P R E S E N T E D B Y: E U N I C E L O R Á N , R U T H C E R P A , K A T I A N M E L E N D E Z

IDENTIFICATION METHODS OF GRAM POSITIVE AND GRAM

NEGATIVE COCCI

OBJECTIVES

• Learn how to perform different methods of detection and differentiation of gram negative and gram positive cocci in the laboratory.

• Learn the mechanism of action of the differentiation and identification methods used.

• Interpret the results from a coccus differentiation and identification method.

HEMOLYSIS TEST

HEMOLYSIS TEST

• This test provides information on what hemolytic enzyme a bacterium possesses.

• The test is performed using blood agar which contains 5% sheep blood.

• It is used to identify Streptococcus pneumoniae.

HEMOLYSIS TEST PROCEDUREStreak culture for isolation on TSA plate with 5% sheep blood.

HEMOLYSIS TEST RESULTS

BACITRACIN TEST

BACITRACIN TEST

• Bacitracin is a mixture of cyclic polypeptides produced by Bacillus subtillis.

• Bacitracin interferes with the dephosphorylation of C 55 – isoprenyl pyrophosphate.

• Differentiates between β-hemolytic Streptococcus.

BACITRACIN TEST PROCEDUREUsing heated forceps, place a bacitracin disk in the first quadrant (area of heaviest growth).

Look for zone of inhibition around disk.

BACITRACIN TEST RESULTS

Inhibition zone

NOVOBIOCIN TEST

NOVOBIOCIN TEST

• Novobiocin interferes with the unpackaging and repackaging of DNA during DNA replication the bacterial cell cycle.

• Novobiocin is obtained from the actinomycete Streptomyces niveus.

• Novobiocin test is a reliable presumptive identification of Staphylococcus Saprophyticus from another coagulase negative Staphylococci spp.

NOVOBIOCIN TEST PROCEDUREApply one 5 µg novobiocin disk onto the inoculated agar surface. Incubate plate aerobically for 18 to 24 hours at 35 to 37° C.

Measure (in millimeters) the diameter of the zone of inhibition around the novobiocin disk, and record as susceptible or resistant.

NOVOBIOCIN TEST RESULTS

Staphylococcus saprophyticus

Staphylococcus epidermidis

PYR HYDROLYSIS TEST

• Differentiates those gram-positive cocci that hydrolyze L-pyrrolidonyl-β-naphthylamide (PYR) from other species. • Streptococcus Group A, β-hemolytic (S. pyogenes)• Enterococcus spp.

• Detects the enzymatic activity of pyrrolidonyl aminopeptidase.

PYR

D-dimethylaminocinnamalde

hyde(reagent)

Red Color

enzymeβ-

naphthylamide

PYR HYDROLYSIS TEST PROCEDURE

Moisten a PYR disk with sterile water.

Add few drops of reagent and observe any color change within 5 minutes.

PYR HYDROLYSIS TEST RESULTS

Interpretation:

Positive Negative

OPTOCHIN TEST

• Identifies Streptococcus pneumoniae from an α-hemolytic streptococcal culture (Viridans spp).

• Optochin is a toxic chemical that harms some bacteria.

• Optochin disks are placed in the culture and are incubated.• Susceptibility = zone of inhibition of 14 mm with a 6 mm disk.• Resistance = visible growth up to the margin of the disk.

OPTOCHIN TEST PROCEDURE

Using a sterile swab, transfer and spread an inoculum of the culture to a SBA plate.

Place an optochin-disk at the center of the plate.

Incubate at 35 - 37° C for 24 hours in a CO2 incubator.

Observe the growth on the surface of the plate.

OPTOCHIN TEST RESULTS

Interpretation:

SusceptibleResistant

BILE ESCULIN TEST

• Differentiates group D streptococci and Enterococcus from other gram-positive, catalase-negative cocci.

• Two-step test:• Growth in the presence of bile.• Hydrolysis of esculin to esculetin and glucose.

Esculin

Glucose

Esculetin Ferric

citrate Black Color

BILE ESCULIN PROCEDURE

Using a sterile loop, inoculate colonies from a blood agar plate to a bile esculin agar plate.

Incubate at 35° C for 18 to 24 hours.

Observe growth and color change in the medium.

BILE ESCULIN RESULTS

Interpretation:

NegativePositivePositive

MOTILITY TEST

• Determines the ability of a bacteria to move in a medium. • Useful in Enterococcus spp.

• Different methods are used:• The Wet Mount – a bacterial sample is observed under

the microscope to identify motility.• The Hanging Drop Method – uses a depression slide to

observe motility. • Culturing Method in a semi-solid medium –

inoculates bacteria in a semi-solid medium using an inoculating needle.

MOTILITY TEST PROCEDURE

The Wet Mount and the Hanging Drop Method:

MOTILITY TEST PROCEDURE

Apply a colony to the end of an inoculating needle.

Insert the needle into the center of the semi-solid medium tube for about one inch.

Incubate at 30° C for 24 to 48 hours.

Observe growth pattern in the tube.

Culturing in a semi-solid medium procedure:

MOTILITY TEST RESULTS

Interpretation:

Very motile

Non-motile

Motile

CAMP TEST

• CAMP factor• Extracellular protein that along with the β-lysin

of Staphylococcus aureus enhances the lysis of red blood cells.

• CAMP test identifies non-hemolytic group B and β-hemolytic streptococci.

• Ej. S. agalactiae

CAMP TEST PROCEDURE

Inoculate a single streak of β-lysin producing S. aureus into a TSA-sheep blood agar plate.

Use an inoculating loop to pick the unknown β-hemolytic streptococcus strain and make a single streak perpendicular to S. aureus.

Incubate overnight at 35° C. Read and interpret results.

CAMP TEST RESULTS

HIPPURATE HYDROLYSIS TEST

• Hippurate hydrolase

hippurate + H2O   benzoic acid + glycinebenzoic acid + ferric chloride • Test detects hippurate hydrolyzing bacterias,

specially from group B streptococci. Example: S. agalactiae

Ferric benzoat

e

HIPPURATE HYDROLYSIS TEST PROCEDURE

Inoculate an hippurate broth with one (1) drop of a fresh Todd-Hewitt broth culture.

Incubate the broth for up to seven (7) days or until turbid growth is seen at 35° C.

Centrifuge the tube of broth to sediment the bacteria.

Pipette 0.8 mL of the clear supernatant to a small clear tube.

Add 0.2 mL of ferric chloride reagent to the supernatant and mix well. Read and interpret results.

HIPPURATE HYDROLYSIS TEST RESULTS

A heavy precipitate that does not clear within 10 minutes

A clear golden-brown liquid

MODIFIED OXIDASE TEST

• Detects enzyme – cytochrome oxidase.

• Modified Oxidase Reagent: 1% tetramethyl-p-phenylenediamine in dimethyl sulfoxide.

• Utilized for differentiating Micrococcus from Staphylococcus.

• Micrococci yields a positive result.• Staphylococci yields a negative result.

MODIFIED OXIDASE TEST PROCEDURE

Use bacteria from a culture grown on blood agar for 24 - 36 hours.

Transfer a Microdase disk from the stock bottle to a petri dish using sterile forceps.

Use an inoculating loop to streak a loopful of bacteria onto the top of the Microdase disk.Read and interpret results.

MODIFIED OXIDASE TEST RESULTS

A blue or purple-blue color change within 2 minutes

No change in color

6.5% NaCl TOLERANCE TEST

• This test differentiates between bacteria that tolerate high (6.5%) concentrations of sodium (NaCl) and those that are inhibited by this salt concentration.• Enterococcus species = positive (growth,

turbid).• Group D Streptococcus = negative (no

growth, clear).• Streptococcus species Viridans group =

negative.

6.5% NaCl TOLERANCE TEST PROCEDURE

Inoculate one (1) or two (2) drops of an overnight Todd Hewitt broth culture into the 6.5% NaCl broth.

Incubate at 37°C in ambient air for a week.

Read and interpret the results.

6.5% NaCl TOLERANCE TEST RESULTS

Positive Negative

6.5% NaCl TOLERANCE TEST RESULTS

• (A) Enterococcus faecalis: Color change from purple to yellow, indicating fermentation of the dextrose and salt tolerance.

• (B) Streptococcus bovis: No color change or growth.

REFERENCES

• Optochin Susceptibility Test. Retrieved September 2014, from http://www.vumicro.com/vumie/help/VUMICRO/Optochin_Susceptibility.htm

• Microbugz. Bile Esculin Test. Retrieved September 2014, from http://www.austincc.edu/microbugz/handouts/Bile%20Esculin%20Test%20Handout.pdf

• Murray, P.R., Baron, E. J., Jorgensen, J.J., Pfaller, M.A., and Yolken, R.H. Manual of Clinical Microbiology, 8th ed Retrieved September 2014, from http://www.cdc.gov/streplab/downloads/general-methods-sections1-2.pdf

REFERENCES

• Novobiocin differentation disk. Retrieved September 2014, from https://catalog.hardydiagnostics.com/cp_prod/content/hugo/NovobiocinDiffDisks.htm

• Bacitracin test: principle, procedure, expected results and quality control. Retrieved September 2014, from http://microbeonline.com/bacitracin-test-principle-procedure-expected-results-and-quality-control/