Cultivation of Streptococcus, Enterococcus, and Similar Organisms
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p253-255
2025-05-17
416
Media of Choice
Except for Abiotrophia and Granulicatella, the organisms discussed in this chapter will grow on standard laboratory media such as 5% sheep blood and chocolate agars. They will not grow on MacConkey agar but will grow on gram positive selective media such as CNA (Columbia agar with colistin and nalidixic acid) and PEA (phenylethyl alcohol agar).
Abiotrophia and Granulicatella will not grow on blood or chocolate agars unless pyridoxal (vitamin B6) is sup plied either by placement of a pyridoxal disk, by cross streaking with Staphylococcus, or by inoculation of vitamin B6–supplemented culture media.
Blood culture media support the growth of all of these organisms, as do common nutrient broths, such as thioglycollate or brain-heart infusion. Blood cultures that appear positive and show chaining gram-positive cocci on Gram stain but do not grow on subculture should be resubcultured with a pyridoxal disk to consider the possibility of Abiotrophia or Granulicatella bacteremia.
Other selective media are available for isolating certain species from clinical specimens. For isolating group A streptococci from throat swabs, the most common medium is 5% sheep blood agar supplemented with trimethoprim-sulfamethoxazole (SXT) to suppress the growth of normal flora. However, this medium also inhibits growth of groups C, F, and G beta-hemolytic streptococci.
To detect genital carriage of group B streptococci during pregnancy, Todd-Hewitt broth with antimicrobials (gentamicin, nalidixic acid, or colistin and nalidixic acid) is used to suppress the growth of vaginal flora and allow growth of S. agalactiae following subculture to blood agar. LIM broth is one medium formulation used for this purpose.
Differentiation of Enterococci and group D streptococci is traditionally based on the ability of the organisms to hydrolyze the glycoside esculin to esculetin and dextrose. The Esculetin reacts with an iron salt to form a dark brown precipitate surrounding the colonies. Enterococcosel agar is a selective differential medium based on the esculin hydrolysis and is also selective by incorporation of inhibitory oxgall (bile salts) for other gram positive organisms and sodium azide for gram-negative organisms.
Incubation Conditions and Duration
Most of the organisms within this group are facultative anaerobes with some preferring a CO2-enriched environment. Laboratories typically incubate blood or chocolate agar plates in 5% to 10% carbon dioxide. This is the preferred atmosphere for S. pneumoniae and is acceptable for all other genera discussed in this chapter. However, visualization of beta-hemolysis is enhanced by anaerobic conditions. Therefore, the blood agar plates should be inoculated by stabbing the inoculating loop into the agar several times (Figure 1, A). Colonies can then grow throughout the depth of the agar, producing subsurface oxygen-sensitive hemolysins (i.e., streptolysin O) (Figure 1, B). Most organisms will grow on agar media within 48 hours of inoculation.
Fig1. Stabbing the inoculating loop vertically into the agar after streaking the blood agar plate (A) allows subsurface colonies to display hemolysis caused by streptolysin O (B). *Based on the reactions of only one isolate. S. bovis variant includes S. infantarius subsp. infantarius, S. lutetiensis, and S. gallolyticus subsp. pasteurianus. Most S. bovis variant strains will be positive for α-galactosidase and S. salivarius will be negative.
Optochin test must be performed in CO2 to avoid misidentification with S. pseudopneumoniae.
Colonial Appearance
Table 1 describes the colonial appearance and other distinguishing characteristics (e.g., hemolysis) of each genus on 5% sheep blood agar. The beta-hemolytic streptococci may have a distinctive buttery odor.
Table1. Colonial Appearance and Characteristics on 5% Sheep Blood Agar
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