1.

Introduction:
Streptococcus is a genus of gram-positive, mostly anaerobic cocci occurring in pairs or
chains. The Greek word "streptos" means "twisted" or "chained", and the word "kokhos"
means a "berry" or "seed", hence the name for this genus of bacteria. Over one hundred
species of Streptococcusare recognized. Of these, about 50% may be found at some time in
the human mouth. The classical name for the oral streptococci is the viridans group. Species
of the viridans streptococci include S. anginosus, S. gordonii, S. intermedius, S. sanguinis,S.
parasanguinis, and the mutans group.
Many species of streptococci may be assigned to Lancefield grouping system . The
system depends on specific antibodies which react with various antigens in the bacterial cell
wall. The groups comprise, A, B, C, D, E, F, G, H, K, L, and R/S. For example, S. pyogenes is
a group A streptococcus (GAS). GAS strains are further distinguished according to their
surface anti-phagocytic M proteins which enable these bacteria to evade the host defense
mechanisms.
Purpose

To identify the different classification or groupings of Family Streptococcaceae

To isolate and identify the different Streptococcal species

To perform diagnostic tests for the identification of Streptococcal species

2. Materials and Methods

a. Materials
1st Laboratory period
1. Alcohol lamp
2. Inoculating loops
3. Sterile cotton swab
4. Blood agar plates (2 pcs)
5. Clinical Specimens (throat swab, dental carries of teeth scrapings,
unpasteurized or stale milk)
6. Broth or plated pure culture of Genus Streptococcus
2nd Laboratory period
7. Nutrient agar slants (2 pcs)
8. Staining racks
9. Microscope
10. Glass slides (2 pcs)
11. Gram staining solutions (crystal violet, iodine, ethyl alcohol, safranin)
12. Cedar wood oil

3rd Laboratory period

13. Glass slides (4 pcs)
14. 3% Hydrogen peroxide
15. Inoculating loops
16. Alcohol lamp
17. BAP- Blood Agar Plates (2 pcs)
18. BEA- Bile esculin Agar (1 pc)
19. Bacitracin disks
20. SXT- Sulfamethoxazole- Trimethoprim disks
21. Optochin disks
b.

Methods
1st Laboratory Period
Clinical Specimens for possible source of Genus Streptococci
1.
2.
3.
4.

Throat swab
Dental caries
Unpasteurized milk or milk allowed to stand at room temperature overnight
Each group will be assigned with specific specimen to collect, from which
possible Streptococcus species may be isolated

Throat swab
5. Ask your patient to seat comfortably, tilt the head back with the mouth wide open.
Collect a throat swab by touching the tip of a sterile cotton swab at the back
portion of the throat near the tonsils. Use a tongue depressor to hold the patients
down during the process.
Dental Caries r teeth scrapings
6. Ask your patient to seat comfortably, tilt the head back with the mouth wide open.
Touching the tip of the sterile cotton swab on a tooth with dental caries of just
one teeth (preferably the back portion)
Unpasteurized milk or milk left standing at room temperature for several hours.
7. The groups which will be assigned with this clinical specimen will be sent home
with NAB (nutrient agar broth) or TSB (trypticase soy broth). Place 0.5 ml of the
rancid milk onto the broth and let stand at room temperature overnight. Then
bring the broth to school the following day.
8. Using the cotton swab with the clinical specimens (use inoculating loop for the
rancid milk), touch the surface of the BAP and do multiple interrupted streaking at
the top of the area A. make as many streak lines as you can without overlapping.

Rotate the swab between the thumb and forefinger as you streak to expose the
entire swab surface to the plate.
9. Inoculate the bacteria into region B by moving a sterile loop once or twice
through region A and then make more streak lines that neither overlap nor enter
region A again. Then stab the agar two or three times.
10. Sterile the loop and inoculate region C using the same technique. If done
carefully, this technique is (almost) guaranteed to produce isolated colonies in
some area of the plate.
11. Do the same with pure culture of Genus Streptococcus.
12. Label the plates and incubate at 37 degrees Celsius for 18-24 hours.
2nd Laboratory Period (Cultural and Microscopic Methods)
1. Examine the cultures for bacterial growth. Study the colonial morphology of the
bacteria particularly the edge, size and elevation of the colony. Observe for any
hemolytic pattern and describe their consistency after a subculture is done. It is
very important that the hemolytic pattern of the organism be identified.
2. Choose a colony most likely similar to the colonial morphology of the genus
Streptococcus and perform gram staining. The group should be able to get gram
(+) cocci in long, short chains or in pairs, the microscopic morphology of genus
Streptococcus.
3. Choose a colony most likely similar to the colonial morphology of the genus
Streptococcus or look-a-like colony which shows gram (+) cocci in chains or pairs
and subculture it on a nutrient agar slant. Incubate at 37 deg for 18-24 hours.
3rd Laboratory Period (Biochemical Methods)
1. Catalase Test
a. Emulsify 1-2 colonies from the NAS (nutrient agar slant) with saline solution
on a clean glass slide.
b. Add 1-2 gtts of 3% hydrogen peroxide.
c. Observe for the formation of rapid and continuous bubbles.
d. Report result.
(+) result = presence of bubbles
(-) result = absence of bubbles
2. Sensitivity Tests to be performed should be based from the patterns of the
cultures.
a. Bacitracin and SXT - Sulfamethoxazole- Trimethoprim Disk Sensitivity
tests
(to be performed with beta hemolytic cultures)

i.

Divide one BAP into 2 portions (to be used for the clinical sx and stock
culture). Select 3-4 beta hemolytic colonies and streak onto the BAP
into two directions to obtain a lawn of growth.
ii.
Using a sterile forceps, place on bacitracin disk and SXT disk in the
center of the streak. The disks should be placed evenly so that zone
diameter can be accurately interpreted. Tap gently with forceps.
iii.
Incubate 18-24 hours at 37 deg C.
iv.
Report results:
a. Susceptible = Any zone of inhibition around the disk
b. Resistant = Growth up to the disk
b. Optochin Disk Sensitivity Test (to be performed with alpha hemolytic
culture)
i.

ii.
iii.
iv.

Divide one BAP into 2 portions (to be used for the clinical sx and stock
culture). Select 3-4 alpha hemolytic cultures and streak onto the BAP
into two directions to obtain a lawn of growth.
Place an Optochin disk in the center streak.
Incubate 18-24 hours at 37 deg C.
Report results:
a. Susceptible = At least 14 mm inhibition (diameter) with 10 ug of P
disk.
b. Susceptible = At least 10 mm inhibition (diameter) with 6 ug of P
disk.
c. Resistant = Growth up to the disk
d. Resistant = Less than 14 mm inhibition (diameter) with 10 ug of P
disk.
e. Resistant = Less than 10 mm inhibition (diameter) with 16 ug of P
disk.

4th Laboratory Period (Identification of Bacterial Species)

1. Report results on all the sensitivity tests performed.
2. Submit isolates to the instructors with the corresponding identity.

3. Experimental results
a. Tables/Graphs
*Colonial Morphology
Clinical Specimens (Throat swab/ Dental caries)

Colonial Morphology:
Throat Swab
Size
pinpoint
Edge/margin
smooth
Elevation
raised
Color
grayish- white
Consistency
mucoid
Hemolytic

pattern

Pure Culture (Genus Streptococcus)

Dental caries
pinpoint
smooth
raised
grayish- white
mucoid

Colonial Morphology:
Size - pinpoint
Edge/Margin - smooth
Elevation - raised
Color - gray
Consistency - mucoid
Hemolytic pattern -

*Microscopic Morphology
Groups culture

Pure culture

Credits: http://quizlet.com/27402796/miml113-flash-cards/

Gram Reaction: Gram positive

Microscopic morphology: Gram positive
cocci in pairs

Gram Reaction: Gram positive

Microscopic morphology: Gram positive
cocci in pairs and chains

*Biochemical Test
Groups culture
(-)

Pure culture
(-)

Credits:
http://iws2.collin.edu/dcain/CCCCD%20Micro/catalase_te
st.htm

Credits:
http://iws2.collin.edu/dcain/CCCCD%20Micro/catalase_test
.htm

Streptococcus salivarius

Streptococcus pyogenes

Biochemical Test
Catalase test

Identify the possible

isolates (based on
the previous tests
performed from day
1 to 3. )

b. Test

4. Discussion
a. Interpretation of results
b. Expectations

5. Answers to Questions for Research

6. Updates

Sources:
http://www.ncbi.nlm.nih.gov/mesh?Db=mesh&term=Streptococcus
http://www.coursehero.com/file/4558370/Lec-3-Streptococcaceae/
http://www.gopetsamerica.com/bio/bacteria/streptococcus.aspx