Practical No.

1 Personal Hygiene

Objective:

To understand and practice proper personal hygiene techniques and assess microbial
contamination on hands, clothing, or surfaces.

Common Activities:

1. Handwashing Techniques:
o Compare microbial load before and after handwashing.
o Use of different handwashing methods: water only, soap, alcohol-based sanitizer.
2. Swab Sampling:
o Collect samples from hands, nails, or equipment surfaces.
o Culture on agar plates (e.g., nutrient agar, MacConkey).
3. Observation:
o Incubate cultures at 35–37°C for 24–48 hours.
o Observe and identify microbial colonies.
o Evaluate effectiveness of hygiene practices.
4. Use of PPE (Personal Protective Equipment):
o Demonstrate correct donning and doffing of lab coats, gloves, masks.

Biosafety Guidelines in the Lab

Biosafety Levels (BSL):

 BSL-1: Basic teaching labs, handling non-pathogenic microbes.

 BSL-2: Moderate-risk agents (e.g., E. coli, Salmonella).
 Higher levels (BSL-3/4) involve dangerous pathogens and are outside most academic
labs.

Key Biosafety Practices:

1. Personal Protective Equipment (PPE):

o Lab coat, gloves, goggles.
o Closed-toe shoes, long pants.
2. Safe Handling of Microorganisms:
o Use aseptic technique to prevent contamination.
 o Work within biosafety cabinets if needed.
3. Waste Disposal:
o Autoclave all biohazard waste.
o Dispose of sharps in puncture-proof containers.
4. Disinfection Protocols:
o Clean benches with 70% ethanol or approved disinfectants before and after lab.
5. Reporting Accidents:
o Any spills, exposures, or injuries must be reported immediately.

6.

Practical No. 2 Dress and safe Laboratory work practice

🧪 Proper Dress for Laboratory Work

1. Lab Coat:
o Wear a lab coat made of flame-resistant material.
o Should be buttoned up and long-sleeved to protect skin and clothing.
2. Gloves:
o Use appropriate gloves (nitrile, latex, etc.) based on the chemicals or biological
agents you're handling.
o Change gloves frequently and never touch your face or phone with them.
3. Eye Protection:
o Safety goggles or glasses must be worn at all times to protect from splashes and
particles.
4. Footwear:
o Closed-toe, non-slip shoes are mandatory. No sandals, flip-flops, or high heels.
5. Hair and Accessories:
o Long hair must be tied back.
 o Avoid dangling jewelry, loose sleeves, or scarves.

✅ Safe Laboratory Work Practices

1. Know the Hazards:

o Read Safety Data Sheets (SDS) for chemicals or materials you’re using.
o Know how to handle spills, fire, or exposure.
2. No Food or Drink:
o Never eat, drink, or store food in the lab.
3. Label Everything:
o All containers must be properly labeled with contents and hazard warnings.
4. Work in a Clean Space:
o Keep your work area tidy and uncluttered.
o Clean up spills immediately using appropriate procedures.
5. Waste Disposal:
o Follow proper protocols for chemical and biological waste.
6. Use Equipment Properly:
o Operate lab equipment only if trained and authorized.
7. Emergency Equipment:
o Know the locations of eye wash stations, showers, fire extinguishers, and exits.

🧬 Biosafety in the Laboratory

1. Biosafety Levels (BSL):

o Labs are classified from BSL-1 to BSL-4 based on the organisms handled.
 BSL-1: Low risk (e.g., non-pathogenic E. coli)
 BSL-2: Moderate risk (e.g., Salmonella)
 BSL-3: High risk (e.g., Mycobacterium tuberculosis)
 BSL-4: Extreme risk (e.g., Ebola virus)
2. Biosafety Practices:
o Always follow institutional biosafety protocols.
o Use biological safety cabinets (BSC) when required.
o Decontaminate surfaces and dispose of biohazardous waste correctly.
3. Personal Hygiene:
o Wash hands before and after lab work, even when gloves are used.
4. Reporting:
o Immediately report accidents, spills, or exposures to your supervisor.
Practical No. 3 Use of Personal Protective Equipment (PPE)
Personal Protective Equipment (PPE) refers to specialized clothing or equipment worn by
employees for protection against health and safety hazards. The correct use of PPE is essential in
minimizing exposure to hazards that cause serious injuries and illnesses in the workplace.

Common Types of PPE:

1. Head Protection – Helmets or hard hats protect against impact, falling objects, or
electrical hazards.
2. Eye and Face Protection – Safety goggles, face shields, or glasses protect against
chemical splashes, flying particles, or harmful light.
3. Hearing Protection – Earplugs or earmuffs reduce the risk of hearing damage from loud
environments.
4. Respiratory Protection – Masks or respirators guard against inhaling harmful
substances like dust, fumes, or chemicals.
5. Hand Protection – Gloves shield against cuts, burns, electrical shock, or chemical
exposure.
6. Body Protection – Coveralls, vests, or full suits protect the skin and body from extreme
temperatures, chemicals, or flames.
 7. Foot Protection – Safety boots or shoes with steel toes and slip-resistant soles help
prevent foot injuries from falling objects or slippery surfaces.
8. Fall Protection – Harnesses and lanyards protect workers at heights by preventing or
arresting falls.

Proper Use of PPE:

 Assessment: Identify hazards and determine appropriate PPE.

 Training: Workers must be trained on when, why, and how to use PPE.
 Fit and Maintenance: PPE must fit properly and be kept clean and in good condition.
 Inspection: Regularly inspect PPE for damage or wear.
 Compliance: Use PPE consistently and correctly according to safety regulations.

Importance:

 Reduces risk of injury or illness.

 Helps employers comply with occupational safety laws.
 Contributes to a culture of safety in the workplace.

Practical No. 4 Lab Facilities and safety equipment

LAB FACILITIES IN A SCIENTIFIC LAB

1. Basic Lab Infrastructure

 Workbenches – Chemical-resistant surfaces for experiments.

 Fume hoods – Ventilation to remove toxic fumes.
 Sink and water supply – For washing hands, equipment, and disposing of liquids.
 Gas supply – For Bunsen burners and gas-based experiments.

2. Storage Facilities

 Chemical storage cabinets – Fire-resistant and labeled by hazard class.

 Refrigerators/Freezers – For temperature-sensitive materials.
 Sharps and biohazard waste containers – Clearly labeled and safely enclosed.

3. Specialized Facilities for Biosafety

 Biosafety Cabinets (BSCs) – For working with infectious materials. Types:

o Class I: Personnel and environmental protection
o Class II: Adds product protection (common in medical labs)
o Class III: Maximum protection (sealed glove boxes)
 Autoclaves – For sterilization of tools and disposal of biohazardous waste.
 Air filtration systems (HEPA filters) – Maintain sterile environment.

🛡️SAFETY EQUIPMENT IN LABS

1. Personal Protective Equipment (PPE)

 Lab coat/gown
 Gloves (nitrile/latex)
 Safety goggles or face shield
 Closed-toe shoes and long pants
 Respirators (in high biosafety or chemical risk settings)

2. Emergency Equipment

 Eyewash station – For chemical splashes to eyes.

 Safety shower – For full-body exposure to hazardous chemicals.
 Fire extinguisher – Different types (A, B, C, D depending on materials).
 First aid kit – Stocked for minor injuries.
  Spill kits – For chemical or biological spills.

3. Monitoring and Communication

 Emergency contact signs

 MSDS (Material Safety Data Sheets) access
 Alarms and ventilation monitors
 Signage for biosafety levels (BSL-1 to BSL-4)

🧪 BIOSAFETY PRACTICES

 Biosafety Levels (BSL): Each level adds more containment:

o BSL-1: Basic microbiological practices
o BSL-2: Moderate-risk agents (e.g., Salmonella, Hepatitis B)
o BSL-3: Aerosol-transmitted agents (e.g., TB)
o BSL-4: Dangerous/exotic agents (e.g., Ebola)
 Containment Practices:
o Use of BSCs
o Decontamination with autoclave
o Controlled lab access
o Vaccination for staff (if applicable)

Practical No. 5 Use of Biosafety Cabinets

Biosafety cabinets (BSCs) are essential equipment in laboratories that handle potentially
infectious agents. They are primarily used to protect laboratory personnel, the environment, and
the materials being handled from exposure to biohazards and cross-contamination.

Uses of Biosafety Cabinets in the Laboratory

1. Personnel Protection:
BSCs create a physical barrier and use airflow systems (HEPA-filtered air) to prevent
exposure to infectious aerosols and droplets.
2. Product Protection:
In Class II and III BSCs, sterile air flows downward over the work surface, protecting
samples from contamination during sensitive procedures such as:
o Cell culture work
o Preparation of sterile solutions
o Genetic manipulation experiments
3. Environmental Protection:
Exhaust air is filtered through HEPA filters before being released into the lab or
outdoors, minimizing the risk of environmental contamination.
4. Handling Risk Group 2 & 3 Pathogens:
Especially when working with agents like Mycobacterium tuberculosis, Salmonella, or
HIV, BSCs are critical for safe handling.
5. Use in Clinical and Research Settings:
In diagnostics, vaccine production, microbiological assays, and molecular biology labs,
BSCs are widely used for safe manipulations.

In biosafety training and practicals, BSCs are used to:

1. Demonstrate Proper Lab Safety Techniques:

o Aseptic technique under a BSC
o Avoiding breaches in airflow by minimizing arm movements or clutter
2. Training on Correct Use of PPE with BSCs:
o Donning gloves, lab coats, and sometimes face shields before use
o Decontamination procedures before and after use (e.g., 70% ethanol wipe-down)
3. Simulating Exposure Scenarios:
o Practicing emergency procedures if airflow fails or spills occur inside the BSC
4. Hands-on Experience with Risk Management:
o Identifying biosafety levels (BSL-1 to BSL-4) and corresponding BSC
requirements
 o Practicing decontamination and waste disposal while using the cabinet

Best Practices When Using a BSC

 Pre-use Checks: Ensure airflow indicators are functional.

 Minimal Movement: Sudden movements disturb airflow.
 Correct Posture: Work at least 4 inches inside the cabinet.
 Avoid Blocking Grilles: Airflow should not be obstructed.
 Regular Maintenance: Annual HEPA filter checks and certifications.

Practical No.6 Use of Laminar Flow hood

A laminar flow hood (also known as a laminar airflow cabinet or clean bench) is a critical piece
of equipment used in laboratories for maintaining a sterile and contamination-free environment.
Here's a breakdown of its practical uses and its role in biosafety:

🔬 Practical Uses in the Lab

1. Sterile Work Area:

 o Provides a constant, unidirectional stream of HEPA-filtered air to prevent
contamination of samples.
o Used extensively in microbiology, molecular biology, and cell culture labs.
2. Sample Preparation:
o Ideal for preparing culture media, sterile solutions, and assembling sterile
equipment.
3. Tissue Culture and Cell Work:
o Protects cells from contamination during handling and incubation setup.
4. Microbiological Testing:
o Ensures that environmental contaminants do not interfere with test results,
especially in pharmaceutical and food labs.
5. Electronic Component Assembly:
o In cleanroom environments, it helps prevent static-sensitive and dust-sensitive
parts from being damaged.

🧫 Biosafety Role

Laminar flow hoods contribute to biosafety but have limitations. Here's how:

✅ What They Protect:

 Product/sample protection only (in horizontal or vertical laminar flow hoods).

 Prevents contamination of work by particles or microbes in the environment.

❌ What They Don't Protect:

 Do NOT protect the user or the environment from biohazardous materials.

 Not suitable for handling infectious agents or hazardous substances.

🔐 For True Biosafety: Use a Biosafety Cabinet (BSC)

 Biosafety Cabinets (Class I, II, III) are designed for both sample and user protection.
 Required for work involving pathogens or human/animal cells.
Summary Table
Feature Laminar Flow Hood Biosafety Cabinet

Protects Sample ✅ ✅

Protects User ❌ ✅

Air Flow Direction Horizontal/Vertical Vertical (filtered/exhausted)

HEPA Filtered Air ✅ ✅

✅ (Class II or III)

Use with Hazardous Agents ❌

Practical No. 7 Use of fire Extinguisher

Using a fire extinguisher in a laboratory setting is a critical safety skill, especially in
environments involving chemicals, flammable materials, and biological agents. Here's how it
connects to both practical use in the lab and biosafety principles:

🔥 Practical Use of Fire Extinguishers in the Lab

1. Understanding Fire Types:

  Class A: Combustibles (paper, wood)
 Class B: Flammable liquids (ethanol, acetone)
 Class C: Electrical fires
 Labs often have ABC fire extinguishers for versatility.

2. PASS Technique for Using a Fire Extinguisher:

 Pull the pin.

 Aim the nozzle at the base of the fire.
 Squeeze the handle.
 Sweep side to side.

3. Practical Steps in a Lab Setting:

 Raise the alarm or alert others.

 Evacuate if the fire is too large or spreading rapidly.
 Use the extinguisher only if trained and it's safe to do so.
 Always keep an exit behind you while extinguishing the fire.

🧫 Biosafety Considerations

1. Fire Risks in Biosafety Labs:

 Flammable chemicals, ethanol-based disinfectants.

 Electrical equipment and Bunsen burners.
 Aerosol generation in fires may spread biological agents.

2. Safety Precautions:

 Fire extinguishers should be accessible and regularly inspected.

 Know the lab’s emergency protocols.
 In BSL-2 or higher labs, extra care must be taken to avoid disrupting containment or
causing biological exposure during a fire.

3. Post-Fire Procedures:

 Decontaminate surfaces if biological materials were involved.

 Report and document the incident.
 Review and update safety protocols.
Practical No.8 Sterilization and Disinfection Methods
Sterilization and disinfection are essential procedures in laboratory and biosafety
practicals to prevent contamination, ensure safety, and maintain integrity in experiments. Here’s
a comprehensive overview of commonly used methods of sterilization and disinfection,
especially in microbiology and biosafety labs:

🔬 Sterilization Methods

(Sterilization = complete destruction/removal of all microorganisms, including spores)

1. Physical Methods
a. Heat Sterilization

 Autoclaving (Moist Heat)

o Temperature: 121°C
o Pressure: 15 psi
o Time: 15–20 minutes
o Used for: Culture media, surgical tools, lab waste, glassware
 Dry Heat (Hot Air Oven)
o Temperature: 160–180°C
o Time: 1.5–2 hours
o Used for: Glassware, metal instruments, powders
b. Filtration

 Removes microbes using filter membranes (0.22 μm pore size)

 Used for: Heat-sensitive liquids like antibiotics, enzymes, sera

c. Radiation

 UV Radiation (Non-ionizing)
o Surface sterilization in biosafety cabinets
 Gamma Radiation (Ionizing)
o Used for: Disposable medical and lab equipment (e.g., petri dishes)

2. Chemical Sterilization

 Ethylene oxide gas: For heat-sensitive equipment (toxic, requires aeration)

 Formaldehyde vapor: Used in fumigation of biosafety cabinets or rooms

🧼 Disinfection Methods

(Disinfection = destruction/removal of most pathogens, not necessarily spores)

1. Chemical Disinfectants
Disinfectant Concentration Used For

70% Ethanol/Isopropanol 70% Surfaces, equipment, skin

Sodium Hypochlorite (Bleach) 0.1–1% Spills, surfaces, biohazardous waste

Glutaraldehyde 2% Heat-sensitive instruments

Hydrogen Peroxide 3–6% Surface disinfection

Phenol 5% General disinfection (limited due to toxicity)

2. Fumigation

 Typically uses formalin + potassium permanganate or hydrogen peroxide vapors for

room decontamination.
🧪 Biosafety Practical Considerations

 Use biosafety cabinets (Class I, II, or III) for handling pathogens.

 Always disinfect before and after working.
 Use autoclave bags for biohazardous waste.
 Perform spillage management using proper disinfectants and protocols.
 Follow biosafety levels (BSL-1 to BSL-4) based on the risk group of the microorganism.

Practical No. 9 Decontamination and Antimicrobial usage

🔬 What is Decontamination?

Decontamination is the process of removing or neutralizing hazardous substances, including

pathogens, from surfaces, materials, or environments to prevent contamination and ensure safety.
✅ Common Decontamination Methods
Method Use Case Procedure

Use 70% ethanol, 0.1–1% sodium hypochlorite, or

Chemical Benchtops, spills,
appropriate disinfectant. Let it sit for at least 10
Disinfection instruments
minutes.

Lab waste, glassware,

Autoclaving 121°C at 15 psi for 15–20 minutes in an autoclave
culture media

UV Light Biosafety cabinets, clean

15–30 minutes exposure with no obstructions
Exposure benches

Whole room or cabinet Use formaldehyde or hydrogen peroxide vapor

Fumigation
decontamination under controlled conditions

Spill Response Use absorbent materials + disinfectant + PPE;

Biohazard spills
Kits follow BSL-specific protocol

🛡️Best Practices for Decontamination

 Always wear PPE (gloves, lab coat, goggles).

 Label and segregate contaminated waste.
 Use biosafety cabinet for handling infectious material.
 Decontaminate tools and surfaces before and after procedures.
 Keep spill kits accessible and know how to use them.

💊 Antimicrobial Usage in Lab Practicals

🧪 Applications in Lab
Purpose Examples

Antibiotic Sensitivity Testing Disc diffusion (Kirby-Bauer), MIC, MBC

Antibiotic Selection in Culture Media Ampicillin in plasmid transformation

Preserving Sterility Antibiotics in tissue culture media

Demonstrating Resistance Mechanisms Beta-lactamase tests, efflux pump assays

🧬 Common Antimicrobials Used
Antimicrobial Target Organism Type Lab Use

Penicillin, Ampicillin Gram-positive bacteria Selective media

Tetracycline Broad-spectrum Sensitivity testing

Streptomycin Gram-negative Resistance studies

Chloramphenicol Broad-spectrum Genetic marker selection

Nystatin, Amphotericin B Fungi Culture contamination control

⚠️Biosafety Considerations

 Avoid misuse to prevent antimicrobial resistance (AMR).

 Dispose of antimicrobial-containing materials as chemical waste.
 Handle all antimicrobials with MSDS (Material Safety Data Sheets) in mind.

Practicals No. 10 Manipulation Techniques for Minimizing

Aerosols
Here’s a concise and practical guide to Manipulation Techniques for Minimizing Aerosols,
especially important in microbiology, molecular biology, and biosafety level (BSL) lab settings:
🌫️Manipulation Techniques for Minimizing Aerosols

🧪 1. Use a Biosafety Cabinet (BSC)

 Always perform aerosol-generating procedures in a Class II BSC.

 Avoid blocking airflow grills.
 Keep all materials at least 4 inches inside the cabinet.
 Disinfect surfaces before and after use.

🧫 2. Open Tubes Carefully

 Open tubes slowly and away from your face.

 Do not snap open tubes or ampoules.
 Wipe rims with disinfectant before opening if contaminated.

🧴 3. Pipetting Techniques

 Use slow and smooth pipetting to avoid splashes.

 Never use mouth pipetting — only use mechanical pipettes.
 Use aerosol-resistant filter tips for infectious or sensitive samples.

🔁 4. Avoid Vortexing & Flicking

 Minimize or avoid vortexing; if necessary, use sealed containers.

 Do not flick tubes to mix; instead, invert gently or use a roller.

🌀 5. Centrifugation Safety

 Always use sealed safety buckets or rotor lids.

 Balance tubes before use to prevent breakage.
 Wait a few minutes after the spin before opening to let aerosols settle.

🧽 6. Decontaminate Spills Immediately

 Cover with absorbent material and apply disinfectant from outside in.
  Wait 10–20 minutes before cleaning up.
 If large or airborne exposure is suspected, evacuate and follow spill protocol.

🧼 7. Waste Disposal

 Use leak-proof, covered containers for contaminated waste.

 Do not overfill sharps or biohazard bins.
 Autoclave infectious waste before disposal.

🧍 8. General Practices

 Work slowly and deliberately — avoid sudden movements.

 Wear full PPE: gloves, gown, face/eye protection.
 Use sealed tubes and containers whenever possible.

Practical No. 11 Practical Cleaning of a Spill

🧪🧹 Practical Cleaning of a Spill (Biohazard or Chemical)
🔴 1. Stop and Secure the Area

 Alert nearby personnel.

 Restrict access to the area to prevent spread.
 Turn off equipment if needed (without endangering yourself).

🧤 2. Put on Personal Protective Equipment (PPE)

 Lab coat or gown

 Disposable gloves (double-gloving recommended for biohazards)
 Face mask or shield
 Eye protection (goggles or face shield)

🧫 3. Cover the Spill

 Gently place absorbent material (e.g., paper towels, spill pads) over the spill.
 Do not wipe or rub at this stage — let the absorbent sit.

🧴 4. Apply Disinfectant

 Pour disinfectant (e.g., 10% bleach or 70% ethanol) around the edges of the spill and
move inward.
 Ensure the absorbent is fully soaked.
 Let it sit for at least 10–20 minutes (contact time is critical).

🗑️5. Clean Up

 Use forceps or gloved hands to carefully remove soaked absorbent.

 Place waste into a biohazard bag (for infectious spills) or chemical waste bag.
 Wipe the area again with fresh disinfectant and clean paper towels.

🧼 6. Final Steps

 Remove gloves last and dispose of them safely.

 Wash your hands thoroughly with soap and water.
  Decontaminate tools or reusable PPE (like goggles).
 Restock the spill kit and report the incident to your supervisor.

✅ Spill Cleanup Checklist (For Practicals)

Item In Spill Kit?

Absorbent paper towels/pads ✔️

Disinfectant (bleach, alcohol) ✔️

Gloves (nitrile/latex) ✔️

Forceps or tongs ✔️

Biohazard bags ✔️

Eye and face protection ✔️

Instruction sheet or SOP ✔️