Lecture 6 & 7 Flashcards
What is preventive maintenance?
Maintenance performed at scheduled intervals to ensure equipment is functioning properly
Every shift, daily, weekly, monthly, quarterly, annually
Maximizes the equipment operational efficiency
Reduces overall cost
What to do when laboratory equipment malfunctions:
- Logs must be kept of when equipment malfunctions
- Keeps record of information like error codes, frequency, if the hotline was called, reference number provided by the tech support, etc
- Must record what corrective actions you’ve taken and the outcome
- If error/malfunction occurs again, you can look back at previous logs for solution
What are centrifuges and what are their components?
One of the most commonly used instruments
It consists of a head or rotor and carriers and sometimes a shield attached to the vertical shaft of a motor – all of this is enclosed within a metal covering
The centrifuge always has a lid and an on/off switch
Some models have a braking system or built in tachometer (Measure the rotational speed)
Some centrifuges are refrigerated
What is centrifugation:
Process used to separate or concentrate materials suspended in a liquid medium
Centrifugation separates on the basis of the particles and density difference between the liquid and solid phases
How do particles separate?
Size, shape, density, viscosity of medium.
What is sedimentation?
Particles/cells in liquid suspension, given time, will eventually settle at the bottom due to gravity
What is the liquid portion called?
Supernatant
What is the sediment called?
Precipitate/pellet
Centrifugal force:
When objects are pulled outwards by the force exerted around the centre of rotation i.e. what causes sample to separate
What is centrifugal force dependant on?
Mass (cells with the liquid medium)
Radius
Speed
What is the speed of the centrifuge expressed as?
Speed is expressed in rpm = revolutions / min
Centrifugal force generated is expressed in terms of relative centrifugal force (RCF) or gravities (g)
What is Relative centrifugal force?
What is it measured by?
Comparison of centrifugal force relative to the speed and radius of the centre of rotation
RCF measured by:
Rotation speed
Diameter of centrifuge
What is a centrifuge is comprised of:
Electric motor
Drive shaft
Rotor to hold tubes
What are the 2 common rotors?
Swinging-bucket rotors
Fixed-angle rotors
Horizontal Type Rotor
Has swinging buckets
Cups are in a vertical position when centrifuge is at rest
When centrifuge begins to spin they swing up to the horizontal position
This type usually operates at 3000 to 5000 rpm
what are the advantages of horizontal type rotors?
- Longer distance of travel may allow better separation
- Easier to withdraw supernatant without disturbing pellet
Angle Head Type Rotor:
The cups are held in a rigid position at a fixed angle – usually 52°
Centrifugation is more rapid
Less chance of disturbing the sediment once centrifuge stops
Produces less heat
This type usually operates at 3000 to 5000 rpm
What are the advantages of Angle Head Type Rotor?
- Sedimenting particles have only short distance to travel before pelleting- shorter run time
- Most widely used rotor type
What are the controls of rotors?
Automatic timer – in minutes
Speed control – governs rpm
Brake – used to stop centrifuge
How to care for a rotor?
- Stress corrosion is thought to be initiated by certain combinations of stress and chemical reaction
- If rotor is not kept clean and chemicals remain on the rotor, corrosion will result
- Any moisture left for an extended time can initiate corrosion.
- It is important that the rotor is left clean and dry. (Wash with mild detergent and warm water, careful use of a nylon bottle brush when necessary).
- Dry the rotor thoroughly and store upside down with the cover and tubes removed
What are the different types of centrifuges?
Table model
Refrigerated
Ultracentrifuge
Cytocentrifuge
Serofuge
Describe a cytocentrifuge:
Spreads monolayers of cells across a slide – used in critical morphological studies
It is used to concentrate cells onto the surface of a glass slide
Can be used for blood, urine, body fluids
The slide is then stained and examined microscopically to generate a differential count and evaluation for the presence of malignant cells
Requires very small samples
Describe a Refrigerated Centrifuge:
Come in different temperature ranges (adjustable)
Specimens protected from heat generated by centrifuge rotors
Temperature must be monitored regularly
(usually documented once a day)
Ex: lactic acid
Describe a Serofuge:
These are the units used in transfusion science
Different heads for different size of tubes
- 10 X 75 mm
- 13 X 100 mm
User friendly
Open system
Describe an Ultracentrifuge:
Used to separate proteins, viruses
Can be used for grossly lipemic samples
Commonly found in research labs
Usually refrigerated
Can reach speeds in excess of 70,000 rpm
Some can reach 90,000 to 100,000 rpm
How to balance a centrifuge?
- Water-filled tube may be used as a balance blank
- Ensure that the centrifuge is PROPERLY BALANCED prior to starting – keeping it free from excessive vibrations – balancing is CRITICAL
- Refers to both number of tubes in the centrifuge AND the volumes in those tubes
- Unbalanced centrifuge will shake and vibrate to the point where you think that it will vibrate right off the bench
Centrifuge maintenance:
Daily maintenance:
- Inspect interior and exterior for spills and contamination
Weekly maintenance:
- Remove buckets and clean with neutral detergent
- Lubricate the bucket pivots and ensure buckets swing freely
- Clean the interior of centrifuge chamber with neutral detergent
Monthly maintenance
- Clean as per weekly maintenance
- Remove and clean rotor with a moist cloth, then dry with gauze
What is the general use for balances?
- Use for mass or weight measurements
- Properly operating is essential in producing high quality reagents and standards
Balances classifications:
Number of pans (single or double)
Mechanical
Electronics
What are the two types of analytical balances?
Mechanical
Electronic
Accuracy:
How close you are to the true value
Tare:
Many balances have the tare mechanism which allows the balance to go back to “0” even when there is an object on the pan – Used to disregard the weight of the weighing container
Capacity:
Target weight that can be weighed accurately on a particular balance – i.e. has the capacity to weigh 300 g – you cannot accurately weigh 301g
SENSITIVITY:
Ability to measure small differences in weight
Describe mechanical balances:
Uses moving parts like springs, fulcrums, beams, etc.
Describe electronic balances:
Uses few if any moving parts – depends on circuit boards and electronics
Mechanical balances have been replaced with electronic balances that are either:
Analytical
Top loading
What is the most accurate and precise balance?
Electronic Analytical Balances
What are the advantages of electronic balances?
3 to 4 times more precise than a mechanical balance
Easy to use
Fewer moving parts – less likely to be damaged
Tare bar – When you tare the balance, it sets it to zero, which then disregards the weight of the weighing container (boat)
Balance may be attached to a computer
What errors can occur during weighing?
- Mechanical errors due to defect in balance
- Forgetting to set the ZERO prior to starting
- Not leveling the balance prior to starting
- Human error – wrong reading – Transcription error
- Hygroscopic objects absorb water from air while being weighed
- Evaporation
- Errors caused by foreign material – use clean balance and weighing containers
What maintenance is require for balances?
Exterior surfaces of the instrument may be cleaned with a cloth dampened with water and a mild detergent or Virex cleaner when a spill occurs
Balance calibration is scheduled maintenance
- Frequency will depend on stability of environment and if the scale moves locations
How do pH meters work?
pH meter detects the amount of hydrogen ions (H+) in a solution and records this on a pH scale
Utilizes a glass electrode, called the probe
The probe has a glass bulb on one end and an electrical wire on the other end
What is an indicating electrode?
- Often referred to as the glass electrode
- Tube is filled with a buffer of known pH usually 0.1M HCl and KCl plus a wire coated with AgCl (silver chloride)
What is a reference electrode?
Has a known stable reference potential which DOES NOT depend on the pH of the solution being tested
How to use a pH meter:
- Standardized at temperature used for testing
- There’s a standby mode for when it’s not being used
- ALWAYS store electrode in distilled H20 or pH 7.0 standard (buffer) when not in use
- Reading – “Use” or pH mode
- Calibrated using standards/buffers of known pH
- ALWAYS rinse with distilled H2O between standards/samples/etc
What maintenance is required for a pH meter?
Daily
- Clean exterior of pH meter with a damp cloth
- Process QCs
Weekly
- Replace buffers used for QCs
Monthly
- pH electrode maintenance is performed, which involves soaking the electrode in HCl for 8 to 12 hours, then conditioning the electrode in a buffer with pH 7.0
- Calibrate after cleaning and conditioning
What is the Spectrophotometer?
Instrument used to measure the light transmitted by a solution to determine the concentration of the light absorbing substance in the solution
BEER’S LAW states – the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light
Colorimetric testing (relies on colour change) use a reagent to react with a solution and causes a color change( darker color = less light emitting through)
What are the 5 main parts of the Spectrophotometer?
- Light Source
- Entrance Slit
- Monochromator
- Sample Compartment
- Detector
What is the function of the light source?
High intensity, which gives off light in a wide range of the spectrum and an optimum amount of light
Incandescent tungsten or tungsten-iodide lamp for work in the visible or near-infrared region
What is the function of the Entrance Slit / Collimator?
Allows narrow band of light to pass through, focusing it and preventing light scattering
What is the function of the Monochromators?
Allows for a narrow band of wavelengths
isolates the wavelength desired and eliminates unwanted wavelengths
Transmit the colour that the analyte you’re measuring can absorb
Can be either Prism or Diffraction grating
What is the function of the Exit Slit?
Concentrates the band of monochromatic light, focusing it on the cuvette
What is the function of the Cuvette?
Light from desired wavelength – from filter or diffraction grating – will pass on to the solution contained in the cuvette
May be round or square