When a specimen, which may be urine, stool, other body fluid (CSF, synovial, pleural), as well as swabs from a wound, surgical incision, or specific area of the body (vaginal, eye, nares)is brought to the micro laboratory for culture it is inoculated on a nutrient plate or in a nutrient broth for growth, as well as specific complex, selective, or differential types of media. Some media (agar plates) contain substances that enhance the growth of certain organisms, while inhibiting the growth of others; some are highly selective for species; and others still can differentiate between species. Inoculating onto these different types of media can then guide the growth and identification of pathogens that are usually prevalent in a specific culture type.
For example, Streptococcus agalactiae, also known as Group B Strep or GBS, is a normal flora of the digestive, urinary, and genital tracts, that is found in 20 - 40% of women. While it usually causes no illness in carriers of the bacteria, a pregnant woman colonized with GBS late in her pregnancy can pass it to her baby with very serious outcomes. Newborn GBS most often infects the lungs, blood , and spinal fluid, causing pneumonia, sepsis, and meningitis, respectively. (ACOG, 2014)
Because a vaginal swab will usually contain a number of mixed bacterial flora, it is important to isolate a pure culture of the organism, which can be used for testing and organism identification. If the culture isn’t pure, the chances of incorrect results of biochemical tests can lead to misinterpretation and misdirection of the investigation process of determining organism identification. And if the micro tech is going down the wrong path, the results will not make sense, costing the tech, hospital, and patient valuable time and money.
So on day one, a vaginal swab for GBS is inoculated in LIM broth, which is a selective, enrichment medium that contains additives to promote the growth of gram positive organisms and inhibit the growth of gram negative organisms. (Beckton-Dickinson, 1/2011) After five hours of incubation an aliquot from the broth is subbed onto Columbia CNA agar with 5% sheep blood agar (SBA),and streaked for isolation. (*see procedure below) Due to its constituents, CNA/SBA agar will further promote the growth of staphylococci and streptococci. (Beckton-Dickinson, 9/2011) The plate is then incubated for 18-24 hours, at which point it will most likely present with mixed colony types, exhibiting different characteristics. Sheep blood is a necessary constituent of the agar because it is the standard medium used for describing colony characteristics. Each of the isolated colony types, or the suspect pathogen to the trained eye, can then be aseptically picked up with an inoculating loop and subbed onto a new CNA/SBA plate . An additional SBA plate may also be subbed with the suspected pathogen, along with Staph aureus in what is called A CAMP test, which distinguishes GBS from other β-hemolytic Streptococcus. (Morello, 2013) After incubation the pure culture of the organism will used for biochemical testing and presumptive identification.
Colony morphology on blood agar includes size, shape, color, margin (edge-pattern), elevation, and texture of colonies. Leboffe and Pierce’s Photographic Atlas for the Microbiology Laboratory (2011) defines bacterial morphology characteristics as follows.
Shape may be described as circular, irregular, or punctiform.
The margin may be entire (smooth with no irregularities), undulate (wavy), lobate (lobed), filamentous, or rhizoid (branched like roots).
Elevations include flat, raised, convex, pulvinate (very convex), and umbonate (raised in the center). Texture may be moist, mucoid, dry, butyrous (butter-like), and viscid (sticky), rough, or rugose (wrinkled) but may also contain other descriptors that are typical of certain species, such as ground-glass appearance (Bacillus anthracis) and fried egg appearance (Yersinia pestis).
Pigment production may be combined with optical properties, such as shiny, glistening, or dull.
Also the type of hemolysis is an important factor for organism identification. Some bacteria produce exoenzymes that lyse red blood cells(RBCs) and degrade hemoglobin. Hemolysis is a zone around the colony where the exoenzymes have caused the sheep red blood cells in the agar to rupture (lyse). The three types are alpha (α-hemolysis), which partially breaks down the RBCs, presenting as a small greenish zone around the colonies; beta (β-hemolysis) is caused by the RBCs being completely broken down, which can be characterized by a large clear zone surrounding the colonies, or by a smaller opaque zone, and anything in between; and gamma (ϒ-hemolysis) is no hemolysis of the organism, although some colonies that do not produce hemolysins will still appear to have a gray area surrounding them. This is not be confused with the greenish zone of α-hemolysis.

S.agalactiae on SBA appear as medium gray-white circular colonies with umbonate darker centers that may be slightly orange in color(kind of like a bulls-eye). They are β-hemolytic , with a small, opaque zone of hemolysis. Some strains of GBS may be ϒ-hemolytic as well.
A simple biochemical test known as a catalase test can be used to differentiate staphylococcus from streptococcus. Staph spp. are catalase positive and Strep spp. are catalase negative. Catalase positive organisms contain catalase, the enzyme that breaks hydrogen peroxide (H2O2) into oxygen (O2) and water (H2O), which causes a bubbling reaction when the two are mixed together.
Catalase negative organisms, such as GBS, do not contain the enzyme , and therefore show no reaction. (Reiner, 2013) Further confirmation of the correct organism selection can be made by a gram stain. Under a microscope S.agalactiae appear as gram positive cocci arranged in pairs or short chains and are non-motile and non-spore forming.
A streak plate method is used for the isolation of colonies on agar, which can then be transferred to another plate for a pure culture. A streak plate is made by placing a drop or a loopful of the specimen, onto the agar at the top part of the plate. You can also take the original swab and roll it back and forth on the agar at the top of the plate. Then using a loop spread the specimen by going back and foth across the agar, but without going overtop of any previously made streak. This is known as the initial streak. Then utilizing only half of the initial streak, make another streak using the same pattern, in what is called the second quadrant. The third streak is made using half of the specimen from the second streak, but now you should begin to open up the area in between each streak made. This process is repeated a fourth time, in the fourth quadrant. If done correctly, there will be easily distinguished isolated colonies available for description and inoculation onto a purity plate. (Katz, 2013)

REFERENCES

American College of Obstetricians and Gynecologists. (2014). Frequently asked questions: Group B Streptococcus and pregnancy. Retrieved April 11, 2014 from https://www.acog.org/~/media/For%20Patients/faq105.pdf?dmc=1&ts=20140413T0112587288

Becton-Dickinson. (Revised: September, 2011). Package insert: BD Columbia CNA Agar with 5% Sheep Blood, Improved II.

Beckton-Dickinson. (Revised: January, 2011). Package insert: BBL Lim broth.

Katz S. (Last update: April 1, 2013). The streak plate protocol. Retrieved April 12, 2014 from http://www.microbelibrary.org/component/resource/laboratory-test/3160-the-streak-plate-protocol

Leboffe MJ. and Pierce BE. (2011). Bacterial growth patterns on agar, In A Photographic Atlas for the Microbiology Laboratory, (4th ed). Englewood: Morton Publishing.

Reiner K. (Last update: April 1, 2013). Catalase test protocol. Retrieved April 12, 2104 from http://www.microbelibrary.org/library/laboratory-test/3226-catalase-test-protocol

Morello J. (2013). Lab Manual and Workbook in Microbiology: Applications to Patient Care. VitalSource Bookshelf . Retrieved April 12, 2014 from http://digitalbookshelf.southuniversity.edu/books/125928896X/epubcfi/6/56