Agar plate

Agar plate

An agar culture ofE. colicolonies
Uses	Microbiological culture Art
Related items	Petri dish Growth medium

Contamination on an agar plate

Anagar plateis aPetri dishthat contains agrowth mediumsolidified withagar,used toculturemicroorganisms.Sometimes selective compounds are added to influence growth, such asantibiotics.

96 pinner used to perform spot assays with yeast, fungal or bacterial cells

Individual microorganisms placed on the plate will grow into individualcolonies,each aclonegenetically 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 cultureor 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,sterileloop 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 performed with pinners (often also called froggers). A third used technique is the use of sterile glass beads to plate out cells. In this technique cells are grown in a liquid culture of which a small volume is pipetted on the agar plate and then spread out with the beads.Replica platingis another technique in order 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 mannerin order to prevent contamination on the agar plates.Plating is thus often done in alaminar flow cabinetor on the working bench next to abunsen burner.

History

In 1881,Fanny Hesse,who was working as a technician for her husbandWalther Hessein the laboratory ofRobert Koch,suggested agar as an effective setting agent, since it had been commonplace in jam making for some time.

Types

An agar plate being viewed in an electroniccolony counter

Example 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 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.

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 growth a particular subset of those organisms.This may take the form of a nutritional requirement, for instance providing a particular compound such aslactoseas the only source ofcarbonand thereby selecting only organisms which canmetabolizethat compound, or by including a particular antibiotic or other substance to select only organisms which areresistantto 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, while 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 on the basis of growth, but are instead distinguished by a color change in some colonies, typically caused by the action of anenzymeon some compound added to the medium.

The plates are incubated for 12 hours up to several days depending on the test that is performed.

Commonly used types of agar plates include:

Red blood cellson an agar plate are used to diagnoseinfection.On the left is a positiveStaphylococcusinfection, on the right a positiveStreptococcusculture.

Blood agar

Hemolyses ofStreptococcusspp.(left) α-hemolysis (S. mitis); (middle) β-hemolysis (S. pyogenes); (right) γ-hemolysis (= nonhemolytic,S. salivarius)

Blood agar plate

Blood agar plates (BAPs) contain mammalian blood (usually sheep or horse), typically at a concentration of 5–10%. BAPs are enriched, differential media used to isolatefastidiousorganisms and detecthemolyticactivity. 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 will 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.BAPs also containmeat extractoryeast extract,tryptone,sodium chloride,and agar.

Chocolate agar

Chocolate agaris a type of blood agar plate in which the blood cells have beenlysedby 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 nochocolateis actually contained in the plate.

Thayer–Martin agar

Thayer–Martin agaris a chocolate agar designed to isolateNeisseria gonorrhoeaeandNeisseria meningitidis.

Thiosulfate–citrate–bile salts–sucrose agar

Thiosulfate–citrate–bile salts–sucrose agarenhances growth ofVibriospp.,includingVibrio cholerae.

General bacterial media

Four types of agar plate demonstrating differential growth depending on bacterialmetabolism

• Bile esculin agaris used for the isolation ofEnterococcusandgroup DStreptococcusspecies.
• CLED agar–cysteine,lactose,electrolyte-deficient agar is used to isolate and differentiate urinary tract bacteria, since it inhibitsProteusspecies swarming and can differentiate between lactose fermenters and nonfermenters.
• Granada mediumis used to isolate and differentiate group BStreptococcus,Streptococcus agalactiaefrom clinical samples. It grows in Granada medium as red colonies and most of accompanying bacteria are inhibited.
• Hektoen enteric agaris designed to isolate and recover fecal bacteria of the familyEnterobacteriaceae.It is particularly useful in isolatingSalmonellaandShigella.
• Lysogeny brothis used to cultureEscherichia coli.
• MacConkey agaris a selective and differential medium used to differentiate betweengram-negativebacteria while inhibiting the growth ofgram-positivebacteria. The addition of bile salts andcrystal violetto the agar inhibits the growth of most gram-positive bacteria, making MacConkey agar selective. Lactose andneutral redare added to differentiate the lactose fermenters, which form pink colonies, from lactose nonfermenters that form clear colonies. An alternative medium,eosin methylene blueserves a similar purpose.
• Mannitol salt agaris also a selective and differential medium. Themannitolindicates 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 agarcontains beef infusion, peptone, andstarch,and is used primarily for antibiotic susceptibility testing. It can be in a form ofblood agar.
• Nutrient agaris 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 growpathogenicbacteria.
• Önöz agarallows more rapid bacteriological diagnosis, asSalmonellaandShigellacolonies can be clearly and reliably differentiated from other Enterobacteriaceae. The yields ofSalmonellafrom stool samples obtained, when using this medium, are higher than those obtained with LEIFSON agar orSalmonella–Shigellaagar.
• Phenylethyl alcohol agarselects forStaphylococcusspecies while inhibiting Gram-negative bacilli (e.g.,Escherichia coli,Shigella,Proteus,etc.).
• 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 ofsoybeanmeal 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-deoxycholateagar is used for the culture ofstoolsamples and contains two indicators. It is formulated to inhibit Gram-positive bacteria, while the growth of Gram-negativebacilliis encouraged. The colonies of lactose fermenters appear yellow. It is also used to culture possibleSalmonellathat may be present in a food sample. MostSalmonellacolonies produce a black centre on it.
• Cetrimide agaris used for the selective isolation of the Gram-negative bacteriumPseudomonas aeruginosa.
• Tinsdale agar containspotassium tellurite,which can isolateCorynebacterium diphteriae.

Fungal media

• Sabouraud agaris used to culturefungiand has a lowpHthat inhibits the growth of most bacteria; it also contains the antibioticgentamicinto specifically inhibit the growth of Gram-negative bacteria.
• Hay infusion agaris specific for the culturing ofslime moulds(which are not fungi).
• Potato dextrose agaris used to culture certain types of fungi.
• Malt extract agar has a high content of peptone and is acidic. It is essentially used in the isolation of fungal microorganisms.
• MMN (Modified Melin-Norkrans) mediumandBAF mediumare used forectomycorrhizalfungi.
• Chromogenic agarscan distinguish some major types of fungal infection.

• 
  Bottom view of a Sabouraud agar plate with a colony ofTrichophyton rubrumvar.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
• 
  Aspergillus nigergrowing on potato dextrose agar

Moss media

• Knop agar is used toaxenicallycultureprotonemaand wholemossplants, such as those ofPhyscomitrella patens,amodel organism.

Yeast media

the yeastCandida albicansgrowing both as yeast cells andfilamentous cellson YPD agar

• YEPDmedia is often used as a general growth media for yeasts likeSaccharomyces cerevisiaeandCandida albicans
• 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.

Mega Plate

• 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. colievolved 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.

See also

• Microbial art
• Viral plaque

Different specific types of agar:

• Casein nutrient agar
• MRS agar
• New York City Agar

External links

• Video of sheep blood agar preparation
• Science Buddies – All About Agar
• Video on aseptic plating methods
• Video on how to use glass beads to plate out samples