EXERCISE 10: ALPHA

HEMOLYTIC STEPTOCOCCI

Purpose of the Experiment

1. To acquaint the student with the
different characteristics of alphahemolytic streptococci.
2. To differentiate Streptococcus
pneumoniae and Streptococcus
viridans

Materials

1. Blood Agar (BA)

plates.
2. Inulin Agar
3. Skimmed Milk
4. 5ug Optochin disk
5. 10% solution sodium
deoxycholate
6. 1% aqueous crystal
violet
7. 20% copper sulfate
8. NSS

9. Inoculating wire loop

and wire needle
10. Alcohol lamp
11. Forceps
12. Cotton Swab

Procedure Day 1
1.) With a cotton swab, acquire a
sample by swabbing the throat of a
classmate exhibiting signs of sore
throat or any abnormality in the throat
region

Procedure Day 1
2.) Inoculate the sample on a BA plate
and observe multiple interrupted
streaking. The plate was incubated at
37C in a candle jar for 24 hours.

Procedure Day 2
1.) Perform a subculture
Divide the secondary plate into two
Inoculate alpha-hemolytic colonies on one side
and beta-hemolytic colonies on the other
Perform uninterrupted multiple streaking for
both sides without flaming in between streaks.
The new BA plate was
incubated at 37C for
24 hours in a candle jar

Procedure Day 2

2.) Make a smear of a colony on a

glass slide for gram staining for the
following meeting.

Procedure Day 3
1.) Do Gram Stain on the previous
smear

2.) Inoculate a tube of skimmed with

an alpha-hemolytic colony from the BA
plate

Procedure Day 3
3.) Perform Optochin sensitivity test
3.1) Divide the BA plate in two. One for
alpha hemolytic colonies and the other for
beta.
3.2) Inoculate both sides heavily with 2-3
corresponding colonies while performing
close
streaking .
3.3) Place an optochin disc at the center of
the inoculated plate
3.4) Incubate the BA plate aerobically at 37C

Procedure Day 3
4.) Perform Inulin Test
4.1) Using a sterile inoculating wire
needle, inoculate an inulin agar by
stabbing the medium until a few mm
from the bottom.
4.2.)Incubate at 37 degrees Celsius
and read the result in 24-48 hours.
Observe color change in the medium.

Procedure Day 4
1.) Perform Capsule Staining (Anthony Method)
1.1) Make a thin smear of a culture in skimmed
milk spreading with a glass slide
1.2) Air Dry. Do not heat fix.
1.3) Stain with 1% aqueous crystal violet
1.4) Wash with a solution of 20% Copper
sulfate
1.5) Air dry in a vertical position, and examine
under oil immersion objective.

Procedure Day 4
2. Bile solubility test
To a tube containing 2ml of NSS, add alphahemolytic colonies until you have a turbid
suspension.
Divide the suspension into 2 test tubes
Mark one tube as CONTROL
To the other test tube, add a few drops of 10%
solution of Sodium deoxycholate
Observe for clearing or lysis which occurs in 510 minutes. Compare the turbidity with that of
the CONTROL tube.

Procedure Day 4
3.) Check results:
A. Inulin fermentation:
Acid- (+) for S. pneumoniae

Procedure Day 4
2.) Check results:
B. Optochin Susceptibility Test:
Zone equal to or greater than 14mm = Bacteria is
susceptible/sensitive = Positive for Streptococcus
pneumoniae

Procedure Day 4
2.) Check results:
C. Bile solubility test
soluble =(+) S. pneumoniae

Results

Results

Conclusion
Streptococcus pneumoniae form alphahemolytic colonies that are optochin sensitive,
bile soluble, ferment inulin, and are capsulated
organisms .
While, Streptococcus viridans form alphahemolytic colonies that are resistant to optochin,
bile insoluble and do not ferment inulin.
therefore; pneumococci can be separately
identified with the following test: Optochin
Sensitivity test , Bile Solubility Test, and Inulin
Test . The capsule is identified through Anthony
Capsular Staining Method.

EXERCISE 11: BETA-HEMOLYTIC

STREPTOCOCCI
PROCEDURE:
1. Similar procedure with Exercise 10;
except you stab at the end of the
first quadrant only, 2-3 times. (Day
1-2)
STAB 23 times

2. Bacitracin Test (Day 3)

2.1 From the same BA Plate, using a
sterile forceps, place a Bacitracin
disc on the center of the inoculated
area.
2.2 Incubate BA Plate aerobically at
37C.
2.3 After 24 hours, read and
examine for a
zone of growth
inhibition around the Bacitracin disc.
** Any zone of growth inhibition is
indicative of Bacitracin susceptibility.

RESULTS
GRAM STAIN:
Gram (+) cocci in chains

RESULTS
BA Plate:
Presence of clear zone of hemolysis around
colonies.

RESULTS
BACITRACIN TEST:
Group A Streptococci:
Bacitracin Sensitive (killed)
- Presence of zone of inhibition
Group B Streptococci:
Bacitracin Resistant (not killed)
- Absence of zone of inhibition

CONCLUSION:
Beta-hemolytic streptococci is gram
(+). Group A beta-hemolytic
streptococci is sensitive to Bacitracin,
while Group B beta-hemolytic
streptococci is resistant.