GTB106 Laboratory Science Tutorial PDF

GTB106 Laboratory Science Tutorial 1 Dr Shuhaila Mat Sharani Physical, Chemical and Biological hazards  Physical Hazards Hazards present in ordinary laboratory equipment and surroundings: Electrical Laboratory Fire Glassware (wiring and...

GTB106 Laboratory Science Tutorial 1 Dr Shuhaila Mat Sharani Physical, Chemical and Biological hazards  Physical Hazards Hazards present in ordinary laboratory equipment and surroundings: Electrical Laboratory Fire Glassware (wiring and instruments appliances) Safety signs are posted in appropriate places as reminders to laboratory users Personal Protective Equipment (PPE) Students are provided with PPE to protect against identified hazards. Safety equipment's include: ▪ Eye, face, and skin: masks, goggles, or protective shield. ▪ Inhalation: respirator or fume hood to protect against inhalation of fumes. ▪ Skin, hands, and arms: gloves (rubber/asbestos). ▪ Body: laboratory coat/apron and appropriate attire (long pants/skirt). ▪ Feet: closed non-slippery shoes. ▪ Hair: tie back long hair  Chemical Hazards Chemicals present a variety of hazards: ▪ Flammable ▪ Toxic ▪ Caustic ▪ Corrosive ▪ Carcinogenic ▪ Mutagenic ▪ Radioisotopes  Chemical Labels Blue Red Yellow White environmentally acute gas 1 hazardous toxicity2 cylinder3 indicates indicates indicates indicates health fire hazard reactive or reactivity hazards or or unstable with water toxics flammability chemicals or contact such as hazards oxidizers explosive 6 flamable corrosive4 5 Colour-coded labeling system for identifying and warning of chemical hazards → NFPA (National Fire Protection Association) health oxidizing serious9health hazard7 8 hazard  Methods of decontamination and disposal Method of decontamination: Methods of disposal: ▪ Boiling (disinfection) ▪ Burial (waste pit or landfill) ▪ Chemical disinfectant ▪ Incineration for solid waste ▪ Autoclaving (sterilization)  Substances recognized as having the potential to transmit pathogens Blood Organs Blood products Sweat Human blood & blood products Semen Breast milk Microorganisms culture & wastes Peritoneal fluid Synovial fluid Animal waste/carcass Human body fluids/tissues Amniotic fluid Vaginal secretions Sharps waste Cerebrospinal Pleural fluid Pathological wastes fluid Unfixed tissue Urine specimens Pericardial fluid Saliva in dental settings where bleeding occurs Standard Precautions handle biological hazards It includes the use of safe work practices: ▪ Wash hand properly before and after ▪ Used of safety needles ▪ Proper PPE (Gown, Mask, Gloves ,Eye and mucous membrane protection ) ▪ Working in Biosafety Cabinet/ laminar flow  Laboratory acquired infections 1 2 Infection through skin & eyes Infection through mouth Cuts and scratches from skin Ingestion during mouth pipetting Hands through contaminated benches Oral-contaminated fingers (eating) & equipment Cigarettes/pipes Hands to face & eyes Eyes through sample splashing  Laboratory acquired infections 3 4 Infection through the respiratory tract Infection of the general public Many common lab procedure release Route of infection – from laboratory or aerosols (infected airborne droplets) hospital settings ‘Droplet's nuclei’ contains bacteria or Contact with patients or hospital staff viruses Inhaled particle size ≤10 µm will not Contact with infectious waste, get trapped discarded or effluent materials  Classification of infective microorganisms Risk group Risk group Present low risk to lab staff & public Present moderate risk to lab staff & public ▪ Unlikely to cause diseases ▪ Can cause serious disease but not serious hazard ▪ Examples: ▪ Effective preventive measures & treatment available Food spoilage bacteria ▪ Risk spread in community is limited Common moulds Bacteria: gram + cocci, enterobacteria, clostridia, Yeasts vibrio Viruses: adenoviruses, polioviruses, hepatitis, coxsackieviruses, coronavirus Fungi: Blastomyces Protozoa: toxoplasma, Leishmania  Classification of infective microorganisms Risk group Risk group ▪ Present high risk to lab workers ▪ Present high risk to lab worker ▪ Present low risk to community ▪ Present high risk to community ▪ Do not spread rapidly among individual ▪ Cause serious diseases ▪ There are effective vaccines and treatment ▪ Disease easily transmitted among individual ▪ Examples: ▪ Effective treatment and preventive Mycobacterium tuberculosis, salmonella measures are not usually available Arboviruses, HIV ▪ Examples: Histoplasma COVID-19 SARS Haemorrhagic fever Glassware and Plasticware  Glass Type and properties Glass Brand Properties Purpose Kimax or Pyrex Borosilicate - inert Multipurpose Borosilicate glass beaker, conical flask, tube test, etc heat resistant until 510˚C Vycor 96% silica, low alkali, high resistant to Optical use Aluminosilicate heat shock Cuvette, thermometer, etc High silica High temperature 900-1200˚C Corex Aluminosilicate glass 6-10 times more Use in stressful condition Aluminosilicate resistant than ordinary 'borosilicate' High thermometer temperature 250˚C, glass; anti scratch and alkali resistant centrifuge tube stable until 670˚C Boron free Soft, not heat resistant, withstand strong Strong alkaline solution storage alkaline solution Low actinic Contains reddish yellow material Storage and handling of light -sensitive substances (eg bilirubin, carotene and vitamin A) Pipette and pipetting technique Meniscus  Pipette classification To Deliver (TD) – Blow-out –has Measuring or Type Design Drainage characteristics deliver exact continuous etched graduated – amount rings (frosted / two Serological, Mohr, Bacteriologic, Bell, To Contain (TC) – thin rings) on top of Kolmer and Kahn and hold particular pipette automatic micropipette volume but not Self-draining – drain Volumetric or Transfer dispense the exact by gravity and do –volumetric/Ostwald- volume not touch liquid in Folin, Pasteur pipette, container automatic macro- pipette (start at 1 ml) or micropipette (between 0.1 and 1000µl)  Micropipette Technique Method 1: Method 2: Rest plunger Rest plunger Forward pipetting First stop A B C Reverse pipetting First stop A D E F Second stop Second stop D E B C C C A E F A B E D D B Normal liquid High viscosity https://www.microlit.us/micropipette-product-guide/  Cleaning and Proper Care of a micropipette Wipe with alcohol or laboratory cleaning agent Solution Types Process to clean by letting on micropipette For aqueous solutions, Rinse the contaminated parts with distilled for 10 minute before wiping organic solvents and water or 70 percent ethanol and air dry at off. proteins approximate 60°F temperature. Place in upright position at specific rack if not use For infectious liquids Autoclave the lower section at a temperature of 120°C for 15-20 minutes then allow it to return at room temperature before reassembling. For radioactive Place the pipette in a solution like Decon substances and then rinse and air dry. Boil lower parts of micropipette in glycine/ For nucleic acids HCI buffer (pH2) for 10 minutes, rinse with distilled water, and air dry. https://www.pipette.com/pipettestands Laboratory Calculation and Reagent Preparation Molarity  Moles and Molarity Origin of mole: the need for a universal unit of measurement to connect the mass of an atom/molecule with the number of atoms/molecules in any given compound. Mole (mol): SI unit of a quantity of a given molecule. A define mass of an element (its atomic weight) contains an exact number of atoms (Avogadro number). Thus, for any given molecule, one mole of a substance has a mass (grams) equal to the atomic mass of the molecule. No. moles (n) = Mass of substance (m) m Relative Formula Mass (RFM) Mr n  Molarity Molar solution: a molar solution is one which contain 1gram molecular weight of (solute) in 1 liter of (solution). n ○ Importance: a fundamental and convenient way of expressing the concentration of a compound in solution (liquid). M V ○ A one molar solution (1M) means: one mole of a given substance in a liter of a solution (1 mol /L). Number of moles (n) = molarity (M) X volume (V) (in liters) ○ Molal solution: a molal solution is one which contain 1gram molecular weight of (solute) in 1 kg of (solvent). Molality  Molarity vs Molality Molarity (M) = moles of solute (n) Molality(m) = mole of solute liters of solution (V) kg of solvent solute and solvent Solvent by itself combined 1 moles CaCl2 = 111.0g 111.0g CaCl2 111.0g CaCl2 1kg water 1L solution Understanding Mass, Weight, Volume and Density Important measures to quantify biomolecules – mass, weight, volume and concentration. Mass is the quantity of matter in each object and independent of gravity. Weight is the force of gravity on a given object and calculated as mass multiplied by gravity. Weight = Mass X Acceleration due to gravity  Volume and Density Volume : the space that mass in each object occupies. Density: a comparison of an object’s mass to its volume. It gives an indication of ‘how big’ something is. Density (ρ) = Mass (m) Volume (v) The formula dictates that objects of high density have a lot of mass occupying a relatively small volume. m ρ v Example: Volume and Density What is the density of an object with a mass of 20 grams and volume 8 cm3 ? m Density = Mass = 20 g = 2.5g/ cm3 Volume 8 cm3 ρ v What is the volume of a liquid with a mass of 10 grams and density 3.5g/ml? Volume = Mass = 10 g = 2.86 ml Density 3.5 g/ml 1 ml = 1 cm3  Example: Volume and Density Q1 : What is the density of an object with a mass of 25 grams and volume 12 cm3 ? Q2: What is the volume of a liquid with a mass of 18 grams and density 2.5 g/ml?  Dilution Adding more solvent without addition of more solute A two-fold dilution → reduces the concentration of a solution by a factor of two that is reduces the original concentration by one half 1:2 1:4 1:8 1:16 Using a Concentrated Solution to Make a Dilute Solution It is sometimes necessary to prepare a dilute solution from a concentrated solution. Example: Preparing 0.1 M HCl from a concentrated HCl solution. Using a general formula; M1 X V1 = M2 X V2 M1 initial molar Q: Prepare 100 ml of 0.1 M HCL using 1.0 M HCL V1 initial volume M2 final molar V2 final volume  Using a Concentrated Solution to Make a Dilute Solution It is sometimes necessary to prepare a dilute solution from a concentrated solution. Example: Preparing 0.1 M HCl from a concentrated HCl solution. Using a general formula; M1 initial molar M1 X V1 = M2 X V2 V1 initial volume Q: Prepare 100 ml of 0.1 M HCL using 1.0 M HCL M2 final molar 1.0M X V1 = 100ml X 0.1M V2 final volume V1 = 100 X 0.1 = 10 ml 1.0 10ml of 1.0M HCL is added to 90ml of H 2O to make 100ml of 0.1M HCL solution Using Percentage to Prepare Solutions Percent solution – one type of expressing concentration. In the lab, percent solutions usually refer to grams of solute per 100 ml of solution. Three way of preparing percent solutions: ○ Weight to volume (w/v): weight in gram of solute per 100 ml of soln. ○ Volume to volume (v/v): mL of solute per 100 ml of soln. ○ Weight to weight (w/w): weight in grams of solutes per 100g of soln. Preparation of the solutions in percentage are given in the next two slides.  Moles, Molarity & Molality No. moles (n) = Mass of substance (m) m Relative Formula Mass (RFM) Mr n Molarity (M) = moles of solute (n) Molality(m) = mole of solute liters of solution (V) solute and solvent kg of solvent Solvent by itself n combined M V  Preparing Molar Solutions Preparing 200 ml of approximately 0.20 M NaOH using solid NaOH  Preparing Molar Solutions Preparing 200 ml of approximately 0.20 M NaOH using solid NaOH Molar (M) = moles (n) Volume in liter (V) Moles (n) = Molar (M) x Volume in liter Volume (V) (V) in liter =MxV = 200 ml = 0.20 M x 0.2 L 1000 ml = 0.04 mol = 0.2 L  Preparing Molar Solutions – Continue … Moles (n) = mass (m) Molar mass (Mr) Mass (m) = Moles (n) x Molar mass (Mr) = 0.04 mol x (23 + 16 + 1) g/mol = 0.04 mol x 40g/mol = 1.6 g m Mr n Balance Analytical Balance Principle Calibration and “Magnetic force restoration” that uses an standardization electromagnet to determine an object’s mass Calibrate to maintain accuracy and precision Extremely Accurate Cleaning The mass can be measuring high degree Clean the weighing pan before and after use to precision and accurately of 0.0001 to 0.00001g ensure that the weighing sample does not get (0.1 mg to 0.01 mg) contaminated.  Weighing using Analytical Balance 1. It is preferable to preheat it for an hour before using it. 2. Set the analytical balance to zero in the no-load condition by pressing the “tare” button. Chamber 3. Place the weigh boat, weigh paper, or other vessel or container in the center of the weighing pan and then shut the glass door of the weighing chamber. 4. Check the value that was displayed after it was stabilized. The appearance of the Chamber Pan Digital stability mark indicates a stable state. door display handle 5. To exclude container mass from the measurement, the ‘TARE’ button is pressed to reset the mass to zero. 6. Add the substance to be weighed after removing the container from the balance. Avoid putting things in the balance pan because doing so can contaminate the Tare button balance. 7. Reset the container’s balance, then wait 5-10 seconds (up to a minute) for the mass Fig 1 Analytical balance reading to stabilize. https://scales-measuring.com/3739-large_default/kern- abj- 220-4nm-analytical-balance.jpg Double Beam Balance Zero indicator Pan Pan 100g beam Zero adjust knob 10g beam Main and supplementary rider Fig 2 Double beam balance http://ecx.images- amazon.com/images/I/41GypGFDPGL._SL500_AA300_.jpg Double Beam Balance Feature Principle Measurement double pan balance, is a With a two-pan design based on the Featuring single weighing scale that acts like a classic Roberval balance principle, unit and application see-saw and gets its Double Beam Balances feature the mode. Angled beams and name from the two additional advantages of magnetic pointer are easy to read pans or balances on damping, beams with sliding and help reduce operator either side of a fulcrum weights and a variety of weighing errors. platforms.  Weighing using Double Beam Balance Zero indicator 1. Setup the main and supplementary rider to utmost left end. Pan Pan 2. Adjust the zero adjust knob (fine-tuning balancing) knob at the right end of the beam, set the pointer of 100g beam Zero adjust the Balance to equilibrium position zero. knob 10g beam 3. To estimate the weight of the object on the left pan and then add suitable standard weights on the right Main and supplementary rider pan. 4. Move the main rider, supplementary rider to make the pointer to equilibrium position zero. Note: If the balance is not in use, don't put anything on the pans.  Medium size (10 - 100g) Triple Beam Balance Largest size (100 - 500g) Smallest (0 - 10g) Rider Beam Pan Balance pointer (0 mark ) Zero adjustment knob Fig 3 Triple beam balance https://tse1.mm.bing.net/th?id=OIP.TlyHOqVPs1mP-em- L7JPnAHaEN&pid=Api&w=1050&h=596&rs=1&p=0 Triple Beam Balance Features Measurement Weighing Has three beams including the 1. The middle beam which is and use 1. Riders on beam is move to leftmost position before measure and twist the largest size (100-500g) measure mass very precisely the zero-adjustment knob until from 0g – 500g with reading error balance pointer with zero mark 2. The front beam which is the of +/- 0.05 gram smallest size (0-10g) 2. Put the object and start weight from middle beam to the front beam 3. The far beam which is the measure mass directly from the (largest to smallest) and stop until medium size (10-100g). objects, find mass by difference the pointer line up with zero mark. for liquid, and measure out a substance.  Triple Beam Balance The triple beam balance is an instrument used to measure mass very precisely. The device has reading error of +/- 0.05 gram. The name refers to the three beams including the middle beam which is the largest size (100-500g), the front beam which is the smallest size (0-10g), and the far beam which is the medium size (10-100g). The difference in size of the beams indicate the difference in weights and reading scale that each beam carry. Riders on beam is move to leftmost position before measure and twist the zero-adjustment knob until balance pointer with zero mark. Put the object and start weight from middle beam to the front beam (largest to smallest) and stop until the pointer line up with zero mark. The triple beam balance can be used to measure mass directly from the objects, find mass by difference for liquid, and measure out a substance. pH meter  The term pH is derived from “p,” the mathematical symbol for negative logarithm, and “H,” the chemical symbol for Hydrogen. pH The formal definition of pH is the negative logarithm of Hydrogen ion activity. pH = -log[H+]  Electrode in pH meter 1. pH Electrode (Measuring Electrode) 2. Reference Electrode Components: Components: Glass Membrane: Allows H⁺ ions to pass, Reference Junction: A porous tip for slow KCl creating a voltage related to the hydrogen ion leakage, creating a stable interface for concentration. measurement. Internal Solution: Contains potassium chloride Internal Solution: Contains KCl for consistent (KCl) for stability. contact with the solution. Internal Electrode: Silver/silver chloride Internal Electrode: Ag/AgCl, generating a (Ag/AgCl), in contact with the internal stable reference voltage. solution and glass membrane, generates Function: potential based on H⁺ concentration. Provides a constant reference potential for Function: comparison, ensuring accurate and stable pH Sensitive to H⁺ ions, generating an electrical readings. potential that varies with pH, enabling pH measurement.  pH meter In a combination pH probe, the most widely used variety, there are actually two electrodes in one body. One portion is called the measuring pH electrode, the other the reference electrode. The potential generated at the junction site of the measuring portion is due to the free hydrogen ions present in solution.  pH meter If the H+ concentration is higher than OH- the material is acidic. If the OH- concentration is higher than H+ the material is basic. 7 is neutral, < is acidic, >7 is basic  Make sure the meter is set to pH mode and adjust the temperature to 25C Rinse the electrode with distilled water in waste beaker Calibration of Prepare buffer solution with 3 range of pH / pH 7, pH 4 and pH 10. pH meter Place the electrode in the buffer solution and press measure button The pH of the buffer appears in the display and make sure to record after the display stabilize Rinse the pH electrode with distilled water before and after use and wipe with tissue to dry the electrode Repeat with different buffer solution and record the pH  Application of pH measurement Agriculture → avoid toxic if pH too low Brewing pH 3.9-4 Corrosion prevention – pH < 4.3 Dairy industry –cheese production pH 4.7-4.9 Fermentation Fertilizer → prevent waste of acid Pharmaceutical → antibiotic produce from molds are grown at precise pH →cause poison