MEDIUM AGAR AND THEIR USES

Bismuth sulfite agar

is one medium used to isolate the typhoid bacterium, the gram-negative Salmonella typhi, from feces.

Sabouraud’s dextrose agar

which has a pH of 5.6, is used to isolate fungi that outgrow most bacteria at this pH.

MacConkey agar

inhibits the growth of Gram-positive bacteria and thus is selective for Gram-negative bacteria

Gram-negative

bacteria able to ferment the lactose (an ingredient of MacConkey agar) produce pink colonies, whereas those unable to ferment lactose produce colorless colonies.

Mannitol salt agar (MSA)

Only salttolerant (haloduric) bacteria can grow on mannitol salt agar (MSA).

Blood agar

is a differential medium because it is used to determine the type of hemolysis that the bacterial isolate produces.

Selenite broth, Rappaport-Vassiliadis broth

elective enrichment media used for the isolation of Salmonellae from samples containing other Gramnegative enteric organisms.

Edwards medium

A blood agarbased selective medium used for the isolation and recognition of Streptococci.

Brilliant Green Agar

An indicator medium for presumptive identification of Salmonella spp. Salmonella colonies and surrounding medium has a pink color.

XLD Agar (Xylose Lysine Deoxycholate agar)

Used for salmonellae isolation, other Gramnegative will also grow; Media turns yellow (lactose-fermenter) or purple (non-lactose fermenter). Black colonies are H2S producers.

Eosin-methylene blue (EMB)

Presumptive identification of E. coli (metallic sheen), other Gramnegative will also grow.

Esculin agar

Differentiating Streptococci from Enterococci. Blackening: Enterococci.

Smith-Baskerville medium

Selective for Bordetella spp. (media turns blue, other turns yellow).

Acidic Dyes

negatively charged chromophore bind to the positively charged molecules in some parts of eukaryotic cells.

Positive staining

involves a positive stain, in which the dye sticks to the cell and give them color.

Negative staining

the dye does not stick to the specimen but settles around its outer boundary, forming a silhouette.

Nigrosin (blue-black) and india ink

black suspension of carbon particles are the dyes most used for negative staining.

Gram Staining

Types of Positive Staining

Simple

requires only one dye.

Differential

2 differential color dyes (primary and counterstain)

Special

used to color and isolate specific parts of microorganisms such as endospore and flagella, reveal the presence of capsules.

Gram Staining

the most widely employed staining method in bacteriology.

Gram Staining

uses crystal violet as stain, iodine as mordant, acetone/alcohol as a decolorizer and safranin red as a counter stain.

Gram Staining • the most wi

1 minute-1 minute-10/30 sec-30 sec

ZIEHL-NEELSEN METHOD

Test to differentiate acid-fast and nonacid-

fast bacteria.

ACID FAST

take up the primary stain (PINK)

NON-ACID FAST

take up the secondary stain (BLUE)

Carbol Fuchsin

Primary Stain- 5 minutes Heat in hot plate)

Acid alcohol

Wash- 15 sec

Methylene Blue

Secondary Stain 1min

Malachite green- Primary stain

5 min (heat in steam)

Water

Wash- 30 sec

ENDOSPORE STAINING (SchaefferFulton or Wirtz- Conklin)

The endospores are stained with malachite green. Heat is used to provide stain penetration. The rest of the cell is then decolorized and counterstained a light red with safranin.

CAPSULE STAINING (Anthony’s Capsule

staining method)

Crystal violet gives bacterial cell

and capsular material dark purple color.

Unlike the cell, the capsule is non-ionic, and

the primary stain cannot adhere. Copper

sulfate is a decolorizing agent and removes

excess primary stain as well as from the

capsule. At the same time, the copper sulfate

acts also as a counterstain by being

absorbed into the capsule and turning it a

light blue.