# Practical: Aseptic Techniques

Aseptic techniques are sterile procedures used to prevent contamination when investigating microbial growth

## Why Aseptic Techniques are Essential - Investigating antimicrobial substances requires sterile working conditions to ensure valid results - Aseptic techniques prevent two major issues: - **Escape** of microbes being studied into the environment - **Contamination** with unwanted, potentially pathogenic microbes from the surroundings - Preventing accidental culture of human pathogens is particularly critical - Human body temperature creates ideal conditions for pathogen growth - Contamination could lead to dangerous microbial populations ## Core Aseptic Techniques The following practices should be implemented when working with microorganisms: - **Wash hands thoroughly** with disinfectant before and after practical work - **No food or drink** permitted in the laboratory - **Disinfect work surfaces** using disinfectant solution or alcohol wipes - **Sterilise all apparatus** including glassware and inoculating loops before use - **Sterilise culture medium** whether using culture broth or agar plates - Boiling agar is an effective sterilisation method - **Use flamed loops or sterile swabs** when transferring cultures - Prevents collection of airborne microbes during transfer - **Flame culture bottle necks** to create upward air current that prevents contamination - **Limit incubation temperature** to below body temperature (typically 25°C maximum in schools) - Reduces risk of culturing human pathogens - **Only remove petri dish lids when necessary** to minimise airborne contamination - **Sterilise or dispose of used equipment promptly** once work is complete

Never incubate bacterial cultures at body temperature (37°C) in school laboratories as this encourages growth of human pathogens

## Example Practical: Testing Bacterial Antibiotic Resistance The disc diffusion method demonstrates aseptic techniques while investigating bacterial susceptibility to antibiotics - Multiple antibiotics can be tested simultaneously on a single plate - Clear zones indicate bacterial vulnerability to specific antibiotics - Larger clear zones suggest greater antibiotic effectiveness ### Apparatus - Sterile agar plates (sterilised by boiling) - Diluted bacterial broth at $1 \times 10^8$ CFU mm$^{-3}$ - CFU = Colony-Forming Unit (a viable bacterial cell capable of division) - Multiple antibiotic solutions at standard concentration - Paper discs - Pipettes - Spreaders - Bunsen burner - Gloves - Goggles - Incubator

Using the same antibiotic concentration across all solutions enables fair comparison of effectiveness

### Method 1. **Pre-soak paper discs** in different antibiotic solutions - Maintain identical concentrations for valid comparison - Each disc represents one antibiotic being tested 2. **Spread bacterial broth** across the sterile agar plate surface using aseptic technique - Use even spreading motion to create uniform bacterial lawn 3. **Position paper discs** on the agar surface with light pressure - Distribute evenly across the plate - Avoid touching plate edges - Ensure discs don't contact each other 4. **Incubate overnight** in a temperature-controlled incubator - Incubator maintains optimal temperature for bacterial growth - Typical school incubation: 25°C for 24-48 hours 5. **Examine results** with petri dish lid remaining on - Never remove lid during observation - Reduces contamination risk ### Results and Interpretation **Antibiotic diffusion pattern:** - Antibiotics diffuse outward from each paper disc creating a concentration gradient - Highest concentration occurs at the disc location - Concentration decreases with distance from the disc **Clear zones (zones of inhibition):** - Indicate bacterial vulnerability to that antibiotic - No bacterial growth occurs where antibiotic concentration exceeds bacterial tolerance - Clear zone ends where antibiotic concentration becomes sub-lethal **Comparing antibiotic effectiveness:** | Observation | Interpretation | |-------------|---------------| | Large clear zone | Highly effective antibiotic - low concentration required to kill bacteria | | Small clear zone | Less effective antibiotic - higher concentration needed | | No clear zone | Bacterial resistance - antibiotic ineffective against this strain |

More effective antibiotics require lower concentrations to kill bacteria, producing larger clear zones

**Example results from a disc diffusion test:** The following chart shows typical zone of inhibition diameters when testing different antibiotics against a bacterial strain:

**Interpreting the results:** - Ampicillin shows the largest zone (28 mm) = most effective against this bacterial strain - Tetracycline also highly effective (24 mm zone) - Penicillin and Streptomycin show moderate effectiveness (18 mm and 15 mm) - Erythromycin shows no zone (0 mm) = bacterial strain is completely resistant **Bacterial resistance:** - Complete absence of a clear zone around a disc indicates total resistance - Bacteria grow right up to the disc edge - This antibiotic provides no antimicrobial effect against this bacterial strain ### Practical Applications This technique allows scientists to: - Identify which antibiotics will effectively treat specific bacterial infections - Detect antibiotic-resistant bacterial strains - Compare relative effectiveness of multiple antibiotics quickly - Guide appropriate antibiotic prescription in clinical settings

Being able to suggest appropriate aseptic techniques for specific experiments is an expected exam skill

## Key Safety Considerations - Always wear appropriate PPE (gloves, goggles, lab coat) - Work near a Bunsen burner flame to create sterile upward air currents - Dispose of bacterial cultures by autoclaving or approved disinfection - Report any spills or contamination immediately - Never open incubating cultures prematurely - Seal petri dishes with tape (not completely - allow gas exchange)