Introduction
Agar plates are essential tools in microbiology, providing solid growth media for cultivating microorganisms in laboratory settings. These versatile surfaces contain nutrients and selective agents that support specific microbial growth while inhibiting others. Proper preparation of agar media is crucial for reliable experimental results, diagnostics, and research applications across fields including clinical microbiology, food safety, and environmental monitoring.
Core Types of Agar Media
General Purpose Media
- Nutrient Agar (NA): Basic medium supporting growth of non-fastidious microorganisms
- Tryptic Soy Agar (TSA): Richer general-purpose medium for a wide range of microorganisms
- Brain Heart Infusion (BHI): Nutrient-rich medium for fastidious organisms
Selective Media
- MacConkey Agar: Selects for Gram-negative bacteria while inhibiting Gram-positive organisms
- Mannitol Salt Agar (MSA): Selects for staphylococci while inhibiting most other bacteria
- Eosin Methylene Blue (EMB): Selects for Gram-negative enteric bacteria
Differential Media
- Blood Agar: Differentiates hemolytic patterns (alpha, beta, gamma)
- Chromogenic Agars: Contain substrates that produce colored colonies for specific organisms
- Triple Sugar Iron (TSI): Differentiates based on sugar fermentation and H₂S production
Standard Agar Plate Recipes
Nutrient Agar
| Ingredient | Amount (per liter) |
|---|---|
| Peptone | 5.0 g |
| Beef extract | 3.0 g |
| Sodium chloride | 5.0 g |
| Agar | 15.0 g |
| Distilled water | 1000 ml |
| Final pH | 7.0 ± 0.2 |
Preparation: Dissolve ingredients in water by heating. Autoclave at 121°C for 15 minutes. Cool to 45-50°C before pouring plates.
Blood Agar
| Ingredient | Amount (per liter) |
|---|---|
| Blood agar base | 40.0 g |
| Defibrinated sheep blood | 50-70 ml |
| Distilled water | 1000 ml |
| Final pH | 7.3 ± 0.2 |
Preparation: Prepare blood agar base according to manufacturer’s instructions. Autoclave at 121°C for 15 minutes. Cool to 45-50°C, add sterile defibrinated blood, mix gently, and pour plates.
MacConkey Agar
| Ingredient | Amount (per liter) |
|---|---|
| Peptone | 17.0 g |
| Proteose peptone | 3.0 g |
| Lactose | 10.0 g |
| Bile salts | 1.5 g |
| Sodium chloride | 5.0 g |
| Neutral red | 0.03 g |
| Crystal violet | 0.001 g |
| Agar | 13.5 g |
| Distilled water | 1000 ml |
| Final pH | 7.1 ± 0.2 |
Preparation: Dissolve ingredients in water by heating. Autoclave at 121°C for 15 minutes. Cool to 45-50°C before pouring plates.
Specialized Media Recipes
Sabouraud Dextrose Agar (SDA) for Fungi
| Ingredient | Amount (per liter) |
|---|---|
| Peptone | 10.0 g |
| Dextrose | 40.0 g |
| Agar | 15.0 g |
| Distilled water | 1000 ml |
| Final pH | 5.6 ± 0.2 |
Optional additives: Chloramphenicol (0.05 g/L) or gentamicin (0.02 g/L) to inhibit bacterial growth.
Mueller-Hinton Agar (for Antimicrobial Susceptibility Testing)
| Ingredient | Amount (per liter) |
|---|---|
| Beef extract | 2.0 g |
| Acid hydrolysate of casein | 17.5 g |
| Starch | 1.5 g |
| Agar | 17.0 g |
| Distilled water | 1000 ml |
| Final pH | 7.3 ± 0.1 |
Preparation: Critical to follow exact protocol for antimicrobial testing. Autoclave at 121°C for 15 minutes. Pour to exact depth of 4 mm for standardized testing.
Chromogenic UTI Medium
| Ingredient | Amount (per liter) |
|---|---|
| Peptone mix | 17.0 g |
| Chromogenic mix | 1.6 g |
| Tryptophan | 1.0 g |
| Sodium chloride | 5.0 g |
| Agar | 15.0 g |
| Distilled water | 1000 ml |
| Final pH | 7.2 ± 0.2 |
Purpose: Rapid identification of urinary tract pathogens based on colony color.
Comparison of Selective Agents
| Selective Agent | Concentration | Target Organisms | Inhibited Organisms |
|---|---|---|---|
| Bile salts | 1-5 g/L | Enteric Gram-negative | Most Gram-positive |
| Crystal violet | 0.001-0.002 g/L | Gram-negative | Most Gram-positive |
| Sodium azide | 0.2-0.3 g/L | Gram-positive | Most Gram-negative |
| Colistin | 10-15 mg/L | Gram-positive | Many Gram-negative |
| Nalidixic acid | 20-40 mg/L | Gram-positive, fungi | Many Gram-negative |
| Cycloheximide | 0.4-0.5 g/L | Bacteria | Most fungi |
| Chloramphenicol | 0.05-0.1 g/L | Fungi, some bacteria | Many bacteria |
| NaCl (high conc.) | 75-100 g/L | Halophilic organisms | Non-halotolerant organisms |
Common Indicators and Their Color Changes
| Indicator | Acidic Color | Neutral Color | Alkaline Color | Application |
|---|---|---|---|---|
| Phenol red | Yellow | Red | Pink-magenta | Carbohydrate fermentation |
| Neutral red | Red | Amber-yellow | Yellow | Fermentation tests |
| Bromothymol blue | Yellow | Green | Blue | pH indicators |
| Bromcresol purple | Yellow | Purple | Purple | Carbohydrate fermentation |
| Methyl red | Red | Orange | Yellow | MR test for acid production |
Step-by-Step Agar Plate Preparation
- Weighing and Mixing:
- Accurately weigh all dry ingredients
- Add to appropriate volume of distilled/deionized water
- Mix thoroughly using magnetic stirrer or heat until completely dissolved
- pH Adjustment:
- Check pH using calibrated pH meter
- Adjust with 1N NaOH (to increase) or 1N HCl (to decrease)
- Verify final pH is within specified range
- Sterilization:
- Transfer to appropriate autoclavable containers (fill to no more than 75% capacity)
- Cap loosely to allow steam penetration
- Autoclave at 121°C (15 psi) for 15-20 minutes (adjust for larger volumes)
- Heat-Sensitive Additives:
- Cool autoclaved media to 45-50°C in water bath
- Add filter-sterilized heat-sensitive components (antibiotics, blood, etc.)
- Mix gently to avoid bubble formation
- Plate Pouring:
- Pour 15-20 ml per standard petri dish (90mm)
- Allow to solidify on level surface
- Store inverted in sterile bags at 2-8°C
- Quality Control:
- Incubate representative plates for 24-48 hours to check sterility
- Test with control organisms to verify performance
Common Challenges and Solutions
| Challenge | Possible Cause | Solution |
|---|---|---|
| Agar doesn’t solidify | Incorrect pH (too acidic) | Adjust pH before autoclaving |
| Insufficient agar concentration | Verify correct measurements | |
| Enzymatic degradation by certain bacteria | Increase agar concentration slightly | |
| Contamination | Improper sterilization | Ensure adequate autoclave time/temperature |
| Poor aseptic technique | Improve technique, use laminar flow hood | |
| Bubbles in plates | Pouring media too hot | Cool to 45-50°C before pouring |
| Agitation during cooling | Mix gently, allow bubbles to rise before pouring | |
| Condensation on lids | Plates poured too hot | Cool media adequately before pouring |
| Rapid temperature change | Allow plates to cool gradually at room temperature | |
| Cracked media | Drying out during storage | Store in sealed bags, maintain humidity |
| Inconsistent results | Variable media composition | Use commercial preparations or precise measurements |
| Improper storage of plates | Follow storage guidelines for each medium type |
Best Practices and Tips
Storage and Shelf Life
- Store prepared plates inverted in sealed plastic bags at 2-8°C
- Label with preparation date, lot number, and expiration date
- General shelf life: 2-4 weeks (plain media), 1-2 weeks (media with antibiotics/blood)
- Check for dehydration, contamination, or color changes before use
Quality Control
- Test each new batch with appropriate control organisms
- Maintain records of QC results and batch information
- Regularly calibrate pH meters and balances
Media Preparation Tips
- Use high-quality, analytical grade reagents
- Measure components precisely using calibrated scales
- Use freshly prepared deionized water
- Follow manufacturer’s instructions for commercial media
- Avoid overheating media (can break down nutrients and agar)
- Pour consistent volumes for standardized testing
Specialized Applications
- For antimicrobial susceptibility testing: maintain exact media depth (4mm)
- For selective isolation: verify selective agents haven’t degraded
- For chromogenic media: follow manufacturer’s reading guides for color interpretation
Resources for Further Learning
Reference Books
- American Society for Microbiology (ASM) Manual of Clinical Microbiology
- Difco™ & BBL™ Manual: Manual of Microbiological Culture Media
- Bergey’s Manual of Determinative Bacteriology
Online Resources
- ATCC Medium Database: www.atcc.org
- CDC Laboratory Procedures: www.cdc.gov/laboratory
- WHO Laboratory Guidelines: www.who.int/publications
Organizations
- Clinical and Laboratory Standards Institute (CLSI)
- American Society for Microbiology (ASM)
- World Health Organization (WHO)
Environment-Specific Media Applications
| Environment | Recommended Media | Target Organisms |
|---|---|---|
| Clinical | Blood agar, MacConkey, Chocolate agar | Pathogens from human samples |
| Food safety | VRBA, XLD, Baird-Parker | Foodborne pathogens |
| Water testing | m-Endo, R2A | Coliforms, heterotrophic bacteria |
| Soil ecology | Soil extract agar, Czapek’s | Diverse soil microbiota |
| Industrial | Specific to application | Process-specific microorganisms |
Remember: Always follow your laboratory’s standard operating procedures and refer to current regulatory guidelines for specific applications.