96 pinner used to perform spot assays with yeast, fungal or bacterial cells
Individual microorganisms placed on the plate will grow into individualcolonies, each aclone genetically identical to the individual ancestor organism (except for the low, unavoidable rate ofmutation). Thus, the plate can be used either to estimate the concentration of organisms in aliquid culture or a suitable dilution of that culture using acolony counter, or to generate genetically pure cultures from a mixed culture of genetically different organisms.
Several methods are available to plate out cells. One technique is known as "streaking". In this technique, a drop of the culture on the end of a thin,sterile loop of wire, sometimes known as an inoculator, is streaked across the surface of the agar leaving organisms behind, a higher number at the beginning of the streak and a lower number at the end. At some point during a successful "streak", the number of organisms deposited will be such that distinct individual colonies will grow in that area which may be removed for further culturing, using another sterile loop.
Another way of plating organisms, next to streaking, on agar plates is thespot analysis. This type of analysis is often used to check the viability of cells and is performed with pinners (often also called froggers). A third technique is using sterile glass beads to plate out cells. In this technique, cells are grown in a liquid culture, in which a small volume is pipetted on the agar plate and then spread out with the beads.Replica plating is another technique used to plate out cells on agar plates. These four techniques are the most common, but others are also possible. It is crucial to workin a sterile manner to prevent contamination on the agar plates.[1] Plating is thus often done in alaminar flow cabinet or on the working bench next to abunsen burner.[2]
In 1881,Fanny Hesse, who was working as a technician for her husbandWalther Hesse in the laboratory ofRobert Koch, suggested agar as an effective setting agent, since it had been commonplace in jam making for some time.[3]
An agar plate being viewed in an electroniccolony counterExample of a workup algorithm of possible bacterial infection in cases with no specifically requested targets (non-bacteria, mycobacteria etc.), with most common situations and agents seen in a New England community hospital setting. Different agar plates are used for different specimen sources, as seen in the upper left quadrant.
Like othergrowth media, the formulations of agar used in plates may be classified as either "defined" or "undefined"; a defined medium is synthesized from individual chemicals required by the organism so the exact molecular composition is known, whereas an undefined medium is made from natural products such asyeast extract, where the precise composition is unknown.[4]
Agar plates may be formulated as either permissive, with the intent of allowing the growth of whatever organisms are present, or restrictive or selective, with the intent of only allowing the growth of a particular subset of those organisms.[5] This may take the form of a nutritional requirement, for instance providing a particular compound such aslactose as the only source ofcarbon and thereby selecting only organisms which canmetabolize that compound, or by including a particular antibiotic or other substance to select only organisms which areresistant to that substance. This correlates to some degree with defined and undefined media; undefined media, made from natural products and containing an unknown combination of very many organic molecules, is typically more permissive in terms of supplying the needs of a wider variety of organisms. In contrast, defined media can be precisely tailored to select organisms with specific properties.
Agar plates may also be indicator plates, in which the organisms are not selected based on growth, but are instead distinguished by a color change in some colonies, typically caused by the action of anenzyme on some compound added to the medium.[6]
The plates are incubated for 12 hours up to several days, depending on the test that is performed.
Blood agar plates (BAPs) contain mammalian blood (usually sheep or horse), typically at a 5–10% concentration. BAPs are enriched, and differential media is used to isolatefastidious organisms and detecthemolytic activity. β-Hemolytic activity will show lysis and complete digestion of red blood cell contents surrounding a colony. Examples includeStreptococcus haemolyticus. α-Hemolysis will only cause partial lysis of the red blood cells (the cell membrane is left intact) and appear green or brown due to the conversion of hemoglobin to methemoglobin. An example of this would beStreptococcus viridans. γ-Hemolysis (or nonhemolytic) is the term referring to a lack of hemolytic activity.[7] BAPs also containmeat extract oryeast extract,tryptone,sodium chloride, and agar.[8]
Chocolate agar is a type of blood agar plate in which the blood cells have beenlysed by heating the cells to 80 °C. It is used for growing fastidious respiratory bacteria, such asHaemophilus influenzae. Chocolate agar is named for its color, and nochocolate is contained in the plate.
CLED agar –cysteine,lactose, electrolyte-deficient agar is used to isolate and differentiate urinary tract bacteria, since it inhibitsProteus species swarming and can distinguish between lactose fermenters and nonfermenters.
Granada medium is used to isolate and differentiate group BStreptococcus,Streptococcus agalactiae from clinical samples. It grows in Granada medium as red colonies, and most of the accompanying bacteria are inhibited.
MacConkey agar is a selective and differential medium used to differentiate betweengram-negative bacteria while inhibiting the growth ofgram-positive bacteria. Adding bile salts andcrystal violet to the agar inhibits the growth of most gram-positive bacteria, making MacConkey agar selective. Lactose andneutral red are added to differentiate the lactose fermenters, which form pink colonies, from lactose nonfermenters that form clear colonies. An alternative medium,eosin methylene blue serves a similar purpose.[11]
Mannitol salt agar is also a selective and differential medium. Themannitol indicates organisms that ferment mannitol: mannitol fermentation produceslactic acid, lowering the pH and turning the plate yellow. The salt is to select forhalophiles; organisms that cannot withstand a high salt content are unable to grow well.
Mueller–Hinton agar contains beef infusion, peptone, andstarch, and is used primarily for antibiotic susceptibility testing. It can be in a form ofblood agar.
Nutrient agar is usually used for growth of nonfastidious organisms and observation of pigment production. It is safe to use in school science laboratories because it does not selectively growpathogenic bacteria.
Önöz agar allows more rapid bacteriological diagnosis, asSalmonella andShigella colonies can be clearly and reliably differentiated from other Enterobacteriaceae. The yields ofSalmonella from stool samples obtained, when using this medium, are higher than those obtained with LEIFSON agar orSalmonella–Shigella agar.
R2A agar, a nonspecific medium, imitates water, so is used for water analysis.
Tryptic (trypticase) soy agar (TSA) is a general-purpose medium produced by enzymatic digestion ofsoybean meal andcasein. It is frequently the base medium of other agar types; for example, blood agar plates are made by enriching TSA plates with blood. TSA plates support growth of many semifastidious bacteria, including some species ofBrucella,Corynebacterium,Listeria,Neisseria, andVibrio.
Xylose-lysine-deoxycholate agar is used for the culture ofstool samples and contains two indicators. It is formulated to inhibit Gram-positive bacteria, while the growth of Gram-negativebacilli is encouraged. The colonies of lactose fermenters appear yellow. It is also used to culture possibleSalmonella that may be present in a food sample. MostSalmonella colonies produce a black centre on it.
Sabouraud agar is used to culturefungi and has a lowpH that inhibits the growth of most bacteria; it also contains the antibioticgentamicin to specifically inhibit the growth of Gram-negative bacteria.
Chromogenic agars can distinguish some major types of fungal infection.
Bottom view of a Sabouraud agar plate with a colony ofTrichophyton rubrum var.rodhaini
CHROMAgar (achromogenic agar) with its distinctive presentation of some major fungal pathogens.
Fungi (ascomycetes) growing inaxenic cultures, each of which is a culture of one selected organism and is free of all other organisms, enabling study of the cultured organism in isolation
Sporulation medium is medium used when spores have to be formed. It can also be used when working with fungi or bacteria depending on whether or not the strain is capable of forming spores.
A 2' x 4' petri plate filled with 14L (liters) of seaweed derived agar medium created by Harvard scientists that was used to see howE. coli evolved to be resistant to antibiotics. The mega plate also helped study more unique concepts of microbiology such as parallel evolution, mutation selection, colonial interference etc.[13]
_cells.jpg)
