Biochemical

Reactions
in
Common Pathogenic Bacterial Isolates Dr.T.V.Rao MD

Dr.T.V.Rao MD

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:

Microbiologists

use biochemical tests, noting a particular microbe's ability to utilize or produce certain chemicals
3 Dr.T.V.Rao MD

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 Dr.T.V.Rao MD

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 Kovacs 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 proskaurs 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

Indole Methyl Red/Voges Proskauer Citrate H2S production Urea hydrolysis Motility Lactose fermentation Sucrose fermentation Glucose fermentation &

gas production
8 Dr.T.V.Rao MD

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 MD catalase Dr.T.V.Rao 9

Catalase test
'Ten vol.' H2O2, is

10

run into a capillary tube, followed by suspension. Gas is usually evolved immediately and only tubes not showing gas within 10 sec. Are Dr.T.V.Rao MD sealed for longer

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-pphenylenediamine OR dimethyl-p-phenylenediamine a redox dye present as the hydrochloride or oxalate salt The dye is reduced to a deep Dr.T.V.Rao MD violet-blue colour in the presence of oxidase enzymes

11

Oxidase test
The oxidase test is a

12

Dr.T.V.Rao MD

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-pphenylenediamine (TMPD) or N,NDimethyl-pphenylenediamine (DMPD), which is also a redox indicator.

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

Positive

control: Pseudomona s aeruginosa Negative control

Enterobacteric ia

14

Dr.T.V.Rao MD

HYDROGEN SULFIDE PRODUCTION

Some bacteria have the

15

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 Dr.T.V.Rao MD to 100 ml nutritive agar).

PROCEDURE and Reading result

Harvest a well

16

Dr.T.V.Rao MD

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

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 Dr.T.V.Rao MD media.

17

Nitrate Medium Nitrate reduction Test

This is a differential

18

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. Dr.T.V.Rao MD

Nitrate reduction Test

After incubation, these

19

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. Dr.T.V.Rao MD

Nitrate reduction Test

The reduction of

20

Dr.T.V.Rao MD

nitrate to nitrite was detected with dimethyl-anaphthylamin (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

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
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.

22

Media and Reagents Used: Tryptone broth contains tryptophan. Kovacs reagentcontains hydrochloric acid, dimethylaminobenzaldehyde, and Dr.T.V.Rao amylMD alcoholyellow in color.

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 is a product of

24

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 Dr.T.V.Rao MD surface of the

Kovacs
(+) Indole test on left --- (-) Indole test on right

25

Dr.T.V.Rao MD

Indole test reactions

26

Dr.T.V.Rao MD

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.
MRtests for acid end products from glucose fermentation. VPtests for acetoin production from glucose fermentation.

Media and Reagents Used:

Glucose Broth Methyl Red indicator for acid Voges Proskauer reagentsA: 5% Alpha-Naphthol, & ethanol,
27 Dr.T.V.Rao MD

B: Potassium Hydroxide, & Deionized Water.

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

The methyl red (MR) and Voges-Proskauer

28

(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 Dr.T.V.Rao MD glucose.

MR/VP continued
Reading Results: MR a + result is red (indicating pH below 6) and a

result is yellow (indicating no acid production) VPA + result is red after VP reagents are added (indicating the presence of acetoin) and a result is no color change.

29

Dr.T.V.Rao MD

Methyl Red: left and right +

Methyl Red
This test is used to

30

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 Dr.T.V.Rao MD below 4.4, a red color will

Methyl Red Test

If the pH indicator

31

Dr.T.V.Rao MD

(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).

reduction
Standardized

32

Dr.T.V.Rao MD

methylene blue in concentrations of 0.1 and 0.01 yo are mixed,with suspension and sealed.

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
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: Simmons Citrate Agar contains sodium citrate (carbon source), ammonium ion (nitrogen source), & pH indicator bromthymol blue. Reading Results:
34 Dr.T.V.Rao MD

A + result is blue (meaning the bacteria metabolised citrate

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

36

Dr.T.V.Rao MD

Left positive and right negative.

Urea Hydrolysis
How to Perform Test: Inoculate Urea broth with inoculating loop.
Property it tests for: This test is done to determine a bacterias 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 yelloworange 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 Dr.T.V.Rao MD ammonia is made. 37change and no

Urease test
This test is used to

38

Dr.T.V.Rao MD

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

Urease Test
Urea is broken down by

39

Dr.T.V.Rao MD

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

Urease test
This test is used to

40

Dr.T.V.Rao MD

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

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 bacterias 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. Dr.T.V.Rao MD

41

Glucose broth with Durham tubes

This is a differential

42

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 Dr.T.V.Rao MD only some of which

Carbohydrate Fermentation tubes Left is (+) -- Middle indicates (+) with gas -- Right is a (-) test

43

Dr.T.V.Rao MD

Sugar Fermentation Tests

Tube 1: Negative acid /Negative gas Tube 2A: Must incubate longer (ambiguous result) Tube 2B: Positive acid /Negative gas Dr.T.V.Rao MD 44 Tube 3A: Positive acid/ Positive gas

45

Dr.T.V.Rao MD

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 bacterias ability to ferment sucrose and production of acid endproduct 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. Dr.T.V.Rao MD

46

Lactose Fermentation
How to Perform Test: Inoculate lactose broth with inoculating loop. Property it tests for: This tests for the bacterias 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. Dr.T.V.Rao MD 47

Lactose fermenter and Non fermenter

48

Dr.T.V.Rao MD

Motility Testing
Motility agar is a

49

Dr.T.V.Rao MD

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

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

50

Dr.T.V.Rao MD

bacterias ability to clot blood plasma using the enzyme coagulase. If the organism has coagulase it will clump rabbit plasma.

Coagulase test
Coagulase is an enzyme

51

Dr.T.V.Rao MD

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
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

Bacteria that ferment any of the three

53

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. Dr.T.V.Rao MD Many bacteria that can ferment sugars

TSI slant
The TSI slant is a

54

test tube that contains agar, a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as sodium thiosulfate and Dr.T.V.Rao MD ferrous sulfate or

TSI - Reactions
Some bacteria utilize thiosulfate anion

55

as a terminal electron acceptor, reducing it to sulfide. If this occurs, the newlyformed hydrogen sulfide (H2S) reacts with ferrous sulfate in the medium to form ferrous sulfide, which is visible as a black precipitate. Examples of sulfideproducing bacteria include Salmonella, Proteus, Citrobacter and Edwardsiella species. The blackening of the medium Dr.T.V.Rao MD is almost always observed in the butt

TSI - Reactions
All lactose

56

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). Dr.T.V.Rao MD Blackening of the

enables to identify the several pathogens

57

Dr.T.V.Rao MD

GELATIN LIQUEFACTION TEST

The purpose of this

58

test is to determine an organism's ability to produce proteolyticlike 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 Dr.T.V.Rao MD species and genra.

GELATIN LIQUEFACTION TEST

Gelatin is to large to

enter a bacterial cell wall and thus extracellular enzymes must catabolize them into smaller components before they can be utilized. Possession of these extracellular gelatinases can aid in the differentiation of bacteria
59 Dr.T.V.Rao MD

INTERPRETING TEST RESULTS:

POSITIVE

60

Dr.T.V.Rao MD

TEST = medium liquefied. NEGATIVE TEST = medium remains

CAMP Test
CAMP factor is a

61

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 Dr.T.V.Rao MD binds to red blood cell

Created by Dr.T.V.Rao MD for e learning by Microbiologists Email doctortvrao@gmail.com

62 Dr.T.V.Rao MD