Question

Biochemical and differential tests can be incorporated into growth media or performed on isolated colonies after they have been cultured. Give three examples of a differential test that is incorporated into media and three examples that are applied after the colonies have been cultured.

Answer 1

Differential test

Explanation:

A differential test helps to distinguish that which species lack or have the particular biochemical process,the three differential test media are:

1) Blood agar: Blood Agar is an enriched medium which is used to culture those bacteria or microbes that do not grow easily

• It is also required to detect and differentiate haemolytic bacteria, especially Streptococcus species
• It is also a differential media in allowing the detection of hemolysis (destroying the RBC) by cytolytic toxins secreted by some bacteria, such as certain strains of Bacillus, Streptococcus, Enterococcus and Staphylococcus

2) Eosin methylene blue agar (EMB): It is a selective and differential medium used to isolate fecal coliforms

• Eosin methylene blue are pH indicator dyes which combine to form a dark purple precipitate at low pH; they also serve to inhibit the growth of most Gram positive organisms
• Sucrose and lactose serve as fermentable carbohydrate sources which encourage the growth of fecal coliforms and provide a means of differentiating them

3) Mannitol Salt Agar (MSA): It 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

Three examples of tests which are done on colonies are:

1) Catalase test: Catalase is an enzyme that breaks hydrogen peroxide (H2O2) into H2O and O2

• Hydrogen  peroxide is often used as a topical disinfectant in wounds, and the bubbling that is seen is due to  the evolution of O2 gas
• A small amount of growth from the culture is placed onto a clean microscope slide
• Few drops of H2O2 onto the smear is added
• A positive result is the rapid evolution of O2 as evidenced by bubbling
• A negative result is no bubbles or only a few scattered bubbles

2) Indole test: The main requirement for a suitable indole test medium is that it contain

s a sufficient amount of tryptophan

• Tryptone broth is commonly used
• The tube of tryptone broth with a small amount of a pure  culture is inoculated
• Incubation is done at 35°C (+/- 2°C) for 24 to 48 hours
• To test for indole production, 5 drops of Kovács reagent is directly added to  the tube
• A positive indole test is indicated by the formation of a pink to red color  in the reagent layer on top of the medium within  seconds of adding the reagent
• If a culture is indole negative, the reagent layer will remain yellow or be  slightly cloudy

3) Urease test: Urease is a constitutively expressed enzyme that hydrolyzes urea to carbon dioxide and ammonia

• The urease test identifies those organisms that are capable of hydrolyzing urea to produce ammonia and carbon dioxide
• Urease test media contain 2% urea and phenol red as a pH indicator
• An increase in pH due to the production of ammonia results in a color change from yellow (pH 6.8) to bright pink (pH 8.2)
• Urea broth is a highly buffered medium requiring large quantities of ammonia to raise the pH above 8.0 resulting in a color change