VMC 311: Systematic Veterinary Bacteriology and Mycology (2+1)

CULTURAL CHARACTERISTICS

• Anaerobic jars with a catalyst, an anaerobic indica­tor and an atmosphere free of oxygen (10% Hydrogen, 5% Co2 and 85% Nitrogen) is essential for the culture of these strict anaerobes.
• Enriched blood agar is highly suitable for these fas­tidious anaerobes.
• Eugon, Columbia, trypticase soy or brain-heart infusion agar enriched with 0.5 per cent yeast extract, vitamin K (10 µg/ml), haemin (5 µg/ml), kanamycin (100 µg/ml) and vancomycin (7.5 µg/ml) are most commoly used.
• Bacteroides spp. (except B. ureolyticus) are resistant to kanamycin but the Fusobacterium spp. is sensi­tive to this antibiotic.
• A 'Fastidious Anaerobe agar' is available commercially with various antibiotic supplements, depending on the anaerobe that is being sought.
• Eugon agar base with 0.2 per cent (w/v) yeast extract, 10 per cent defibrinated horse blood and one µg/ml lincomycin is the selective medium for Bacteroides nodosus.
• Lemco agar containing pulverized hoof powder can also be used. Members of the B. fragilis group will grow on bile aesculin medium with 5 per cent sheep blood.
• Agar media should be pre-reduced in an anaerobic jar for 6-24 hours before use. The plates are incubated at 35-37°C immediately after streaking for 4-8 days.
• Liquid media such as Cooked meat broth with 0.4 per cent glucose or thioglycollate medium is also suitable with the addition of the vitamin K-haemin supple­ment.

Colonial morphology and microscopic appearance

• The cellular morphology, and sometimes the colonial morphology, can be very variable depending on the strain, medium and cultural conditions.

Bacteroides nodosus

• B. nodosus in a Gram-stained smear from enriched blood agar, appears as straight or slight­ly curved rods with the characteristic terminal knobs on one or both ends of the cells
• Three basic colonial types are described
  • B-type: papillate or beaded (most pathogenic) from ovine foot rot.
  • M-type: mucoid (less pathogenic) from non-invasive infections of sheep and cattle.
  • C-type: circular (non-pathogenic) and resulting from repeated passage in media.
  • The colonies, generally, are greyish-white and 0.5-3.0 mm, diameter, in 3-7 days.

Bacteroides melaninogenicus

• Circular, entire, convex and shiny colonies, 0.5- 2.0 mm in diameter.
• Colonies become darker after 5-14 days, being black in the centre with a grey-brown periphery.
• Haematin (black or brown) pigment is seen best on media containing blood.
• A few strains are haemolytic on rabbit blood agar. The colonies fluoresce under ultra -violet light.

Bacteriodes asaccharolyticus

• Colonies are 0.5-1.0 mm in diameter, round, convex, opaque and light grey after 48 hours' incubation.
• In 6-14 days the colonies may become black. Some strains are haemolytic on rabbit blood agar

Bacteroides fragilis

• Colonies are circular, entire, low convex, translucent to semi-opaque.
• They tend to have concentric rings of growth. Less than 1 % of strains are haemolytic.

Fusobacterium necrophorum

• F. necrophorum produces grey to yellowish shiny colonies on blood agar, that are about 2-3 mm in diameter after 48 hours' incuba­tion.
• Haemolysis is variable.
• A Gram-stained smear from the colonies shows long Gram-negative filaments that are less characteristic than those from direct microscopic exam­ination of specimens.
• Lipase, but not lecithinase, activi­ty is exhibited by many strains of F. necrophorum on egg yolk agar.