Mic 224 Lab 7

Selective and Differential Media I:

MSA, SBA, STA, and GEL

Media Types

• Media is divided into 4 general categories:– General purpose: supports growth of a wide

range of organisms– Selective: has ingredients that prevent the

growth of unwanted organisms while providing nutrients for those that are desired

– Differential: contains reagents/indicators that can differentiate between the organisms growing on the media

– Selective and Differential

Mannitol Salt Agar (MSA)

• Mannitol Salt Agar (MSA) is a selective and differential medium. The high concentration of salt (7.5%) selects for members of the genus Staphylococcus, since they can tolerate high saline levels. Organisms from other genera may grow, but they typically grow very weakly.

• MSA also contains the sugar mannitol and the pH indicator phenol red.  If an organism can ferment mannitol, an acidic byproduct is formed that will cause the phenol red in the agar to turn yellow. Most pathogenic staphylococci, such as Staphylococcus aureus, will ferment mannitol.  Most non-pathogenic staphylococci will not ferment mannitol.

MSA Results

MSA Procedure

• Plate 1: Create a streak plate of S. aureus

• Plate 2: Create a streak plate of a nose swab- use a sterile swab to transfer the sample into section one of the MSA plate. Use a sterile loop and the streak plate method to complete

Sheeps Blood Agar (SBA)• Blood agar contains general nutrients and 5% sheep blood.  It is

useful for cultivating fastidious organisms and for determining the hemolytic capabilities of an organism. 

• Some bacteria produce exoenzymes that lyse red blood cells and degrade hemoglobin; these are called hemolysins.

• Bacteria can produce different types of hemolysins.  – Beta-hemolysin breaks down the red blood cells and hemoglobin

completely. This leaves a clear zone around the bacterial growth. Such results are referred to as β-hemolysis (beta hemolysis).

– Alpha-hemolysin partially breaks down the red blood cells and leaves a greenish color behind.  This is referred to as α-hemolysis (alpha hemolysis).  The greenish color is caused by the presence of biliverdin, which is a by-product of the breakdown of hemoglobin. 

– If the organism does not produce hemolysins and does not break down the blood cells, no clearing will occur.  This is called γ-hemolysis (gamma hemolysis). 

SBA Procedure

• Plate 1: Create a streak plate of S. aureus

• Plate 2: Take a throat sample with a sterils swab and streak into section one of the SBA. Use a sterile loop and the streak plate method to complete

SBA Results

Starch Agar (STA)• Starch agar is a differential medium that tests the ability of an

organism to produce certain exoenzymes, including a-amylase and oligo-1,6-glucosidase, that hydrolyze starch.  Starch molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized by the organism.

• Starch agar is a simple nutritive medium with starch added.  Since

no color change occurs in the medium when organisms hydrolyze starch, we add iodine to the plate after incubation. Iodine turns blue, purple, or black (depending on the concentration of iodine) in the presence of starch. A clearing around the bacterial growth indicates that the organism has hydrolyzed starch.

STA Procedure

• On one STA plate: separate the plate into 2 sections. On one half of the plate streak in a single line Bacillus subtilis on the other half streak in a single line E. coli

Starch Results

Hydrolysis-Bacillus No Hydrolysis-E.coli

Gelatin Agar (GEL)• Nutrient gelatin is a differential medium that tests the ability of an

organism to produce an exoenzyme, called gelatinase, that hydrolyzes gelatin.

• Gelatinase allows the organisms that produce it to break down gelatin into smaller polypeptides, peptides, and amino acids that can cross the cell membrane and be utilized by the organism.

• When gelatin is broken down, it can no longer solidify.  If an organism can break down gelatin, the areas where the organism has grown will remain liquid even if the gelatin is refrigerated.

• In order to determine if an organism contains this enzyme the reagent 1%HCL is added to the plate after incubation. If the enzyme is produced you will see a halo or zone of clearing

GEL Procedure

• On one GEL plate: separate the plate into 2 sections. On one half of the plate streak in a single line Bacillus subtilis on the other half streak in a single line E. coli

GEL Results

Left: E.coli –does not contain gelatinase. Right: Bacillus- contains gelatinase

Halo or zone of clearing