Transcript Centrifugation

Welcome to the Clinical Lab
Instrumentation in the Lab
Definitions
 While you’re looking at these slides, watch out for
the underlined words. Do you know what they mean?
If you don’t, stop
and look them up.
Introduction
 List of most common instruments that you’ll want to
be aware of:
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Centrifuge
Microscope
Spectrophotometer
Balance
pH meter
Autoclave
Laboratory Glassware
Safety Cabinets and Hoods
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The Centrifuge
 The centrifuge is an instrument designed to spin test
tubes, with their bottoms tilted outward, around a
central axis. The centrifugal force this creates pushes
the heavier matter in the tube toward the bottom.
This step is necessary to, for example, separate the
components of blood in order to test it.
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The Centrifuge (cont.)
 Centrifugation is a process that involves the use of
centrifugal force to separate two components of
liquids.
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More-dense components of the mixture drop to the bottom
and are called the precipitate.
The less-dense components of the mixture migrate towards the
top of the tube are is called the supernate.
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So, if we consider whole blood (basically cells, and serum) which
component will drop to the bottom of a centrifuged tube?
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The Centrifuge (cont)
 The main setting on a centrifuge is the one that determines how fast it spins. This
can be measured by the amount of acceleration to be applied to the sample
(RCF), rather than specifying a rotational (RPM).
 This distinction is important because a larger centrifuge will apply more
acceleration to a sample than a small centrifuge spinning at the same
rotations per minute. So, two centrifuges with different diameters running
at the same rotational speed will subject samples to different accelerations.
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RPM: revolutions per minute; is the speed expression.
Radius of Rotation: (r) is the distance from the center of rotation to the tubes spinning, expressed in cm
RCF: relative centrifugal field; force acting on the sample(s)
𝑹𝑷𝑴 2
• RCF= 11.2 X R (
𝟏𝟎𝟎𝟎
)
This is important because some tests ask for a particular centrifugal force while others
require certain RPMs. To be able to go back and forth between the two measurements
makes you a good laboratory scientist!
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The Centrifuge
(cont.)
Safe use of a centrifuge
 There are two very important realities when safely
using a centrifuge
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1) The tubes being spun must be balanced
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For tubes to be balanced, their weight must be placed equally
around the circumference of
bucket
The Centrifuge (cont.)
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2) The lid must be closed
A closed lid is so important that a
latch on the lid has become a required
safety feature for centrifuges.
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The Centrifuge (cont.)
Below is a microfuge which generally
holds tubes 0.5 - 2 mL (much less
volume than normal tubes) of liquid,
and are spun at maximum speeds of
12,000–13,000 rpm ( the centrifuge
on the left spins 40-50 times slower).
This means that they can create
pellets at the bottom of the tube, not
just a more dense layer of liquid.
This centrifuge, though larger
than the microfuge on the right,
spins slower. It’s advantage
though is that it has “boats”
which hold multiple tubes.
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The Centrifuge (cont.)
Please watch this short video on how to use a
centrifuge!
• SAFE USE OF A CENTRIFUGE
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The Microscope
 The goal of using a microscope is to obtain increased
magnification, resolution and contrast of a specimen.
 There are several types, but we’ll focus on the
binocular (pun!)
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The Microscope continued
The microscope you use may not be exactly like this one, but all the
parts should be close to this diagram. On the next slides the parts
of the microscope that are critical to efficient use will be
highlighted.
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The Parts of a Microscope – refer to the
preceding picture
 The oculars are the two lens at the top that you look through. They
are usually 10x or 15x (magnify the image 10 or 15 times its normal
size)
 Illuminator – light source
 Condenser lens focus the light onto the specimen
 The diaphragm (rotating disc under the stage) has different sized
holes and is used to vary the intensity and size of the cone of light
that is projected upward into the slide. There is no set rule
regarding which setting to use for a particular power. Rather, the
setting is a function of the transparency of the specimen, the degree
of contrast you desire and the particular objective lens in use.
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The Parts of a Microscope (cont.)
 The objective lenses are located on the nose piece
above the stage. Each objective lens is labeled with
its magnification power
 The low power objective lens is the shortest of these
lenses.
 Higher power objective lenses are longer, and the
longest of these is often an oil immersion lens that
provides maximum magnification.
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Microscope
 Can you label this microscope?
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Using a Microscope
1) While looking through the eyepiece of the
microscope, lower the stage to bring the image of the
specimen into focus by turning the coarse adjustment
knob.
2) Move the location of the slide on the stage to bring
an item of interest into the field of view.
3) Adjust the condenser so that the light is sharply
focused on the specimen, then adjust the diaphragm to
control the brightness of the light.
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The Spectrophotometer
 The Spectrophotometer allows one to measure the
concentration of a substance within a test sample. It
does this by measuring the amount of light of a
specified wavelength which passes through a
medium.
 This all works because of a principle called Beer's
law, the amount of light absorbed by a chemical in
solution is proportional to the concentration of the
absorbing material or chemical present.
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Spectrophotometer
(cont.)
 Spectrophotometer can be used for tests like:
 determining how much glucose is in a specimen of blood, or
 how much cardiac enzyme is present after a heart attack, or
 how much bacteria is in spinal fluid
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Please watch this video on how to use the
spectrophotometer.
Spectrophotometer Video
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Top-Loading Scale
 Although the terms mass and weight are often used interchangeably,
they are different properties.
 Mass is a measurement of the amount of matter something contains.
 Weight is the pull of gravity on an object’s mass.
 Practically, we use scales to measure both mass and weight, but
technically, the raw numbers they give us are for weight alone.
 Simply, a scale measures weight and works by measuring the downward
pressure on its pan.
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Top-Loading Scale (cont.)
 Scales are available with a variety of maximum
capacities and with different precisions, sensitivities
and tolerances. The top-loading scales in your labs
are used to determine masses to ±0.01 g
 Analytical scales are sensitive to ± 0.0001 g. In
general, top-loading balances can measure materials
with masses up to several hundred grams whereas
the analytical balances are limited to a maximum
mass of one hundred grams.
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Top-Loading Scale (cont.)
 The sensitivity of the laboratory scales makes it
necessary to adhere to some strict rules when
making measurements.
 All scales are affected by vibrations of the bench
top and by the movement of air currents around
the balance pan.
 Top-loading scales are usually used with the balance
pan open to the air.
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It is necessary to place these scales out of the direct path
of room ventilation. They cannot be used in a hood
because the drafts are too strong causing the
measurements to fluctuate significantly.
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Precautions when using the top-loading scale
1. DO NOT ATTEMPT TO MAKE ADJUSTMENTS TO THE
SCALE YOURSELF. If the scale is not functioning properly,
notify your instructor.
2. Be sure the scale pan is clean. If not, remove any solid debris
with a soft brush. If a liquid spill occurs, notify the instructor
immediately.
3. Do not lean on, rest hands or arms on, or write lab reports on
the balance tables.
4. Determine the weight of clean, dry, room-temperature objects
only.
5. Clean up after yourself! Sweep off the top pan with the brush.
7. Re-zero the balances between uses and turn off the balances
when all work is completed
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How to use the top-loading balance video
How to Use a Top-Loading Balance
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pH Meter
 Ph Meters quickly determine the pH of a solution.
 A pH meter measures a voltage created by the ions in
the solution that pass through the meter's
membrane, and associate this voltage with a
particular pH.
pH probe
pH Meter
 Most pH meters are calibrated by using a set of
standard solutions that are guaranteed to be at a
particular pH. Three of these points are the
minimum number needed to establish a calibration
curve with which the meter will work to associate
each voltage reading with a particular pH.
 Watch the videos on the following slide. Take good
notes! Refer to your specific pH meter for detailed
tips for use.
First video: How to calibrate a pH meter
Calibrating a pH Meter
Second video: How to use a pH meter
Correct use of a pH Meter
The Autoclave
 Autoclaves sterilize equipment and supplies,
killing biological contamination and
denaturing proteins. Autoclaves will not
remove chemical contamination like acids or
stains.
Autoclave (cont.)
Autoclaves generally have two cycles:
1. Fast Exhaust cycle - For dry goods, glassware,
etc. This cycle charges the chamber with steam and
holds it at a set temperature for a set period of time. At
the end of the cycle a valve opens and the chamber
rapidly returns to atmospheric pressure.
2. Slow exhaust cycle - Used to prevent sterilized
liquids from boiling, steam is exhausted slowly at the
end of the cycle, allowing the liquids (which will be
super-heated) to cool.
Sterilization requires
• temperatures of at least 121-124 C
• pressure of 15 psi
• time of 15 minutes
Safe Use of the Autoclave
 Not all plastics can be autoclaved. Polypropylene and
polycarbonate will survive, but polyethylene and
high density polyethylene will not. Different types of
plastic can be identified by looking for initials
imprinted on the bottom of containers.
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PP=polypropylene
PC=polycarbonate
PE=polyethlylene
HDPE=high density polyethylene)
 If you are unsure about a new container, place it in
an autoclave safe container the first time.
General Precautions for Autoclave Use
Do not open any autoclave until the pressure gauge
labeled “chamber” is at zero, stand back and allow
steam to escape through the open door before
reaching in.
 Never open an autoclave set for “slow exhaust” until the
cycle is complete.
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Superheated liquids can boil over, possibly damaging both autoclave
and operator. After the cycle is complete, let liquids stand 10 minutes
 Pyrex bottles, empty or full, should have their caps
placed on loosely, to prevent explosion due to expansion.
 Use tinfoil to cover non-safety-glass bottles (non-Pyrex).
How to Use an Autoclave Video
Safe Use of an Autoclave
Biological Safety Cabinets and Hoods
 Biological Safety Cabinet (BSC) is an enclosed, HEPA
filter ventilated workspace used to:
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safely work with materials that maybe susceptible to
contamination with other external elements.
create a barrier between a pathogenic substance and the
laboratory personnel.
BSCs and Hoods continued
The safety level designation (BSL2, BSL3, BSL4) of the
laboratory will determine the type of BSC used.
These safety levels were created to protect the
scientists working with different dangerous materials as
well as those outside this environment (It wouldn’t be
safe to ventilate some viruses into the
atmosphere. These safety levels
address that!) The levels are explained
In following slides.
BSL-2
 Biosafety Level 2 builds upon BSL-1. BSL-2 is
suitable for work involving agents that pose
moderate hazards to personnel and the environment.
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Laboratory personnel have specific training in handling
pathogenic agents
Access to the laboratory is restricted when work is being
conducted
Materials used in BSL-2s must be handled and disposed of as
biohazards
BSL-3
 Biosafety Level 3 is much the same as BSL-2, but
the infectious agents studied will cause serious or
lethal diseases
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Laboratory workers need special training to work in these
safety cabinets
Waste is considered hazardous
BSL-4
 BSL-4 is required for work with dangerous and
exotic agents that pose a high individual risk of
aerosol-transmitted laboratory infections, and which
cause severe to fatal disease in humans for which
vaccines or other treatments are not available
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This is the environment you see on the movies where the
scientists are wearing suits and air packs.
Ebola virus, Marburg virus, various other hemorrhagic
diseases are studied here
Great Video on BSL-4 Labs
Fume Hood
 Hoods or fume hoods are also designed to prevent
contamination, but are not totally enclosed. They are used when
potentially harmful reagents are made, poured, and discarded so
that aerosols and/or fumes are not released into the laboratory
environment.
Laboratory Glassware
 Glassware should always be cleaned with caution as to not break, crack, chip or
scratch the glassware.
 Glassware should be properly cleaned and sanitized according to the level of
cleanliness required for a specific test.
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If your test requires all procedures to be done within the hood then you should use
autoclaved glassware.
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Always rinse your glassware prior to autoclaving.
Cover the top with foil.
If it is a bottle, make sure the cap is not tight or cover top with foil.
Always use autoclaved tape to insure the glassware was properly autoclaved.
Date your autoclaved glassware.
If your test does not require any procedures be done in a hood then you can use aseptic
washed glassware.
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All glassware must be rinsed.
Soak in a 10% bleach bath for at least one hour.
Washed with a cleaning solution, rinsed thoroughly and dried upside down on a clean drying
rack.
Laboratory Grade Water
 Water is probably the most common laboratory reagent, so its quality is
very important.
 Water is processed to reduce cells, bacteria, minerals, chemicals that in
contaminate tests or instruments .
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Milli-Q water is water that has been sterilized by passing it through a
filter with VERY small pores (like 2 microns in diameter). It can still
have minerals and/or chemicals in it.
Dionized water (DI water) has been converted into steam by heating it.
The steam is then condensed back into water. This removes dissolved
materials as well as killing any bacteria, resulting in pure water.
Laboratory Grade Water (cont.)
 There are four types of laboratory water.
Type 1: highest class of purity i.e mili-q or deionized water.
 Type 2: has impurities in the water but is still used in lab
assays.
 Type 3 and Type 4: not pure and are used for glassware or
laboratory cleaning of non sterile surfaces.
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NOTE: Do not consume water processed for the laboratory.
Review Questions
 List two safety rules for centrifuges.
 What is the difference between a microfuge and a
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regular bench-top centrifuge?
When thinking about a microscope, what is the name
of the lens you look through?
When thinking about a microscope, what is the name
of the lens at the bottom of the turret – the ones that
can be turned?
When thinking about top-loading scales, that does it
mean to tare?
True or false: All plastics will melt in the autoclave.
Review Questions (2)
 True or false: Autoclaves even remove chemical
stains
 Why are Biosafety cabinets used? Are they the same
as fume hoods?
 Is DI water potable water?