BMS103 S2

Professional Ethics and Laboratory Safety

Objectives + Prac. Qs

1. Understand professional ethics in scientific practice

2. Identify the core ethical principles in science and recognise ethical dilemmas

3. Define the requirements for working safely in a science laboratory

Practice:

• What is the difference between personal and professional ethics?

• List the 6 core ethical principles in Science.

• Explain why plagiarism is an ethical breach.

• Provide examples of PPE worn in the laboratory.

• What are the four categories of safety signs?

• Which equipment should you use when you work with dangerous chemicals?

• Which equipment should you use when you work with dangerous microorganisms?

• Laboratories are classified into how many Physical Containment (PC) levels?

• Which equipment is typically used to dispose of biological wastes at high temperature and pressure?

• Why is laboratory safety essential?

Ethics

Set of moral principles that govern an activity - responsibilities and rights.

Personal vs Professional Ethics

Core Ethical Principles in Science

1. Honesty - Report data truthfully. Do not fabricate data. Acknowledge limitations.

2. Integrity - Act with sincerity. Honour agreements

3. Accountability - Follow institutional and governmental policies and regulations.

4. Respect - Colleagues, human and animal subjects, the environment (sustainability)

5. Confidentiality - Protect private information and not disclose to unauthorised parties.

6. Beneficence - act for the benefit of others, promoting their wellbeing

Ethics in research

Australian code for the responsible conduct of research

• Principles of responsible research conduct:

  • • Honesty, Rigour, Transparency, Fairness, Respect, Recognition, Accountability, Promotion.

• Responsibilities of institutions

• Responsibilities of researchers

Ethics in Human

National statement on ethical conduct in human research

• Values and principles of ethical conduct

• Conditions for consent

• Ethical issues in recruitment of research participants who may experience increased risk

• Research governance and ethics review

Common Ethical Issues

• Data fabrication

  • Violates honest

  • Wastes resources

  • Potential harm to society

• Plagiarism

  • Violates intellectual honesty

  • Disrespects other's work

  • Misleads public

• Conflicts of interest

  • Violates transparency

  • Undermines fairness

  • Disrespects colleagues

• Coerced consent

  • Violates beneficence

  • Invalidates consent

  • Erodes trust in research

• Misuse of forensic evidence

  • Violates scientific integrity

  • Compromises justice

Laboratory Safety

Laboratory Safety

Set of rules, procedures, and practives designed to protect people, property and environment in laboratory setting.

Applies to students, teachers, researchers, technicians

• Personal safety

• Chemical and biological safety

• Equipment safety

• Emergency procedures

• Behavioural expectations

• Training

Personal Safety

Wear personal protective equipment (PPE)

• Lab coats - Do NOT wear lab coats outside laboratory

• Gloves

• Goggles (safety glasses)

• Closed-toe shoes

• Long hair must be tied back

Laboratory Hazards

Safety Signs

1. Prohibitory signs

2. Warning signs

3. Mandatory signs

4. Emergency escape and first aid signs

Chemical Hazards

Global harmonised system (GHS) hazard pictohrams for chemical labelling

Found on    

• Chemical containers

• Labels

• Safety Data Sheets (SDS)

Working with Dangerous Chemicals

Use a fume hood

• Reduces exposure to hazardous fumes

• Provides a barrier of protection against uncontrolled reactions

Working with Infectious Microorganisms

• Use a biological safety cabinet

• Laboratories are classified into physical containment (PC) levels

  • AKA biosafety levels (BSLs)

  • Four levels - PC1/2/3/4

Waste Disposal

It is important to dispose of chemical and biological wastes properly

• Protect the environment (sustainability)

Chemical wastes are disposed in proper waste containers

• According to safety data sheet

Biological wastes are typically autoclaved

• High temperature and high pressure

Some biological wastes are incinerated

Emergency Procedures

• Know locations of eyewash, safety shower, fire extinguisher, first aid kits, exits

• Follow institution’s emergency plan

• Contact appropriate emergency services

Laboratory staff will guide you in case of emergency

Laboratory Safety is Essential

• Prevents - injuries, exposure, contamination

• Protect - expensive and sensitive equipment

• Maintain - reliable and valid scientific results

• Comply - legal and institutional safety regulations

Monitoring Ethics and Safety

Each institution has specific committees that monitor ethics and safety.

Committees at MU:

• Human Ethics Committee

• Animal Ethics Committee

• Institutional BioSafety Committee

• Safety in Research and Teaching Committee

Quantitative literacy

Objectives

• Understand decimal places, significant fugures and scientific notation

• Be able to convet units

• Be able to perform dilution calculations

• Be able to perform calculations for specific laboratory techniques including    

  • Serial dilution

  • Viable counts

  • Microscope magnification

• Be able to differentiate between accuracy and precision

• Be able to calculate percentage error in measurement

• Be able to differentiate between sensitivity and specificity of a diagnostic test

Definition

Ability to understand, interpret, communicate numbers/mathematical information to:

• Solve problems

• Make informed decisions

Important in:

• Research

• Medical treatments

• Diagnostic tests

• Forensic tests

Math

Decimal Places

• Position digits to right of decimal point

• Two decimal places means two digits to the right of decimal point

Significant Figures

What:

• Figures or digits required for certain accuracy level

• Starts from first non-zero digit

• Number written to three significant figures will have maximum of three non-zero digits

Rules:

• Non-zero digits are significant

• Zeros between non-zero digits are significant

• Zeros to the right of a decimal point are significant

• Zeros to the left of the first non-zero digit are not significant

• Zeros at the end of a whole number (i.e. no decimal point) may or may not be significant

Round to SF

• Identify last SF

• If next digit is 5 or greater, +1 to last SF

• If next digit less than 5, leave last SF as is

Scientific Notation

Consider 0.000000234

• To 2 dp = 0.00

• To 3 SF = 0.000000234

  • Too long to write

→ 2.34 × 10^-7

Units of Measurement

Important when quanitifying measurements

Measuring ‘a time of 20’ has non meaning without its unit

• A time of 20 secs

• A time of 20 mins

• A time of 20 hrs

We MUST report units of measurement

All units have a symbol

Conversion of units

Moles to Mass

Molarity

Solute + solvent = solution

• Measure of number of moles of solute per unit volume of solution

• Molarity = concentration of solution in molL^-1

• molL^-1 can also be written as M

  • 1 molL^-1 = 1 M

Dilutions and Concentration

Dilution questions:

1. What is the final concentration of Reagent X when 200 µL of water is added to 50 µL of 10 mM Reagent X.

2. What volume of 10 M glucose solution must be used to make a 250 mL solution of 50 mM glucose solution?

Serial Dilutions:

Concentration of Solutions

• Mole per unit volume (aka molarity) – in molL-1 or M

• Mass per unit volume – in gL-1

• Percentage weight by volume – % (w/v)

• Percentage volume by volume – % (v/v) • Parts per million – in ppm

• Parts per billion – in ppb

Viable Counts Calculation

To calculate the concentration of viable bacteria

Each living bacterial cell forms a colony on agar plates

Concentration can be calculated in CFU/mL

    CFU = colony-forming units

To calculate concentration of bacteria (in CFU/mL) in the original suspension, use formula:

Magnification on Microscope

Accuracy vs Precision + Errors

Errors

• Measured as percentage errors

• Errors provide information about accuracy of measurement

  • High % error = low accuracy

  • Low % error = high accuracy

Diagnostic Test accuracy

• True positive = sick person diagnosed sick

• False positive = healthy person diagnosed sick

• True negative = healthy person not diagnosed sick

• False negative = sick person not diagnosed sick

Sensitivity vs Specificity

Sensitivity

• Ability to correctly identify true positives

  

Specificity

• Ability to correctly identify true negatives

Example

• PCR is golden standard for diagnosing COVID-19

• New lateral flow test for c19 is currenly on the market table below shoes performance compared to PCR

Prac Q:

1. Calculate the sensitivity and specificity of the new lateral flow test

Spectrophotometry

Objectives

• Explain principles of spectrophotometric analysis

• Identify main features of spectrophotometer + describe how absorbance is measured

• Define Beer-Lambert law + explain how it is used to determine concentration of substances in solution

Use

Used by scientists for qualitative and quantitative analysis of substances

• Clinical diagnostics

  • Concentration of clinical markers     

    • Haemoglobin

    • Glucose

    • Cholesterol

• Molecular research

  • Concentration of DNA and RNA

  • Concentration of cellular protein extracts

• Biochemistry research

  • Measure enzymatic activity over time

Principles

Measures intensity of light absorbed by substance in solution

Spectro

• Latin - specere - to look at, appearance

Photo

• Greek - phos - light

Metry

• Greek - metron - to measure

Different substances absorb different wavelengths of light

Electromagnetic Radiation (Light)

Light (EMR)

• Form of energy

• Made of photons

Energy of one photon is

• Directly proportional to frequency (f)

• Inversely proportional to wavelength (λ)

7 types of EMR (most to least energetic)

• Radio waves

• Microwaves

• Infrared

• Visible light

• Ultraviolet

• X-rays

• Gamma rays

Reading Down the List (Radio → Gamma): Wavelength decreases, while frequency and energy increase.

Reading Up the List (Gamma → Radio): Wavelength increases, while frequency and energy decrease.

(Short wavelength = more energy)

Visible Light

• White light has seven colours (ROYGBIV)

• Colour depends on energy/frequency/wavelength

Colour of a Solution

• All substances contain molecules + electrons that absorb, transmit and reflect certain wavelengths of light

• Colour visible represents the reflected light

Absorption Spectrum

Shows which wavelengths of light is absorbed by a substance

Colours absorbed are the colours we do NOT see

Measured using spectrophotometer

• SP was set at 400 nm, 401 nm, 402 nm…to 700 nm - recorded absorbance

Spectrophotometer

What?

A spectrophotometer passes monochromatic light through a solution and measures its absorbance

• Monochromatic light = one specific wavelength of light (e.g. 600 nm)

• Absorbance = measure of amount of light absorbed

Which wavelengths are absorbed = substance identity

How much light is absorbed = substance quantity

1. Put solution in cuvette

2. Open the top hatch on SP and place cuvette inside

3. On SP press measure after selecting wavelength

4. SP shines light on cuvette

5. Some light gets absorbed by solution in cuvette

6. There will be lower intensity of light passing the cuvette

7. Which wavelengths are absorbed = substance identity,

   How much light is absorbed = substance quantity

Components + Process + Calculations

SP is built up w two parts

• Spectromenter

• Photometer

1. White light (or other light - depends on type of SP) passes through collimator

2. Collimator concentrates light in one direction through monochromator

3. Monochromator splits the light into each of its colours (different wavelengths)

4. The wavelengths selectively pass the wavelength selector - the selector slit has a hole to allow which wavelength to pass

5. After wavelength selection, it passes the sample solution (cuvette). It is usually less intense than I0

6. Passing light will hit the detector (photocell) and convert it to an absorbance value

t = intensity of transmitted light

0 = intensity of incident light

Transmittance = fraction of incident light transmitted

E.g.

• If 90% of light is transmitted, transmittance is 0.9

• If solution absorbed 20% of incident light, transmittance is 0.8

Transmittance is always value - amount absorbed

Identifying a Substance

• Measure absorbance at different wavelengths

  • 400-700nm, 25nm intervals

• Plot absorbance vs wavelength

  • wavelength = independent variable (x-axis)

  • absorbance = dependent variable (y-axis)

• Compare to known absorption spectra

Beer-Lambert Law

E = constant physical property of analyte

To find E, we can find graph absorbance vs concentration

Determining Concentration

Example Prac:

• Standard curve shows absorbance of solutions containing different amounts of analyte P.

• Question: give absorbance of solution containing analyte P is 0.70, what is the concentration of analyte P in this solution?

Applications of Spectrophotometry

• Determine concentratin of nucleic acids (DNA and RNA)

  • Both DNA and RNA have a high absorbance at 260m, (UV light)

• Determine purity of DNA, RNA, and proteins

  • A260/A280 is used

• Enzyme kinetics

  • By measuring the concentration of a coloured product over time

Practice Qs

• Which colour of light has the longer wavelength – red or green?

• What is the meaning of ‘monochromatic’?

• How do we calculate Absorbance?

• When can we use the Beer-Lambert Law to determine concentration?

• What is the wavelength of light at which DNA absorbs maximally?

• What is the wavelength of light at which RNA absorbs maximally?

• What is the wavelength of light at which proteins absorb maximally?

Agarose Gel Electrophoresis

Objectives

• Explain principles of agarose gel electrophoresis

• Describe how agarose gel electrophoresis separates DNA Fragments

• Outline steps of gel preparation, loading and running

• Interpret agarose results

What?

• A technique which uses electricity ti separate nucleic acids and proteins based on their size and charge

• Relies on properties of charged particles

  • Unlike charges attract/like charges repel

Charge of DNA

DNA:

• Double helix

• Direction (5’ to 3’)

• DNA made of:

  • Nitrogenous bases (ATCG)

  • Deoxyribose sugar

  • Phosphate backbone

DNA affected by electric field

• Due to DNA molecules having same mass to charge ratio

• DNA movement depends on size only

Agarose

• Linear polymer (polysaccharide) obtained from red algae

• Agarose obtained as white powder

• Agarose powerder dissolves in near-boiling TAE buffer

  • TAE = tris-acetate EDTA

• Forms a gel when cooled

• Gel contains pores

TAE Buffer

• Tris-acetate-EDTA - used as solvent

• Contains ions to conduct electricity during electrophoresis

• Buffer = maintains constant pH

  • Usually pH 8.3

  • Can be purchased as 50x TAE

• Must dilute using water to 1X TAE before use

  • 1 in 50 dilution

    • 100 mL of 50X TAE in a final volume of 5L

Concentration of Agarose Gel

Given as % (w/v) = mass of agarose in 100 mL of 1X TAE buffer

• Pore size depends on agarose concentration

  • Higher = thick gel = smaller pores = separate shorter DNA fragments

  • Lower = thin gel = larger pores = separate larger DNA fragments

Preparing Agarose Gel

1. Weigh agarose powder

2. Add to 1x TAE buffer

3. Heat to dissolve (in microwave)

4. Cool and add DNA stain

   • Ethidium bromide (EtBr) or SYBR green

   • Allows DNA to be visible

5. Pour into mold with comb

6. ALlow gel to set (30-45)

7. Remove comb to create wells

Preparing and Loading DNA samples

Preparation:

1. Mix DNA samples with loading dye

   • LD contains glycerol allowing DNA to sink to bottom of well

   • LD contains dye allowing monitoring of the run

2. Mix DNA ladder (DNA marker) with loading dye

   • DNA ladder contains different DNA

   • fragments of known sizes (=standard curve)

Loading:

1. Place gel in gel tank in correct orientation

   1. DNA samples closer to black (-) electrode

2. Add 1x TAE buffer until gel is immersed

3. Load DNA ladder in first well

4. Load each DNA sample in a separate well

Running and Visualising Gel

Running:

1. Connect electrodes to a power supply

2. Allow gel to run at 80-100V for 40-60 minutes

DNA will migrate towards positive electrode (anode)

Visualising:

• UV light or blue light required    

  • Use transilluminator

• DNA stain (in gel) binds to DNA and fluoresces

• DNA appears as bright bands

Polymerase Chain Reaction

DNA Sequencing

Mass Spectrophotometry

Flow Cytometry

Biomedical Science

Clinical Laboratory Science

Microbiology

Genetics and Molecular Biology

Genomics

Immunology

Forensic Biology

Forensic Chemistry and Toxicology

Pharmacology and Toxicology