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/