Biotech Midterm

Unit 1: Health and Safety

Health and Safety

What are health and safety regulations?

• Legal guidelines that minimize risk and help to protect participants from hazards in the workplace.

What is Occupation Health and Safety

• An area of public health focusing on illness and injury in the workplace

• Guide health and safety regulations

Health and Safety Risks

• These risks include

  • Chemical Hazards (chemicals used in experiments or specific work)

  • Physical Hazards (radiation, heat, noise)

  • Biological Hazards (blood-borne pathogens, other infectious diseases)

  • Ergonomical Hazards (repetitive motions, heavy lifting)

Precautions

• Precautions that can be taken to ensure safety are:

  • Vaccines

  • Wearing proper Personal Protective Equipment

    • Minimizes exposure to hazards that can lead to serious injuries in the workplace.

    • Use of disinfectants and cleaners (still chemical hazard)

Radiation

• Radiation protection is used to reduce the unnecessary exposure, minimizing the harmful effects of ionizing radiation

• Follows ALARA: As low as reasonably achievable

  • Time

  • Distance

  • Shielding

GHS Pictograms and NFPA Diamond

• OSHA adopted NINE pictograms providing recognition of the hazards of certain substances.

• The national fire association (NFPA) has a color-coded diamond containing four quadrants in which numbers (4-0) are used in the upper three quadrants to signal the degree

  • Health hazard (blue)

  • Flammability hazard (red)

  • Reactivity hazard (yellow)

  • Bottom quadrant is used for special hazards

Reporting and Understanding Hazards

Documenting and Reporting Hazards and Compliance

• OSHA has identified SIX action items to identify and assess hazards:

  • Collect existing information about workplace hazards

  • Inspect the workplace for safety hazards

  • Identify health hazards

  • Conduct incident investigations

  • Identify hazards associated with emergency and non routine situations.

  • Characterize the nature of identified hazards, identify interim control measures (reduces human exposure), and prioritize the hazards of control.

Maintain the Equipment

• Part of safety is maintaining your equipment.

  • Is the UV light in the Goggles’ cade working?

  • Is the fire extinguisher up to code?

  • Is the autoclave working appropriately?

• Routine maintenance checks will ensure that your labs run smoothly and will ensure your safety!

Safety Data Sheets

• Formerly called MSDS, new SDS are required to be used friendly.

  • Sections 1-8: General information (easy to get to)

  • Sections 9-11 and 16: technical and scientific information

  • Sections 12-15: Should be consistent with the UN Globally Harmonized System of Classification and Labeling of Chemicals (GHS); not enforced by OSHA

• Safety Data sheets include information such as:

  • Properties of each chemical.

  • The physical, health, and environmental health hazards

  • Protective measures

  • Safety precaution for handling, storing, and transporting the chemical

• Safety Data Sheets must be in English but may appear in other languages as well.

Personal Protective Equipment (PPE)

• PPE is specialized clothing or equipment used to ensure safety in a place of work against possible hazards.

• In biotechnology labs, possible PPE may be 

  • Safety goggles or glasses

  • Gloves

  • Lab coats or aprons

  • In some instances, something as specialized as a Biosafety Lab 4 “Positive Pressure Suit”

Emergency Equipment

• Shower: In case a chemical gets ON you, you are to stand under the shower for 20 minutes or until paramedics arrive.

• Eye wash: If a chemical gets in your eye(s), you are to use the eye wash for 20 minutes or until paramedics arrive

• Fire blanket: If a classmate or teacher catches on fire, quickly unfold the blanket and throw it on top of them to extinguish the flame.

• Fire extinguisher: teacher use only! Point the extinguisher at the base of the flame.

• Shut off button: in case the gas or power needs to be shut off immediately, press the button!

• Broken glass container: Dispose of broken glass here!

Aseptic Technique and Decontamination

Aseptic Technique and Standard Operating Procedures (SOPS)

• Aseptic technique is a method used to prevent the contamination of microorganisms.

  • Require multiple barriers such as sterile gowns, sterile gloves, sterile drapes, or masks.

  • Require environmental controls such as keeping doors closed and limiting traffic.

  • Only sterile to sterile contact is allowed in aseptic technique.

• Often, aseptic techniques follow a Standard Operating Procedure. SOPs are a set of written instructions that describe the step by step process that must be taken to perform a routine activity.

• Gloves should be sprayed with alcohol. 

Decontamination Lab Techniques

• Sterilization: The process by which something is made to be free of bacteria or other living microorganisms. These techniques include:

  • Autoclaving

  • Heating

  • Chemicals/solvent

  • Radiation

• Disinfection: The process by which the number of microorganisms is reduced or eliminated from inanimate objects. These techniques include:

  • Washing with soap and warm water

  • Chemicals

  • The use of steam

Chemical Safety, ISO, and Biosafety

Reagents and Compounds

• It is important to know the hazards of the reagents and the compounds with which you are working.

• Reagents can be classified by their hazards- corrosive, irritant, flammable, etc.

• Just as reagents are hazardous, so are compounds. Examples include

  • Arsenic

  • Formaldehyde

  • Asbestos

  • Mercury

• Be sure to read the SDS, look at the GHS pictogram, identify NFPA safety ratings, and read all labels.

Chemical Storage

• Chemical storage is an important aspect of safety that may easily be overlooked.

• All chemicals should be stored on level, sturdy, and secured shelves that are at eye level.

• Chemicals should be separated according to their specific hazards

  • There should be an acid cabinet for acids, a base cabinet for bases, and a flammable cabinet for flammables.

  • Oxidizers should be kept away from anything flammable or combustible.

  • Compatibility MUST be considered when organizing a chemical storage room.

Chemical Disposal

• To determine how to dispose of chemicals, one should always refer to SDS.

• In high school, MOST chemicals can be poured down the sink.

• However, there are certain chemicals that must be stored until a professional disposal company can pick them up and dispose of them in a more controlled manner. The company we use in Forsyth county is called MKC enterprises.

International Organization for Standardization (ISO)

• Ensures that the products and services you use daily are safe, reliable, and high quality.

  • Their goal is to achieve consistent universally recognized standards

• They also focus on sustainability and ethical practices

• ISO standards for biotechnology support innovation while ensuring safety, efficacy, and ethical considerations in fields like gene editing and bio-manufacturing

Biosafety Levels (BSL)

• Biosafety levels are used to identify the protective measures needed in a laboratory setting to protect workers, the environment, and the public

• These levels are outlines in the Biosafety in Biomedical Laboratories 

• The BMBL outlines specific practices and safety and facility requirements

Biosafety Level Meanings

• Biosafety Level 1 - ex. Nonpathogenic strain of E. coli, chicken pox.

  • Used to study infectious against or toxins known to consistently cause disease

  • Follow basic safety procedures called standard microbial practices

• Biosafety level 2 - ex. Staph infections

  • Used to study moderate-risk infectious agents or toxins that pose a risk if accidentally inhaled, swallowed, or exposed to the skin

  • Must have access to handwashing sinks and eye wash stations

  • Must have an incinerator, an autoclave, and/or another sterilization technique

• Biosafety level 3 - ex. Tuberculosis, west nile virus

  • Used to study infectious agents or toxins that may be transmitted through the air and cause potentially lethal infection through inhalation exposure

  • Experiments are performed in biosafety cabinets

  • BSL-3 labs should be easy to decontaminate, should have 2 self-closing doors, sealed windows and wall surface, and filtered ventilation systems

• Biosafety level 4 - ex. Ebola, Monkey Pox

  • Used to study infectious agents or toxins that pose a high risk of aerosol-transmitted laboratory infections and life-threating disease for which no vaccine or therapy is available 

  • Laboratories are located in safe, isolated zones or housed in separate buildings.

  • Personnel are required to wear a full-body, air-supplied suit.

  • All personnel shower before exiting that laboratory and go through a series of procedures to decontaminate

Unit 2: Buffers, Solutions, Calculations

Buffers Notes

What is pH?

• pH is a logarithmic calculation of the number of H+ in a solution.

  • Acids have more H+ than -OH and range from 0-6.5

  • Bases have more -OH than H+ and range from 7.5-14

  • Neutral solutions have a pH of 7

Buffers

• A buffer is a solution that acts to resist change in pH when the hydrogen ion concentration is changed.

• A buffer solution is made up of some kind of salt or organic molecule added to deionized water

• The buffering salt of molecule ionizes and interacts with H+ and -OH ions removing them from the solution

Why are the buffers used?

• Buffers have many uses in Biotechnology

  • Proteins and nucleic acids are stored in buffers as they are sensitive to changes. The buffer helps to preserve the structure and function of these molecules.

  • Serves as the ingredient to resist pH change in cell culture growth media

  • The liquid phase of column chromatography

  • Conducting electricity in a gel box

Solutions and Calculations Notes

Solutions

• A solution is a homogenous mixture of two or more substances

• There are two parts of a solution

  • The solute is the substance BEING dissolved

  • The solvent is the substance DOING the dissolving

• An important skill in Biotechnology is to make solutions for research

• It is an important to be BOTH accurate and precise when making solutions

• Solutions are in mass/volume units 

Mass/Volume

• Most common measurement are mg/mL, g/L, and ug/uL

  • Concentration desired x Volume desired = Amount of solute (weight)

% Mass/Volume

• Units MUST be in g and mL. Remember 1% = 1 g in 100 mL

• Convert % value to decimal value (move the decimal two places to the left)

  • Concentration desired (decimal form) x Volume desired = Amount of solute to be used

Molar Solutions

• For this, you will need the Formula Weight (molecular weight). If using stock chemicals, you can find this on the bottle and in the SDS. This can also be calculated using a periodic table.

• 1 mole of NaCl weighs 58.4 g. A molecule of NaCl weighs 58.44 atomic mass units (amu). This is because Na= 22.99 amu and Cl= 35.45 amu

• Concentration desired (Mol/L) x Volume desired (L) x Molecular Weight (g/mol) = Amount of solute (g)

Stock Solutions and Serial Dilutions

• A stock solution is a concentrated solution of a substance that can be used to create serial dilutions.

• Serial dilutions are a series of dilutions that reduce the concentration of a solution from a concentrate. 

Dilutions

• To calculate how much stock solution you should use to make a desired solution, use formula C1V1=C2V2

  • C1: Concentration of stock solution

  • V1: Volume to use of the Stock

  • C2: Desired concentration of diluted stock

  • V2: Desired volume of diluted stock

Serial dilutions

• A serial dilution is a solution that is repeatedly diluted by the same dilution factor

  • Ex: An insulin solution is diluted so that each vial has 10 times less insulin than the previous vial

Homeostasis, Normality, and Molarity

• In biochemical reactions, homeostasis, normality, and morality are all related but represent different aspects of the chemical environment within living organisms.

  • Homeostasis: Refers to the ability of the organism to maintain stability or balance.

  • Normality: a measure of concentration used in chemical reactions, particularly acid base reactions; less commonly used in biochemical reactions than molarity. It is based on the number of exchangeable protons, hydroxide ions, electrons, etc. that can undergo a chemical reaction

  • Molarity (M): a measure of concentration representing the number of moles a solute dissolved in one liter of solution; typically used to express the concentration of reactants and products.

Spectrophotometry

• A spectrophotometer is an instrument which measures the absorbance of light (written Ax, where x is the absorbance) by a solution or a particular wavelength/ pigmentation. 

• By taking this measurement, one can quantify how much of something a sample contains or its purity

• The concentration of a protein is determined by first generating a standard curve of the absorbance of KNOWN protein concentrations.

• Then, using the standard curve, one can determine the concentration of unknown samples by comparing absorbance values.

• Colorimetric tests include the biuret test, the Lowry assay, and the bradford assay

Electrophoresis

• Gel electrophoresis uses electrical current to move charged particles based on size. 

• Commonly used for DNA analysis. Specifically it can be used in DNA fingerprinting and separation of RNA or proteins.

How does it work?

• An agarose gel should be mixed and poured

• The power sources is added and causes negativity charged molecules to move down the gel

• The smaller the molecules, the faster they will move

• To find size, a “ladder”, known length of samples, is loaded for comparison

How to interpret electrophoresis

• Depending on the use, this may vary

• If it is a criminal or body identification, the match should be identical

• If it is paternity, the match will be a mixture of mom and dad.

• In biotechnology, electrophoresis is used to separate samples (DNA, RNA, or protein structures)

• This is based on charge and size.

Chromatography

• A chemical tool used in biotechnology to bind proteins based on particular properties. It can be used to analyze the purity of a substance.

• Different chromatography techniques are based on properties like size, ion exchange, hydrophobic interactions and affinity.