Question

Biochemical Tests I and II

1. Identify some major differences between bacteria species.

2. Define selective and differential media. Know which media we used to belong in each category.

3. For each type of selective media: know what type of bacteria are able to grow on that media.

4. For each type of differential media or test: know which characteristic is being differentiated and

how to interpret the results.

5. By comparing known species on selective and differential media, be able to identify the species

in an unknown sample

Answer 1

                                          Answer-                                                        

According to the given question-

Question 1-

We know that the Bacteria are classified mostly on the basis of Phenotypic Characteristics such as –

Morphologic Characteristics – based on gram staining they are divided into gram-positive and gram-negative, based on shape, based on motility, based on acid-fast reaction, based on the presence of spores, based on arrangement of flagella, and based on presence of capsules.

Growth Characteristics – based on the growth such as the bacteria is growing aerobically, or anaerobically, or microaerobically, or facultatively.

Biochemical Characteristics – based on their reaction such as – utilization of fermentation of carbohydrates, nitrate reduction, oxidase, as well as enzymes that are capable of degrading amino acid.

Question 2-

Selective media – are defined as the media, in which only particular types of the organism can grow, and this media also inhibit the growth of other types of microorganism. Selective media is used to isolate or identify particular types of microorganisms.

In selective media particular types of sugar, antibiotics, dyes, salts, as well as inhibitors, are used which allow the growth of certain microorganisms and inhibit the growth of other microorganisms. E.g. Eosin Methylene Blue Agar for isolation of fecal coliforms and it inhibit the growth of gram-positive organism due to presence of Eosin Y and methylene blue, they work as an inhibitor as well as a dye in the medium.

Differential media - is defined as the media used for the differentiation of species that are closely related or belongs to the same group of organisms. E.g. SIM medium - this medium is a combination of three tests, having three different parameters such as -

S = Sulfur Reduction – for differentiation of enteric microorganisms.

I = Indole Production - for differentiation of Enterobacteriaceae

M = Motility -

This test is mainly used for differentiating Shigella and Salmonella.

Question 3-

Eosin Methylene Blue Agar for isolation of fecal coliforms and it inhibit the growth of gram-positive organism due to presence of Eosin Y and methylene blue, they work as an inhibitor as well as a dye in the medium.

Dye Eosin Y and methylene blue combine and produces a complex of dark purple color and generally precipitate when the pH of the medium decreases or remains low.

In this medium lactose as well as sucrose used as a fermentable carbohydrate, responsible for promoting the growth of microorganisms which are fecal coliforms, and based on them we can easily differentiate them.

Microorganisms which are Vigorous fermenter of both the sugar sucrose and lactose, they produce an acidic byproduct in a large amount and due to which a dark purple complex formation takes place with the binding of dye.

e.g. Escherichia coli is a vigorous fermenter, observed as a green metallic sheen. While the microorganism which is weak or Slow carbohydrates fermenter, they produce a pink color mucoid colonies. While those microorganism which is unable to ferment the sugar remains normal colored or colorless and they do not belong to fecal coliform.

Question 4-

SIM medium - this medium is a combination of three tests, having three different parameters such as -

S = Sulfur Reduction – for differentiation of enteric microorganisms.

I = Indole Production - for differentiation of Enterobacteriaceae

M = Motility -

This test is mainly used for differentiating Shigella and Salmonella.

SIM medium composition – iron, sodium thiosulfate, and nutrients such as peptone having several amino acids such as tryptophan.

When a microorganism reduces the sulfur present in the media into hydrogen sulfide, then the hydrogen sulfide reacts with the iron and form a complex of ferric sulfide in the form of a black precipitate. This is a positive result for the reduction of sulfur

Some microorganisms have the capability to produce a tryptophanase enzyme, responsible for hydrolyzing the amino acid tryptophan present in peptone. pyruvic acid, indole, and ammonia are formed due to the action of this enzyme in the medium by the process of delamination. After that, the Kovac’s reagent is added to the medium for testing the indole.

The Kovac’s reagent contains DMABA or p-dimethylaminobenzaldehyde , Hcl or hydrochloric acid, as well as n-amyl alcohol. The indole reacts with DMABA and leads to the production of a quinoidal compound of red color, this is a positive result for the Indole test.

e.g. Salmonella typhimurium - positive for motility as well as sulfur reduction but negative for indole while Escherichia coli is negative for sulfur reduction and positive for indole production.

Question 5-

So based on the above describes tests we can easily identify our unknown microorganism, first based on morphological characteristics followed by the biochemical tests as well as selective and differential tests.