306 (= CCM
-
-
-
884 = NCDO 777 = T. Gibson strain G33), the
neotype strain of Micrococcus vavians..
Spheres, 0.9 t o 1.1 pm in diameter, occurring
in pairs and tetrads. Nonmotile. Nonsporeforming. Gram positive.
The cell wall contains peptidoglycan of the
lysine-alanine3.4 type ( 1 7).
Agar colonies: Circular entire, 2 t o 3 mm in
diameter, slightly convex, smooth with glistening surface, yellow.
Agar slant: Good growth with yellow, waterinsoluble pigment.
Nutrient broth: Moderate turbidity and sediment are formed.
Chemoorganotroph: Metabolism respiratory.
Strictly aerobic.
The biochemical characteristics and the GC
composition of the DNA of this strain are given
in Table 2.
Susceptibility t o antibiotics: Susceptible to
penicillin,
streptomycin,
chloramphenicol,
tetracycline, erythromycin, oleandomycin, and
novobiocin. Resistant t o lysozyme.
Micrococcus varians can be distinguished
from other species of the genus Micrococcus by
means of the characters listed in Table 3 .
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