Bordetella

Bordetella pertussis is the etiologic agent of whooping cough. This very fastidious organism grows best on special media. The two most common are Bordet-Gengou (BG) agar, which is enriched with glycerin, potato, and 30% defibrinated sheep blood, and Regan-Lowe (RL) agar, which consists of charcoal agar, defibrinated horse blood, and an antimicrobial agent to inhibit growth of normal respiratory flora. The charcoal is present to adsorb toxic substances that might be present in the agar. Visible colonies are produced only after three to five days incubation in a microaerophilic atmosphere. On BG medium, the colonies are raised, rounded, and glistening (resembling mercury droplets or a bisected pearl), and usually have a hazy zone of hemolysis. On RL medium, the colonies are round, domed, shiny, and may run together slightly.

B. pertussis is a gram-negative bacillus resembling Haemophilus species, with which it was once classified. When whooping cough is suspected, the best specimen for laboratory diagnosis is a nasopharyngeal swab, but throat swabs may be used in addition.

Purpose	To observe Bordetella pertussis in demonstration and to examine a throat culture on Bordet-Gengou (BG) and Regan-Lowe (RL) media
Materials	Prepared Gram stains of B. pertussis Projection slides, if available Bordet-Gengou and Regan-Lowe agar plates

Procedures

1. Examine the prepared Gram stains and record your observations.
2. Observe colonial morphology as demonstrated.
3. Collect a throat specimen as in Experiment 21.5 and inoculate the Bordet-Gengou and Regan-Lowe plates. Incubate the plates at 35°C in a candle jar or CO₂ incubator for 24 hours.

Results

1. Describe the microscopic morphology of B. pertussis.
2. Describe your observations of demonstration material.
3. Describe the appearance of your Bordet-Gengou and Regan-Lowe throat culture plates.
• What is the total number of colonies? BG _______________ RL _______________
• How many colony types can be distinguished? BG _______________ RL _______________
• How does the flora compare with that of your throat culture in Experiment 21.5?