Granada Medium
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Granada medium is a selective and differential culture medium designed to selectively isolate '' Streptococcus agalactiae'' (Group B streptococcus, GBS) and differentiate it from other microorganisms. Granada Medium was developed by Manuel Rosa-Fraile et al. at the Service of Microbiology in the Hospital Virgen de las Nieves in Granada (Spain). Identification of GBS on granada medium is straightforward and relies on detection of granadaene, a red polyenic pigment specific of GBS. Granada medium is marketed in the US by Hardy Diagnostics and in the European Union and UK as a trade mark (®) by Biomerieux and
Becton Dickinson Becton, Dickinson and Company (BD; also Becton Dickinson or Becton) is an American multinational medical technology company that manufactures and sells medical devices, instrument systems, and reagents. BD also provides consulting and analytic ...
.


Composition

pH 7.45±0.1


Background and principles

Granada medium was developed for selective isolation and identification of GBS from clinical specimens. Production of a red pigment ( granadaene) on granada medium is unique to β-
hemolytic Hemolysis or haemolysis (), also known by several other names, is the rupturing (lysis) of red blood cells (erythrocytes) and the release of their contents (cytoplasm) into surrounding fluid (e.g. blood plasma). Hemolysis may occur in vivo ...
group B streptococci isolated from humans. Granadaene is a non-
isoprenoid The terpenoids, also known as isoprenoids, are a class of naturally occurring organic chemicals derived from the 5-carbon compound isoprene and its derivatives called terpenes, diterpenes, etc. While sometimes used interchangeably with "terpene ...
polyenic pigment (ornithinrhamnododecaene) with a
conjugated system In physical organic chemistry, a conjugated system is a system of connected p-orbitals with delocalized electrons in a molecule, which in general lowers the overall energy of the molecule and increases Chemical stability, stability. It is Reson ...
of 12 double bonds. β-hemolysis and pigment production are encoded in GBS by a
gene cluster A gene cluster is a group of two or more genes found within an organism's DNA that encode similar peptide, polypeptides or proteins which collectively share a generalized function and are often located within a few thousand base pairs of each othe ...
of 12 genes, the ''cyl'' cluster. Moreover, it has been suggested that GBS pigment and hemolysin are identical or closely related molecules and it has also been reported that they are important factors contributing to GBS virulence.


Components

Granada agar consists primarily of a
proteose A proteose is any of various water-soluble compounds that are produced during in-vitro or in-vivo hydrolytic breakdown of proteins a little before producing amino acids Amino acids are organic compounds that contain both amino and carbox ...
peptone
starch Starch or amylum is a polymeric carbohydrate consisting of numerous glucose units joined by glycosidic bonds. This polysaccharide is produced by most green plants for energy storage. Worldwide, it is the most common carbohydrate in human diet ...
agar buffered with
MOPS MOPS (3-(''N''-morpholino)propanesulfonic acid) is a buffer solution, buffer introduced in the 1960s, one of the twenty Good's buffers. It is a structural analog to MES (buffer), MES, and like MES, its structure contains a morpholine ring. HEPES ...
(a Good's buffer) and phosphate and supplemented with
methotrexate Methotrexate, formerly known as amethopterin, is a chemotherapy agent and immunosuppressive drug, immune-system suppressant. It is used to treat cancer, autoimmune diseases, and ectopic pregnancy, ectopic pregnancies. Types of cancers it is u ...
and antibiotics. Proteose peptone, horse serum, glucose and sodium
pyruvate Pyruvic acid (CH3COCOOH) is the simplest of the alpha-keto acids, with a carboxylic acid and a ketone functional group. Pyruvate, the conjugate base, CH3COCOO−, is an intermediate in several metabolic pathways throughout the cell. Pyruvic ...
provide nutrients for the growth of ''Streptococcus agalactiae'', sodium pyruvate provide also protective effect against
reactive oxygen species In chemistry and biology, reactive oxygen species (ROS) are highly Reactivity (chemistry), reactive chemicals formed from diatomic oxygen (), water, and hydrogen peroxide. Some prominent ROS are hydroperoxide (H2O2), superoxide (O2−), hydroxyl ...
(ROS). MOPS and phosphate buffer the medium. Methotrexate triggers pigment production and starch stabilizes the pigment. The selective supplement contains the antibiotics, colistin (inhibitory for gram-negative bacteria) and
metronidazole Metronidazole, sold under the brand name Flagyl and Metrogyl among others, is an antibiotic and antiprotozoal medication. It is used either alone or with other antibiotics to treat pelvic inflammatory disease, endocarditis, and bacterial vagino ...
(inhibitory for anaerobic bacteria), and
crystal violet Crystal violet or gentian violet, also known as methyl violet 10B or hexamethyl pararosaniline chloride, is a triphenylmethane, triarylmethane dye used as a histological stain and in Gram staining, Gram's method of classifying bacteria. Crystal ...
to suppress the accompanying gram-positive bacteria. A key component of granada medium is Proteose Peptone N3 (Difco & BD). This pepsic peptone was developed by DIFCO (Digestive Ferments Company) during the
First World War World War I or the First World War (28 July 1914 – 11 November 1918), also known as the Great War, was a World war, global conflict between two coalitions: the Allies of World War I, Allies (or Entente) and the Central Powers. Fighting to ...
for producing bacterial toxins for vaccine production. Fort development of red-brick colonies of GBS in granada medium it is necessary the presence of the peptide Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe in the culture medium. This peptide only is produced during the hydrolysis with pepsin of mammal albumin. For optimal production of pigment it is also necessary the presence in the peptone of other substances (uncharacterized at present) from mammal gastrointestinal wall tissues used to prepare some peptones. The presence of starch is a basic requirement to stabilize the pigment allowing the development of red colonies of GBS. Nevertheless, if soluble starch is used it results in a culture medium that deteriorates quickly at room temperature because soluble starch is hydrolysed by serum (added as supplement) amylase. This drawback can be addressed either not using serum or using unmodified starches to prepare the culture medium, because unmodified starches are more resistant to the hydrolytic action of amylase.


Uses

GBS grows on granada agar as pink-red colonies after 18–48 hours of incubation (35–37 Â°C), better results are obtained in anaerobiosis (culturing in an anaerobic environment). Granada agar is used for the primary isolation, identification and screening of β-hemolytic GBS from clinical specimens. This culture medium is selective for GBS, nevertheless other microorganisms (such as
enterococci ''Enterococcus'' is a large genus of lactic acid bacteria of the phylum Bacillota. Enterococci are Gram-positive cocci that often occur in pairs (diplococci) or short chains, and are difficult to distinguish from streptococci on physical charac ...
and
yeasts Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitu ...
), resistant to the selective agents used, can develop as colorless or white colonies. Granada agar is useful for the screening of pregnant women for the detection of vaginal and rectal colonization with GBS to use intrapartum antibiotic prophylaxis to avoid early-onset GBS infection in the newborn. It has also been suggested that GBS pigmentation on Granada agar can help to identify pregnant women and newborns at increased risk for developing invasive GBS disease


Procedure

The specimens can be directly streaked on a plate of granada agar or after an enrichment step to obtain maximum isolation. Specimens should be streaked as soon as possible after they are received in the laboratory. If material is being cultured from a swab (e.g.- from a vaginal or vagino-rectal swab), roll swab directly onto the agar plate to provide adequate exposure of the swab to the medium for maximum transfer of organisms. Place the culture in an anaerobic environment, incubate at 35-37 Â°C, and examine after overnight incubation, and again after approximately 48 hours. To increase recovery of GBS, swabs can also be inoculated previously into a selective enrichment broth medium, such as the Todd-Hewitt broth supplemented with
gentamicin Gentamicin is an aminoglycoside antibiotic used to treat several types of bacterial infections. This may include bone infections, endocarditis, pelvic inflammatory disease, meningitis, pneumonia, urinary tract infections, and sepsis amo ...
or colistin and
nalidixic acid Nalidixic acid (tradenames Nevigramon, NegGram, Wintomylon and WIN 18,320) is the first of the synthetic quinolone antibiotics. In a technical sense, it is a naphthyridone, not a quinolone: its ring structure is a 1,8-naphthyridine nucleus that ...
and incubated for 18–24 hours at 35-37 Â°C.


Results

Colonies of β-hemolytic GBS appear on granada medium as pink or red colonies, and they are easily distinguished from other microorganisms that may have also grown on the plate. Any degree of orange development should be considered indicative of a GBS colony, and further identification tests are not necessary. Non-β-hemolytic GBS develops on granada agar as white colonies that, if necessary, can be further tested using latex agglutination or the
CAMP test The CAMP test (Christie–Atkins–Munch-Petersen) is a test to identify group B β-hemolytic streptococci ('' Streptococcus agalactiae'') based on their formation of a substance, CAMP factor, that enlarges the area of hemolysis formed by the Π...
.


Variant

Granada agar plates can also be incubated aerobically provided that a coverslip is placed over the inoculum on the plate. Granada medium can also be used as liquid media (granada broths) such as Strep B carrot broth When using granada media liquids anaerobic incubation is not necessary.


Granadaene and ''Streptococcus agalactiae''

β-hemolysis and pigment (granadaene) production are encoded in GBS by a
gene cluster A gene cluster is a group of two or more genes found within an organism's DNA that encode similar peptide, polypeptides or proteins which collectively share a generalized function and are often located within a few thousand base pairs of each othe ...
of 12 genes, the ''cyl'' cluster. Moreover, it has been suggested that GBS pigment and hemolysin are identical or closely related molecules and it has also been reported that they are important factors contributing to GBS virulence. Nevertheless, 1–5% of GBS strains are non-hemolytic and do not produce pigment. However these non-hemolytic and non-pigmented GBS strains (lacking pigment and hemolysin) are considered less virulent.


References

{{Reflist Microbiological media Cell culture media