Pipettes / Automatic Pipet / Basic Separation Techniques Flashcards
(56 cards)
1
Q
- The tip should be immersed in the liquid to be transferred to a level that will allow it to remain in the solution after the volume of liquid has entered the pipet
A
DESIGN / CALIBRATION
2
Q
- Holds a particular volume but does not dispense that
exact volume - Delivers the amount by washing it and emptying
- For viscous sample
A
To contain (TC)
3
Q
To contain (TC)
Calibrated by:
Drainage characteristic:
A
Mercury
Blowout
4
Q
- Will dispense the volume indicated
- Transfer exact amount
- Do not blow the last drop
- Rate of flow is gravity
- For Non - Viscous samples
A
To deliver (TD)
5
Q
To deliver (TD)
Calibrated by:
Drainage characteristic:
A
Water
Self-draining
6
Q
DRAINAGE CHARACTERISTICS
- Contains two etched rings with colored rings near the top where you place the rubber aspirator. Before, mouth pipetting was used but was discontinued a few years ago
- Markings are absent; Liquid is allowed to drain by gravity
A
- Blow out
- Self-draining
7
Q
MEASURING OR GRADUATED PIPETTE
Capable of dispen different volumes
A
Serologic
Ball, kolmer or khan
Micropipet
8
Q
MEASURING OR GRADUATED PIPETTE
- Graduated up tsing severalo the tip; Blow - out pipet
- Types of micropipettes; Other types are Sahli hellige, Lang-Levy, RBC & WBC pipette.
- With total holding of less than 1 mL; May serologic or Mohr
A. Ball, kolmer or khan
B. Micropipet
C. Serologic
A
CAB
9
Q
TRANSFER PIPETTE
Designed to dispense one volume without further
subdivision
A
Pasteur pipet
Volumetric pipet
Ostwald folin
Micropipet/automated pipet
10
Q
TRANSFER PIPETTE
For volumes less than 1 mL; Automatic implies that the mechanism that draws up liquid and dispense it
A
Micropipet/
automated
pipet
11
Q
TRANSFER PIPETTE
● Bulb is near the mouthpiece or tip
● With etch marked near the delivery tip
● Drainage characteristic: blow out
● Calibration : TC and TD
● Used to transfer viscous liquids
A
Ostwald folin
12
Q
TRANSFER PIPETTE
● Bulbs in the middle
● For preparation of Control, Reagent
and Standards (Calibrators)
● Drainage characteristic: always self
draining
● Calibration: TD
● With the greatest degree of
accuracy and precision ( Most
accurate pipes)
● Non-viscous or aqueous samples
● When dispensing, always upright.
A
Volumetric
pipet
13
Q
● Do not have calibration marks
● Transfer solution or biological fluids
without consideration of specific
volume
● Maybe disposable or not
● Maybe calibrated or not
● Not used in clinical chemistry
A
Pasteur pipet
14
Q
TYPES OF AUTOMATIC PIPET
A
- POSITIVE DISPLACEMENT
- DISPENSER PIPETTE
- AIR DISPLACEMENT
15
Q
● Uses a suction & dispense sample
through a polypropylene tip.
● 1 or 2 stop
○ the 2nd stop button is
depressed to 2nd stop to
“blow out”.
● Tips can be used once.
● Seals require periodic lubrication.
A
AIR DISPLACEMENT
16
Q
AIR DISPLACEMENT
- Piston-mechaism: Relies on a piston for suction creation to draw the sample into a disposable tip
- Sample used:
A
- Air movement
- Non-viscous samples
17
Q
Method
- To aspirate the sample in the tip, press the plunger to the first stop and immerse the micropipette tip vertically in the sample
- Slowly release the plunger while the tip is immersed. The sample will be aspirated into the micropipette tip
- To dispense the sample, place the tip on the inner wall of the receiving vessel at a steep angle
- Slowly press the plunger again to the first stop to
dispense the sample into the receiving vessel - To empty the tip completely, press the plunger to the
second stop - Wipe the tip on the inner wall while taking the tip out to the receiving vessel
A
FORWARD PIPETTING
18
Q
- To Aspirate the sample in the tip, press the plunger to the second stop and immerse the micropipette tip vertically in liquid
- Slowly release the plunger while the tip is immersed. The sample will be aspirated into the micropipette tip
- To dispense the sample, place the tip on the inner wall of the receiving vessel at a steep angle
- Slowly press the plunger to the first stop
- Wipe the tip on the inner wall while taking the tip out of the receiving vessel
A
REVERSE PIPETTING
19
Q
● Uses a glass capillary tip fitted with a Teflon-tipped plunger.
● No carryover.
● Tips are reusable.
● The plunger setting must be checked & Teflon tip replaced periodically.
A
POSITIVE DISPLACEMENT
20
Q
POSITIVE DISPLACEMENT
- Piston-Mechanism
- Sample used
A
- Syringe like
- Viscous samples
21
Q
PIPET CALIBRATION
Calibrated every?
A
3 months (quarterly, 3-4x)
22
Q
● Done by delivering and weighing a solution of known specific gravity such as water;
● The most desirable method for pipet calibration
A
Gravimetric
Method
23
Q
PIPET CALIBRATION
For automatic pipetting devices: Spectrophotometer and uses potassium dichromate
A
Photometric Method
24
Q
Photometric Method
- Molar extinction coefficient of a compound is obtained or comparison of different dilutions of potassium dichromate and its absorbance
- a primary standard that is readily available and has sharp absorption peak at 350 nm.
A
- Spectrophotometer
- Potassium dichromate
24
Process whereby centrifugal force is used to separate
solid matter from a liquid suspension
CENTRIFUGATION
25
# CENTRIFUGATION
Parts include:
○ Head/rotor
○ Carrier
**○ Shields**
**○ Motor**
○ Chamber
**○ Control Panel**
26
# Centrifugation
1. Rule of thumb
2. Purpose of the HOLE in centrifuge machine:
3. force acting in the sample
4. actual speed
5. RPM can be converted into RCF
1. Placing the tube by exact positioning or by the exact opposite
2. Speed verification; using a tachometer
3. Relative Centrifugal Force (RCF)
4. Revolutions per minute (RPM)
5. RCF= 1.12 x 10^5 x radius x RPM^2
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1. How many minutes do you stand a sample?
2. What will happen to the sample if centrifuged without a completed clot?
3. What is the allowable hours before processing the specimen?
4. What analytes should be processed immediately or else results will be affected?
5. How many mg/dL does Glucose decrease every hour?
6. How long does a sample needs to be centrifuged?
7. How to obtain better separation of sample?
8. What part of the centrifuge influences speed (RCF/RPM)?
1. 30 minutes
2. Hemolyzed
3. within 2 hours
4. Electrolytes, potassium, glucose
5. 5-10 mg/dL (RT)
6. 10-15 minutes at 1000-1300 g (RCF) (tube with gel separator); same with tube without gell separator but only 1000g
7. Increase time and speed
8. Motor, rotor
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# OPERATIONAL VERIFICATION (according to CLSI)
1. For critical applications , RPM is checked and recorded
2. For routine applications, RPM is checked and
recorded
1. at least monthly
2. at least quarterly
28
* The cups are held in a rigid position at a fixed angle
* This position makes the process of centrifuging more rapid than horizontal-head centrifuge
* Less chance that the sediment will be disturbed when the centrifuge stop
* Used when rapid centrifugation of solutions containing small particle is needed up to 7000 rpm (slower than ultracentrifuge)
* Angle: 25-40
* Ex. For microhematocrit (10,000-15,000 rpm)
FIXED ANGLE / VERTICAL CENTRIFUGED
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* Protects biological specimen damage when heating during centrifugation
* -10°C or -20°C (-50°F or -68°F)
* For Thermally labile analytes (easily denatured, damaged at high temperature)
* Ex. lipoprotein (chylo -> VLDL -> LDL -> HDL)
REFRIGERATED CENTRIFUGE / ULTRACENTRIFUGED
29
* have holders that swing outward in response to centrifugal force, so that the tube is held vertically when placed into or taken out of the holder, but held horizontally during centrifugation.
* At rest, the tubes are in vertical position
* During centrifugation the tubes are in horizontal position
* Not capable of higher speed
* Max: 3000 rpm
* Ideally used for gel separator
SWINGING BUCKET / HORIZONTAL CENTRIFUGE
30
* Not used in clinical chemistry
* Used in histopathology
* Uses a very high-torque and low inertia motor to spread the monolayers of cells rapidly across a special slide for clinical morphologic studies.
* Only small amount of sample is used
* Body fluids like: Urine, Pleural fluid, etc
CYTOCENTRIFUGE
31
Which of the following is not a characteristic of
a Horizontal centrifuge?
A. Specimen cups are in vertical position when at rest
B. The cups move to a horizontal position during centrifugation
C. Capable of higher speed
D. As the sample is centrifuged, the particles being sedimented travel down through the liquid to the bottom of the tube
C. Capable of higher speed
32
What is the most commonly used centrifuge?
fixed angle centrifuge
33
Which centrifuge is used when rapid
centrifugation of solution is needed? (board
exam question)
A. Horizontal-head
B. Cytcentrifuge
C. Fixed-angle
D. None of the items
Answer: C. Fixed-angle
34
Which is the reference moethod for lipoprotein analysis?
Answer: Ultracentrifugation
35
# MAINTENANCE OF CENTRIFUGE
* Depending on usage: **Clean** interior components with soap and water followed by freshly made **10% v/v bleach solution**, including sample buckets. Wearing protective gloves, wipe interior sides and bottom, taking care when removing broken pieces of glass.
Weekly or
biweekly
36
* Place two equally balanced containers into the centrifuge, cover, and operate at the most commonly used speed, listening for unusual vibrations. Check the braking mechanism to ensure a smooth, gradual stop.
* Check the timer of the centrifuge at 15 minutes, 10 minutes, 5 minutes, and 1 minute for the time the centrifuge motor is spinning (reaches the desired rpm until the motor shuts off) using a stopwatch
Monthly
36
* Inspect gasket and check for wear and defects , and inspect cover latch for appropriate seal.
* Inspect head, head shaft, and coupling for evidence of wear, cracks in fitting, corrosion, uneven wear, and signs of fatigue. Inspect brushes for wear and replace according to manufacturer’s instructions.
* Check the revolutions per minute at several commonly used speeds, including 3000 and 1500 rpm, while centrifuging a balanced load (after it has reached stable speed) using a tachometer aimed at the reflective strip viewed through the top of the centrifuge.
* If the revolutions per minute vary by more than 5%, consult the manufacturer’s instructions for solving the problem.
* If an analog scale is provided for speed, the scale can be relabeled to match the actual speed. Lubricate the centrifuge shaft according to manufacturer’s instructions, if applicable.
Quarterly
36
* The second separation process used to separate serum from a blood clot-used in place of centrifugation
* filter material is made of paper cellulose and its derivatives, polyester fibers, glass and a variety of resin column material
* filter paper differs in terms of pore size and should be collected according to separation needs
* filter paper should not be used when using strong acids or bases.
* liquid that passes through the filter paper
FILTRATION
37
method for separating macromolecules from a solvent or smaller substances
DIALYSIS
38
What are the three methods of separation?
Centrifugation, Filtration, and Dialysis
39
For critical applications, RPM is checked and
recorded
a. weekly or biweekly
b. at least monthly
c. at least quarterly
b
40
For routine applications, RPM is checked and
recorded
a. weekly or biweekly
b. at least monthly
d. at least quarterly
at least quarterly
41
What are the two operational verification activities for centrifuge set by CLSI?
1. Check timer against standardized time source (stopwatch)
2. Verification of speed
42
Verification of centrifuge's speed is done using
using a tachometer or strobe
light
43
Weekly or biweekly cleaning uses soap and water followed by
freshly made 10% v/v bleach
solution
44
Inspection of gasket and checking for wear and
defects, and inspecting cover latch for
appropriate seal is done
a. weekly
b. monthly
c. quarterly
quarterly
45
Checking the braking mechanism to ensure a smooth, gradual stop is done
a. weekly
b. monthly
c. quarterly
monthly
46
Checking the revolutions per minute is done
a. weekly
b. monthly
c. quarterly
quarterly
47
What do you call the product of filtration?
Filtrate
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