IDENTIFICATION
METHODS OF GRAM
POSITIVE AND GRAM
NEGATIVE COCCI
PRESENTED BY: EUNICE LORÁN, RUTH CERPA,
KATIAN MELENDEZ
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 PROCEDURE
Streak culture for isolation on TSA plate with
5% sheep blood.
Incubate the plate for 24 hours at 35° C.
Observe the results.
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 PROCEDURE
Using an inoculating loop, streak two or three
suspect colonies of a pure culture onto a
blood agar plate.
Using heated forceps, place a bacitracin
disk in the first quadrant (area of heaviest
growth).
Incubate the plate for 18 to 24 hours at 35° C
RT.
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 PROCEDURE
From a pure culture transfer isolated colonies
of aerobic, catalase-positive, coagulasenegative gram-positive coccus aseptically to
a sterile plate of nutrient agar.
Apply 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.
enzyme
PYR
βnaphthylamide
Ddimethylaminocinnamalde
hyde
(reagent)
Red
Color
PYR HYDROLYSIS TEST PROCEDURE
Moisten a PYR disk with sterile water.
Using a sterile loop, rub colonies on the
disk.
Incubate disk at room temperature for 10
minutes.
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 αhemolytic 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:
Resistant
Susceptible
BILE ESCULIN TEST
• Differentiates group D streptococci and
Enterococcus from other gram-positive, catalasenegative cocci.
• Two-step test:
• Growth in the presence of bile.
• Hydrolysis of esculin to esculetin and glucose.
Esculin
Esculetin
Glucose
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:
Positive
Negative
Positive
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
Culturing in a semi-solid medium procedure:
Apply a colony to the end of an inoculating
needle.
Insert the needle into the center of the semisolid medium tube for about one inch.
Incubate at 30° C for 24 to 48 hours.
Observe growth pattern in the tube.
MOTILITY TEST RESULTS
Interpretation:
Very
motile
Nonmotile
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 βhemolytic streptococci.
• Ej. S. agalactiae
CAMP TEST PROCEDURE
Inoculate a single streak of β-lysin
producing S. aureus into a TSAsheep 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 + glycine
benzoic acid + ferric chloride
Ferric
benzoate
• Test detects hippurate hydrolyzing bacterias,
specially from group B streptococci.
Example: S. agalactiae
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-pphenylenediamine 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_Su
sceptibility.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-methodssections1-2.pdf
REFERENCES
• Novobiocin differentation disk. Retrieved September 2014,
from
https://catalog.hardydiagnostics.com/cp_prod/content/hug
o/NovobiocinDiffDisks.htm
• Bacitracin test: principle, procedure, expected results and
quality control. Retrieved September 2014, from
http://microbeonline.com/bacitracin-test-principleprocedure-expected-results-and-quality-control/