Practical 6 Identification of Gram Ve Bacteria

7/30/2019 Practical 6 Identification of Gram Ve Bacteria

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Practical 6Ms. Banan A. Atwah

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IDENTIFICATION OF GRAM POSITIVE BACTERIA

bjectives:O1. To know the major biochemical tests used in microbiology. To be familiar with the basic principles of those biochemical tests. To be able to read the positive and negative tests results.

To know the application of those tests in identification of gram positivebacteria.

ackground:B2.A variety of biochemical tests can be performed directly using single

colonies seen on primary isolation plates. Depending on the nature of

biochemical reactions, some reactions become positive within a minute or

two, other take less than an hour, and most require incubation for 4-18

hours. Such tests are help in placing an isolate of known morphology into afurther subdivision, thus directing the additional procedures needed for

identification. Frequently, an isolate can be identified to a level that is

clinically useful based on these assessments.

3. Materials:1.Set of Gram stain2.Glass slides3.Wire loop & wooden sticks4.Normal saline5.Cultures of (Staphylococcus aureus, S. epidermidis, S.

saprophyticus) on blood agars.

6.Cultures of [Streptococcus pyogenes "group A", S.agalactiae"group B"] on blood agars.

7.Culture ofEnterococcus faecalis (group D) on blood agar8.Cultures of (Streptococcusviridians, S. pneumoniae) on blood

agars9.3% H2O2 (hydrogen peroxide) reagent10.Human plasma and Agglutination paper


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4. Methods: Describe the gross colonial morphology of all given plates. Perform Gram staining of the available organisms and then comment on

the results.

Then biochemical tests will be demonstrated, you should know the testname, understand the principle and be able to differentiate between

positive and negative results. The tests demonstrated as follows:

1.Catalase test: Fig.1This test is used to differentiate those bacteria that produce the enzyme

catalase, such as staphylococci, from non-catalase producing bacteria

such as streptococci.

Method of the test:

1.With a wooden stick take a part of single colony and put it on a slide.2.Add one small drop of H2O2 to the bacteria.3.Look for immediate bubbling by the production of gas

Results:

Gas production (bubbles) Catalase positive (all Staphylococcusspecies)

No Gas production (No bubbles) Catalase Negative (allStreptococcus species)

2 H2O2 2 H2O + O2 (gas)

Figure 1: Catalase test

Catalase Enz.


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2. Coagulase test:

This test is used to differentiateStaphylococcus aureus which produces

the enzyme coagulase, from S. epidermidis and S. saprophyticus

which do not produce coagulase.

Principle:

Coagulase causes plasma to coagulate (clot) by converting the plasmafibrinogen to fibrin.

Two types of coagulase are produced by most strains of S. aureus:Free coagulase: converts fibrinogen to fibrin by activating a

coagualse-reacting factor present in plasma. It is detected by

clotting in the tube test(Fig. 2)Bound coagulase (clumping factor): converts fibrinogen directly

to fibrin without requiring a coagulase-reacting factor. It can be

detected by clumping of bacterial cells in the rapid slide test.

Method for slide test (bound coaglulase):Fig.3

1.Add one drop of either human plasma or latex reagent on to paperslide

2.Using a plastic or wooden stick, take part of test colony to the slide

3.Mix well and look for clumping within 10 seconds.Results:

Clumping or clots formed (plasma coagulated) within 10seconds Coagulase produced (coagulase positive) the organism

is S. aureus.

No clumpimg within 10 seconds No coagulase produced(coagulase negative) the organism may be (other staphylococciS. epidermidis or S. saprophyticus)See chart on page 8

Fibrinogen fibrin (clot) coagulationBacterial coagulase


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Figure 3: Slide coagulase test

+v

Figure 2: Tube coagulase test

_

+v

_


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Unknown culture identification (1)

table1Cultural characteristics (Colony gross morphology):-1

Colony morphologyReaction on the plate

ObservationsColony characteristicsNo.

Very small, Small, Medium, large, very

largeSize1

Punctiform, circular, filamentous,irregular,

rhizoid, spindle

Form2

Flat, raised, convex, pulvinate,umbonateElevation3

Entire, undulate, lobate, erose,

filamentous, curledMargin4

White, grey, yellow, black, orange, pink,red, etc

Color5

haemolysis, orHaemolysis6

Color of the pigment productionPigment production7

Fruity, freshly cut apple, fishy, fecal orputrid, bleach, pungent

Odor8

Transparent, Opaque, TranslucentOpacity9

Smooth, Glistening, Rough, dullSurface10

Buttery, viscid, Brittle, mucoidConsistency11

Table 1: Gross Colony Characteristics


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Fig.4Gram Staining:-2

Gram reaction

Shape

Arrangement

GGrraamm nneeggaattiivveeGGrraamm ppoossiittiivvee

CCooccccii BBaacciillllii SSppiirraall

SSiinngglleePPaaiirrss

((ddiipplloo--))CChhaaiinn

((ssttrreeppttoo--))CClluusstteerr

((ssttaapphhyylloo--))iirrrreegguullaarr

Figure 4: Main bacterial shapes & arrangements


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:Biochemiacal test-3

(write name of organism)Differential test

Reaction results

Negative (or resistant)Positive (or sensitive)

CatalaseCoagulaseMannitol salt agar

DNAse

Novobiocin sensitivity test

Bacitracin disk

CAMP test

optochin sensitivity diskBile esculin hydrolysis


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Cocci

Bacilli

Catalase

Staph. Strept.

S.Aureus (non-Aureus)

Novobiocin

disk

S.Epi S.sapro.

Bacitracin

disk

St. gp A B,C,G

Optochin

disk

St.pneu. St.Viri

Bile Esculin

hydrolysis

Endo -

Spores

Non-

sporingSSppoorriinngg

CAMPtest

St.gp B St.gpC,G

+ --

+

+S -R

-

-

+S -R-R+S

+

Coryne.diphtheria

Type of

Haemolysis

Presence of

air

Aerobic Anaerobic

Bacillus

Spp.Clostridium

G +ve Bacteria

Lactobacilli

nterococcus

Faecalis

OtherSt.gp D

+-

Coagulase

DNase

+

e.g. Bacillus cereus

Catalase +ve

Streptococcus pyogenes

Streptococcus agalactiae

Streptococcus pneumoniaeStreptococcus viridans

Practical 6 Identification of Gram Ve Bacteria

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