Bacitracin and Optochin Tests

Bacitracin Test

Beta-hemolytic Streptococcus
are spherical bacteria that
produce hemolysins capable of completely
lysing (bursting open) red blood cells. When grown on sheep blood agar,
colonies of beta-hemolytic Streptococcus are encircled by visible areas of clearing where beta hemolysis has occurred.

Beta-hemolytic streptococci may be pathogens or non-pathogens. Group A Streptococcus (Streptococcus pyogenes) causes
"strep throat". Strep throat may lead to delayed sequelae such as
rheumatic fever, glomerulonephritis, and/or scarlet fever. The bacitracin susceptibility
test is used to distinguish Group A streptococci, which cause ninety
percent of acute streptococcal infections in humans, from other
streptococci. When grown on blood agar, Group A streptococci are
sensitive to (killed by) the antibiotic bacitracin . A sterile disk
impregnated with bacitracin is placed on the first sector of an
isolation plate before incubation. A zone of inhibition (area with no
growth) will be seen around the disk after incubation if the organism
is a Group A beta-hemolytic Streptoccus. Other beta-hemolytic
streptococci are resistant to (not killed by) bacitracin. Their
colonies will thus grow right up to the disk of bacitracin.




Blood agar plate
Group A Streptococcus and Group C Streptococcus
with bacitracin disks

Optochin Sensitivity and Bile
Solubility


Alpha-hemolytic Streptococcus are spherical
bacteria that produce hemolysins capable of partially lysing (bursting) red blood cells.
When grown on sheep blood agar, colonies of alpha-hemolytic Streptococcus
are encircled by green areas of partial clearing where alpha hemolysis
has occurred.

Alpha-hemolytic Streptococcus may be pathogens,
such as Streptococcus pneumoniae, or part of the normal flora of
the upper respiratory tract ("viridans Strep", e.g. Streptococcus mitis).
Streptococcus pneumoniae is the major cause of community-acquired
bacterial pneumonia and the most frequent cause of otitis media (middle
ear infections) and bacteremia in infants and children. Thus it is important
to distinguish Streptococcus pneumoniae from other alpha-hemolytic
streptococci that may be normal flora.
Two tests are commonly employed for distinguishing
Streptococcus pneumoniae from the viridans strep it resembles: the
optochin test and the bile solubility test.








The Optochin Test

Optochin (ethylhydrocupreine hydrochloride), a quinine
derivative, has a detergent-like action and causes selective lysis of pneumococci.
A sterile disk impregnated with optochin is placed on the first sector
of an isolation plate before incubation. A zone of inhibition (area with
no growth) of 14 mm. or more in diameter will be seen around the disk after
incubation if the organism is Streptococcus pneumoniae. Other alpha-hemolytic
streptococci are resistant to (not killed by) optochin. Their colonies
will thus grow right up to the disk of optochin or have zones of inhibition
less than 14 mm. in diameter.



The Bile Solubility Test

Bile will selectively lyse colonies of Streptococcus
pneumoniae while other strep are immune to bile's activity. When a
bile salt such as desoxycholate is added directly to Streptococcus
pneumoniae growing on an agar plate or in a broth culture the bacteria
will lyse and the area become clear. Other alpha-hemolytic streptococci
are resistant to (not lysed by) bile and will stay visible or turbid (cloudy).