Agarose gel

By Levi Clancy for Student Reader on
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Agarose gel (aka agar) is a very firm jelly that is laced with nutrients to grow colonies of microbes.

A colony is a single cell which has reproduced to form a visible dot of cells. Note that fungal colonies are oftentimes fuzzy, while bacterial colonies are typically compact and gummy. Agar nomenclature is very simple. For example, YTA-enriched GMA is GMA enriched with a tiny amount of YTA.

Also, please review types of microbes since it is crucial to create an optimal environment for the microbe you are selecting or screening. Lastly, remember that antibiotics can be added to agar to inhibit the growth of a particular type of microbe.

Agar nameAbbreviationOverview

Minimal agar

MA

A simple medium containing a few salts.

Glucose minimal agar

GMA

Minimal agar with glucose.

Yeast tryptone agar

YTA

A complex agar containing yeast extract and tryptone.

Blood agar

Usually just a mixture of sterile sheep blood and agar, blood agar is useful for screening based on α- or β-hemolysis. Many bacteria secrete enzymes that destroy blood cells, causing a clear lytic zone to form around the colony on an otherwise red plate. α-hemolysis is caused by overall harmless bacteria that degrade hemoglobin to greenish-yellow bile pigments. However, β-hemolysis is caused by pathogens such as Streptococcus and causes completely clear lytic zones by a massively destructive battery of lytic enzymes.

Hektoen-Enteric agar

HE

A selective and differential agar containing bile salts, bromothymol blue and acid fuschin. Bile select for Salmonella and Shigella by killing gram-positive and coliform species. Bromothymol blue and acid fuschin are indicators: coliforms appear orange/salmon; and non-lactose fermentors form blue-green (Salmonella) or green (Shigella) raised and moist colonies.

Levine's EMB agar

A selective & differential media for isolating gram-negative enteric bacteria. EMB agar contains eosin Y, methylene blue and lactose. This selects for gram-negative cells because methylene blue inhibits gram-positive growth. Also, this agar is differential because eosin Y is an indicator: lactose fermentors like Enterobacter appear pink/brown; E. coli (also a lactose fermentor) appears metallic green; and non-lactose fermentors are translucent and amber or colorless.

Sabouraud agar

SA

Saboraud's agar (SA) is a complex media that selects for molds and yeasts via acidity (pH 5.6) and high sugar content (2% glucose).

Glucose, yeast, CaCO3 agar

GYC

Contains glucose, yeast extract and calcium carbonate (GYC). GYC is an indicator for glucose fermentation; lactic acid, a byproduct of glucose fermentation, reacts with calcium carbonate for form clearings.

Starch agar

Secreted amylase breaks down starch into maltose and other sugars, forming a clearing around amylase-secreting colonies. Pouring iodine onto a starch agar plate will reveal undigested starch (blue) and digested starch (reddish brown). A common positive control is Bacillus subtilis.

Gelatin agar

Secreted proteolytic enzymes hydrolyze gelatin (a protein) into constituent amino acids. Trichloroacetic acid precipitates undigested gelatin -- if poured on the plate, hydrolyzed gelatin appears as a clearing amidst an opaque precipitate of undigested gelatin. A common positive control is Bacillus subtilis.

Egg yolk plate

Secreted phospholipases hydrolyze lecithin, the major phospholipid in egg yolk. Lecithin hydrolysis releases long chain fatty acids, visible as an insoluble waxy buildup around colonies. A common positive control is Pseudomonas fluorescens.

O/F media

Usually used with agarose, oxidation/fermentation media (O/F media) consists of tryptone (an amino acid source), 1% glucose and bromothymol blue (an indicator dye). Despite its name, bromothymol blue is green at neutral pH. Anaerobic conditions are created by pouring oil over the agar; aerobic conditions are created by not pouring oil. Oxidation and fermentation of glucose both result in acid byproducts which turn bromothymol blue a yellow color. Deamination of amino acids leads to alkaline byproducts which turn bromothymol blue a blue color.

King's B medium

King's B Medium (KBM) with 0.2% KNO3 is one of many agars used for distinguishing pseudomonads. Positive controls such as P. fluorescens fluoresce in KBM, while negative controls such as P. aeruginosa do not. Also, a nitrite reagent can be added after incubation to assay for reduction of nitrate to nitrite.