Lab Skills

1
Lab Skills

• Unit B Summary

2
Safety Rules

• No eating or drinking in the laboratory. No gum
  chewing. No makeup application.
• Wear safety apparel, such as safety glasses,
  gloves, lab coats, and other protective clothing
  as necessary. Tie hair back if using Bunsen
  burners.
• Know the location of fire exits, fire
  extinguishers, and safety showers.
• Wash hands regularly, especially after working
  with microorganisms or chemicals.
• Be aware of potential dangers. Before using
  products or equipment, carefully read labels,
  experimental protocols, and equipment
  instructions and read literature. Know the
  location of and how to read Material Safety Data
  Sheets (MSDS).
• Contaminated samples (chemical, biological, and
  glass) must be disposed of in appropriate
  containers. Do not pick up broken glass with
  your hands. Learn the specific methods from your
  lab supervisor.
• Label all samples and reagents clearly with the
  name of the item, the name of the person who
  prepared the sample, and the date of preparation.
• Know emergency phone numbers and the best way to
  contact facility safety officer.
• Report spills and accidents to your lab
  supervisor or safety officer immediately.

3

• MSDS sheets
• Chemical Name
• Stability
• Reactivity
• Physical Data
• Toxicity
• Health Effects and First Aid
• Storage and disposal
• What does each area mean?
• Red Flammability
• Yellow Reactivity or instability
• White Special hazard
• Blue Health and Hazard

4

• Occupational Safety HealthAdministration
  ensures worker Safety and protection
• Environmental Protection Agency is responsible
  for protecting the environment
• Department of Transportation need to know what
  they are transporting

5

• Personal Protection Equipment
• Lab coat
• Safety Glasses / Goggles
• Gloves
• Face shield
• Closed toe shoes
• No contacts
• No loose or hanging clothes such as ties
• Minimal jewelry
• Lab coat, safety glasses and gloves most common
• Glasses required for all liquid experiments

6

• Prokaryotic cells (bacteria) no nucleus or
  membrane-bound organelles
• Eukaryotic cells (all others) may have the
  following organelles

7
Cell Structures

• Nucleus controls activities in the cell
• Cytoplasm solution outside nucleus but inside
  cell
• Ribosomes makes proteins
• Er (endoplasmic reticulum) passageway for
  protein transport
• Golgi packages the proteins
• Mitochondria converts food to energy for cell
• Chloroplast coverts sunlight to food in plant
  cells
• Vacuole storage of water, enzymes, waste
• Lysosomes digests foreign material or bad cell
  parts
• Cell (plasma) membrane controls what comes in /
  out of cell
• Cell wall external support for plant cells

8
Viruses

• Nonliving
• Composed of Nucleic acid and protein
• Grouped according to Presence of Capsid and
  envelope shape AND RNA or DNA, single or
  double stranded structure
• Can replicate only by invading host cell and
  using its enzyme and organelles.
• Bacteriophage viruses that infect bacteria
  Used to study viruses
• Lytic Cycle
• Viral genome is released into the host cell
• Replication follows immediately
• Cellular components used to make new viruses
• Viral enzyme kills cell.
• Lysogenic Cycle
• Nucleic acid of virus becomes part of the host
  cells chromosome
• Nucleic acid remains in the cell in this form for
  many generations

9
Biomolecules

• Water - polar, good solvent, sticks together,
  resistant to heat change, inorganic
• Organic made of carbon
• Carbohydrate used for energy, sugars, starch,
  C6H12O6, building block is monosaccharide
• Lipid used as barrier in membrane, fats, oils,
  long carbon chain, building block is fatty acid
• Protein used for enzymes and structure,
  building block is amino acids
• Nucleic acids DNA and RNA, building block is
  nucleotides

10
Metrology

• Units define measurements give the numbers
  value
• Accuracy is how close an individual value is to
  the true or accepted value
• error True value measured value X 100
• True value
• Precision is the consistency of a series of
  measurements
• Take an average of the deviation, so it is the
  average deviation from the mean
• Standards are Measurements made in accordance
  with an external authority

11
Metrology

• Verification - Check of the performance of an
  instrument or method without adjusting it.
• Calibration - Bringing a measuring system into
  accordance with external authority, using
  standards
• Tolerance - Amount of error that is allowed in
  the calibration of a particular item.
• Traceability - The chain of calibrations,
  genealogy, that establishes the value of a
  standard or measurement
• Error is responsible for the difference between a
  measured value and the true value
• Three types of error
• Gross (blunders)
• Random
• Systematic

12
Volume

• Graduated cylinders over 10 ml
• 10 ml serological pipets
• 5 ml serological pipets
• 2 ml serological pipets
• 1 ml serological pipets
• P1000 micropipets
• P100 micropipets
• P10 micropipets
• Multichannel pipets

13
Pipets

• Use the right instrument to make the correct
  measurements
• Verify and calibrate micropipets with water which
  has a density of 1 g for every ml
• Maintenance micropipets by cleaning and storing
  properly and recording.

14
Terminology for weighing

• Range
• the span from lightest to heaviest weight that a
  balance is able to measure
• Capacity
• the heaviest sample that a balance can weigh
• Sensitivity
• the smallest value of weight that will cause a
  change in the response of the balance.

15
Proper weighing procedure

• Make sure the balance is level
• Adjust the balance to zero
• Tare the weighing container or weigh the empty
  container
• Place the sample in a the weighing container and
  read the weight
• Remove the sample
• Clean the balance and surrounding area

16
Proper weighing techniques

• Always use a calibrated weight to verify the
  scale is in proper working order (daily)
• Always use a weigh boat or weigh paper do not
  place materials directly on the pan
• Do not touch the chemicals or material being
  weighed
• Do not return unused chemicals to their storage
  bottle (unless you use a sterile spatula or
  spoon)

17
Calibration of Balances

• First step is to zero the balance. It should
  read zero every time you press the zero button
• Second calibration point is taken at the upper
  end of the capacity of the balance
• Place a certified weight on the balance and
  verify it reads the correct weight
• Some scales will prompt you to enter the weight
• A third reading can be made using a lighter
  calibrated weight and verifying it reads the
  proper weight

18
Equipment Log Books

• Notebooks or binders used to maintain operating
  procedures, calibration records, verification
  checks
• Example the equipment log book in the back of
  the room
• Incubator temp charts
• Refrigerator temp charts
• Pipet calibration records
• Balance calibration and verification charts

19
Solutions

• Solution a homogeneous mixture in which one or
  more substances are dissolved in another.
• Solute substances that are dissolved
• units are often g, mg, or µg
• Solvent substances in which solutes are
  dissolved (often times this is water or a buffer)
• units are often L, ml, or µl
• Concentration amount per volume mass/vol
• units are g/L, g/ ml, mg/ml, molar

20
Solution Prep

• Mass/volume
• mass/volume
• Molarity
• molarity (M) is equal to the number of moles of
  solute that are dissolved per Liter of solvent
• Dilution - C1xV1 C2xV2

21
Acids

• Definition electrolyte that donates hydrogen
  ions
• Properties
• Acids in water conduct electricity
• The stronger the acid the stronger the
  conductivity
• Acids react w/metals to produce H2 gas
• Acids are indicators they cause reversible color
  changes
• Phenolphthalein and litmus are two examples of
  acid-base indicators
• Acids react w/hydroxide compounds to form water
  and salt this type of reaction is called
  neutralization
• Strong acids completely dissociate in water to
  release hydrogen ions H
• i.e. hydrochloric acid (HCl) HCL in water ? H
  Cl-
• Tastes Sour

22
Bases

• Definition electrolyte that yields hydroxide
  ions or accepts hydrogen ions
• Properties
• Bases in water conduct electricity
• The stronger the base the stronger the
  conductivity
• Bases react with acids in neutralization
  reactions to form water and a salt
• Bases cause reversible color changes in acid-base
  indicators (color is pH dependent)
• Bases in water solution are slippery to the touch
• Caution even dilute bases can be caustic!
• Strong bases completely dissociate in water to
  release hydroxide ions OH-
• NaOH in water ? Na OH-
• The OH- ions react with H to form water,
  thereby ? the concentration of hydrogen ions
• Tastes bitter

23
pH is

• A way to express hydrogen ion concentration in a
  solution
• Measurement of the acidity/alkalinity of an
  aqueous solution
• pH is the log of the H concentration
• pH is measured on a scale
• Ranges from 0 to 14
• Pure water
• H concentration is 1x10-7 mole/L
• The log of 1x10-7 -7
• The log of 7 7
• The pH of pure water 7

24
Buffer ?

• Substance(s) that when in aqueous solution
  resists a change in H concentration even if
  acids or bases are added
• Some buffers change pH as their temperature
  and/or concentration changes
• Tris buffer is widely used in molecular biology
  it is very sensitive to temperature and the pH
  will vary greatly at various temperatures.

25
Measuring pH

• Indicators
• Phenophthalein, phenol red, bromothymol blue,
  universal indicator to name a few
• pH Paper
• pH Meters

26
pH Meter

• Meter / electrode system for measuring pH in
  laboratory
• Provides greater accuracy, sensitivity than
  chemical indicators
• Can measure pH of a solution to the nearest 0.1
  unit
• Can be used with variety of aqueous solutions
• Consists of
• Voltmeter measures voltage
• Two electrodes connected to one another (sensor
  probe)
• When immersed in the sample they develop an
  electrical voltage that is measured by the
  voltmeter
• Calibration recommended with each use, when
  battery replaced and when fluid in sensor is
  changed

27
Microscope

• Important Lab instrument

28
Why use a microscope?

• To view objects and detail too small to see with
  human eye. Improves resolution of object
• List examples of how what you used it for
• Blood cell detail
• rbc, wbc, platelets
• Bacteria
• Cocci, rod, bacillus
• Protozoa
• Trichomonas
• Giardia

29
Types of Microscopes

• Compound microscope
• Bacteria, fungi and protozoa
• Electron microscope
• Required for viruses
• Fluorescence
• Used as a diagnostic tool for immunofluorescence
  tests

30
Parts of a microscope

• Coarse adjustment
• 1st step in focusing to change the distance
  between specimen and lens
• Fine adjustment
• To fine tune the picture
• Used particularly with 100x and oil objective
• Stage
• Holds the slide, moved up and down with coarse
  and fine adjustment

31
Parts of a microscope

• Objectives
• Common objectives are 4x, 10x, 40x, 100x, oil
• Total magnification is eye piece magnification
  multiplied times objective you are viewing with
• 40x objective and 10x eye piece is 400x
  magnification
• Oil objective is labeled and is always used with
  immersion oil
• Oil increases the resolving power by focusing the
  light rays

32
Proper care for microscope

• Always start with lowest objective to focus
• Always store with lowest objective locked in
  place
• Carry with two hands
• Cover with dust cover
• Be sure to clean oil off of oil objective
• Use fine adjustment with 100x and oil objective

33
Spectrophotometers

• When light shines on a solution, it can bounce
  off of the molecules (reflect), pass through the
  solution (transmittance), or some of the energy
  be absorbed by the solution.
• Spectrophotometers are instruments that measure
  the interaction of light with materials in
  solution
• Spectrophotometers compare the light transmitted
  through a sample to the light transmitted through
  a blank.
• The blank contains everything
• except the analyte

34
Chromatography

• Physical properties that can be used to separate
  molecules
• Size
• Shape
• Density/gravity
• Charge
• State (solid, liquid, gas)
• Phase changes (mp, bp, evap)

35
Chromatography Key Terms

• Chromatography techniques for the separation of
  complex mixtures that rely on the differential
  affinities of substances
• Stationary phase what you pack the column with
  or the plate/paper
• Mobile phase solvent/phase moving in the bed
  fraction or sample being separated
• Effluent the mobile phase leaving the column
• Types of Chromatography
• Paper
• Thin Layer
• Ion Exchange or Affinity
• Size Exclusion
• High Pressure Liquid Chromatography (HPLC)

36
Gel Electrophoresis

• Definition the process of separating molecules
  based on size and charge
• Agarose highly purified agar, heated and
  dissolved in buffer. Forms a matrix of pores for
  molecules to travel through.
• Smaller molecules travel further
• Molecules migrate towards the
• positive (red) end of the chamber

37
Gel Electrophoresis

• Process
• Make Agarose gel
• Thinner gels (0.8) yield better results for
  larger DNA
• Prepare samples
• Restriction enzymes used to cleave at specified
  sites
• Apply samples to gels, apply current
• If samples run from positive end they will run
  off the gel
• Stain gels to see bands
• Would not be able to see bands if we did not stain

38
Gel Electrophoresis

• DNA molecules have a negative charge
• This allows them to migrate towards the positive
  end of the chamber
• The samples and the electrophoresis chamber use
  specialized buffers. Using
• TAE/TBE buffer helps stabilize the sample
• and allows the reaction to occur quicker in
• the chamber.
• If water were in the chamber instead of TAE/TBE
  buffer the reaction would take much longer or
  migration may not occur at all
• Stains ethidium bromide will cause the bands to
  glow orange under UV light. Fast stain will
  result in blue bands

39
Uses for Gel Electrophoresis

• DNA fingerprinting or profiling
• Paternity testing
• Crime scene sample analysis
• Identification of bacteria and other pathogens
• Who is credited with discovering the DNA
  profiling process?
• Alec Jefferies in 1985

40
Cell Culture

• Definition the in vitro growth of cells isolated
  from multi-cellular organisms
• Process Cells will continue dividing until they
  fill up the container cell to cell contact stops
  cell division
• Uses vaccines, research of all kinds including
  stem cell, recombinant DNA, production of
  antibodies

41
Types of Cell used

• Bacterial cells
• Yeast cells
• Mold cells
• Plant cells
• Insect cells
• Mammalian cells

42
Growing Bacterial Cells

• Choose bacteria E. coli most common
• Make media
• Petri plates use agar in media (Luria Broth,
  nutrient agar)
• Liquid cultures use broth (LB, nutrient broth)
• Sterilize media in autoclave
• Pour media plates
• Innoculate media
• Grow cells in incubator (37oC)