Microbiology Laboratory: Biosafety Levels, Practices, and Microscopy Fundamentals

Overview of Laboratory Biosafety Levels (BSL)

• Definition of Biosafety Levels (BSL): A classification system consisting of four levels (one through four) designated to protect personnel and prevent the escape of microorganisms from the laboratory environment through engineering designs and construction protocols.
• BSL-1 (Safest Level):     * Scope: Appropriate for work involving well-characterized agents not known to cause disease in healthy, immune-competent adult individuals.     * Risk Profile: These microorganisms do not normally cause diseases in healthy people. However, they may pose a risk to immune-compromised individuals.     * Containment: Basic level of containment with no major engineering controls required.     * Examples: $E. ext{ Coli}$ and Saccharomyces cerevisiae (also referred to as cicaromyces cerevisiae in the transcript).
• BSL-2 (Moderate Hazard):     * Scope: Used for work involving agents of moderate potential hazard to personnel, the environment, and the agents themselves.     * Risks: Infectious agents or toxins can cause disease if inhaled, swallowed, or if there is skin exposure. Potential effects include skin irritation, diarrhea, or vomiting.     * Requirement: All equipment must be autoclaved, decontaminated, and disposed of properly.
• BSL-3 (Severe/Lethal Potential):     * Scope: Work performed with indigenous or exotic agents that may cause severe or potentially lethal disease specifically through inhalation or aerosol formation.     * Risk Profile: High potential for infection by aerosols; diseases can have lethal consequences.     * Engineering Controls: These laboratories require specific engineering controls to prevent the escape of dangerous agents into the environment.
• BSL-4 (Highest Danger):     * Scope: Used for managing the most dangerous infectious agents, known as Risk Group 4 pathogens.     * Risk Profile: Extremely high risk of life-threatening disease transmission via aerosol or unknown routes. Often involves agents with no known vaccines or therapies.     * Safety Infrastructure:         * Typically located in a separate building or isolated from communities.         * Utilizes specialized ventilation and waste control systems.     * Examples: Ebola virus and Smallpox virus.         * Ebola Outbreak Case Study: An outbreak occurred in the early $2000s$. Contracting Ebola causes severe illness where the virus induces bleeding from the eyes, nose, and anus, eventually leading the individual to bleed out and die.

Clinical Microbiology and Common Hospital Pathogens

• Patient Context: Students in clinical tracks will deal with patients who have contracted specific bacteria in hospital settings.
• Clostridium difficile (C. Diff):     * Proper name: Clostridium difficile.     * Common nickname: C. Diff.     * Symptoms: Causes severe diarrhea in patients and can be very dangerous.

Essential Biosafety Practices and Laboratory Conduct

• Hand Hygiene:     * Wash hands with soap and dry with paper towels whenever entering or leaving the laboratory.     * Wash hands immediately if leaving for a restroom break to avoid contaminating outside objects like phones or faces.
• Workspace Maintenance:     * Keep the work area clean and disinfected to prevent spreading bacteria to yourself or taking it home to loved ones.
• Personal Item Restrictions:     * Backpacks and purses must never be placed on bench tops due to potential contamination from previous lab sessions.     * Electronic devices (cell phones) should not be placed on tabletops, especially when working with bacteria like Strep or Staph, to prevent taking bacteria home in your pocket.
• Personal Protective Equipment (PPE) and Dress Code:     * Lab Coats: Must be worn at all times to prevent contamination of personal clothing and protect against accidental staining (e.g., from Gram stains).     * Footwear: Closed-toe shoes are mandatory to protect feet from spills of caustic or acidic reagents that could cause burns.     * Eye Protection: Goggles must be worn to prevent microbes or chemicals from entering the eyes.     * Gloves: Must be worn while handling materials. They should be removed and disposed of before washing hands and exiting the lab.     * Hair: Long hair must be tied back. This prevents hair from catching fire when leaning over open flames (Bunsen burners or alcohol lamps).
• Food and Drink:     * Strictly prohibited. No water bottles or food permitted.     * Nothing should be placed in the mouth or eyes while in the lab.
• Instrument Handling and Sterilization:     * Transport: Always use test tube racks to move cultures; never carry individual tubes by hand to avoid breakage.     * Placement: Do not place contaminated instruments (inoculating loops, needles, pipettes) directly on bench tops.     * Sterilization: Loops and needles must be sterilized using alcohol lamps.     * Disposal: Pipettes must be disposed of properly at all times.     * Removal: Do not remove media, equipment, or bacterial cultures from the laboratory.
• Spill Procedures:     * Immediately cover spilled cultures or broken tubes with paper towels.     * Saturate the area with disinfectant (e.g., $10\%$ bleach).     * Notify the instructor (Joe, Professor Pringle, or Dr. Rangel) immediately.

Introduction to Microscopy

• Etymology: "Micro" means small; "Scope" means to observe tiny or small things.
• Definition: Microscopy is the technique of utilizing an instrument (microscope) to view objects not visible to the naked eye.
• Types of Optical (Light) Microscopes:     * Simple Microscope: Uses a single lens.     * Compound Microscope: Utilizes two or more lenses working in a system to magnify images using light.
• Parfocal Capability: A parfocal objective lens stays in focus even when the magnification is changed (e.g., switching between objectives).

Anatomy of the Compound Microscope

• Base: Supports the microscope and ensures it is solid on the tabletop.
• Arm: Used for carrying. Correct transport involves grabbing the arm with one hand and supporting the base with the other.
• Rheostat: Controls the light intensity from the illuminator.
• Illuminator: The actual light source at the base.
• Condenser: Condenses and focuses light through the sample to make it more visible.
• Diaphragm: Controls the specific amount of light entering the condenser.
• Mechanical Stage: The platform where the slide is placed. Includes knobs to move the slide left, right, forward, and backward.
• Rotating Nosepiece: Holds and rotates the objective lenses.
• Objective Lenses: Lenses that provide different levels of magnification.
• Coarse Focus Knob: Moves the stage up and down rapidly to find the specimen.
• Fine Focus Knob: Used for sharp focusing to make the image clear.
• Body Tube: Transmits the image from the objective lens to the ocular lens.
• Ocular Lenses (Eyepieces): Where the specimen is viewed. These typically have a constant magnification of $10 \times$.

Magnification and Scientific Principles of Microscopy

• Calculating Total Magnification: Multiply the objective lens power by the ocular lens power ($10 \times$).     * Scanning Lens (Red): $4 \times$ objective. Total magnification: $4 \times 10 = 40 \times$.     * Low Power Lens (Yellow): $10 \times$ objective. Total magnification: $10 \times 10 = 100 \times$.     * High Power Lens (Blue): $40 \times$ objective. (Note: The blue lens was referred to as $40 \times$ in practice, though once mentioned as a possible $100$. Total is objective $\times 10$).     * Oil Immersion Lens (Black): $100 \times$ objective. Total magnification: $100 imes 10 = 1000 \times$.
• Resolution (Resolving Power):     * The ability of a lens to reveal fine details or distinguish two points as distinctly separate.     * Lower resolution results in two objects appearing as one large object.
• Field of View (FOV):     * The bright circular area visible when looking through the eyepieces.     * Inverse Relationship: As magnification increases, the field of view diameter decreases.     * At higher magnification, you see more detail of a smaller area (e.g., seeing details of a single blood cell vs. seeing many cells at once).
• Working Distance:     * The physical distance between the objective lens and the slide.     * Inverse Relationship: As magnification increases, the working distance decreases (the lens gets closer to the slide).
• Light Intensity:     * As magnification increases, light intensity naturally decreases.     * Adjustments must be made using the rheostat, condenser, or diaphragm.
• Oil Immersion Technique:     * Purpose: Oil is used specifically with the $100 \times$ (black-coded) lens to prevent light loss due to refraction and to prevent scratching the lens.     * Index of Refraction: Oil has a refractive index similar to glass (not air).     * Maintenance Rule: Never use the coarse focus when using the oil immersion lens. Once oil is applied, never rotate back to the $40 \times$ lens, as it will be damaged.

General Microscope Operating Procedures

1. Transport: Carry using two hands (arm and base).
2. Cleaning: Use chem-wipes or lens paper to clean lenses before or after use to remove smudges or bacteria.
3. Initial Setup: Place the slide on the mechanical stage and center it. Start with the smallest ($4 \times$) scanning lens.
4. Focusing: Use the coarse focus first to bring the image into view, then the fine focus to sharpen.
5. Increasing Magnification: Rotate the nosepiece to the $10 \times$ lens. Use only the fine focus from this point onward to sharpen the image.
6. Storage: Remove the slide, turn off the light, disconnect the cord, wrap it around the microscope, and place it back in the cabinet.

Questions & Discussion

• Question (Student): Regarding the tests on Thursdays and Fridays, since electronics aren't allowed in the lab, do we take the tests outside the lab before proceeding in?
• Response (Dr. Rangel/Joe): During lab activities, devices cannot be out because bacteria are present. However, on exam or quiz days, no bacteria are out on the benches. We take the quizzes/exams at the beginning of the period in the lab room before any bacteria or activities are started. Once activities finish, bacteria are put back in the incubator and the area is cleaned. Devices are needed for the quizzes/exams, but must be put away once the lab work begins.
• Question (Student): Will the laboratory biosafety questions be multiple choice?
• Response (Dr. Rangel): Yes, they will be multiple choice questions.