BIOCHEMICAL TEST

Prof. Dr. Marlina, MS, Apt.

Laboratory Investigation of Microbial infections : 

Laboratory Investigation of Microbial infections Examining specimens to detect isolate and identify pathogens: 1- Microscopy 2- Culture techniques 3- Biochemical reactions 4- Serological identification: 5- Molecular biology techniques 6- Bacteriophage typing

 

Identification of an Unknown Bacterium: : 

Identification of an Unknown Bacterium: Dr.T.V.Rao MD 3 Microbiologists use biochemical tests, noting a particular microbe's ability to utilize or produce certain chemicals

 

Biochemical tests help in Identification of several Bacterial isolates : 

Biochemical tests help in Identification of several Bacterial isolates EVERYTHING that a living organism does is the result of the activity of an ENZYME, the SUMMATION of the activities of all an organism's enzymes equals its BIOCHEMICAL FINGERPRINT.

That is, an organism is the totality of its enzymes, so by determining which enzymes are present in an unknown organism one can DESCRIBE & IDENTIFY that organism 4 Dr.T.V.Rao MD

 

BIOCHEMICAL REACTION

Biochemical Reaction Use of substrates and sugars to identify pathogens:

a- Sugar fermentation: Organisms ferment sugar with production of acid only Organisms ferment sugar with production of acid and gas Organisms do not ferment sugar

b- Production of indole: Depends on production of indole from amino acid tryptophan Indole is detected by addition of Kovac’s reagent Appearance of red ring on the surface e- H2S production: Depends on production H2S from protein or polypeptides Detection by using a strip of filter paper containing lead acetate

 

BIOCHEMICAL REACTION (CONT.)

c- Methyl red reaction (MR): Fermentation of glucose with production of huge amount of acid Lowering pH is detected by methyl red indicator

d- Voges proskaur’s reaction (VP): Production of acetyl methyl carbinol from glucose fermentation Acetyl methyl carbinol is detected by addition KOH Color of medium turns pink (positive) e-

 

BIOCHEMICAL REACTION (CONT.) : 

f- Oxidase test: Some bacteria produce Oxidase enzyme Detection by adding few drops of colorless oxidase reagent Colonies turn deep purple in color (positive)

g- Catalase test: Some bacteria produce catalase enzyme Addition of H2O2 lead to production of gas bubbles (O2 production)

h- Coagulase test: Some bacteria produce coagulase enzyme Coagulase enzyme converts fibrinogen to fibrin (plasma clot) Detected by slide or test tube method

i- Urease test: Some bacteria produce urease enzyme Urease enzyme hydrolyze urea with production of NH3 Alklinity of media and change color of indicator from yellow to pink

 

COMMON TESTS TO IDENTIFY BACTERIAL ISOLATES

Common Tests To identify Bacterial isolates : Indole Methyl Red/Voges Proskauer Citrate H2S production Urea hydrolysis Motility Lactose fermentation Sucrose fermentation Glucose fermentation Gas production

CATALASE TEST

Catalase test . This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas. The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result .

Catalase test :  Catalase test 'Ten vol.' H2O2, is run into a

capillary tube, followed by suspension. Gas is usually evolved immediately and only tubes not showing gas within 10 sec. Are sealed for longer observation

Oxygen is sometimes toxic. Small amounts of superoxide free radicals are

formed during the normal respiration of organisms that use oxygen as the final electron acceptor.

Obligate anaerobes from some oxygen free radicals that are toxic to the cell. Hence, if bacteria wants to grow in oxygen environment, enzymes like catalase and superoxidase dismutase must be present for neutralization of the toxic form of oxygen(oxygen radical)

During normal aerobic respiration, hydrogen ions are produced and have to be removed by bacterial cell. The electron transport system (ETS)  in cellular respiration (a part of glycolysis) involves these H+ ions and combines them with oxygen to form water. Water is harmless. Energy is given off and stored in the form of Adenosine Triphosphate.

AEROBES-ANAEROBES

During normal aerobic respiration, hydrogen ions are produced and have to be removed by bacterial cell. The electron transport system (ETS)  in cellular respiration (a part of glycolysis) involves these H+ ions and combines them with oxygen to form water. Water is harmless. Energy is given off and stored in the form of Adenosine Triphosphate.

What is toxic is Hydrogen Peroxide that is formed by the cytochromes in ETS. Water being harmless is not required to be removed by the bacteria. So, what is harmful to bacteria cell that requires it to be removed instantly??

answer: H2O2.

Functions of catalase Protects bacteria from toxic hydrogen

peroxide (H2O2) accumulation, which can occur during aerobic metabolism. If hydrogen peroxide accumulates, it becomes toxic to the organism.

Since Catalase breaks H2O2 down into water and O2, the presence of oxygen can be characterized by bubbles which indicates a (+) result.

What bacteria could mostly likely be detected?

most aerobic organism make catalase.

catalase +

OXIDASE TEST OXIDASE TEST The Oxidase test (also known as the Cytochrome

Oxidase test) is used to look for oxidase enzymes produced by certain bacteria. Oxidases catalyse electron transport between substrates acting as electron donors in the bacterium and tetramethyl-p-phenylenediamine OR dimethyl-p-phenylenediamine - a redox dye present as the hydrochloride or oxalate salt The dye is reduced to a deep violet-blue colour in the presence of oxidase enzymes.

  Oxidase test :  Oxidase test The oxidase test is a test used in

microbiology to determine if a bacterium produces certain cytochrome c oxidases. It uses disks impregnated with a reagent such as N,N,N′,N′- tetramethyl-p-phenylenediamine (TMPD) or N,N-Dimethyl-p-phenylenediamine (DMPD), which is also a redox indicator.

FILTER STRIP METHOD

Filter strip method Soak strips of filter paper in a fresh dye

solution, drain and freeze dry. Strips should be stored in an air-tight bottle and kept in a cool dark environment. Strips prepared in this manner will keep for several months, and have a faint pastel-violet color. To use, take a strip and soak in distilled water. Pick the colony to be tested with a loop and rub onto moistened strip. A color change within 10 seconds indicates a positive reaction.

Oxidase testing needs controls :  Oxidase testing needs controls Positive

control: Pseudomonas aeruginosa Negative control Enterobactericia E.coli. Klebsiella spp.

HYDROGEN SULFIDE PRODUCTION   HYDROGEN SULFIDE PRODUCTION Some bacteria have the enzymatic

capability to degrade amino acids  (cysteine, cystine etc.) that contain sulfhydryl group (-SH) producing hydrogen sulfide. Hydrogen sulfide reacts with heavy metals such as lead or iron forming a black precipitate. You can use TSI medium (contains iron) or prepare a nutritive agar with lead acetate (1g Pb acetate to 100 ml nutritive agar).

 

PROCEDURE and Reading result :  Harvest a well isolated colony and inoculate

a TSI tube by stabbing the medium. Incubate at 37 °C, 24 hours. Reaction is positive if a black color appears. Bacteria growing in TSI degrade amino acids  forming ferrous sulfide which blackens the medium

 

Hydrogen sulphide production :  H2S production: Depends on production H2S

from protein or polypeptides Detection by using a strip of filter paper containing lead acetate H2S production. H2S production, either via cysteine catabolism or thiosulfate reduction

NITRATE MEDIUM – NITRATE REDUCTION TEST

Nitrate Medium – Nitrate reduction Test This is a differential medium. It is used to determine if an organism is capable of reducing nitrate (NO3-) to nitrite (NO2-) or other nitrogenous compounds via the action of the enzyme nitratase (also called nitrate reductase). This test is important in the identification of both Gram-positive and Gram-negative species.

 

Nitrate reduction Test :  Nitrate reduction Test After incubation, these

tubes are first inspected for the presence of gas in the Durham tube. In the case of non fermenters, this is indicative of reduction of nitrate to nitrogen gas. However, in many cases gas is produced by fermentation and further testing is necessary to determine if reduction of nitrate has occurred.

 

Nitrate reduction Test :  The reduction of nitrate to nitrite was

detected with dimethyl-a-naphthylamin (Wallace & Neave, 1927) and sulphanilic acid. The reaction was rapid with all the species tested; at 30 min. the results were consistent with the usual cultural method.

I M VI C TESTS

I M Vi C Tests I M Vi C is an acronym that stands for :indole ,

methyl red, Voges-Proskauer , and citrate . To obtain the results of these four tests

three test tubes are inoculated: tryptone broth (indole test), methyl red – Voges Proskauer broth (MR-VP broth), and citrate test.

INDOLE TEST Indole Test How to Perform Test: Inoculate Tryptone broth with inoculating loop.

Property it tests for: This test is performed to help differentiate species of the family Enterobacteriaceae. It tests for the bacteria species’ ability to produce indole. Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan, which makes by-products, of which, indole is one. Media and Reagents Used: Tryptone broth contains tryptophan. Kovac’s reagent—contains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcohol—yellow in color. Reading Results: Kovac’s reagent reacts with indole and creates a red color at the top part of the test tube. 22 Dr.T.V.Rao MD

 

PRINCIPLES OF INDOLE TEST :  Principles of Indole Test The test organism is inoculated into tryptone

broth, a rich source of the amino acid tryptophan.

Indole positive bacteria such as Escherichia coli produce tryptophanase, an enzyme that cleaves tryptophan, producing indole and other products.

 

When Kovac's reagent (p-dimethylamino benzaldehyde) is added to a broth with indole in it, a dark pink colour develops.

The indole test must be read by 48 hours of incubation because the indole can be further degraded if prolonged incubation occurs.

The acidic pH produced by Escherichia coli limits its growth.

CONT: Indole Test :  Indole Test Indole is a product of the

breakdown of another amino acid, tryptophan by the enzyme TRYPTOPHANASE.

To test for indole Kovacs reagent is added to the SIM medium following growth.

If indole is present a red ring forms around the surface of the medium.

 Tryptone Broth after addition of Kovacs(+) Indole test on left   ---   (-) Indole test on right : 

Tryptone Broth after addition of Kovacs(+) Indole test on left   ---   (-) Indole test on right

METHYL RED/VOGES PROSKAUER (MR/VP)

Methyl Red/Voges Proskauer (MR/VP) How to Perform Tests: Inoculate 2 glucose broths

with inoculating loop. After 48 hours of incubation, add a few drops of

MR to one tube, and VP reagents to the other tube. Properties they test for: Both tests are used to help differentiate species of

the family Enterobacteriaceae. MR—tests for acid end products from glucose

fermentation. VP—tests for acetoin production from glucose

fermentation.

 

Media and Reagents Used: Glucose Broth Methyl Red

indicator for acid Voges Proskauer reagents A: 5% Alpha-Naphthol, & ethanol, B: Potassium Hydroxide, & Deionized Water.

METHYL RED (MR) AND VOGES-PROSKAUER (VP) TESTS

Methyl red (MR) and Voges-Proskauer (VP) tests The methyl red (MR) and Voges-Proskauer (VP)

tests are read from a single inoculated tube of MR-VP broth.

After 24-48 hours of incubation the MR-VP broth is split into two tubes.

One tube is used for the MR test; the other is used for the VP test.

MR-VP media contains glucose and peptone. All enterics oxidize glucose for energy; however

the end products vary depending on bacterial enzymes.

Both the MR and VP tests are used to determine what end products result when the test organism degrades glucose.

MR/VP CONTINUED : 

MR/VP continued Reading Results: MR— a + result is red (indicating pH below

6) and a – result is yellow (indicating no acid production)

VP—A + result is red after VP reagents are added (indicating the presence of acetoin) and a – result is no color change.

Methyl Red: left – and right +

METHYL RED

This test is used to determine which fermentation pathway is used to utilize glucose.

In the mixed acid fermentation pathway, glucose is fermented and produces several organic acids (lactic, acetic, succinic, and formic acids).

The stable production of enough acid to overcome the phosphate buffer will result in a pH of below 4.4.

If the pH indicator (methyl red) is added to an aliquot of the culture broth and the pH is below 4.4, a red color will appear (first picture, tube on the left

 

METHYL RED TEST

Methyl Red Test If the pH indicator (methyl red) is added to

an aliquot of the culture broth and the pH is below 4.4, a red color will appear (first picture, tube on the left).

If the MR turns yellow, the pH is above 6.0 and the mixed acid fermentation pathway has not been utilized (first picture, tube on the right).

METHYLENE- BLUE REDUCTION

Methylene- blue reduction Standardized methylene blue in concentrations of

0.1 and 0.01 yo are mixed,with suspension and sealed. Readings are made after 4 and 24 hr. at 37".

  Voges-Proskauer (VP) test :  The reagents used for the VP test are :

Barritt's A (alpha-napthol) and Barritt's B (potassium hydroxide).

When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a pink-burgundy colour (a positive VP test).

This colour may take 20 to 30 minutes to develop. E. coli does not produce acetyl methyl carbinol, but Enterobacter and Klebsiella do.

CITRATE TEST :  Citrate Test How to Perform Test: Inoculate slant

with inoculating loop. Property it tests for: This test is used to help differentiate species of the family Enterobacteriaceae. It is selective for bacteria that has the ability to consume citrate as its sole source of carbon and ammonium as sole nitrogen source. Media and Reagents Used: Simmon’s Citrate Agar contains sodium citrate (carbon source), ammonium ion (nitrogen source), & pH indicator—bromthymol blue. Reading Results: A + result is blue (meaning the bacteria metabolised citrate and produced an acid end product) and a – result remains green 34 Dr.T.V.Rao MD

 

SIMMON'S CITRATE AGAR : 

Simmon's citrate agar Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color. Growth of bacteria in the media leads to development of a Prussian blue color (positive citrate). Enterobacter and Klebsiella are citrate positive while E.coli is negative. 35 Dr.T.V.Rao MD

  Citrate Test :  Citrate Test Left positive and right negative. 36

Dr.T.V.Rao MD  

Urea Hydrolysis :  Urea Hydrolysis How to Perform Test: Inoculate

Urea broth with inoculating loop. Property it tests for: This test is done to determine a bacteria’s ability to hydrolyze urea to make ammonia using the enzyme urease. Media and Reagents Used: Urea broth contains a yeast extract, monopotassium phosphate, disodium phosphate, urea, and phenol red indicator. Reading Results: Urea broth is a yellow-orange color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made. 37 Dr.T.V.Rao MD

 

Urease test :  Urease test This test is used to identify bacteria capable of

hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus Proteus

from other enteric bacteria. The hydrolysis of urea forms the weak base, ammonia, as

one of its products. This weak base raises the pH of the media above 8.4 and

the pH indicator, phenol red, turns from yellow to pin.   Urease Test :  Urease Test Urea is broken down by the enzyme UREASE into carbon

dioxide and ammonia. Ammonia turns the medium alkaline; that is it raises the

pH to above 7.0. In this test the bacteria are inoculated into urea broth

which contains the pH indicator (phenol red) which changes from yellow to red/pink as the pH increases (Atlas pg. 79).

After 24 to 48 hours of incubation the tubes are observed for a color change indicative of urea digestion.

UREASE TEST

Urease test This test is used to identify bacteria capable of

hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus

Proteus from other enteric bacteria. The hydrolysis of urea forms the weak base,

ammonia, as one of its products. This weak base raises the pH of the media

above 8.4 and the pH indicator, phenol red, turns from yellow to pink

GLUCOSE FERMENTATION & GAS PRODUCTION

Glucose Fermentation & Gas Production How to Perform Test:

Inoculate broth with inoculating loop. Property it tests for:

This test is done to help differnetiate species of the family Enterobacteriaceae.

This tests for the bacteria’s ability to ferment glucose and produce gas and/or an acid end-product.

 

MEDIA AND REAGENTS USED

Glucose broth contains beef extract, gelatine peptone, and glucose.

A phenol red indicator is added to indicate an acid enproduct.

A Durham tube is added to indicate gas production.

Results A positive result for acid is yellow after indicator is added (indicating glucose fermentation) A positive result for gas is a bubble in the Durham tube.

A completely negative result has no color change or reddish color and no bubble

GLUCOSE BROTH WITH DURHAM TUBES

Glucose broth with Durham tubes This is a differential medium. It tests an organism's ability to ferment the

sugar glucose as well as its ability to convert the end product of glycolysis, pyruvic acid into gaseous byproducts.

This is a test commonly used when trying to identify Gram-negative enteric bacteria, all of which are glucose fermenters but only some of which produce gas.

Carbohydrate Fermentation tubesLeft is (+)  --  Middle indicates (+) with gas  -- Right is a (-) test : 

Carbohydrate Fermentation tubesLeft is (+)  --  Middle indicates (+) with gas  -- Right is a (-) test Dr.T.V.Rao MD 43

 

Sugar Fermentation Tests :  Sugar Fermentation Tests Tube 1: Negative acid /Negative gas Tube 2A: Must incubate longer (ambiguous

result) Tube 2B: Positive acid /Negative gas Tube 3A: Positive acid/ Positive gas

SUCROSE FERMENTATION

Sucrose Fermentation How to Perform Test: Inoculate sucrose broth with inoculating loop.

Property it tests for: This test is done to help differentiate species of

the family Enterobacteriaceae. This tests for the bacteria’s ability to ferment

sucrose and production of acid end-product Media and Reagents Used: Sucrose broth contains

beef extract, gelatin peptone, and sucrose. Phenol red indicator is added to indicate an acid

end-product. Results A positive result is yellow after indicator is

added (indicating sucrose fermentation) A negative result has no color change or is

reddish.

LACTOSE FERMENTATION

Lactose Fermentation How to Perform Test: Inoculate lactose broth with inoculating loop.

Property it tests for: This tests for the bacteria’s ability to ferment

lactose. Media and Reagents Used: Lactose broth contains beef extract, gelatin

peptone, and lactose. A phenol red indicator is added to indicate acid

production from fermentation. Results A positive result is yellow after indicator is

added (indicating lactose fermentation) A negative result will have no color change or will

be redish.

MOTILITY TESTING

Motility Testing Motility agar is a differential medium used to determine whether an organism is equipped with flagella and thus capable of swimming away from a stab mark.

The results of motility agar are often difficult to interpret.

Generally, if the entire tube is turbid, this indicates that the bacteria have moved away from the stab mark (are motile).

The organisms in the two tubes pictured on the right are motile

COAGULASE TEST

Coagulase Test How to Perform Test: Inoculate rabbit plasma with one single colony. Break up colony and stir until blended in

plasma. Incubate at 37 degrees C for 24 hours. Property it tests for: This tests for the

bacteria’s ability to clot blood plasma using the enzyme coagulase.

If the organism has coagulase it will clump rabbit plasma.

Media and Reagents: This media contains rabbit plasma dissolved in buffer .

 

COAGULASE TEST

Coagulase test Coagulase is an enzyme that clots blood plasma by catalyzing the conversion of a soluble protein (fibrinogen) to an insoluble protein (fibrin).

This test is performed on Gram-positive, catalase positive species to identify the coagulase positive Staphylococcus aureus.

Coagulase is a virulence factor of S. aureus. The formation of clot around an infection caused

by this bacteria likely protects it from phagocytosis.

Coagulase Results :  Coagulase Results Reading Results: If the organism is has coagulase it will clump the

plasma. If the organism does not have coagulase it will

not clump the plasma.

TRIPLE SUGAR IRON AGAR

Triple Sugar Iron Agar Bacteria that ferment any of the three sugars in the medium will produce by products .

These byproducts are usually acids, which will change the color of the red pH-sensitive dye (phenol red) to a yellow color.

Position of the color change distinguishes the acid production associated with glucose fermentation from the acidic byproducts of lactose or sucrose fermentation.

Many bacteria that can ferment sugars in the anaerobic butt of the tube are enterobacteria.