Laboratory Safety: Guidelines and Procedures

Safety in the Laboratory

• Laboratory safety is crucial for the well-being of personnel and students, environmental protection, and the accuracy of results
• Labs involve potential hazards such as chemicals, high temperatures and pressures, and electrical equipment
• A strong safety culture and guidelines are essential to minimize risks

Laboratory Safety Guidelines

• Safety goggles must be worn when working with chemicals or near others doing so
• A laboratory gown, apron, gloves, and hairnet are required; PPE is mandatory for entry
• Bags and books are prohibited in chemical use areas
• Long hair must be confined, preferably with a hairnet during experiments
• Food, drinks, and gum are not allowed in the laboratory at any time
• Only scheduled instructors and students may stay in the laboratory
• Know the location of fire alarms, extinguishers, fume hoods, safety showers, and eyewash stations
• Unauthorized experiments, work, and presentation are prohibited
• Chemicals should never be taken without proper authorization
• Read labels to understand hazards and first aid

Chemical Handling

• Use the indicated amount of chemicals efficiently
• Add water to acid to control the reaction and prevent splattering
• Maintain cleanliness and organization in the workspace
• Never taste chemicals due to serious health consequences
• Never pipette by mouth for health and accuracy, use proper equipment
• Never return unused chemicals to avoid hazardous reactions and contamination
• Clean spills immediately and report them to the teacher

Glassware and General Practices

• Check glassware for cracks before use to prevent cuts and exposure
• Wash all glassware after each use with appropriate solvents or deionized water
• Wash arms and hands with soap and water before leaving
• Report any spills, fire, explosion, or injury immediately

Importance of Safety and Chemical Hazards

• Safety protocols protect against accidents, injuries, and long-term health effects, and maintain the integrity of experiments
• Laboratory chemicals can be flammable, toxic, corrosive, reactive, and more; awareness is vital for risk minimization

Corrosive and Toxic Substances

• Corrosive substances cause visible destruction or irreversible tissue damage at contact
• Example corrosive chemicals are acetic acid, hydrochloric acid, hydrogen peroxide
• Toxic substances are divided into causing poisoning by ingestion, absorption and inhalation
• Example toxic chemicals are formaldehyde, hydrogen chloride, hydrogen peroxide and iodine

Flammable Chemicals and First Aid

• Flammable chemicals include gases, volatile liquids, and dusts that form explosive mixtures in air
• Common examples are acetic acid, acetone, ethyl alcohol, kerosene, and xylene

Handling Burns

• Immerse thermal burns in cold water until pain stops, then cover with a sterile dressing
• For fume inhalation, move the victim to fresh air, keep them lying down, and lightly covered

Common Laboratory Signage

• Flammable materials should be kept away from oxidizing substances, flames, or sparks, use eye protection when working with highly flammable substances.
• Examples include ethanol and isopropanol
• Oxidizing agents transfer oxygen to other substances and should be stored separately from flammables
• Always wear gloves, eye protection, and a lab coat

Additional Chemical Hazards

• Corrosive materials can damage skin or other substances
• One drop can cause serious eye damage, so use non-corrosive gloves, eye protection, and lab coats
• Toxic materials are highly harmful and can cause death through swallowing, inhalation, or absorption
• Eye protection, gloves, and a face mask should always be used

Remaining Lab Hazzards

• Irritants cause itchiness, soreness, redness, and blistering
• Exercise caution and proper protection when preparing solutions with irritants
• Chemicals in the health hazard category can cause reproductive toxicity, respiratory problems, mutagenicity, and carcinogenicity

Biosafety and Biosecurity

• Biohazard, this sign indicates the presence of biological materials and specifies waste disposal areas
• Biological agents of concern can cause significant public health impact, morbidity, mortality, social disruption, and panic, especially when deployed as biological weapons

Hazards and Classifications

• Hazard: Anything with the potential to cause harm
• Risk: The likelihood that a hazard will occur
• Laboratory Hazards: Includes electric shock, toxic vapors, compressed gases, flammables, etc.

Categorizing and Agent Examples

• Category A agents pose high infectivity and danger to infected individuals and laboratory personnel
• Examples include Anthrax, Botulism, Plague, Smallpox, Tularemia
• Viral Hemorrhagic Fevers examples include Filoviruses like Ebola and Arenaviruses like Lassa

Agent Examples

• Category B includes agents that are extremely toxic but have difficulties with dissemination or lower infectivity
• Examples include Brucellosis, Epsilon toxin, Food safety threats like Salmonella, Q fever and Ricin toxin
• Viral encephalitis caused by alphaviruses, water safety threats like Vibrio cholerae
• Category C agents represent "emerging” agents and potential future infective threats e.g. Nipah Virus

Biosafety and Its Importance

• Biosafety is about applying safety precautions to reduce the risk of exposure to infectious microbes and limit contamination
• There are 4 biosafety levels with specific controls for containment, based on infectivity, disease severity, transmissibility, nature of work, and route of exposure.
• Biosafety levels range from BSL-1 to BSL-4, each building on the controls of the level before

Biosafety Level 1

• Microbes are not known to consistently cause disease in healthy adults
• Standard microbiological practices are followed, and work can be done on an open bench or table
• PPE (lab coats, gloves, eye protection) are worn
• A sink must be available for hand washing, and the lab should have doors and fitted with screens

Biosafety Level 2

• Microbes pose moderate hazards to laboratorians and the environment
• Microbes are typically indigenous and associated with diseases of varying severity
• Personnel are trained, and access is restricted during work
• Procedures with aerosols or splashes are done in a biological safety cabinet (BSC)

Biosafety Level 3

• Builds upon the containment requirements of BSL-2
• Microbes can be either indigenous or exotic and can cause potentially lethal diseases through respiratory transmission
• Laboratorians require medical surveillance and may get immunization

BSL3 Safety Equipment

• Personnel must receive specific training in handling pathogenic/lethal agents
• Appropriate PPE must be worn and respirators may be
• Air flow must be directional by drawing air into the laboratory from clean areas to potentially contaminated areas
• Stainless steel is used

Biosafety Level 4

• Involves dangerous and exotic microbes, posing a high risk of aerosol-transmitted infections
• Infections caused by these microbes are frequently fetal
• Ebola and Marburg are 2 examples of this type of virus

Biosafety Precautions

• Change clothing before entering
• Shower upon exiting and decontaminate all materials
• Work must be performed within an appropriate Class III or wearing a full-body suit

Biological Agents of Concern

• Anthrax is a zoonotic disease, generally found in herbivores that ingest spores from contaminated soil.
• It has been present for centuries, and was previously identified as “wool sorters' disease"
• The spores are extremely resilient
• Botulism is a primarily foodborne illness described in 1897
• Causative Agent: Clostridium botulinum which produces dangerous toxins under low-oxygen conditions

Plague Types

• Symptoms of botulism: Botulinum toxin is the most poisonous substance known; less than 1 mcg is a fatal dose for an adult
• There are five major types of botulism: foodborne botulism, infantile botulism, wound botulism, iatrogenic and adult intestinal botulism.
• There is 94% of foodborne botulism cases
• Plague affects humans and other mammals . Plague occurs in three forms: bubonic, septicemic and pneumonic
• All the main functions of this diseases are
  • 1 Bubonic plague which is named after the swollen lymph nodes that typically develop in the first week after you become infected.
  • 2 Septicemic plague which occurs when plague bacteria multiply in your bloodstream.
  • 3 Pneumonic plague which affects the lungs and can be easily spread

Tularemia and Small Pox

• Tularemia is a highly infectious zoonotic disease typically found in animals such as rabbis and rodents
• Transmission of tularemia from person to person has NOT been reported.
• Smallpox was one of the most severe infectious diseases, it was eradicated in 1980
• The first use of smallpox as a biological weapon was during the French and Indian Wars

Biosecurity, Biorisk and Microscopy

• Person-to-person transmission of the disease occurs by droplet spread from infected persons
• Smallpox is caused by the variola virus. Smallpox can only be spread by humans
• Biosecurity deals with the prevention of misuse of pathogens and toxins
• Biorisk is the probability or chance that a particular adverse event will occur.
• Used to magnify objects too small to be visualized with the naked eye

Types of Microscopes

There are two major types of microscopes:

• Light microscope- Visible light is passed through the specimen and then through series of lenses. Also, forms a dark image against a brighter background
• Electron microscope which can be be used to visualize small objects

Microscopy parts

Magnification is used with an Ocular lens - 10x the 4 primary objective lenses are:

• Scanner
• LPO
• HPO
• Oil immersion objective

Lenses and Stages

• Ocular Lens / Eyepiece which re-magnifies the image form by the objective lens
• Is held by the arm
• Mechanical Stage holds the objective slide in postion

Fine and Course Adjusments

• Diaphragm: Controls the amount of light entering the condenser
• Coarse and Fine Adjustment Knob: Can move either the stage or the nosepiece to focus the image
• Illuminator, light source
• Phase-Contrast Microscopy allows examination of internal structures

Microscopy

• Fluorescent Microscopy, uses fluorochromes to stain microorganisms and to identify medically important fungi grown in culture
• Dark Field Microscopy uses darkfield condenser that blocks light that would enter and directs the light to hit the specimen at an oblique angle to detect spirochetes

Resolution

• Terminologies
• Refractive Index, it a measure of the relative velocity at which light passes through a material
• Resolution, it is the ability of a lens to distinguish small objects that are adjacent or close together
• Resolving Power, refers to the ability of the lenses to separate closely distant objects
• Contrast, to make objects stand out from the background
• To Identify Microorganisms the following preperation is needed: Wet Mount Preparation Hanging Drop Preparation and Staining