BIOCHEMICAL TEST TO IDENTIFY BACTERIA - PowerPoint Presentation

Slide1

BIOCHEMICAL TEST TO IDENTIFY BACTERIA

Prepared by:

Miss

Norzawani

Jaffar

Bsc

(

Hons

) Biomedical SciencesSlide2

Learning Outcomes

Able to understand the principle and function of biochemical test;

Beta-

glucuronidase

Bile solubility

Catalase

Citrate utilization

Coagulase

DNA-

ase

Indole

Litmus milk

decolorization

Lysine

decarboxylase

Oxidase

UreaseSlide3

Introduction

While several commercial system for identifying bacteria are available, these are often difficult to obtain or too expensive to use in developing countries.

This subunit includes a range of conventional biochemical test and tablet identification test which most district laboratories will be able to perform.Slide4

TEST

PURPOSE

Beta-

glucuronidase

To identify

E.coli

Bile

Solubility

To differentiate S.

pneumoniae

from other alpha-

haemoytic

streptococci

Catalase

To differentiate staphylococci from streptococci

Citrate

utilization

To differentiate

enterobacteria

Coagulase

To identify S.

aureus

DNA-

ase

To help

identify S.

aureus

Indole

To differentiate

Gram negative rods, particularly

E.coliSlide5

TEST

PURPOSE

Litmus

milk

decolorization

To help identify

Enterococcus

and some

clostrodia

Oxidase

To help identify

Neisseria

,

Pasteurella

,

Vibrio

, Pseudomonas

Urease

To help identify Proteus,

Morganella

, Y.

enterocolitica

, H. pylori

Lysine

decarboxylase

To assist in the identification

os

Salmonella and

shigellaSlide6

Bile Solubility Test

To differentiate

S

.

pneumoniae

, which is

soluble in bile and bile salt,

from other alpha-hemolytic streptococci (

viridans

streptococci) which are insoluble.

PRINCIPLE:

A Heavy

inoculum

of the test organism is emulsified in physiological saline and the bile salt sodium

deoxycholate

is added. This dissolves

S.

pneumoniae

as shown by a clearing of the turbidity with in 10-15 minutes.

Viridans

and other streptococci are not dissolved and therefore there is no clearing of the turbidity.Slide7
Slide8

Catalase

test

Used to differentiate those bacteria that produce the enzyme

catalase

, such as

staphylococci

, from

non-

catalase

producing bacteria such as streptococci.

PRINCIPLE:

Catalase

act as a catalyst in the breakdown of

hydrogen peroxide to oxygen and water

. An organism is tested for

catalase

production by bringing it into contact with hydrogen peroxide. Bubbles of oxygen are released if the organism is a

catalase

producer. The culture should not be more than 24 hrs old.Slide9

Most aerobic

organsims

will display (+) results.

e.g.

Staphyloccocus

aureus

.

Some anaerobic organisms will display (-) results, indicating that they do not produce

catalase

to prevent oxygen accumulation. Why?

Because since oxygen is totally

not used for survival

of these organisms, they

do not have the ability

to produce

catalase.Slide10

Citrate Utilization Test

Purpose:

The citrate utilization test is used to determine the ability of an organism, using the enzyme

citrase

, to use citrate as its sole carbon source

Used occasionally to assist in the identification of

enterobacteria

.Slide11

Coagulase

Test

This test is used to identify S.

aureus

which

produces the enzyme

coagulase

.

PRINCIPLE:

Coagulase

cause plasma to clot by converting fibrinogen to fibrin. Two types of

coagulase

are produced by most strains of

S.aureus

Free

coagulase

which converts

fibronogen

to fibrin by activating a

coagulase

-reacting factor present in plasma. Free

coagulase

is detected by clotting in the test tube.

Bound

coagulase

(clumping factor) which converts fibrinogen directly to fibrin without requiring a

coagulase

-reacting factor. It can be detected by the clumping

pf

bacterial cells in the rapid slide test.Slide12
Slide13
Slide14

A tube test must be performed when the result of a slide test is not clear, or when the slide test is negative and Staphylococcus has been isolated from a serious infections.

Before performing a

coagulase

test, examine a Gram stained smear to confirm that the organism is a Gram positive

coccus

.Slide15

DNA-

ase

test

Used in the identification of

S.aureus

which produces

deoxyribonuclease

(DNA-

ase

) enzymes. The DNA-

ase

test is particularly useful when plasma is not available to performed a

coagulase

test or when the results of a

coagulase

test are difficult to interpret.

PRINCIPLE:

Deoxyribonuclease

hydrolyzes deoxyribonucleic acid(DNA). The test organism is cultured on a medium which contains DNA. After overnight incubation, the colonies are tested for DNA-

ase

-production by flooding the plate with a weak hydrochloric acid solution. The acid precipitates

unhydrolyzed

DNA. DNA-

ase

producing colonies are therefore surround by clear areas due to DNA hydrolysis.Slide16

Note there is breakdown of the DNA in the agar. There is a clear zone (arrow) around the bacterial growth where there is no longer any DNA left in the agar to precipitate out of solution after the

HCl

was added. Slide17

Indole

Test

Testing for

indole

production is important in the identification of

enterobacteria

. Most strains of

E. coli, P.

vulgaris

, P.

rettgeri

, M.

morganii

,

and

Providencia species break down the amino acid tryptophan with the release of

indole

.

PRINCIPLE:

The test organism is cultured in a medium which contains tryptophan.

Indole

production is detected by

Kovac’s

or Ehrlich’s reagent which contains 4 (p)-

dimethylamino-benzaldehyde

. This reacts with the

indole

to produce a red

coloured

compound.

Kovac’s

reagent is recommended in preference to Ehrlich’s reagent for the detection of

indole

from

enterobacteria

.Slide18

Result: Red

surfeca

layer------------ positive

indole

test

No red layer ------------------- negative

indole

test.Slide19

Litmus Milk

Decolorization

test

A rapid and inexpensive technique to assist in the identification of

enterococci

.

It is based on ability of most strains of

Enterococcus

sp to reduce litmus milk by enzyme action as shown by

decolorization

of the litmus.

PRINCIPLE

A heavy

inoculum

of the test organism is incubated for up to 4 hours in a tube containing litmus milk. Reduction of litmus milk is indicated by a change in

colour

of the medium from mauve to white or pale yellow.Slide20

Result: White or pale yellow-pink

colour

---------------suggestive of

Enterococcus

No change or a pink

colour

-------------- probably not

EnterococcusSlide21

Oxidase

Test

The

oxidase

test is used to assist in the identification of Pseudomonas,

Neisseria

,

Vibrio

,

Brucella

,

Pasteurealla

species, all of which produce the enzyme

cytochrome

oxidase

.

PRINCIPLE

A piece of filter paper is soaked with a few drops of

oxidase

reagent. A colony of the test organism is then smeared on the filter paper.

When the organism is

oxidase

-producing, the

phenylenediamine

in the reagent will be oxidized to a deep purple

colour

.Slide22

Result:

Blue-purple

colour

------------------

positve

oxidase

test(within 10 seconds)

No blue-purple

colour

--------------Negative

oxidase

test (within 10 seconds)

Note!: Ignore any blue-purple

colour

that develops after 10 seconds.Slide23

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. If, however, the stab mark is clearly visible and the rest of the tube is not turbid, the organism is likely

nonmotile

(tube pictured on the left). Slide24
Slide25

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.

Proteus mirabilis

is a rapid

hydrolyzer

of urea (center tube pictured here). The tube on the far right was inoculated with a

urease

negative organism and the tube on the far left was

uninoculated

.Slide26
Slide27

END

Q&ASlide28

Take note from

http://norazlicucst.weebly.com

MICROBIOLOGY II: TOPIC 2