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
What is the function of the Detector?
A photoelectric cell that detects the light which passes through the specimen into the cuvette
They have a light sensitive surface which is capable absorbing detected light and emitting electrons – electrons produced in proportion to the amount of light hitting it
What is the function of the Galvanometer?
The electrons are then passed on to the galvanometer
Electrons emitted from detector collected by galvanometer and records this electrical energy on a scale as (A) absorbance or (T) transmittance
Galvanometer readings are lower for concentrated solutions than they are for dilute solutions
What Care and handling is needed for Spectrophotometer?
Always follow manufacturer’s recommendations
Spillage may damage photoelectric cell
If filters are used (versus grating), never operate without filters
Stable, non-vibrating bench
What maintenance is needed for Spectrophotometer?
Maintenance will follow the instructions provided by the manufacturer for the analyzer housing the
spectrophotometer
Cleaning of the exterior
Replacing the lamp when required
Running QC
What are the 6 temperature controlled equipment?
Ovens – hot air, convection, microwave
Waterbaths
Incubators
Refrigerators
Freezers
Autoclave
What are the 3 different types of ovens used in lab?
Hot Air Ovens
Convection Ovens
Microwave Ovens
Hot Air Ovens:
Commonly used to dry glassware
Never dry volumetric glassware in hot air ovens
Convection Ovens
Used for heating or drying
Temperature range 40°C to 300°C
Microwave Ovens:
Used primarily in Histotechnology
Accelerates/enhances cell fixation to slides, tissue embedding and immunohistochemical procedures
Waterbaths:
For procedures requiring 37°C incubation
May be used for thawing
Top up regularly due to evaporation
Temperature MUST be monitored and recorded as part of Quality Assurance (QA) programs
Water-bath Maintenance:
Daily
- Record temperature of water bath
- Add water to reach the fill line
Weekly
- Drain water from water bath
- Clean interior (and baskets if present)
- Replace water
- Add a conditioner to the water bath to prevent microbial growth
Incubators:
Used in microbiology to control temperature and humidity in bacterial cultures
Adjustable temperature ranges for specific organisms – 30°C, 35°C
Temperatures must be monitored and recorded for quality assurance process
Can be connected to CO2 supply to create specific atmosphere
Refrigerators:
Usually set at 4°C +/- 2°C
Have temperature recording capabilities
7-day temperature chart
Always keep a separate thermometer inside
Freezers
Usually set at –20°C or cooler (can be as low as –80°C)
Can be used for frozen blood products (ex FFP), lyophilized materials, specimens, etc.
Temperature recording capabilities
Connected to an alarm system
Maintenance for temperature-controlled equipment:
Daily
- Record temperature on log
Weekly
- Replace temperature charts if equipment has a 7-day recording chart
As-needed
- Clean interior and exterior of equipment
What to do when temperature-controlled devices malfunction?
Must investigate when temperature is out of range and/or equipment is alarming
Door ajar, malfunctioning thermometer, malfunctioning equipment
Corrective action log must be initiated
Monitor every 4 hours
If reagents, products, samples, etc are in the fridge/freezer/room, must move them to an alternative fridge/freezer/room
Hot Plates:
RT to several 100°C
Used for heating or boiling liquids
Incinerators:
Used in microbiology
Insert loop all the way into the heating
element for maximum 5 seconds
All demonstrable microorganisms will
be destroyed within that 5 seconds
What is water level of purity is required for:
reagent preparation
solution preparation
reconstituting lyophilized materials
sample dilutions
What are the 3 levels of water used in lab?
Type I
Type II
Type III
What is the most pure form of water?
Type I Reagent Water
How is type 1 Reagent Water created?
Usually undergoes at least two procedures i.e. distillation followed by deionization
What is the maximum colony count for type 1 water?
Maximum colony count less than or equal to 10 CFU/mL (colony forming unit/mL)
What should type 1 water be used in?
Preparation of standards
Preparation of buffers
Control preparation
Quantitative procedures
Electrophoresis
Toxicology
Chromatography
Type II Reagent Water
Used for general laboratory testing that does not require type I water
Maximum colony count is 1000 CFU/ml
Used primarily for qualitative procedures in all clinical lab disciplines
Type III Reagent Water:
Least pure
Maximum colony count is not specified for this type
Silicate content 1.0 mg/ml
pH 5 to pH 8
May be used for some qualitative tests (i.e. Those done in urinalysis)
What are the four methods used in the preparation of laboratory water?
Distillation
De-ionization
Combination de-ionization/distillation
Reverse osmosis
Distilled Water:
Achieved by using a still and a rather simple procedure:
- Boil water to produce steam
- Cool the steam to condense it
- Collect the water produced – known as the distillate
Water produced by a single distillation is commonly used in labs – It contains no organic materials and very few minerals
CO2, chlorine, and ammonia are not removed
Double-distilled Water:
Is not necessarily pure water
May contain dissolved gases – from distillation process or storage container that may cause interference in certain tests that require water devoid of nitrogen (ammonia-free)
Deionization:
Water is filtered through beds of synthetic ion exchange resins
Ion exchange resins contain positively charged and negatively charged particles
They combine with the ions present in the water and removes them
The resin is made up of two kinds of beads:
- Cation beads contain hydrogen ions (H+)
- Anion beads contain hydroxide ions (OH-)
Combination Deionized/Distilled H2O:
Higher purity water is produced using special units that first deionize then distill water
Some systems will distill first then deionize
Reverse Osmosis Water:
Water is passed through a semi-permeable membrane under pressure leaving behind all large molecules
The semi-permeable membrane separates the pure water into one stream and the mineralized water into another stream
Other Processes:
Ultra filtration, ultraviolet oxidation, sterilization – used only POST purification processes
Testing Purity of Water
Periodic testing should include:
- Microbial monitoring
- Resistivity
- pH
- Pyrogens
- Silica
- Organic contaminants
Specific Resistance of Laboratory Water
- The greater the resistance, the purer the water
- Pure water is a poor conductor of electricity
- The more impurities it contains (these would be ions not removed), the greater the conductivity OR the lower the resistivity
pH of Laboratory Water
- Pure water typically has a pH around 7.0
- pH can be one of the quickest indicators of process contamination in the production of pure water
- Carbon dioxide acidifies water lowering the pH
Grades of Chemicals
- Analytical Reagent (AR)
- Chemically Pure (CP)
- USP or NF – United States Pharmacopia and National Formulary – generally less pure than CP grade
ANALYTICAL GRADE REAGENTS (AR):
- Have a high degree of purity
- Often used in reagent preparation
CHEMICALLY PURE GRADE REAGENTS (CP):
- Pure enough for many analyses
- Limits of tolerate impurities is not known
- Not acceptable for research use and certain clinical lab procedures – procedure will specify the need for AR reagents
Reagent Container Labeling:
All labels should include:
Name and concentration of reagent
Date prepared
Initials of the individual preparing reagent
Commercially Prepared Reagents:
Always follow manufacturer’s instructions
When a new vial, bottle, kit, etc. is opened the “OPEN” or “IN USE” date is always recorded on the vessel including the initials of the individual who opened it
A reagent’s expiration date on the vial is valid unless opened – Oftentimes once a vial is open, it is only ”good” for 24 hours, 7 days, etc.
What are the Six components to the infection cycle?
What is an Infectious Agent?
Any disease-causing microorganism (pathogen)
- Bacteria, viruses, fungi, parasites
The greater the virulence, invasiveness, and pathogenicity, the greater the possibility that the organism will cause an infection
What is a Reservoir Host?
- “Individual” in which the infectious microorganism resides
- Animals, water, air, soil, humans
- Animals or humans not showing outward signs or symptoms of disease but are capable of transmitting disease are called carriers
What is a portal of exit?
- Route by which the pathogen leaves the reservoir host
- Breaks in the skin
- Respiratory secretions
- Reproductive secretions
- Blood
- Feces
What is mode of transmission?
- Method by which the pathogen gets from the reservoir host to the new host
- Way it moves or is carried
- May occur through direct contact, air, food, body fluids, insects, etc
Ex: Hands of HCW may carry bacteria from one person to another - Aerosols and inhalation are the most hazardous and the most difficult to contain
What is portal of entry?
- Place on the body of a susceptible host that the pathogen uses for entry
- An opening allowing the organism to enter the host
- Mucous membranes, wounds, mouth, nose, skin, vascular system, urinary catheters, IV, etc
What is a susceptible host?
- Person who cannot resist a microorganism invading the body, multiplying, and resulting in infection
- Lacking immunity or physical resistance to overcome the invasion by the pathogenic organism
- Factors affecting susceptibility: Age, immune competence, medication, nutritional status, pregnancy, metabolic disorders, malignancy
What are nosocomial infections?
What are they primary due to?
Primarily due to:
1. High prevalence of pathogens in hospitals
2. High prevalence of compromised hosts (patients)
3. Efficient mechanisms of transmission from patient to patient (chain of transmission)
What are the classifications of nosocomial infections?
Exogenous: Caused by pathogens in healthcare environment
Endogenous: Normal microbes that become pathogens due to opportunistic factors within the healthcare setting
Iatrogenic: Direct result of a medical procedure (ie surgery, catheterization, antibiotic therapy
What microorganisms are extremely resistant?
Prisons
What microorganisms are Highly Resistant?
Protozoal & Bacterial endospores
What microorganisms are Resistant?
Mycobacteria & Non-enveloped viruses
What microorganisms are Susceptible?
Fungal spores
Gram-negative bacteria
Enveloped viruses
Gram-positive bacteria
What microorganisms are Highly Susceptible?
Mycoplasmas
What are precautions of Automated Analyzers?
- Ensure that all reagents are stored according to requirements
- Wear gloves and use gauze to carefully wipe sample probes
- Use closed sampling instruments whenever possible
- Avoid over-filling specimen cups; transfer the required aliquot using a pipettor
What are precautions of Microtomes?
- Use automated microtomes when possible
- Make sure the microtome is in the locked position when positioning paraffin blocks
- Handle microtome knives only by the knife handles
- Avoid distraction while working with a microtome
- Avoid using the fingers to remove sections from the knife; use a brush, forceps and a microscope slide instead
- Sterilize knives and section flattening devices after use by autoclaving
What are precautions of Cryostats?
- Keep the cover closed during cutting
- Wear gloves when preparing frozen sections
- Decontaminate the cryostat contents frequently
- Defrost and decontaminate the cryostat weekly
- Handle knives cautiously
What are precautions of Electrophoresis apparatus?
- Use only electrophoresis equipment that is properly equipped with electrical interlocks to interrupt current flow when box is opened
- Verify the integrity of the tank on a regular basis to detect leaks or damaged areas of the tank
- Discourage accidental contact with the apparatus by locating it away from high traffic areas and provide physical barriers to the unit
4.Display proper warning of high voltage
What are precautions of Flame Photometers?
- Securely attach the tubing connecting the instrument to the gas cylinder
- Chain the gas cylinder securely to a solid support (wall, bench, etc)
- Frequently inspect all hoses, joints and connections
- Be familiar with requirements for handling compressed gases
What are precautions of Mass spectrophotometers?
- Verify gas, pump, exhaust and drain system tubing connections are correct before use
- Ensure that pumps are vented outside the laboratory
- Pump exhaust may contain traces of the samples being analyzed, solvents or reagent gases
- Avoid contact with heated parts while in operation
- Used pump oil may also contain traces of analytes and should be handled as hazardous waste
What Biohazards arise from Flow cytometers?
- Sample handling
- Flow-generated aerosols and/or droplets
- Waste management and equipment maintenance
What has the highest level of potential infection exposure in surgical pathology?
Cryostats: the frozen section laboratory
What are the 5 components of the laboratory security program?
- Physical security
- Personnel suitability and reliability
- Inventory
- Information management and security
- Incident and emergency response
Describe Physical Security:
- Limit lab access
- Visible ID badges
- Card or code access
- Lock places where hazardous materials are stored
- Never leave hazardous materials unattended or unsecured
- Provide workers ready access to telephones or emergency alert devices
Describe Personnel Suitability and Reliability?
- Ensure personnel are adequately qualified, trained, and have experience
- Perform reference checks on new employees
- Restrict off hours access to individuals authorized by supervisor
- Avoid working alone
5.Monitor ongoing suitability by paying attention to behaviour
Describe Inventory?
- Record what hazardous materials are being ordered
- Use an access control system to obtain and replace highly hazardous materials
- Review and revise inventories
- Get rid of surplus hazardous materials
- Track and document disposal
Describe Information Management and Security?
- Determine level of risk and align with the level of information security required
- Control access to sensitive or confidential information
Describe Incident and Emergency Response?
1.Train lab staff on security procedures
2. Provide emergency responders with information on serious hazards
3. Establish a protocol for reporting incidents or suspicious activities
4. Ensure investigation of incidents and implementation of corrective actions
What are risks groups?
The classification of biological material based on its inherent characteristics, including pathogenicity, virulence, risk of spread, and availability of effective prophylactic or therapeutic treatments
What is risk group 1:
low individual risk, low community risk
What is risk group 2:
moderate individual risk, low community risk
What is risk group 3:
high individual risk, low community risk
What is risk group 4:
high individual risk, high community risk
What are the containment levels?
Describe the minimum physical features and operational practices needed for the safe handling and storage of biohazards within an identified area, such as the laboratory.
Containment Level 1
Containment Level 2
Containment Level 3
What does commissioning a laboratory mean?
Refers to the systematic process of verifying that each component of the laboratory has been installed according to regulations, safety standards, engineering codes, and the policies of the organization
What does decommissioning a laboratory mean?
Refers to the steps involved in changing activities, moving out of a lab, or closing a lab altogether
What aspects of the lab are subject to the commissioning process?
- Fire protection features of the laboratory
- Laboratory ventilation system
- Overall lab design and quality control of materials
- Verification of biosafety containment levels
- Verification of radiation safety requirements
- Lab equipment which requires specialized installation or calibration
- Testing and certification of emergency wash equipment
- Training and orientation of staff to changes
Pathogenicity:
the property of causing disease
Virulence:
the severity or harmfulness of a disease or poison.
