use of differential, selective and enriched medias:
Bacterial cultivation:
Cultivation is a technique of growing microbes in culture from the site of infection (in vivo) on artificial media in the laboratory (in vitro) to aid in the diagnosis and treatment of disease. The goals of cultivation include the following:
- Grow and isolate all microbes present in an infection
- Determine which of the microbes are the causative agent of infection, and which ones are simply colonizers or contaminants
- Gain sufficient growth of any clinically relevant microbes so that they can be identified and characterized
media types:
Microbes need to eat! They need nutrients! Because of this, a wide variety of microbial nutrients or "food" are provided to encourage growth of a wide variety of microbes. The most important microbial nutrients include sugars and proteins, and these are incorporated into culture media. "Picky" microbes that need complex, exceptional media in order to grow are referred to as fastidious. Those that are more basic and grow easily are referred to as non-fastidious.
Growth media are either liquid (broth) or solid (agar). If liquid broths change in appearance from clear to turbid or cloudy, this indicates bacterial growth. Solid agar is made from water, nutrients, and agarose gel, a solidifying agent. The petri dish containing the agar is referred to an agar plate. Bacterial growth occurs from a single bacterial cell rapidly dividing to form a single, visible colony. If all of the colonies are the same, the culture is considered to be a pure culture. If there are mixed colonies, and an iinfection is suspected, the suspect colony will be subcultured on another agar plate and incubated in attempt to isolate a pure culture.
Growth media are either liquid (broth) or solid (agar). If liquid broths change in appearance from clear to turbid or cloudy, this indicates bacterial growth. Solid agar is made from water, nutrients, and agarose gel, a solidifying agent. The petri dish containing the agar is referred to an agar plate. Bacterial growth occurs from a single bacterial cell rapidly dividing to form a single, visible colony. If all of the colonies are the same, the culture is considered to be a pure culture. If there are mixed colonies, and an iinfection is suspected, the suspect colony will be subcultured on another agar plate and incubated in attempt to isolate a pure culture.
classification of media:
SUPPORTIVE MEDIA: Supportive media contain nutrients that will enable a wide variety of most non-fastidious microbes to grow.
SELECTIVE MEDIA: Selective media is used to select or isolate specific types of bacteria. Selective media contains antimicrobials and biochemicals which encourage the growth of one type of bacteria while inhibiting others, enabling bacterial isolation. Examples include Phenylethyl alcohol agar (PEA), Crystal violet agar, Mannitol salt agar (MSA).
SELECTIVE MEDIA: Selective media is used to select or isolate specific types of bacteria. Selective media contains antimicrobials and biochemicals which encourage the growth of one type of bacteria while inhibiting others, enabling bacterial isolation. Examples include Phenylethyl alcohol agar (PEA), Crystal violet agar, Mannitol salt agar (MSA).
- PEA Agar: This medium is used for the isolation of most Gram-positive cocci. Phenylethyl alcohol partly inhibits Gram-negative bacteria.
- Crystal violet agar, such as MacConkey: This medium is selective for most Gram-negative microbes. Crystal violet dye and bile salts inhibit most Gram-positive organisms.
- MSA Agar: This medium, containing 7.5% NaCl, inhibits most organisms other than salt-loving microbes (halophilic) such as Staphylococcus.
- MSA Agar: A high salt concentration of 7.5% NaCl inhibits growth of most bacteria other than staphylococci. Because of the sugar D-mannitol, in combination with a pH indicator designed to detect acid build up due to mannitol fermentation, a change in the coloration of the agar surrounding the colonies permits differentiation between staphylococcal species. S. aureus ferments mannitol, acidifying the agar, and phenol red pH indicator detects this. Staphylococcus aureus produces a yellow zone surrounding the colonies, whereas staphylococci that do not ferment mannitol do not produce a change in coloration.
- MacConkey Agar: Crystal violet and bile salts in the media prevent the growth of Gram-positive organisms while allowing the isolation of Gram-negative ones. The sugar lactose, bile salts, and the pH indicator neutral red enable differentiation between enteric bacteria based on their ability to ferment lactose. Lactose fermentation acidifies the medium, producing a pink-purple discoloration around the colonies, and the precipitation of the bile salts results in a hazy/cloudy appearance. Coliform bacilli ferment lactose, which acidifies the medium and produces a pink-red-purple coloration on the surface. Dysentery, typhoid and paratyphoid bacilli are non-lactose fermenters and do not produce acid, so colonies remain transparent or clear and uncolored on MacConkey agar.
- Blood Agar Plate (BAP): The 5% or 10% sheep's blood that is incorporated into this agar is designed to enrich the media for the purpose of cultivation of fastidious organisms such as Streptococcus spp. It also enables the display of hemolysis patterns of some microorganisms, especially staphylococci and streptococci. Hemolysis patterns are defined as follows:
- Alpha hemolysis: This is incomplete lysis of red blood cells, resulting in a reduction of hemoglobin to methemoglobin, resulting in a greenish zone around the bacterial colonies. Streptococcus pneumoniae and viridans streptococci are examples.
- Beta hemolysis: This is complete lysis of red blood cells, resulting in complete destruction and utilization of hemoglobin, resulting in a clear zone surrounding the colonies. It is produced by 2 types of beta hemolysins: streptolysin O (antigenic, oxygen-labile enzyme) and streptolysin S (a nonantigenic, oxygen-stable enzyme). The hemolysis is enhanced when the blood agar plates are both streaked and stabbed, which may show subsurface hemolysis by streptolysin O due to the reduced oxygen tension underneath the surface of the agar.
Trypticase Soy Agar with 5% sheep's blood (Bap/blood agar plate)
This type of agar, referred to as TSA II agar, is used to cultivate fasidious microbes and for the observation of hemolytic reactions. It is a general purpose media that allows a vast array of microbes to grow. Nutrients such as amino acids are provided (enzymatic digests of casein [tryptone] and soybean meal), as well as glucose (sugar and energy), NaCl to provide a homeostatic osmotic balance, and a pH buffer to maintain neutrality (dipotassium phosphate). The base is agar, which is the gel in which the nutrients are dissolved. 5% sheep's blood is added to the agar to encourage the growth and recovery of fastidious organisms and to provide a basis for interpretation of hemolytic reactions.
To read the plate properly, hold it up to the light and observe the plate with the light coming through from behind (transmitted light).
Hemolysis: lysing of the red blood cells in the agar by extracellular enzymes produced by certain bacteria
To read the plate properly, hold it up to the light and observe the plate with the light coming through from behind (transmitted light).
Hemolysis: lysing of the red blood cells in the agar by extracellular enzymes produced by certain bacteria
- Beta-hemolysis: complete clearing of the red blood cells around the bacterial colony, producing a clear zone of inhibition of growth around the colony
- Alpha-hemolysis: partial lysis of the red blood cells around the bacterial colony, producing a greenish discoloration around the colony
- Gamma-hemolysis: nonhemolysis
chocolate agar with 10% horse's blood
Chocolate agar is one of the non-selective and enriched agars used to cultivate fastidious organisms that have special requirements and needs for special nutrients in order to grow. It is used to isolate pathogens such as Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catharralis, Haemophilus influenzae, Francisella tularensis. It contains 10% horse's blood in which the red blood cells have been lysed by slowly heating the agar to 80 degrees Celsius. Additionally, chocolate agar contains Factor V (NAD) and Factor X (hemin), both of which are needed by H. influenzae in order to grow. With addition of a Bacitracin antibiotic disk, the media also becomes selective for the H. influenzae, which shows resistance to the disk. The peptone base supplies proteins to the bacteria.
Chocolate plated medium is usually incubated at 37 degrees Celsius in aerobic conditions enriched with CO2. Subcultures can be grown on slanted media or subculture plates.
Chocolate plated medium is usually incubated at 37 degrees Celsius in aerobic conditions enriched with CO2. Subcultures can be grown on slanted media or subculture plates.
macconkey agar
This type of agar is both selective and differential and is the most commonly and frequently used primary selective and differential agar. It is selective for enteric Gram-negative bacilli (Enterobacteriaceae, which normally grow in the gut) and it is differential because it aids in the differentiation between lactose-fermenting organisms and non-lactose-fermenting organisms. Gram-positive organisms are inhibited because crystal violet dye and bile salts are added to the agar base, which prevents them from growing. Enteric bacteria that ferment lactose, the sugar in the medium, appear to pinken the agar because fermentation results in changes in the pH (<6.8), which are indicated by the pH indicator neutral red. The hazy appearance produced by some organisms are due to the precipitation of the bile salts in the medium. Those that do not ferment lactose appear as clear/transparent, because they do not utilize the lactose in the agar. Other nutrients included in the agar include peptone (protein source) and NaCl to maintain osmosis. Non-lactose-fermenters utilize peptone in the agar instead of lactose, which produces ammonium as a metabolic byproduct, raising the pH, leading to the formation of transparent colonies on the agar.
Examples of lactose + organisms: E. coli, Klebsiella spp, Enterobacter spp, Citrobacter spp (slow), Serratia spp (slow)
Examples of lactose - organisms: E. coli (some strains), Acinetobacter, Salmonella, Shigella, Proteus, Pseudomonas
Examples of lactose + organisms: E. coli, Klebsiella spp, Enterobacter spp, Citrobacter spp (slow), Serratia spp (slow)
Examples of lactose - organisms: E. coli (some strains), Acinetobacter, Salmonella, Shigella, Proteus, Pseudomonas
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hektoen (HEK) agar:
Hektoen agar is both selective and differential. Its main purpose is isolation of pathogenic bacteria such as Salmonella and Shigella species. It contains lactose, sucrose, salicin, peptone, pH indicators yellow and bromthymol blue, H2S indicators, and inhibitors of Gram-positive bacteria. Because Salmonella and Shigella utilize peptone in the media instead of the sugars, this alkalizes the media, producing a transparent to blue-green color, whereas normal gut bacteria able to utilize one or more of the sugars acidify the media, causing it to turn a yellow-orange color. Salmonella, an H2S-producer, results in the production of black-centered colonies. This is due to the presence of ferric ammonium citrate or thiosulfate in the medium, which precipitates in the presence of H2S gas (hydrogen sulfide). Bile salts prevent the growth of other H2S-producing organisms. Poor or no grwoth on the medium means that the organism is inhibited by the bile and/or one or more of the dyes included in the agar. Good growth means that the organism is not inhibited by the bile or any of the dyes in the agar. Green or blue-green growth means that the organism does not ferment lactose. Pink to orange growth means that the organism produces acid from lactose fermentation. If the growth has a black center, the organism produces H2S from sulfur reduction.
- Blue-green colonies: Salmonella, Shigella, Proteus
- Black Centers: Salmonella, Proteus
macconkey agar with sorbitol
This agar is a variation of traditional MacConkey agar in that it contains the sugar sorbitol instead of lactose. It is selective for the highly pathogenic Escherichia coli O157:H7 serotype, a non-sorbitol-fermenter on SMAC agar. It utilizes the peptone in the agar instead of the sorbitol, producing clear colonies on the agar.
yersinia cin agar/Cefsulodin-irgasan-novobiocin
This agar is selective for isolation of Yersinia spp, such as Y. enterocolitica, a causative agent of gastroenteritis. It is a peptone base with yeast extract, mannitol, and bile salts supplemented with cefsulodin, irgasan and novobiocin, along with a neutral red indicator and crystal violet indicators.
Yersinia enterocolitica producing "bull's eye" or "target" colonies on CIN agar.By CDC/ Dr. Todd Parker. Ph.D.; Assoc. Director for Lab. Science/DPEI(Acting) and LRN Training Coordinator - This media comes from the Centers for Disease Control and Prevention's Public Health Image Library (PHIL), with identification number #17249.Note: Not all PHIL images are public domain; be sure to check copyright status and credit authors and content providers.English | Slovenščina | +/−, Public Domain, https://commons.wikimedia.org/w/index.php?curid=32979849
tcbs agar (thiosulfate citrate-bile sucrose):
This agar is a highly selective media utilized to isolate Vibrio cholerae and Vibrio parahaemolyticus. Sodium citrate and sodium thiosulfate inhibit the growth of most Enterobacteriaceae and the sodium thiosulfate provides a sulfur source, along with ferric citrate, for the detection of hydrogen sulfide. Bile salts and sodium cholate (ox gall) prevent the growth of Gram-positive organisms. Sucrose is the sugar included in the medium, because Vibrio spp are fermenters of this sugar, and the pH indicators thymol blue and bromothymol blue are added, which upon fermentation of sucrose acidifies the medium, resulting in the producing of yellowing of the medium. Peptone supplies the proteins.V. cholerae colonies grow as large, yellow colonies on TCBS agar. E. faecalis colonies are also yellow, but they are much smaller than those of V. cholerae.
campylobacter agar/skirrow agar
This medium consists of a peptone and soy protein base lysed with horse's blood. Antibiotics such as vancomycin inhibit the growth of Gram-positive organisms. Polymixin B and trimethoprim inhibit most Gram-negative organisms.
Other formulations may contain charcoal, and CVA formulation contains cefoperazone, cephalothin, vancomycin and amphotericin B in a Brucella agar base with sheep's blood in order to inhibit the growth of most Gram-positive and Gram-negative bacteria and yeast.
Campy-blood agar contains vancomycin, trimethoprim, polymixin B, amphotericin B, and cephalthin in a Brucella agar base with sheep's blood.
Other formulations may contain charcoal, and CVA formulation contains cefoperazone, cephalothin, vancomycin and amphotericin B in a Brucella agar base with sheep's blood in order to inhibit the growth of most Gram-positive and Gram-negative bacteria and yeast.
Campy-blood agar contains vancomycin, trimethoprim, polymixin B, amphotericin B, and cephalthin in a Brucella agar base with sheep's blood.
mannitol salt agar
This agar is a selective and differential medium used for the isolation of S. aureus. The high concentration of NaCl at 7.5% provides nutrients for Staphylococcus spp, while inhibiting most other types of bacteria that cannot survive in such a high salt concentration. It contains the sugar D-mannitol, which is fermented by S. aureus, and the pH phenol red indicator, which detects acid produced by mannitol-fermenting staphylococci. Casein and beef peptone provide the nutrients for non-mannitol fermenters. Poor or no growth indicates that the organism is inhibited by the salt in the medium. Good growth means that the organism is not inhibited by the salt in the medium. Yellow growth means that the organism produces acid from the fermentation of mannitol in the medium. Red or pink growth means that the organism does not ferment the mannitol in the medium.
endo agar:
Endo agar is used to detect fecal contamination of water or dairy products. It contains color indicators such as basic fuchsin and sodium sulfite. Lactose is is the agar as well, which can be fermented by certain bacteria like E. coli. Lactose-fermenters will grow as red or pink colonies that darken the medium surrounding them. This is due to the reaction between sodium sulfite and acetaldehyde. Non-fermenters will grow as colorless or light pink colonies. The lovely metallic sheen produced by E. coli and K. pneumoniae is a result of the large amounts of acid they produce when they ferment lactose.
eosin methylene blue agar:
Eosin Methylene Blue Agar is used to isolate fecal coliforms such as E. coli and other members of the Enterobacteriaceae. This medium contains a peptone base, lactose, sucrose, and eosin Y and methylene blue dyes. The sugars provide the fermentable carbohydrates to promote growth, whereas the dyes inhibit Gram-positive organisms. When large amounts of acid are produced during the fermentation process, a beautiful dark purple complex with a metallic green sheen is produced. This is an indicator of heavy lactose and/or sucrose fermentation typical of fecal coliforms like E. coli. Those that do not ferment as much or produce as much acid grow as pink-colored colonies on the agar. Nonfermenters remain colorless, their original color, or the color of the medium.
cna agar with blood:
Columbia Colistin-Nalidixic Acid Agar is a nutritionally rich agar containing 5% sheep's blood blood agar and 10 mg of colistin per liter and 15 mg of nalidixic acid per liter, which promotes the growth of Gram-positive organisms and prevents the growth of Gram-negative ones. Three peptone (protein) sources are included in the agar mix, and it is a type of supportive and selective and differential media used to help differentiate bacterial colonies based on their hemolysis patterns.
martin-lewis agar
This agar contains antimicrobials that are selective for the growth of Neisseria spp
new york city (nyc) agar:
NYC agar is selective for Neisseria gonorrhoeae. It consists of a peptone base with cornstarch, supplemented with yeast dialysate, 3% hemoglobin, and horse plasma. Antibiotics include vancomycin, colistin, amphotericin B, and trimethoprim.
thayer-martin agar:
This agar is a Mueller-Hinton based agar containing 5% sheep's blood plus antimicrobials that prevent growth other than Neisseria spp bacteria, including the pathogenic Neisseria gonorrhoeae and Neisseria meningitidis. It contains the antibiotics Vancomycin, Colistin, Nystatin, and Trimethoprim, by which other Gram-negative, Gram-positive and fungal organisms are inhibited. Martin-Lewis agar is very similar, but it is modified in that it substitutes Ansamycin for Nystatin and has an increased concentration of Vancomycin. Modified Thayer-Martin agar is called MTM agar.
ssa agar
Streptococcal Selective Agar is selective for S. pyogenes (Group A strep) for the isolation of the pathogens which cause strep throat. Antibiotics promote the growth of Gram-positive organisms and prevent the growth of Gram-negative ones. This media contains crystal violet, colistin, and trimethoprim-sulfamethoxazole in 5% sheep's blood agar base.
neomycin agar
This blood agar is enriched with neomycin antibiotic to encourage the growth of Group B streptococci (S. agalactiae) and prevent the growth of Gram-negative organisms
cdc ana agar
This all-purpose, non-selective agar is useful for the initial recovery of most anaerobic organisms.
cdc ana agar with pea
This anaerobic agar contains blood, phenylethyl alcohol, and antibiotics which prevent the growth of Gram-negative organisms and promote the growth of Gram-positive ones, especially Gram-positive cocci. There are a few Gram-negatives, however, that are only partly inhibited and may still grow slowly on this medium.
cdc kv-laked agar
This anaerobic media is laked with Kanomycin and Vancomycin in order to prevent the growth of Gram-positive organisms and encourage the growth of Gram-negative ones
xld agar:
Xylose Lysine and Desoxycholate (XLD) agar is selective and differential for Shigella species and for Salmonella species. The salts included in the agar will block many non-enteric pathogens and Gram-positive organisms from growing on the plate. A phenol red indicator is included, which detects increased acidity from fermentation of sugars. Non-fermenters like Shigella and Salmonella will remain colorless (pink-to-red). Salmonella colonies will produce a black center due to the production of H2S gas. Nonpathogens produce yellow colonies.
Brain-heart infusion (bhi) agar:
This all-purpose fungal media is used for the initial isolation of yeasts and molds and contains antibiotics such as gentamicin to prevent the growth of bacteria. It is rich in nutrients and is available as a broth or as an agar. It may or may not contain blood. Several key ingredients are infused together for optimal growth, and they include several animal tissue sources, peptone (protein), phosphate buffer, and dextrose (sugar), which gives the bacteria the energy they need.
BHI broth is often used to culture a patient's blood for bacterial growth and identification.
BHI broth is often used to culture a patient's blood for bacterial growth and identification.
bhi agar with blood
This all-purpose fungal media is used for the initial isolation of yeasts and molds and contains sheep's blood and the other components listed above.
phenylethyl alcohol agar (pea):
PEA agar is 5% sheep's blood agar supplemented with phenylethyl alcohol to prevent the growth of Gram-negative bacteria. Its purpose is to isolate staphylococci and streptococci from mixtures of bacterial flora. Gram-positive anaerobes can be isolated using this medium. It is undefined and selective.
sabouraud agar
This media is used for the isolation of yeasts
potato flake agar
This media is used to encourage sporulation and pigmentation and growth of molds for the identification of mold morphologies
cornmeal agar
This media is used to differentiate the types of yeast based on pseudohyphae, conidia and blastoconidia formation and patterns
chromagar:
CHROMagars are agar plates containing color indicators for selective growth of certain organisms like MRSA, yeasts, or S, agalactiae.
clotest
This is for the isolation of H. pylori and will turn bright pink/fuschia if positive, due to the production of the urease enzyme. This is also called the Rapid Urease Test.
jembec plate
The JEMBEC system is a plate/tray containing modified Thayer-Martin medium intended for the recovery of Neisseria gonorrhoeae. This plate is incubated in the CO2 incubator and should be slightly opened to enable a higher amount of CO2 to enter and enhance the growth of this fastidious organism.
haemophilus quad plate
This is for the isolation of Haemophilus spp. H. influenzae only grows in quadrants III and IV, because it requires factor X and V (hemin and NAD) and will grow in the presence of blood (it utilizes hemoglobin).
nocardia quad plate
This is for the isolation of Nocardia and Streptomyces spp
egg yolk agar (lipase test)
This egg yolk agar is used to determine which organisms produce lipase to aid in their identification. If there is clearing in the agar, then lipase is present. If there is no clearing in the agar, then lipase is absent. This is useful for differentiation of Clostridium spp and Bacillus spp.
Clostridium botulinum, causative agent of botulism, on egg yolk agar, producing lipase.By CDC/ Courtesy of Larry Stauffer, Oregon State Public Health Laboratory - This media comes from the Centers for Disease Control and Prevention's Public Health Image Library (PHIL), with identification number #1930.Note: Not all PHIL images are public domain; be sure to check copyright status and credit authors and content providers.English | Slovenščina | +/−, Public Domain, https://commons.wikimedia.org/w/index.php?curid=21110497
tsb broth
This is an all-purpose enrichment broth that is good for the isolation of many fastidious and nonfastidious microorganisms since it can support the growth of a vast array of microbes.
gn (gram-negative) broth:
This is a selective for the growth of stool organisms (Enterobacteriaceae). This broth is peptone-based with glucose and mannitol, sodium citrate and sodium desoxycholate (a bile salt) for the recovery of enteric pathogens. The bile salt inhibits growth of Gram-positive organisms.
thioglycollate broth (thio):
This enrichment broth is designed for the recovery of anaerobes. If an anaerobe is growing, the media will become cloudy/hazy. The broth contains yeast extract, casitone, glucose (dextrose), NaCl, sodium thioglycollate, L-cystine, agar, resazurin and distilled or DI water.
lim broth
This is for the recovery of Streptococcus agalactiae (Group B strep) from vaginal specimens
bhi broth
This is for the recovery of fungal (yeast and molds) organisms
middlebrook 7h9 broth
This is for the recovery of Mycobacterium spp
glycerol
This is used for the preserving and freezing of microbes for QC, projects, unknowns, and research
sugars slant agars (dextrose/glucose, lactose, maltose, sucrose)
This is used for the differentiation of Neisseria spp
This is the sugars test. Tube 1 contains maltose. Tube 2 contains dextrose (glucose). Tube 3 contains lactose. Tube 4 contains sucrose. Whenever the bacterium ferments a sugar, it causes the medium to become acidic, so it turns yellow due to a color indicator. In the medium shown above, the bacterium only ferments maltose and dextrose, so the tops of the mediums turned yellow.
triple sugar iron slant
This is used to differentiate organisms that are acid-producers via the fermentation of various sugars such as glucose, alkaline-producers via the fermentation of peptones versus sugars, H2S gas producers, and gas producers. These are used on the stool bench when identifying the Enterobacteriaceae. The media slant on the far left is uninoculated, and the original color is pink-orange. The 2nd tube on the left contains Pseudomonas aeruginosa. The top half of the slant has changed color (red/alkaline) and the bottom half has stayed the same. No CO2 gas has been produced but a little H2S is being produced. The tube in the middle contains E. coli and has completely turned yellow (acidic/fermentation in top and bottom half of slant), and has produced CO2 gas (cracking, movement, spaces in the medium). The 4th tube (right) contains S. typhimurium and has changed color on the top (red/alkaline) and bottom (yellow/acidic), is an H2S gas producer (black/middle), and is motile. The last tube (#5, right) contains Shigella flexneri and has changed color on the bottom (yellow/acidic) and top (red/alkaline) and does not produce H2S or CO2 gas.
urea agar
This is used to determine which organisms are urease-producers, such as Proteus spp, E. coli, H. pylori, C. neoformans, Klebsiella, Corynebacterium species, Brucella species, Ureaplasma, Nocardia, S. saprophyticus, Morganella, Providencia, Serratia species, etc...
lysine iron agar
This is used to see which organisms utilize lysine decarboxylization and utilize iron.
- purple slant/yellow butt = LSI Negative
- turbid, purple butt = LSI Positive
- black precipitate = H2S Positive
Motility indole ornithine (mio) agar or sulfur indole motility agar (sim)
This media is used to aid in the determination of which organisms are motile, non-motile, indole-producers, and which ones decarboxylize ornithine
bile esculin agar (bile esculin test)
Bile esculin agar (BEA) contains a nutrient agar base enriched with ferric citrate. Hydrolysis of esculin occurs by Group D streptococci (enterococci), which have the ability to hydrolyze the glycoside esculin in the presence of bile, blackening/browning the surrounding agar. This reaction occurs with the iron salts in the medium. In addition, Group D enterococci can also be distinguished from other nonenterococci by their ability to grow in 6.5% sodium chloride broth. Sodium desoxycholate blocks many other types of bacteria from growing on the agar medium. Some formulations also add vancomycin to select for MDR enterococci.