How to Prepare & Stain a Peripheral Blood Smear | Hematology Lab Guide

In BSL-2 laboratories, students must wear mandatory personal protective equipment including safety glasses, gloves, lab coats, closed-toed shoes, and long pants at all times; they must follow strict contamination prevention protocols such as avoiding food and personal items in the lab, properly labeling all containers with initials and dates, and using designated biohazard waste containers for all contaminated materials; before leaving, students must disinfect their bench tops, turn off and unplug equipment, empty biohazard bags, remove and store lab coats, dispose of gloves in biohazard bins, and wash hands thoroughly; in case of emergencies, students should remain calm, use the nearest evacuation exit, and meet instructors outside the building.

Biosafety Level 1 (BSL-1) applies to agents posing no harm to workers or environment, using standard open benches without special containment. Practices include hand hygiene and prohibition of eating/drinking. PPE includes eye protection, gloves, and lab coats. BSL-2 handles pathogenic/infectious organisms, requiring all BSL-1 practices plus: puncture-resistant sharps containers, no direct glass handling, decontamination before disposal, controlled access excluding immunocompromised persons, Class II biosafety cabinets, autoclaves, and eye wash stations. BSL-3 handles agents causing serious disease via aerosol transmission (e.g., yellow fever, West Nile virus), requiring BSL-2 practices plus: Tyvek gowns, respirators, self-closing double doors, and specialized ventilation.

Biosecurity combines 'bio' (life) and 'seguridad' (security), referring to quality of life free from damage, risk, or danger. It encompasses norms, measures, and protocols applied in scientific research to prevent infections from biological, chemical, or physical hazards. Universal precautions (1985, CDC) treat all patients' fluids as potentially infectious. Standard precautions (1996) apply to all patients regardless of diagnosis. CDC established four biosafety levels: BSL-1 handles minimal-risk agents with no special equipment; BSL-2 handles moderate-risk agents with restricted access and biological safety cabinets; BSL-3 handles exotic, potentially fatal agents in clinical and research labs; BSL-4 handles high-risk agents (Ebola, Smallpox) with full-body suits and negative-pressure facilities. PPE materials are non-woven cellulose and polypropylene, liquid-repellent, including caps, gowns, masks, shoe covers, and boots.

This video presents comprehensive safety protocols for working in Biosafety Level 2 laboratories, emphasizing that personal protective equipment (safety glasses, gloves, lab coats, closed-toed shoes, and long pants) is the first line of defense against biological and chemical agents; students must follow strict procedures including proper disposal of biohazard waste in designated containers, knowing emergency equipment locations (eye wash, safety shower), understanding UV light safety, and completing seven essential end-of-lab steps (bench disinfection, equipment shutdown, biohazard bag management, PPE removal and disposal, and hand washing) to ensure a safe laboratory environment.

This extensive section covers biosafety levels, personal protective equipment (PPE) protocols, and hand hygiene techniques. Key topics include: (1) BSL-1 uses standard laboratory attire with contained work areas; (2) BSL-2 requires laboratory coats, gloves, and eye protection; (3) BSL-3 requires full-body suits, face shields, and respiratory protection with airlock access; (4) BSL-4 requires complete protective suits with sealed interfaces and maximum containment; (5) PPE must be donned in specific sequence: gown, mask, face shield and goggles, then gloves; (6) PPE must be removed in reverse order: gown, mask, face shield and goggles, then gloves; (7) Hand hygiene must be performed at four critical times: before and after handling biological samples, before and after eating or drinking, and before and after touching the face; (8) Proper hand hygiene requires thorough washing with soap and water, including all surfaces: palms, backs of hands, between fingers, and under fingernails; (9) Alcohol-based hand rubs are used when hands are not visibly soiled, requiring 3-5 ml application and thorough rubbing until dry; (10) Soap and water washing is required when hands are visibly soiled or after exposure to certain microorganisms; (11) In resource-limited settings, paper towels or tissues can be used to remove visible contamination.

A compound microscope consists of multiple interconnected components working together for specimen visualization. From top to bottom, these include oculars (10x magnification) with adjustable thumb screws, a revolving nose piece containing four objectives (4X scanning, 10X low power, 40X high dry, 100X oil immersion), coarse and fine adjustment knobs, a mechanical stage with clips, a condenser with iris diaphragm, and a field diaphragm at the base. The microscope achieves total magnification by multiplying objective magnification by ocular magnification (e.g., 40X objective × 10X ocular = 400X total). Light travels upward from the base through the field diaphragm, condenser, specimen, objective, internal lenses, and finally through the oculars for viewing.

Different objective magnifications allow progressively less light to pass through specimens. Lower magnifications (4x, 10x) permit maximum light penetration, providing better resolution. Higher magnifications (40x, 100x) restrict light due to smaller lens apertures. The condenser diaphragm controls light intensity and contrast—fully open provides minimal contrast, while partially closed increases contrast by restricting light to a narrower cone. Higher magnifications require careful balance between illumination and contrast. The 100x objective requires oil immersion to eliminate the air gap between specimen and lens, matching refractive indices and preventing light scattering. Without oil, image construction is impossible. These principles explain why high-magnification microscopy demands specialized techniques and equipment optimization.

A compound microscope uses multiple lenses to magnify specimens. Key components include: the eyepiece (optical lens) for viewing, diaphragm for light control, nosepiece holding four objective lenses (4x scanning, 10x low power, 40x high power, 100x oil immersion), stage for specimen placement, mechanical stage for precise movement, coarse and fine adjustment knobs for focusing, light source and condenser for illumination, and arm connecting to the base. Total magnification equals eyepiece magnification multiplied by objective lens magnification (e.g., 10x eyepiece × 40x objective = 400x). Low power objectives (4x, 10x) are used for initial specimen location, while high power objectives (40x, 100x) provide detailed observation. The 100x oil immersion lens requires immersion oil for optimal resolution.

The total magnification of a compound microscope is the product of the magnification of the objective lens and the magnification of the eyepiece. Angular magnification is the ratio of the angle subtended by the final image at the eye to the angle subtended by the object at the unaided eye. When viewing through the microscope, the final image appears larger and at a greater distance, resulting in a larger angular size compared to viewing the object directly. The magnification depends on the focal lengths of both lenses and the tube length.

A compound microscope uses two convex lenses: an objective lens close to the object and an eyepiece (ocular lens) at the other end. The objective forms a real, inverted, magnified image (a1b1) of the object, which then serves as the object for the eyepiece. The eyepiece forms the final virtual, magnified image at LDDV or infinity. Total magnification is the product of objective and eyepiece magnifications. For image at LDDV: m = (v1/u1) × (d/u2). For image at infinity: m = (v1/u1) × (d/f2). In terms of tube length L: m = (L/f1) × (d/f2), where f1 and f2 are focal lengths of objective and eyepiece respectively.

Staining methods are based on chemical properties: acidic structures bind basic dyes (hematoxylin stains nuclei blue), while basic structures bind acidic dyes (eosin stains cytoplasm pink). Hematoxylin and eosin (H&E) is the most common staining combination. Acidophilic structures appear pink/red, while basophilic structures appear blue/purple. This principle allows visualization of different cellular components by exploiting their chemical affinities for specific dyes.

Common dyes include Methylene blue, Safranin, Crystal violet, Iodine (mordant), and Nigrosin (Indian ink). Basic dyes (positively charged) bind to negatively charged bacterial components like nucleic acids and proteins: Crystal violet, Methylene blue, Safranin, Malachite green. Acidic dyes (negatively charged) bind to positively charged backgrounds: Eosin, Nigrosin, Rose Bengal. Basic dyes are used in direct staining, while acidic dyes are used in indirect staining. Chromogen is the color-generating component in stains.

Cells consist of nucleus and cytoplasm with distinct chemical properties. The nucleus contains DNA and RNA, which are acidic in nature. The cytoplasm is basic in nature. Hematoxylin is a basic dye that stains acidic structures (nucleus) blue, while eosin is an acidic dye that stains basic structures (cytoplasm) pink. Hematoxylin serves as the primary stain, and eosin acts as the counterstain. Mordant (aluminum potassium sulfate) is essential as it bridges hematoxylin and tissue, enabling proper dye binding. Without mordant, hematoxylin cannot effectively stain tissue.

Basic dyes (hematoxylin) have positive charges and bind to negatively charged cellular components: phosphate groups in nucleic acids (DNA, RNA), sulfate groups in glycosaminoglycans, and carboxyl groups in proteins. This produces basophilic (blue-purple) staining of nuclei, ribosomes, and extracellular matrix. Acidic dyes (eosin) have negative charges and bind to positively charged components, producing eosinophilic (pink) staining of cytoplasm and intracellular membranes. pH affects ionization of these groups.

Histological staining increases contrast of cellular structures, making it easier to identify nuclei and cytoplasm. A stain is a chemical substance that binds to cellular structures through acid-base affinity: acidic dyes bind to basic structures, while basic dyes bind to acidic structures. Hematoxylin (basic, purple) and eosina (acidic, pink) are the most commonly used stains. Hematoxylin stains acidic structures like DNA and RNA (nucleus, rough endoplasmic reticulum), while eosina stains basic structures like proteins (cytoplasm, extracellular matrix). Basophilia refers to affinity for basic dyes (purple appearance), and acidophilia refers to affinity for acidic dyes (pink appearance). The cytoplasm color varies based on protein content and rough endoplasmic reticulum abundance. Cell density and extracellular matrix presence also affect staining intensity.