Biochemical Reactions in : Common Pathogenic Bacterial Isolates
Dr.T.V.Rao MD Biochemical Reactions in 1 Dr.T.V.Rao MD Laboratory Investigation of Microbial infections : Laboratory Investigation of Microbial infections Examining specimens to detect isolate and identify pathogens:
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.) : 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.) : 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
Glucose fermentation & gas production 8 Dr.T.V.Rao MD 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. 9 Dr.T.V.Rao MD 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 10 Dr.T.V.Rao MD 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 11 Dr.T.V.Rao MD 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. 12 Dr.T.V.Rao MD 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. 13 Dr.T.V.Rao MD Oxidase testing needs controls : Oxidase testing needs controls Positive control: Pseudomonas aeruginosa
E.coli. Klebsiella spp 14 Dr.T.V.Rao MD HYDROGEN SULFIDE PRODUCTION : HYDROGEN SULFIDE PRODUCTION Dr.T.V.Rao MD 15 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 : PROCEDURE and Reading result Dr.T.V.Rao MD 16 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 : Hydrogen sulphide production Dr.T.V.Rao MD 17 e- 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, produces a black precipitate in the media. 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. 18 Dr.T.V.Rao MD 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. 19 Dr.T.V.Rao MD Nitrate reduction Test : 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 20 Dr.T.V.Rao MD 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. 21 Dr.T.V.Rao MD 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. 23 Dr.T.V.Rao MD 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. 24 Dr.T.V.Rao MD 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 Dr.T.V.Rao MD 25 Indole test reactions : Indole test reactions 26 Dr.T.V.Rao MD 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:
Methyl Red indicator for acid
Voges Proskauer reagents—A: 5% Alpha-Naphthol, & ethanol, B: Potassium Hydroxide, & Deionized Water. 27 Dr.T.V.Rao MD 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. 28 Dr.T.V.Rao MD 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 + – 29 Dr.T.V.Rao MD Methyl Red : 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). 30 Dr.T.V.Rao MD 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). 31 Dr.T.V.Rao MD 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". 32 Dr.T.V.Rao MD Voges-Proskauer (VP) test : 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. 33 Dr.T.V.Rao MD 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.
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 38 Dr.T.V.Rao MD 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. 39 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 pink 40 Dr.T.V.Rao MD 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.
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. 41 Dr.T.V.Rao MD 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. 42 Dr.T.V.Rao MD 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 44 Dr.T.V.Rao MD Slide 45: 45 Dr.T.V.Rao MD 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.
A positive result is yellow after indicator is added (indicating sucrose fermentation)
A negative result has no color change or is reddish. 46 Dr.T.V.Rao MD 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.
A positive result is yellow after indicator is added (indicating lactose fermentation)
A negative result will have no color change or will be redish. 47 Dr.T.V.Rao MD Lactose fermenter and Non fermenter : Lactose fermenter and Non fermenter 48 Dr.T.V.Rao MD 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 49 Dr.T.V.Rao MD 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. 50 Dr.T.V.Rao MD 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. 51 Dr.T.V.Rao MD 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. 52 Dr.T.V.Rao MD Triple Sugar Iron Agar : Triple Sugar Iron Agar Bacteria that ferment any of the three sugars in the medium will produce byproducts 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. 53 Dr.T.V.Rao MD TSI slant : TSI slant The TSI slant is a test tube that contains agar, a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate. 54 Dr.T.V.Rao MD TSI - Reactions : TSI - Reactions Some bacteria utilize thiosulfate anion as a terminal electron acceptor, reducing it to sulfide. If this occurs, the newly-formed hydrogen sulfide (H2S) reacts with ferrous sulfate in the medium to form ferrous sulfide, which is visible as a black precipitate. Examples of sulfide-producing bacteria include Salmonella, Proteus, Citrobacter and Edwardsiella species. The blackening of the medium is almost always observed in the butt (bottom) of the medium. 55 Dr.T.V.Rao MD TSI - Reactions : TSI - Reactions All lactose fermenters result in yellow slant/yellow butt (acid/acid reaction), whereas non-lactose fermenters may result in pink/yellow or yellow/yellow (if sucrose is fermented). Blackening of the butt due to H2S production may mask the acid reaction (yellow) in the butt.. 56 Dr.T.V.Rao MD Reading the results on TSI enables to identify the several pathogens : Reading the results on TSI enables to identify the several pathogens 57 Dr.T.V.Rao MD GELATIN LIQUEFACTION TEST : GELATIN LIQUEFACTION TEST Dr.T.V.Rao MD 58 The purpose of this test is to determine an organism's ability to produce proteolytic-like enzymes and liquefy gelatin. This test is used to differentiate between species in the genera Staphylococcus and Clostridium as well as aid in the identification of other species and genra. GELATIN LIQUEFACTION TEST : GELATIN LIQUEFACTION TEST Dr.T.V.Rao MD 59 Gelatin is to large to enter a bacterial cell wall and thus extracellular enzymesmust catabolize them into smaller components before they can be utilized. Possession of these extracellular gelatinases can aid in the differentiation of bacteria INTERPRETING TEST RESULTS: : INTERPRETING TEST RESULTS: Dr.T.V.Rao MD 60 POSITIVE TEST = medium liquefied.NEGATIVE TEST = medium remains solid. CAMP Test : CAMP Test Dr.T.V.Rao MD 61 CAMP factor is a diffusible, heat-stable protein produced by group B streptococci. This is a synergistic test between Staphylococcus aureus and Streptococcus agalactiae. S. agalactiae produces CAMP factor. S. aureus produces sphingomyelin C, which binds to red blood cell membranes. The two bacteria are streaked at 90o angles of one another. They do NOT touch Slide 62: Dr.T.V.Rao MD 62 Created by Dr.T.V.Rao MD for ‘e” learning by Microbiologists