1. Biochemical Activities of Bacteria
Bacteria accomplish their various biochemical activities (growth and multiplication) using raw materials (nutrients) obtained from the environment. The biochemical transformations that occur both inside and outside of bacteria are governed by biological catalysts called enzymes.
Fermentations are energy-producing biochemical reactions in which organic molecules serve both as electron acceptors and donors. The ability of microorganisms to ferment carbohydrates and the types of products formed are very useful in identification.

2. For example, if fermenting bacteria are grown in a liquid culture medium containing the carbohydrate glucose, they may produce organic acids as by-products of the fermentation. These acids are released into the medium and lower its pH. If a pH indicator such as phenol red or bromcresol purple is included in the medium, the acid production will change the medium from its original color to yellow

4. Gram Negative Bacilli-Enterobacteriacae
Oxidase test :
Used to distinguish fermenters (Oxidase negative) from non-fermenters(Oxidase positive).
Testing for the presence of the enzyme indophenol oxidase.
The reagent (Tetramethyl-para-phenylenediamine) will be oxidized in the presence of oxygen by the enzyme indophenol oxidase producing a dark-purple product called indophenol.
Production of a dark purple product (within 10 – 30 seconds) is a positive ( + ) test for oxidase.

5. Urease test : Testing for the presence of the enzyme urease.
This enzyme will break down urea to ammonia and carbon dioxide.
CO(NH2) ® NH CO2
The ammonia produced increases the pH of the media (becomes more alkaline) and causes the indicator present in the media (Phenol red) to change from a yellow-orange color to a hot pink color.
A hot-pink color is an indication of a positive test for Urease.
000

6. Triple Sugar Iron (TSI) Test
Test conditions:
Provides information about sugar fermentation and the possible by-products produced by the organisms tested.
Three sugars are detect, glucose, lactose, and sucrose are present in the media at different concentrations (Glucose at 0.1%, Lactose at 1.0%, and Sucrose at 1.0%).
Fermentation is an anaerobic process.
When sugar is fermented there may be an acid end-product produced.
Phenol red is again used in the media as a pH indicator and will change from red to yellow under acidic conditions.
Some organisms also may produce hydrogen gas (H2) as a by-product of fermentation.
Also present in the media is ferrous sulfate, which will react with any hydrogen sulfide (H2S) produced as a another possible by-product to form a black precipitate (ferrous sulfide). H2S production is an anaerobic process so the black precipitate produced will only appear in the butt of the tube.

7. Procedure: Work in pairs You will be testing three organisms:
Pseudomonas aeruginosa
Escherichia coli
Proteus vulgaris
You will need one TSI agar slant for each organism/group (3 tubes).
The tests require anaerobic conditions, therefore the organisms must be stabbed into the agar media. For this reason you will use sterile inoculating needles (not loops) for inoculating the tubes.
The best method is to obtain one of the organisms, preferably on a nutrient agar plate, and roll the tip of a sterile inoculating needle in the organism.
Next stab the agar slant nearly to the bottom of the agar tube and when removing the inoculating needle, lightly streak the surface of the slant.
Follow the same procedure for each organism tested.
The tubes are placed in the incubator for hours at 37 °C.

8. Interpretation of Results:
Many of the enteric organisms will ferment glucose with the production of acids which will change the color of the medium in the butt and along the slant from red to yellow because of a reduction in the pH (within the first few hours).
However, since the glucose is present in small amounts (0.1%), the supply is soon exhausted and the organisms growing on the surface of the slant in the presence of oxygen are forced to catabolize peptones and amino acids in the media for their energy supply. Alkaline end-products ( NH4OH) are produced from these substances which revert the pH of the slant to an alkaline pH and thus change the color of the agar slant back to red (after hours).
Organisms such as Salmonella spp. or Shigella spp. and other organisms which attack glucose but do not ferment lactose or sucrose will produce an alkaline slant and acid butt in TSI slants in 18 to 24 hours. Since metabolism is progressing at a slower rate in the butt, this reversion does not usually take place in the butt until 48 hours or longer.
If the glucose is metabolized to CO2, the gas will be seen as bubbles or cracks in the agar butt. If hydrogen sulfide is formed during growth, a gray or black streak of iron sulfide is seen originating where the inoculating needle entered and throughout the agar butt.
Organisms which attack lactose and/or sucrose, such as Escherichia, will produce acid slants and acid butts usually with the formation of gas. In these cases, the acid slants do not revert to an alkaline status because lactose (1%) and sucrose (1%) are being fermented and are present in concentrations ten times that of glucose.
Some organisms (e.g., Pseudomonas, Acinetobacter) fail to ferment even glucose, and because they are strictly aerobic, they fail to grow in the butt of the tube. In these cases, the butt will be unchanged in color, and the slant either alkaline or unchanged.

9. TSI Test Results Control Eschericihia Pseudomonas aeruginosa
Klebsiella
Enterobacter
Pseudomonas aeruginosa
Proteus vulgaris
Shigella
Salmonella enteritidis

10. Recording the Results of the TSI Tests
K = alkaline = Red;  A = acid = Yellow;  NC = No change;  G = gas produced; 
 H2S = hydrogen sulfide produced
Acid or alkaline results in the slant are reported first, followed by the butt results (e.g., K/A would be read as "K over A" or "alkaline over acid" and refers to an alkaline slant and acid butt).
Slant result / Butt result
K/A ® Only Glucose fermented; peptone utilized
A/A ® Glucose and Lactose/Sucrose fermented
K/K ® No sugars fermented, only peptone utilized
K/NC ® No sugars fermented, Peptone used aerobically only
NC/NC ® No growth, neither sugars or peptone used
A/AG ® Glucose and Lactose/Sucrose fermented, gas produced
A/A + H2S ® Glucose and Lactose/Sucrose fermented, H2S produced
A/AG + H2S ® All sugars fermented and both H2S and gas produced
K/AG ® Only Glucose fermented; peptone utilized, gas produced
K/A + H2S ® Only Glucose fermented; peptone utilized, H2S produced
K/AG + H2S ® Only glucose fermented; Peptone utilized, both gas and H2S
produced

11. Motility Test
To test for bacterial motility a diluted agar is used. Normally the agar concentration is 1.5 g of agar per mL of nutrient media broth. Motility agar is usually 0.7 g agar per mL of nutrient broth.
When the lower concentration agar solidifies it is less gel-like and more fluid allowing bacteria with flagella to move into the surrounding media. Additionally, a special reagent, Triphenyltetrazonium chloride, or TTC, is used to make the motility more visual in the tube. TTC is colorless and soluble in it’s oxidized form, but becomes insoluble and dark red in color when in it’s reduced state. Any metabolic process, such as the expenditure of energy for flagellar movement, will reduce the TTC and cause the appearance of the red, reduced TTC in areas where motile bacteria are present.

12. Identification of GNB – IMViC Tests
Mnemonic for the four Biochemical tests performed on the Gram Negative Bacilli being studied
Indole test
Methyl Red test
Voges-Proskauer test
Citrate utilization test
These tests divide the Enterobacteriaceae into two major groups:
Escherichia coli group
Enterobacter-Klebsiella group

13. Indole test:
Tests for the production and secretion of the enzyme tryptophanase.
If the organism can produce tryptophanase and break down tryptophan to Indole the Indole will react with Kovac’s reagent (p-dimethylaminobenzaldehyde) forming a pink-colored chemical complex.
We use commercially prepared slides impregnated with Kovac’s reagent

14. Methyl Red Test
Some organisms produce acid from the metabolism of glucose (fermentation) in a sufficient quantity to alter the pH of the media to about 4.4. These are stable acids and are not further metabolized.
Methyl red indicator is used to detect the presence of these acids in the MRVP broth medium. Methyl red indicator at this pH (~ 4.4) changes to a bright cherry red color.

15. Voges-Proskauer Test
Some microorganisms produce organic acids from glucose metabolism, but further metabolize the acid produced to various neutral end products, like acetoin, and 2,3-butanediol.
There is an initial pH drop in the MRVP broth, but the neutral end product raise the pH so that the methyl red test will be negative.
The presence of acetoin, and 2,3-butanediol is tested for using a-naphthol which reacts with these two neutral products to produce a mahogany red color.

16. Citrate Test
Citrate may be used as a carbon source by some microorganisms.
The Citrate test uses an agar medium with citrate and the pH indicator Bromothymol blue present. At the pH of the un-inoculated medium the color is Blue-green.
If the organism can utilize Citrate as a carbon source the breakdown of citrate releases bicarbonate ions (HCO3-) into the medium. The bicarbonate ions raise the pH of the medium above 7.4 pH. This causes the Bromothymol blue indicator to turn dark blue in color.

17. Catalase test Principles
Some bacteria contain flavoproteins that reduce O2, resulting in the production of hydrogen peroxide (H2O2) or superoxide (O2 –). These are extremely toxic because they are powerful oxidizing agents and destroy cellular constituents very rapidly. A bacterium must be able to protect itself against such O2 products or it will be killed. Many bacteria possess enzymes that afford protection against toxic O2 products. Obligate aerobes and facultative anaerobes usually contain the enzymes superoxide dismutase, which catalyzes the destruction of superoxide, and either catalase or peroxidase, which catalyze the destruction of hydrogen peroxide as follows:

18. Most strict anaerobes lack both enzymes and therefore cannot tolerate O2.
Catalase production and activity can be detected by adding the substrate H2O2 to an appropriately incubated (18- to 24-hour) tryptic soy agar slant culture. If catalase was produced by the bacteria, the above chemical reaction will liberate free O2 gas. Bubbles of O2 represent a positive catalase test; the absence of bubble formation is a negative catalase test. Catalase activity is very useful in differentiating between groups of bacteria. For example, the morphologically similar Enterococcus (catalase negative) and Staphylococcus (catalase positive) can be differentiated using the catalase test

19. Coagulase test Principles
Coagulases are enzymes that clot blood plasma by a mechanism that is similar to normal clotting. Although coagulase activity is not required for pathogenicity, this enzyme is a good indicator of the pathogenic potential of S. aureus. Coagulase-producing staphylococci (termed coagulase positive) form a fibrin clot around themselves and avoid attack by the host’s defenses. In the coagulase test, coagulase positive staphylococci will cause the plasma to clot by using coagulase to initiate the clotting cascade. Citrate and EDTA are usually added to act as anticoagulants and prevent false-positive results. Cultures should be considered coagulase negative if they are unclotted after 4 hours.

20. Oxidase test
Principles
Oxidase enzymes play an important role in the operation of the electron transport system during aerobic respiration. Cytochrome oxidase (aa3 type) uses O2 as an electron acceptor during the oxidation of reduced cytochrome c to form water and oxidized cytochrome c. The ability of bacteria to produce cytochrome oxidase can be determined by the addition of the oxidase test reagent or test strip (an Oxidase Disk) to colonies that have grown on a plate medium. Or, using a wooden applicator stick, a bacterial sample can either be rubbed on a Dry Slide Oxidase reaction area, on a KEY test strip, or filter paper moistened with the oxidase reagent. The light pink oxidase test reagent (Disk, strip, or Slide) serves as an artificial substrate, donating electrons to cytochrome oxidase and in the process becoming oxidized to a purple and then dark purple compound in the presence of free O2 and the oxidase. The presence of this dark purple coloration represents a positive test. No color change or a light pink coloration on the colonies indicates the absence of oxidase and is a negative test.