The facultative streptococci are the largest group of bacteria isolated from the oral
cavity. They comprise almost 50% of the organisms isolated from plaque and the
gingival sulcus. The most abundant oral streptococci are the alpha-hemolytic (Viridans)
streptococci. Gamma-hemolytic streptococci are usually present but in smaller
amounts. The beta-hemolytic streptococci are frequently present in small numbers or
as transient flora from the oropharynx and seldom produce infection in the oral
The purpose of this exercise is to demonstrate the difference in colonial morphology
of facultative oral streptococci grown on blood agar and Mitis-Salivarius (M-S) agar.
M-S agar is a differential medium containing dyes, nutrients, 5% sucrose and
growth inhibitors for organisms other than streptococci.
In addition, the demonstration at the front table represents an experimental model
which demonstrates the formation of dextran by cariogenic streptococci. This
extracellular polysaccharide will accumulate on the glass culture tube and wire
insert and form a gelatinous mass. In the patient, dextran combines with
components of saliva to form an insoluble complex which adheres to the surface of
teeth. The in-vitro production of dextran by Streptococcus mutans permits analysis
of "pure" plaque without interference from other oral microorganisms and saliva.
II. LAB WORK
PART I: ORAL STREPTOCOCCI (Tues., 9/12)
Materials supplied: (work in groups of 4)
- 3 Mitis-Salivarius agar plates (M-S) (one blue and one green stripe)
- 3 pipets, 1 ml
- 3 sterile cotton swabs
- Use the same plaque dilution tubes you used in Exercise 14 to inoculate the
Mitis-Salivarius plates. Use the same technique.
- Label the plates with your name and either 5, 50, 500 and pipet 0.1 ml of each
dilution to the M-S plates.
3.Use a sterile swab to spread the liquid over the entire plate.
- Put these plates in the assigned anaerobes petri dish racks - these are
different from the ones used in Exercise 14.
SECOND LAB PERIOD (after 72 hours): (Fri., 9/15)
Materials supplied: (work in groups of 4)
Pure cultures on BAP and M-S plates:
- Streptococcus mutans (red dot)
- Streptococcus salivarius (pink dot)
- Streptococcus mitis (yellow dot)
- Streptococcus sanguis (green dot)
- Examine each stock culture of oral streptococci on BAP for isolated colonies.
Note size, shape, texture and color of colonies. Determine the type of
hemolysis produced. Record results in the chart.
- Each group will prepare Gram stains from BAP of each stock culture. Select
isolated colonies for best results. Record gram reaction, morphology, and
arrangement of organisms in the chart. If in your practice, you make Gram
stains of oral lesions, etc. these organisms will be among the contaminants.
It seems reasonable then that you should be experienced in their cellular
morphology. Gram Stain Results
- Examine each stock culture of streptococci on M-S agar. Use the dissecting
microscopes at the front bench to better observe the colonial morphology of
individual colonies. Observe size, color of the colony, whether raised or flat
to the agar surface, and consistency. Streptococcus mutans colonies are
frequently surrounded by a clear gelatinous "puddle" consisting of large
amounts of dextran. Record your observations.
- Now that you are familiar with the stock cultures, compare these to the
various colonial morphologies you observe on the plaque dilution plates.
Do you observe any typical Streptococcus mutans colonies, with puddles?
- Compare the numbers of colonies obtained on the M-S agar with the same
dilutions of the anaerobic blood agar plates from Exercise 14. Do the
numbers show the selective nature of M-S agar?
PART II: DEXTRAN DEMONSTRATION (at the front table - Fri., 9/15)
Streptococcus mutans (cariogenic) and Streptococcus salivarius (noncariogenic)
individually inoculated into culture broth tubes containing 5% sucrose. A sterile
wire insert was suspended in the broth of each tube and incubated at 37°C. At daily
intervals each wire insert was transferred into a new 5% sucrose tube containing a
fresh inoculum of the appropriate streptococcus species and reincubated. This
procedure was continued for 7 days. The tubes on demonstration
represent each 24
hour period of both Streptococcus species.
- Beginning with Day 1, observe the culture tubes of each Streptococcus
species for the production of dextran.
- If dextran is present, note the Streptococcus species responsible. Note the
increase in amount of dextran from day to day.
- Record your observations using the following scale:
- 0 = no dextran produced
- + = scant dextran produced
- ++ to +++ = moderate dextran produced
- ++++ = maximum dextran produced
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Publication Policy, 9/23/96