Legend:  + = positive > 90%,     - = negative > 90%, d = variable, w = weak positive reaction
 
Indole
production
Lecithinase
Esculin
hydrolysis
Starch
hydrolysis
H2S
Amygdalin
Galactose
Glucose
Glycogen
Group I
-
-
+
-
+
-
-
+
-
Group II
-
-
-
d
-
dw
dw
+
dw
Group III
d
d
-
-
d
-
dw
+
-
Group IV
-
-
-
-
+
-
-
-
-
Taxonomy
Morphology
Cultural characteristics
Biochemical characters
Ecology
Pathogenicity
References
Phylum Firmicutes, Class Clostridia, Order Clostridiales, Family Clostridiaceae, Genus Clostridium, Cluster I (Clostridium sensu
stricto),
Clostridium botulinum  (van Ermengem 1896) Bergey, Harrison, Breed, Hammer and Huntoon 1923.
Historical synonyms:
Bacillus botulinus van Ermengem 1896,   Ermengemillus botulinus (van Ermengem 1896) Heller 1922,  
Botulobacillus botulinus (van Ermengem 1896) Orla-Jensen 1909.
7
Clostridium botulinum toxin types: A,B,C,D,E,F,G.
There are four metabolic groups, and other species that are phenotypically similar:
Group I - strains of type A, proteolytic strains of types B and F, and
C. sporogenes;
Group II - strains of type E and saccharolytic strains of types B and F;
Group III - strains of types C and D and Clostridium novyi type A;
Group IV - strains of type G and
Clostridium subterminale.
Group I - type A and proteolytic strains of types B and F
Gram-positive, straight to slightly curved rods, 0.6-1.4 x 3.0-20.2 µm. Motility is variable.
Spores are oval, subterminal, and swell the cell; sporulation occurs most readily on
egg-yolk agar plates incubated for 2 days or on chopped-meat agar slants incubated
at 30 ºC for 1 week.
Group II - type E and saccharolytic strains of types B and F
Gram-positive, straight rods, 0.8-1.6 x 1.7-15.7 µm, occurring singly or pairs. Motile by
peritrichous flagella. Spores are oval, eccentric to subterminal, and usually swell the
cell; sporulation occurs readily in broth and on solid media. Type E spores have characteristic appendages and exosporium; spores
of type F have an exosporium.
Group III - type C and type D
Gram-positive, straight rods, 0.5-2.4 x 3.0-22.0 µm, occurring singly and in pairs. Motile by peritrichous flagella. Spores are oval,
subterminal, swelling the cell. Sporulation of most strains occurs most readily on chopped-meat agar slants incubated at 30 ºC for 1
week.
Group IV - type G
Gram-positive, straight rods, 1.3-1.9 x 1.6-9.4 µm , occurring singly and in pairs. Motile by peritrichous flagella. Spores are oval,
subterminal, and swell the cell. Spores are rarely seen and they are not detected in any medium.
Group I
Surface colonies on blood agar plates are 2–6 mm in diameter, beta-hemolytic, flat to raised, translucent to semiopaque, gray,  
circular to irregular, scalloped or rhizoid margin, often with a mottled or crystalline internal structure. Cultures in PYG broth are turbid
with a smooth or flocculent sediment and have a pH of 5.6–6.2 after incubation for 7 days. Ammonia produced from the deamination
of amino acids often masks acid production from carbohydrates. Abundant  growth in nutrient broth or cooked meat broth. Growth is
inhibited by 6.5% NaCl, 20% bile, pH of 8.5 and  some strains of
C. perfringens and C. sporogenes isolated from soil. Optimum
temperature 30-40 ºC. Growth at 25 or 45 ºC is variable. Products in PYG broth: acetic and butyric acids.
Group II
Surface colonies on blood agar plates are beta-hemolytic, 1–5 mm diameter, irregular with lobate or scalloped margins, raised,
translucent to opaque, gray-white, with a mottled or mosaic internal structure. Cultures in PYG broth are turbid with a smooth
sediment, and have a pH of 5.2–5.5 after incubation for 1–2 days. Poor to moderate  growth in nutrient broth or cooked meat broth;
abundant growth in broth with fermentable carbohydrate. Growth is inhibited by 6.5% NaCl,  20% bile,  pH of 8.5 and  strains of
C.
perfringens
isolated from soil. Optimum temperature 25-37 ºC. Growth at 5 or 45 ºC is weak and variable. Pproducts in PYG broth:
acetic and butyric acids, hydrogen.
Group III
Surface colonies on blood agar plates are beta-hemolytic, 1–5 mm in diameter, circular to slightly irregular, slightly scalloped or
lobate, flat to raised, translucent, gray-white, with a mottled or mosaic internal structure. Cultures in PYG broth are turbid with a
smooth or flocculent sediment and have a pH of 5.2–5.7 after incubation for 1–2 days. Growth is inhibited by 6.5% NaCl, 20% bile, pH
of 8.5. Growth is stimulated by a fermentable carbohydrate. Optimum temperature 30-37 ºC. Growth at 25 or 45 ºC is weak and
variable. Products in PYG broth: acetic, propionic and butyric acids, hydrogen.
Group IV
Surface colonies on blood agar plates are beta-hemolytic, 1–4 mm in diameter, circular to irregular, lobate to filamentous, raised,
translucent, smooth, and shiny. A spreading film may cover the entire plate. Large, rough, fried-egg colonies may be formed.
Cultures in PYG broth are turbid with a smooth white sediment and have a pH of 6.2–
6.3 after 5 days incubation. Growth is inhibited by 6.5% NaCl, 20% bile. Moderate gas
production detected in PYG deep agar cultures.
Optimum temperature 30-37 ºC. Can grow at 25 or 45 ºC. Products in PYG broth: acetic,
isovaleric, isobutyric, phenylacetic and butyric acids, hydrogen.
Isolated from soil,  marine and lake sediments,  animal, bird, and fish intestines and carcasses, feces, food (particularly improperly
preserved vegetables, meat, and fish), rotting vegetation. Susceptible to chloramphenicol, tetracycline, penicillin (strains of group I)
Susceptible to chloramphenicol, clindamycin, erythromycin, penicillin, metronidazole, rifampin, cefoxitin and tetracycline (groups II, III
& IV). Resistant to nalidixic acid and gentamicin.
All strains produce neurotoxins with similar effects on an affected host, but the toxins of the different types are serologically distinct.
Some toxins, particularly those of the nonproteolytic strains, require trypsin activation for effectiveness in laboratory toxin testing.
Human disease (botulism) is caused by toxins elaborated under anaerobic conditions usually by colonization of food, less often by
colonization of wounds, or colonization of the intestinal tract as in infant botulism. The toxin of
C. botulinum type C is inactivated by
bacteria in the rumen of cattle and sheep. Toxin production by type C and D (group III) is phage-mediated.
Pathogenic for laboratory animals.
  1. N.A. Logan and P. De Vos, 2009. Genus I. Clostridium Prazmowski 1880. In: (Eds.) P.D. Vos, G. Garrity, D. Jones, N.R. Krieg, W.
    Ludwig, F.A. Rainey, K.-H. Schleifer, W.B. Whitman. Bergey’s Manual of Systematic Bacteriology, Volume 3: The Firmicutes,
    Springer, 738-828.
  2. Smith L.D.S. and Hobbs G., 1975. Genus III. Clostridium Prazmowski 1880. In: (Eds.) Buchanan R.E. and Gibbons N.E., Bergey’s
    Manual of Determinative Bacteriology, Eighth Edition , The Williams & Wilkins Company, Baltimore, 551-572.
  3. Macovei A., 2009. Identificarea bacteriilor anaerobe. In: Tratat de Microbiologie Clinica (Ed. Buiuc D. si Negut M.), editia a IIIa,
    Editura Medicala, Bucuresti, 900-927.
  4. Secasiu V., 2001. Boli produse de germeni din genul Clostridium. In: Boli infectioase ale animalelor, Moga Manzat R., Ed. Brumar,
    Timisoara, 481-612.
H2 is produced very abundantly. Milk reaction and meat digestion are positive for
group I, negative for group II, and variable for groups III and IV.

Positive results for H
2 production, casein hydrolysis (group IV) and gelatin hydrolysis.
Negative results for nitrate reduction, urease, Voges-Proskauer test, substrate utilized
and/or acid produced from: arabinose, cellobiose, dulcitol, lactose, mannitol,
raffinose, rhamnose, salicin, sorbose & xylose.
Variable results (but negative for group IV) for: fructose, glycerol and maltose.
Clostridium botulinum
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Differential characters of the Clostridium botulinum biotypes:
 
Inositol
Inulin
Mannose
Melibiose
Ribose
Sorbitol
Starch
Sucrose
Trehalose
Group I
-
-
-
-
-
dw
-
-
-
Group II
dw
dw
dw
-
d
dw
d
dw
dw
Group III
dw
-
dw
dw
d
-
-
-
-
Group IV
-
-
-
-
-
-
-
-
-