Transcript Powerpoint Summary

Lab Skills
Unit B Summary
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.
MSDS sheets
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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
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
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
Prokaryotic cells (bacteria) – no nucleus or membranebound organelles
Eukaryotic cells (all others) – may have the following
organelles:
Cell Structures
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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
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
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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 cell’s chromosome
– Nucleic acid remains in the cell in this form for many generations
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
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
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
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
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.
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.
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
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)
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
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
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
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
Acids
Definition: electrolyte that donates hydrogen
ions
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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”
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 = OHNaOH in water  Na+ + OHThe OH- ions react with H + to form water, thereby  the
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
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H+ concentration is 1x10-7 mole/L
The log of 1x10-7 = -7
The – log of –7 = 7
The pH of pure water = 7
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.
Measuring pH
Indicators
– Phenophthalein, phenol red, bromothymol
blue, universal indicator to name a few
pH Paper
pH Meters
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
Microscope
Important Lab instrument
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
Types of Microscopes
Compound microscope
– Bacteria, fungi and protozoa
Electron microscope
– Required for viruses
Fluorescence
– Used as a diagnostic tool for
immunofluorescence tests
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
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
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
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
Chromatography
Physical properties that can be used to
separate molecules
– Size
– Shape
– Density/gravity
– Charge
– State (solid, liquid, gas)
– Phase changes (mp, bp, evap)
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
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Paper
Thin Layer
Ion Exchange or Affinity
Size Exclusion
High Pressure Liquid Chromatography (HPLC)
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
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
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
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
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
Types of Cell used
Bacterial cells
Yeast cells
Mold cells
Plant cells
Insect cells
Mammalian cells
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)