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Predicated on their metabolic characteristics, i.e., their metabolic demand for oxygen, bacteria are classified as aerobic, facultative, or anaerobic. Morphologically, they are characterized as cocci or bacilli (rods). Based on Gram’s Method of staining (Box 1), bacteria are further classified as gram-positive or gram-negative. The distinct staining properties of bacteria are related to their architectural and biochemical differences.7
| Step 1. | Apply a thin film of the specimen to a glass slide and allow it to dry. |
| Step 2. | Fix the slide in methanol for 1 minute or fix by quickly passing the slide through a flame several times. |
| Step 3. | Flood the slide with crystal violet stain for 30 seconds. |
| Step 4. | Rinse gently with running water. |
| Step 5. | Flood the slide with Gram’s iodine wash for 30 seconds. |
| Step 6. | Rinse gently with running water. |
| Step 7. | Apply acetone decolorizer so it runs over stained areas until no more color washes out. |
| Step 8. | Rinse gently with running water. |
| Step 9. | Flood the slide with safranin counterstain for 30 seconds. |
| Step 10. | Rinse gently under running water and allow the slide to air dry. |
Gram-positive bacteria possess a thick peptidoglycan cell wall interspersed with lipoteichoic acid underlain by the cytoplasmic membrane (Figure 1).8 Gram-negative bacteria have an outer membrane with lipopolysaccharides and a lipoprotein layer underlain by a thin peptidoglycan layer and the cytoplasmic membrane (Figure 2).8 The ability of antibacterial agents to diffuse into bacteria is also affected by these structural differences.
During staining, crystal violet interacts with iodine forming a complex. Acetone extracts lipids from the outer membrane, cell wall, and cytoplasmic membrane of bacteria.7 The damage to gram-negative organisms is more extensive and they lose their crystal violet-iodine complexes, i.e., they are decolorized; and when counterstained with safranin, they appear red (Figure 3).7 Gram-positive bacteria retain their crystal violet-iodine complexes and appear deep purple (Figure 4).7
An average adult harbors at least 300 oral bacterial species and more than 700 strains of bacteria have been isolated from test cases.9-11 Most odontogenic infections are polymicrobial. The number of strains per infection ranges from 1 to 10 with an average number of 4 isolates.9,12-24 The predominant flora creates an ecosystem of synergism by elaborating a more favorable acidic environment and decreased oxygenation to support the growth and proliferation of its members. Ultimately, facultative and anaerobic gram-positive and gram-negative cocci and bacilli predominate in all types of odontogenic infections (Table 1).9-31
| Gram-staining and morphologic characteristics | Facultative anaerobes | Obligate anaerobes |
|---|---|---|
| Gram-positive cocci | Streptococcus Enterococcus |
Streptococcus Peptostreptococcus |
| Gram-positive bacilli | Actinomyces Lactobacillus |
Actinomyces Lactobacillus Proprionibacterium Bificobacterium Eubacteria |
| Gram-negative cocci | Neisseria | Veillonella |
| Gram-negative bacilli | Capnocytophaga Eikenella |
Porphyromonas Prevotella Fusobacterium Campylobacter Bacteroides |
| Spirochetes | Treponema | |
| Crescent-shaped | Selenomonas |
