Overview
This lecture focuses on isolation and inoculation techniques used in microbiology, along with different types of culture media and their purposes.
Isolation Techniques
- Isolation separates different bacterial species in a mixed sample using plating methods.
- Colonies are groups of identical bacteria; appearance helps distinguish species.
- Streak plate technique spreads bacteria over plate quadrants to dilute and isolate colonies.
- Pour plate technique involves diluting bacteria in broth, then plating to separate species.
- Spread plate technique uses a small sample spread with a “hockey stick” to isolate colonies.
- Isolated colonies can be picked to obtain pure cultures for further analysis.
Culture Types and Inspection
- Pure culture has only one bacterial species; all colonies look alike.
- Mixed culture contains multiple bacterial species with varied colony appearances.
- Contaminants are unwanted organisms, usually visible as odd or outlier colonies.
- Agar slants are solid media in tubes used to grow and observe pure cultures.
Microbial Identification Methods
- Identification uses colony and cell morphology, staining techniques (simple, Gram), DNA sequencing, and biochemical tests.
- Biochemical testing checks how bacteria react to chemicals/environments and is key for species identification.
- Immunological testing is another method but not covered in lab.
Media Types and Uses
- Media classified by physical state: liquid (broth), semi-solid (agar), or solid.
- Agar is a solidifying agent; it provides structure but no nutrients.
- Nutrient broth (liquid) and nutrient agar (semi-solid) contain beef extract and peptone as nutrients.
- Chemical classification: synthetic (chemically defined) or complex (contains undefined ingredients).
- General purpose media supports broad microbial growth; usually non-synthetic.
- Enriched media has added substances (e.g., blood) for fastidious organisms.
- Blood agar is enriched and used to detect hemolytic activity (breakdown of red blood cells).
- Selective media promotes growth of some microbes while inhibiting others.
- Differential media allows growth of several species with differing visible reactions (e.g., color).
- Some media, like MacConkey agar, are both selective (for Gram-negative bacteria) and differential (lactose fermentation: pink vs. white).
- Reducing media (e.g., thioglycollate/THIO) absorbs oxygen for growing anaerobic bacteria.
- Carbohydrate fermentation media indicates acid production by color change due to sugar breakdown.
Key Terms & Definitions
- Colony — a visible mass of identical bacterial cells grown from a single cell.
- Streak Plate — a method to isolate bacteria by spreading them over an agar surface in quadrants.
- Pure Culture — contains only one species of microorganism.
- Contaminant — any unwanted microorganism in a culture.
- Agar — a gelatin-like substance used to solidify culture media; provides structure, not nutrients.
- Synthetic Media — culture media with precisely known chemical composition.
- Enriched Media — contains extra nutrients for fastidious bacteria.
- Selective Media — inhibits some microbes while supporting others’ growth.
- Differential Media — distinguishes microbes by changes (often color) in the medium.
- Reducing Media — absorbs oxygen to grow anaerobic microbes.
Action Items / Next Steps
- Practice streak plate and other isolation techniques in lab.
- Review textbook sections on the six “I’s” of microbiology and media types for homework.
- Read about immunological and carbohydrate fermentation media in the textbook for further understanding.