Diagnosis, Therapy, and Prevention of Bacterial Diseases
I Laboratory Identification of Bacteria
• Most medically important bacteria can be classified as gram-positive or gram-negative and further identified based on their shape and various metabolic properties as summarized in Figure 8-1.
• Step 1: treat smear with crystal violet primary stain.
• Step 2: add iodine solution, which precipitates primary stain within the peptidoglycan layer of the cell wall.
• Step 3: rinse with solvent that dissolves the outer membrane and washes out the crystal violet from the thin peptidoglycan layer of gram-negative bacteria but not from the thick peptidoglycan layer of gram-positive bacteria.
• Step 4: counterstain with red safranin, which is taken up by gram-negative organisms, allowing them to be visualized.
• Purple (positive reaction): organisms with thick peptidoglycan cell wall
• Red (negative reaction): organisms with thin peptidoglycan layer and an outer membrane
• Gram-variable or gram-resistant: modified cell wall old cultures or cells treated with β-lactam antibiotics in which the peptidoglycan is weakened—therefore, poor or no color retention; modified cell wall (Box 8-1)
B Growth and isolation of bacteria
• Most bacteria will grow on blood agar or other nonselective media.
• Selective medium inhibits growth of some bacteria (e.g., EMB agar inhibits gram-positive bacteria).
• Differential medium incorporates an identifying test.
a. MacConkey agar is both selective (inhibits gram-positive bacteria) and differential (tests for lactose use).
• Special medium incorporates particular metabolites or provides specific culture conditions required by certain bacteria.
1. Metabolic tests for fermentation of various sugars and production of byproducts (e.g., acid or gases)
• Lactose fermentation differentiates the more and less pathogenic Enterobacteriaceae.
a. Lactose fermenting includes more benign Escherichia coli and Klebsiella species.
b. Nonlactose fermenting includes more pathogenic Shigella and Salmonella species.
• Glucose and maltose fermentation differentiate Neisseria species.
