EXERCISE 15

ORAL STREPTOCOCCI

I. INTRODUCTION

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 cavity.

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)

• 3 Mitis-Salivarius agar plates (M-S) (one blue and one green stripe)
• 3 pipets, 1 ml
• 3 sterile cotton swabs

1. Use the same plaque dilution tubes you used in Exercise 14 to inoculate the Mitis-Salivarius plates. Use the same technique.
2. 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.
3. 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)

• Streptococcus mutans (red dot)
• Streptococcus salivarius (pink dot)
• Streptococcus mitis (yellow dot)
• Streptococcus sanguis (green dot)

1. 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.
2. 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
3. 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.
4. 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?
5. 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) were 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.

1. Beginning with Day 1, observe the culture tubes of each Streptococcus species for the production of dextran.
2. If dextran is present, note the Streptococcus species responsible. Note the increase in amount of dextran from day to day.
3. Record your observations using the following scale:
   • 0 = no dextran produced
   • + = scant dextran produced
   • ++ to +++ = moderate dextran produced
   • ++++ = maximum dextran produced