BIOCHEMICAL TEST

PRINCIPLE

REAGENT

RESULT

CONTROL ORGANISM

1. Oxidase Test

to detect the presence of cytochrome oxidase in a particular bacteria. The principle of oxidase test also takes the transportation of electrons in consideration. Electron donors and redox are the catalyze 2. Nitrate Reduction used to determine the ability Test of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase. It also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen. 3. O-Nitrophenyl to differentiate late lactose Beta-Dfermenters from lactose Galactophyranoside nonfermenters in the Test ( OPNG) familyEnterobacteriaceae. In order for bacteria to ferment lactose, they must possess two enzymes: -galactoside permease, a membranebound transport protein, and -galactosidase, an intracellular enzyme that

Cytochrome oxidase Tetramethyl p-phenylene diamine dihydrochloride filter paper method

( + ) Bluish purple

(+) P. aeruginos a

(-) E.coli

Sulfanilic Acid and Alpha Napthylamine

(+) Red color

Colorless: add zinc dust/ powder

Orthonitrophenol

Turns colorless: Positive (+) Yellow

Turns red: Negative ( unreduced nitrate ) (-) Change in color

hydrolyzes the disaccharide into -glucose and galactose 4. LOA Test Lysine- OrnithineArginine test useful for differentiating the Enterobacteriaceae. Each decarboxylase enzyme produced by an organism is specific to the amino acid on which it acts. Therefore, we test the ability of organisms to produce arginine decarboxylase, lysine decarboxylase, and ornithine decarboxylase using three different but very similar media. Moellers decarboxylase Medium Mineral oil ( anaerobic ) Bromcresol Purple ( indicator) ( + ) Purple ( - ) Yellow

5. Triple Sugar Iron

is designed to differentiate Ph indicator: Phenol Red among the different groups or Ferrous Sulfate : Black genera of the Enterobacteriaceae, which are all gram negative bacilli capable of fermenting glucose with the production of acid, and to distinguish them from other gram negative intestinal bacilli. This differentiation is based on the differences in carbohydrate fermentation patterns and hydrogen sulfide production by the various groups of intestinal

A/A : 2-3 sugars fermented K/A : glucose fermented K/K: No sugars fermented

6. Sugar Fermentation Test

organisms. Carbohydrate fermentation is indicated by the presence of gas and a visible color change of the pH indicator, phenol red. The production of hydrogen sulphide in the medium is indicated by the formation of a black precipitate that will blacken the medium in the butt of the tube. A metabolic process Andrade Indicator performed by almost all types of bacteria is known as Durhams tube: Gas fermentation. This will result indicator in the production of ATP, the ultimate energy source of the organism. This will happen either in the presence or absence of atmospheric oxygen. Bacteria utilize the nutrients in their environment to produce ATP for their biological processes such as growth and reproduction. The enzyme systems in bacteria allow them to oxidize environmental nutrient sources. Bacteria will use different energy sources in the medium depends on the specific enzymes of each bacteria. Many bacteria

( + ) Pink

possess the enzymes system required for the oxidation and utilization of the simple sugar, glucose. Some bacteria have the ability to degrade complex carbohydrates like lactose, sucrose or even polysaccharides. Such bacterium should possess the enzymes that should cleave the glycosidic bonds between the sugar units and the resulting simple carbohydrate can be transported into the cell. Lactose is a disaccharide consisting of the glucose and galactose connected by glycosidic bond. The bacteria which produce the enzyme lactase will break this bond and thus release free glucose that can be easily utilized by the organism. The characteristics feature of the enzyme production in the bacteria enables them to use diverse carbohydrates and this will aid in the identification of unknown bacteria. Fermentation is best described by the degradation of glucose by EmbdenMeyerhof pathway or

Glycolytic pathway

7. Lysine Iron Agar

8. Indole Test

9. Rapid Spot Indole test ( Blue)

s used to distinguish bacteria which are able to decarboxylate lysine and/or produce hydrogen sulfide from those that cannot. This test is particularly useful for distinguishing different Gramnegative bacilliespecially among the Enterobacteriaceae. is a biochemical test performed on bacterial species to determine the ability of the organism to convert tryptophan into the indole. This division is performed by a chain of a number of different intracellular enzymes, a system generally referred to as "tryptophanase." to detect the presence of indole, which is one of the degradation products of the bacterial metabolism of tryptophan.

a. Lysine decarboxylation b. Lysine Deamination c. H2S: Ferric NH4 Citrate d. Brom Cresol Purple: indicator Kovacs/ Ehrlichs Reagent Pdimethylaminobenzaldehy de

(+) Purple: LIA K/K ( + ) red ( + ) Black

(-) Yellow: LIA K/A ( - ) Purple

( + ) Red ring : SIM medium

Filter paper strips impregnated with Pdimethylaminobenzaldehy de

Screening for indole production 10. Methyl Red test Methyl Red: indicator ( + ) Red ( pH below 4.4 ) ( - ) yellow

is anindicator dye that turns red in acidic solutions. It is an azo dye, and is a dark red crystalline powder. Methyl red is a pH indicator; it is red in pH under 4.4, yellow in pH over 6.2, and orange in between, with a pKa of 5.1.

11. Vogues Proskauer Test

12. Citrate Utilization Test

determines the capability of some organisms to produce non acidic or neutral end products, such as acetyl methyl carbinol, from the organic acids that result from glucose metabolism. distinguish between members of the Enterobacteriaceae family based on their metabolic by-products. Citrate utlilization test is used to determine the ability of bacteria to utilize sodium citrate as its only carbon source and inorganic(NH4H2PO4) is the sole fixed nitrogen source.

Alpha napthol and KOH Barrits method Alpha napthol and 40% KOH in creatinine Coblentz method Simmons Citrate and Bromthymol Blue ( + ) Red ( - ) Yellow

( + ) Blue

( - )Green

13. Acetate Utilization Test

14. Acetamide Utilization Test

15. Phenylalanine Deaminase Test

Acetate agar is used to test an Acetate organisms ability to utilize acetate. The medium contains sodium acetate as the sole carbon source and inorganic ammonium salts as the sole source of nitrogen. Growth is indicative of a positive test for acetate utilization. Acetamide agar is used to test Acetamide an organisms ability to utilize acetamide by deamidation. The medium contains acetamide as the sole carbon source and inorganic ammonium salts as the sole source of nitrogen. Growth is indicative of a positive test for acetamide utilization. Phenylalanine This test determines 10% FeCl3 whether the microbe produces the enzyme phenylalanine deaminase, which is needed for it to use the amino acid phenylalanine as a carbon and energy source for growth.

( + ) Blue, ( - ) Green, alkaline pH Acidic pH

( + ) E. Coli

(- ) Shig. Flexne ri

( + ) Blue

(+) P.aerugin osa

(-) S.malt ophilia

( + ) Green

16.