Biochemical Testing

7/28/2019 Biochemical Testing

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UNIVERSITY OF PORT HARCOURT

FACULTY OF PHARMACEUTICAL SCIENCES

DEPARTMENT OF PHARMACY

AN

ASSIGNMENT

ON

BIOCHEMICAL TEST

PRESENTED BY

NAME: NWACHUKWU ONYEDIKACHI R

MAT NO: U2009/4725253

COURSE: PHARMACEUTICAL MICROBIOLOGY

CODE: PMB 341.1

COURSE LECTURER: PHARM A. AWANYE

APRIL 2013


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INTRODUCTION

Biochemical testing is used to identify bacteria and yeast because different

species produce different enzymes. Such biochemical test are designed to detect

the presence of enzymes.

Examples of biochemical tests are:

1. Oxidase test2. Indole test3. Methylated test4. Galactosidase test5. Citrate test6. Urease test7. Production of hydrogen sulphide (H2S)8. Agglutination of trivalent serum9. Dnase test10.Coagulase test

GALACTOSIDASE TEST

Lactose utilization requires a couple of enzymes, one of which is beta-

galactosidase. In this test, you use a molecular decoy called ONPG (Ortho-nitrophenyl--D-galactopyranoside that will turn to a yellow colour in the

presence of this enzyme.

ONPG is an analog of lactose that the enzyme can break down to produce a

yellow coloured end-product O-nitrophenol. Since this enzyme is made only in

the presence of the lactose substrate, you need to be sure to grow this organism

on media high in lactose from lactose broth on TSIA agar to name a couple.

Note, the test is generally for gram bacteria and also to identify some

staphylococcus species

OBJECTIVE

Identify the ability of the bacterium to make the enzyme beta-galactosidase

. Materials needed are:

-

ONPG Disc- 0.85- 0.99 Nad


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- 1ml pipettes and pi-pumps- Sterile small tubes and cups

PROCEDURE

1. First, your organism needs to be growing in any medium with lactose (toinduce the production of galactosidase enzyme)

2. Pipette 0.5ml of the saline into a sterile tube.3. Inoculate with the bacterium and add the ONPG disc in a sterile manner

(for cups dipped in alcohol and flame) to the tube.

4. Incubate at 3C for 4 hours. There is so little fluid in the tube, it willdehydrate if you do not read it in a few hours (alternatively you can coverit with paraffin)

RESULT

Fluid and the disc will turn any shade of yellow if positive for galactosidase

enzyme.

TEST ORGANISM ONPG HYDROLYSIS

CitrobacterFreyndii +

EnterobacterGerogenes +

Escherichia Coli +

Proteus Vulgaris

Salmonella Serotype Arizone +

Salmonella Serotype Typhirium


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OXIDASE TEST

INTRODUCTION

Oxidase test can be defined as a test carried out in microbiology to

determine whether a bacterium produces certain cytochrome c oxidase. This

test uses disks that are impregnated with either of the reagents;-N,N,N,N-

Tetramethyl-p-phenylenediamine(TMPD) or

N,N/diamethylphenylenediamine(TMPD).

These reagents are redox indicators. They give dark blue colour to Maroon

colour when being oxidized and colourless when being reduced.

Bacteria which are oxidase positive possess cytochrome oxidase or indolepheno

l oxidase. These both catalyse the transport of electron from donor

compounds(NADPH) to electron acceptors (usually oxygen).TMPD

dihydrochloride acts as an artificial electron donor for the enzyme oxidase.

N

H2N

N

H3C CH3

N

H3C CH3

TMPD

CH3

CH3

DMPD


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CLASSIFICATIONS

Strains are found to be either oxidised positive or oxidase.

OXIDASE POSITIVE BACTERIA

Bacteria which are oxidase positive contain cytochrome c oxidase. These

bacteria use oxygen for their energy production with an electron transfer chain.

Examples of oxidase-positive bacteria are:

a. The pseudomonadaceaeb.Neisseria Gonorrhoeaec. Legionella pneumophilad. Gram-negative, spiral curved rods-Helicobacter phylori,Vibro cholera

and Campylobacter jejuni.

OXIDASE-NEGATIVE BACTERIA

Bacteria in this case do not also use oxygen for their energy production

with an electron transfer chain. Examples are:

a. Enterobacteria

b. Escherichia coli

COLOR CHANGE

1. An oxidase positive test will give purple colour through Maroon and

then black within 10-30 seconds.

2:An oxidase negative test will give no colour change at all.

PROCEDURES

-Moisten filter paper with the substrate 9%tetramethyl-p-

phenylenediaminedihydrochloride or select a commercially available

paper impregnated with the substrate.

-Use a platinum wire to remove a small portion of a bacterial colony from

the agar surface and rub the sample on the filter paper.

-Observe inoculated areas of the paper or disk for colour change to deep

blue or purple within 10seconds.

INDOLE TEST

The indole test can be defined as a biochemical test carried out on

bacterial species to determine the organisms ability to split indole from


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the amino acid called tryptophan. This division is performed by a chain of

a number of different ultra cellular enzymes referred to as trytophanase.

CLASSIFICATION

Strains are found to be either indole-positive bacteria or indole-

negative bacteria

INDOLE-POSITIVE BACTERIA

Bacteria that test positive for clearing indole from tryptophan

includes

a. Aeromona punctata

b. Bacillus Alvei

c. Aeromonas Hydrophilia

d. Pasteurella Multocida

e. Streptococcus Faecalis etc

INDOLE-NEGATIVE BACTERIA

Bacteria which test negative with the indole test includes:

a. Pasteurella Ureae

b. Most Bacillus sp

c. Bordetella sp

d. Pseudomonas sp

COLOUR CHANGES

- A positive test gives a red or red-violet colour in the surface alcohol

layer of the broth.

- A negativ e test gives a yellow colour. A variable result can also occur,

showing an orange colour as a result, due to the presence of Skatole also

known as Methyl Indole or Methylated Indole.

PROCEDURE

A pure bacteria culture was grown in sterile tryptophan or peptone

broth and allowed to incubate for 24-48 hours at 32C.After incubation, 5

drops of Kovac's reagent which contains isoamyl alcohol paradimethyl

aminobenzaldehyde and concetrated hydrochloric acid is then added to

the broth culture.

A red colour indicates an indole positive bacteria and a yellow

colour indicates an indole-negative test.


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METHYL RED TEST

Methyl Red with the molecular formula C15H15N3O2 and also with

the molecular structure below is an indicator dye that turns red in acidic

solutions. It is an azodye and a dark-red crystalline powder. It gives red in

pH under 4-4 yellow in pH over 0-2 and orange between, with a PKA of

5.1

Methyl Red is a test in microbiology used to identify bacteria

producing stable acids by mechanisms of mixed acid fermentation of

glucose. It is also a test carried out to identify enteric bacteria based on

their pattern of glucose mechanism. All enteric bacteria will produce

pyruvic acid from glucose metabolism.

CLASSIFICATION

Enteric bacteria may be found to be either methyl red positive or

negative.

METHYL RED POSITIVE

The enteric bacteria that are found here makes use of the mixed acid

pathway to metabolize pyruvic acid to other acids like lactic, acetic, and

formic acids. These bacteria are referred to as methyl Red-positive

bacteria. Examples are:

a. Escherichia coli

b. Proteus vulgaris

METHYL RED NEGETIVE

The bacteria make use of the butylenes glycol pathway to metabolize

pyruvic acid to neutral end-products. They are referred to a methyl Red

negative bacteria. Examples are

a. Serratia marcescens

b:Enterobacter aerogenes

RESULTS OR COLOUR CHANGES

The Enteric bacteria that tend to metabolise pyruvic acid to other

acids lower the pH of the medium to 4.2 thereby giving out a red colour

which is positive to the test. While those that metabolize pyruvic acid to


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neutral end products tend to lower the pH of the medium to 6.0,there by

giving out a yellow colour which is negative to the test.

PROCEDURE

An isolate is inoculated into a tube with sterile transfer loop. The tube

is incubated at 35c for 2-3 days. After incubation,2.5ml of the medium is

between palms of the hands to disperse the methyl red.

CITRASE TEST

This type of test is used to detect an organism ability to use citrate

as its sole source of carbon and energy.

Bacteria are incubated in a medium containing sodium citrate and a pH

indicator like bromothymol blue. The medium alone contains inorganic

ammonium salts used as the sole source of nitrogen.

CITRATE POSITIVE BACTERIA

There will be growth on the medium and the colour change from

green to blue. Examples are:

a. Frateuria aurantia

b. Escherichia coli

CITRATE NEGETIVE BACTERIA

No growth is observed or takes place on the medium and no colour

changes.

PROCEDURE

Bacteria colonies are picked up using a loop and inoculated into

Simmon's citrate agar and incubated overnight at 3c for up to days.

The medium changes colour from green to blue.

UREASE TEST

This is a rapid test for the diagnosis of H.pylori. It is also a test

to detect the ability of H.pylori to secrete enzymes which catalyses the

conversion of urea to ammonium and bicarbonate. This test is performed

at the of gastroscopy.


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PROCEDURE

A biopsy of mucosa is taken from the antrum of the stomach and

placed into the medium containing urea and phenol red. The urease that

is being produced by H.pylori hydrolysis urea to ammonia and colour

changes of the specimen from yellow to red are being observed.

The specimen that gives a colour change of red is positive to the test

while the specimen that gives a yellow colour is negative to test.

COAGULASE TEST

Coagulate test is a protein that can or be produced by several micro

organism that enable the conversion of fibrinogen to fibrin. Coagulase is

also produced by Yersinia pestis.

Coagulase test is carried out to differentiate Staphylococcus aureaus from

coagulase negative staphylococci. S.aureus always produce to forms of

coagulase which are either bound coagulase or free coagulase.

Bound coagulase, otherwise known as ''clumping factors'' csn also be

detected by doing a tube coagulase test.

SLIDE TEST

Two drops of saline are put on to the slide which is labelled test(t)

and control(c).The two saline are emulsified with the test organism by

using a wire loop.

Place a drop of plasma on the inoculated saline drop corresponding

to the test an mix well with wooden applicator stick. Rock the slide

gently for losses.

RESULT

Macroscopic clumping would be observed in the plasma within 10

seconds and no change in the saline drop, if the test is positive

If the test is negative,no clumping will be observed

TUBE TEST

This test uses rabbit plasma that has been inoculated with a

staphylococcus colony (i.e gram positive cocci which is catalase

positive)the tube is then incubated at 3C for one hour thirty minutes.if

negative then continue incubation up to 18hours.

RESULT


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- the serum will coagulate resulting in a clot if the test is positive.

- if negetive,the plasma remains liquid.The negative result may be

S.epidemides.

example of coagulase positive are;

a:Staphylococcus aureus

b:Staphylococcus delphini

c:Staphylococcus hyicus

example coagulase negetive are;

a:S.caprace

b:S.epidermis

c:S.warneri

d:S.hominis

DNASE TEST

This principle is a test use to determine the ability of an organism

to hydrolyze DNA.The medium is pale pale green because of the DNA-

methyl green complex.If the organism growing on the medium

hydrolyses DNA,the green colour fades and the colony is sorrounded by a

colourless zone.

METHOD

- inoculate the DNASE agar with the organism to be tested and streak

eus for isolation .

- incubate aerobically at 35c for 3-24 hours.

RESULT

Positive when DNA is hydrolyzed and combined with highly

polymerized DNA at a pH of 7.5,turning the medium coloured around the

test organism.

Negetive if there is no degradation of DNA,the medium remains

green.

QUALITY CONTROL

positive-staphylococcus aureus

Negetive-staphylococcus epidermis

PRODUCTION OF HYDROGEN SULPHIDE

Principle

this is used to detdct hydrogen sulphide librated as aresult of the

degradation of sulphur containing amino acid

METHOD


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- Inoculate two sim tubes,one with E.coli and the other with proteus

vulgaris.Use an inoculating needle and stab the agar with a single in and

out motion.

- Examine each sim tube for the presence of a black colour which

indicates the production of hydrogen sulphide which combines with the

peptonised iron in the sim medium and represents a positive test.the

absence of black is a negetive test.

EXPECTED RESULT

Proteus vulgaris is positive with E.coli

AGGLUTINATION

This is a suspension of the specific antibody mixed with the

insoluble antigen, such as red blood cells,bacteria or fungi.In

agglutination reactions, however relatively large insoluble particles are

involved rather than soluble molecules.Streptococcus species are used.

EXPECTED RESULT

- Readily visible clump will form if the test is positive

- No clumping if result is negetive

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Biochemical Testing