Identification of Gram-Positive Bacteria 1-Streptococci

DISTINCTIVE CULTURE CHARACTERISTICS
The Streptococci require more nutrients than the Staphylococci. Primary plating of oropharyngeal swab samples should be on BAP (Blood Agar) and CA (Chocolate Agar: heated blood agar). The Nutrient Agar plates (NA) that were adequate for the growth of the Staphylococci are not sufficient to support the group of many of the Streptococci.
β-hemolysis on BAP will be accentuated by stabbing the agar after streaking (increases activity of streptolysin O, which is oxygen labile). Colony morphology will likely be a pinpoint colony appearance. Viridans Streptococci and S. pneumoniae will produce α-hemolysis. Gram staining will reveal purple-staining cocci that are smaller than the Staphylococci. S. pneumoniae is lancet-shaped and typically occurs in pairs. Streptococci grown in broth culture will appear as long chains of cocci upon Gram-staining. Older cultures may appear Gram-negative due to autolysis.
Most members of this group also require low oxygen tension. This means that they will grow best in the presence of 5% CO2, a condition that mimics the human body. This can be accomplished either by using a CO2 incubator or a candle jar placed in a normal incubator. A candle jar is a chamber in which oxygen is depleted by lighting a candle, closing the lid, and allowing the candle to be extinguished when it has consumed the available oxygen. This does not establish an anaerobic condition, merely a microaerophilic one. Stabbing the agar with the innoculating loop after the streak is another technique that facilitates Streptococcal growth.
No special considerations are required for specimen collection and transport when Streptococci are the suspected etiologic agent of infection. Antigen detection methods are most frequently used for identification of patient swab samples, including latex agglutination, and ELISA. The "C" substance recognized by the Lancefield grouping sera is a polysaccharide, and this permits the arrangement of the streptococci into a number of antigenic groups identified as Lancefield group A, B, C, D and so forth. In most clinical laboratories only groups A, B, and D are routinely identified since these groups are responsible for most infections. Culture should be done with negative rapid antigen testing.
APPROACH TO IDENTIFICATION OF THE STREPTOCOCCI
Several methods allow discrimination among the Streptococcus spp. The catalase test distinguishes Staphylococci from Streptococci because Streptococci do not produce catalase. Go to Staphylococcal Identification for the catalase test procedure. Once test methods have indicated that the bacteria in question is a Streptococcus, a handful of presumptive tests that correlate highly with serological identification methods are typically used to identify bacterial species.
HEMOLYSIS ON BLOOD AGAR
Blood agar is considered to be both a nutritive and differential medium. Many bacteria produce extracellular enzymes that lyse red blood cells present in the agar (hemolysis). The appearance of hemolysis on blood agar plates (BAP) is characteristic for many types of bacteria, especially the pyogenics. Complete clearing in a zone around bacterial colonies is termed βhemolysis. Partial hemolysis produces a zone of greenish tint around the colony ( α hemolysis). Bacteria lacking hemolysin produce no halo around the colonies ( γ hemolysis or nonhemolysis).
MATERIALS:
• Blood agar plate (BAP) plates
• Sterile cotton swabs in sterile saline
Use the moistened swab to rub gently back and forth with a sweeping motion over the oropharyngeal mucosa. You will probably active the gag reflex, so be quick and careful. Use the swab to deposit the material collected onto the surface of the sterile BAP. Use a flamed and cooled innoculating loop to spread the sample over the plate and accomplish isolating dilution. Incubate for 48 hours at 37oC with a CO2 environment.
PRESUMPTIVE TESTS FOR GROUP A STREPTOCOCCI
BACITRACIN SUSCEPTIBILITY TEST
The growth of Group A streptococci is inhibited by a low concentration (0.02 to 0.04 units) of bacitracin in paper disks on blood agar medium, but most other streptococci are not inhibited. Use of low-concentration bacitracin or "Taxo A" disk was previously employed in clinical laboratories for the presumptive Group A streptococci identification. The diameter of a zone of inhibition around the disc indicates sensitivity (>10 mm = sensitive). However, recently it has been recognized that several group C and G Strep. are also sensitive to bacitracin.
The PYR test uses L-pyrrolidonyl-α-naphthylamide substrate to detect the presence of the enzyme L-pyrroglutamyl-aminopeptidase. This test is considered to be more reliable than bacitracin sensitivity. It is performed in much the same way as the oxidase test, using a colony smeared on filter paper impregnated with the substrate. A drop of indicator reagent should produce a red color within 5 min. if the colony is Streptococcus pyogenes.
The laboratory identification of Group B hemolytic streptococci has been simplified by implementation of the CAMP test which is easy to perform and well within the capabilities of small laboratories.
PRINCIPLE:
The hemolytic activity of staphylococcal-hemolysin on red blood cells (RBC) is enhanced by an extracellular factor produced by Group B streptococci, called the CAMP factor. Therefore, wherever the two reactants overlap, an accentuation of the ß-hemolytic reaction will be noted.
PROCEDURE:
The CAMP test is performed by making a single streak of the streptococcus perpendicular to a strain of Staphylococcus aureus that is known to produce ß-hemolysin. The two streak lines must not touch one another. The inoculated plate must be incubated with an ambient atmosphere (room temperature). The plate should not be incubated in an anaerobic environment because many Group A streptococci are positive in the absence of O2. Any Bacitracin-negative, CAMP-positive, bile-esculin-negative streptococcus can be reported as: Group B streptococcus, presumptive by CAMP.
Positive control: S. agalactiae.
PRESUMPTIVE TESTS FOR GROUP D STREPTOCOCCI
Lancefield Group D streptococci are divided into two groups: (1) Enterococci, and (2) nonenterococci. Enterococcus faecalis and Enterococcus faecium are the species of Enterococcus most commonly isolated in laboratories which identify Enterococci. However, many laboratories do not attempt to differentiate them into species, but simply report "Enterococcus". Similarly, Strep. bovis and Strep. equinus may not be differentiated, but reported as "Group-D streptococci, not Enterococci".

Bile-Esculin Hydrolysis Test
The purpose of this test is to determine the ability of an organism to hydrolyze the glycoside esculin to esculatin and glucose in the presence of bile (10 - 40%). This test aids in the differentiation of group D streptococci from other "not group D streptococci".
PROCEDURE:
1. Inoculate the organism to be tested into the bile esculin
medium. Incubate at 37oC for 24 hours (stab into medium,
then streak on slant).
INTERPRETATION:
Positive Test: Presence of a black to dark brown color on the slant (half or more of the medium is blackened) -- (Enterococcus faecalis)
Negative Test: No blackening of the medium OR blackening of less
than half the tube after 72 hours of incubation -- (Streptococcus agalactiae)
Salt Tolerance Test for Enterococci
This test is based on the ability of the enterococci to grow in 6.5% NaCl and separates them from the "Group-D streptococci, not enterococci".
PROCEDURE:
Inoculate a loopful of organism to be tested into the 6.5% NaCl broth and incubate at 37oC for 18 hours (overnight).
Positive control: Enterococcus faecalis