Unit 2: Microscopy, Equipments & Instruments Used & Unit 3: Sterilisation Flashcards
What is a microscope?
An instrument used to see objects that are too small for the naked eye.
What distinguishes optical microscopes from electron microscopes?
Optical microscopes use light, while electron microscopes use electrons to image samples.
What is the role of magnification in microscopy?
Magnification is the degree of enlargement of an object’s dimensions.
What are the differences between bright field and dark field microscopes?
Bright field microscopes produce dark images against light backgrounds; dark field produces bright images against dark backgrounds.
Who invented phase contrast microscopy and what was its significance?
Frits Zernike invented it, allowing the observation of living cells and won a Nobel Prize in 1953.
What is a scanning electron microscope (SEM)?
A SEM is an electron microscope that images samples using a high-energy electron beam in a raster scan pattern.
What information does a scanning electron microscope provide?
SEMs provide data on surface topography, morphology, composition, and crystallographic information of a sample.
How does a transmission electron microscope (TEM) work?
A TEM uses a focused beam of electrons to transmit through a specimen, allowing visualization of internal structures.
What are the limitations of light microscopes?
They have a maximum magnification of 500x to 1000x and a resolution limit of 0.2 micrometers.
Who developed the transmission electron microscope and when?
Max Knoll and Ernst Ruska developed the TEM in Germany in 1931.
What fields utilize transmission electron microscopes?
TEMs are used in cancer research, virology, material science, pollution studies, nanotechnology, and semiconductor research.
Why was there a demand for electron microscopes in the 1930s?
There was a need to observe fine details of organic cell structures, requiring magnifications over 10,000x
Who discovered the electron and when?
J.J. Thompson discovered the electron in 1897.
What are the primary differences between optical and electron microscopes?
Optical microscopes use glass lenses and low magnification; electron microscopes use magnetic lenses and high magnification.
What is the fundamental principle behind electron microscopic functionality?
Electron microscopes utilize a beam of energetic electrons to examine objects at a very fine scale.
What is the initial process in Transmission Electron Microscopy?
A thin specimen is irradiated with a uniform current density electron beam.
How is the electron beam focused onto the specimen in TEM?
A two or three stage condenser lens system is used to focus the electron beam.
What are the main differences in electron interaction between SEM and TEM?
SEM is based on scattered electrons, while TEM relies on transmitted electrons.
What is necessary for specimen preparation in TEM compared to SEM?
TEM requires the sample to be cut thinner, while SEM does not have this requirement.
How are Clostridium tetani spores used to test sterilization efficacy?
Paper strips containing 10 spores are placed in an oven for sterilization.
What indicates failure of the sterilization process using Clostridium tetani spores?
Any growth in thioglycollate broth after incubation indicates sterilization failure.
What is a hot air oven?
An oven that provides a temperature higher than atmospheric pressure, operating between 50 - 250°C.
What are the common sterilization settings for hot air sterilization?
170°C for 30 mins, 160°C for 60 mins, or 150°C for 150 mins or longer.
What are the main uses of a hot air oven?
It is used for rapid evaporation, rapid drying, and sterilization of heat-resistant materials.
Why is slow cooling important post-sterilization?
It prevents glass cracking and avoids air entry that can introduce contaminants.
What are the environmental benefits of dry heat sterilization?
It is nontoxic and does not harm the environment.
How long does dry heat sterilization typically take?
Dry heat sterilization requires longer exposure times of 1.5 to 3 hours.
Which materials are suitable for hot air sterilization?
Glassware, forceps, swabs, and water impermeable oils, waxes, and powders.
What is a major disadvantage of dry heat sterilization?
The method is time-consuming due to slow heat penetration.
What is the mechanism of dry heat sterilization?
Sterilization occurs through conduction, where heat penetrates from the outer surface to the center.
Why is dry heat sterilization suitable for metals and sharp instruments?
It is noncorrosive, making it safe for these materials.
What preparation steps are necessary for glassware before sterilization?
Ensure dryness, plug with cotton wool, wrap in Kraft paper, and avoid oven overload.
How can sterilization effectiveness be monitored?
Use chemical controls and Browne’s tubes, noting the color change from red to green.
What is the primary function of centrifugation?
It separates components of a mixture based on their density using centrifugal force.
What are the main components of a centrifuge?
A centrifuge consists of an electric motor and a rotor to hold samples.
What is density gradient centrifugation?
A procedure for separating particles like viruses, ribosomes, or DNA.
How are centrifuges categorized?
Centrifuges are categorized by speed and application, including low-speed, high-speed, and ultracentrifuges.
How does density gradient centrifugation work?
Samples are placed on a preformed gradient and centrifuged using rate zonal or equilibrium methods.
What is the maximum speed of low-speed centrifuges?
Low-speed centrifuges typically range from 4000 to 5000 rpm.
What is centrifugation?
A procedure using centrifugal force for sedimentation of mixtures.
What occurs during the centrifugation process?
Macromolecules band within the gradient, allowing for collection as pure fractions.
What rotor types are used in low-speed centrifuges?
Low-speed centrifuges use fixed angle and swinging bucket rotors for efficient separation.
What is the main purpose of low-speed centrifuges in biochemistry?
They are used for rapid sedimentation of coarse precipitates or red blood cells.
How does density affect separation in centrifugation?
More dense components move away, while less dense ones move towards the axis.
What are the main applications of density gradient centrifugation?
It separates multicomponent mixtures and measures the sedimentation coefficient of macromolecules.
How are low-speed centrifuges used to separate components of a sample?
The sample is spun to pack particles into a pellet, with supernatant removed by decantation.
What does the equation F = m * ω² * r represent?
It defines the centrifugal force experienced by sedimenting particles.
What are the two methods of centrifugation in this technique?
Zonal centrifugation uses a preformed gradient; isopycnic centrifugation forms a self-generating gradient.
What are the key features of high-speed centrifuges?
They have higher speeds, temperature control, and typically operate at near 4°C.
What is Relative Centrifugal Force (RCF)?
A measurement of centrifugal force reported as a multiple of gravity (g).
List two applications of high-speed centrifuges.
They are used for sedimentation of cell debris and separation of cellular organelles.
In which fields is centrifugation commonly applied?
Biotechnology, clinical laboratories, and research.
What distinguishes ultracentrifuges from other centrifuges?
They achieve very high speeds while requiring refrigeration and high vacuum to reduce heat.
What are the maximum speeds and RCF for low-speed centrifuges?
Low-speed centrifuges operate at 1-6000 rpm with a maximum RCF of 6000 g.
Which components can be pelleted by an ultracentrifuge?
Ultracentrifuges can pellet cells, nuclei, organelles, ribosomes, and macromolecules.
Describe the procedure for preparative centrifugation in biochemistry.
Sample is spun, separating pellet and supernatant, followed by decantation for further analysis.
What is the role of homogenization in the centrifugation process?
Homogenization breaks cell membranes to mix contents before centrifugation.
How does particle size and density affect centrifugation outcomes?
Larger, denser particles pellet at lower centrifugal forces during centrifugation.
What tasks are included in the quarterly maintenance checklist?
The checklist includes cleaning filters, filling the water tank, checking manometers, verifying temperature accuracy, and activating safety valves.
What are the key steps in the sterilization process?
Record pressure, temperature, and time; verify equipment functionality; listen for abnormal sounds; clean the chamber; check earth leakage current.
How is limescale defined and removed?
Limescale is a chalky deposit from hard water, removed by heating the chamber with acid to dissolve it.
What is the purpose of sterilization cassettes?
Sterilization cassettes hold instruments during sterilization and may include markers for monitoring the process.
Why is it important to activate safety valves during preventive maintenance?
Activating safety valves ensures they operate correctly and helps prevent equipment malfunctions.
What is the primary purpose of an autoclave?
To prepare materials for bacteriological cultures, sample collection, and sterilization of contaminated items.
What are the main safety risks associated with operating an autoclave?
High pressure can cause cracks or leaks; operators risk being scalded by hot steam.
How does pressure affect temperature in an autoclave?
Increasing pressure raises the temperature; at 15 Psi, it reaches 121°C necessary for sterilization.
What are the main components of an autoclave?
Key components include heating elements, temperature controller, pressure sensor, chamber, door gasket, solenoid valve, and water level sensor.
What materials are used in the construction of an autoclave?
Autoclaves are typically made of gun metal and include features like a controller and safety valves.
Why is temperature management important in autoclave operation?
Industrial autoclaves operate at higher temperatures, requiring careful preparation for safety.
How does the sterilization mechanism differ between dry and wet sterilization?
Dry sterilization uses oxidation at higher temperatures, while wet sterilization uses steam to coagulate proteins at lower temperatures.
What are key safety features included in an autoclave?
Safety features include low water level sensors, alarms, automatic cut-off mechanisms, and pressure gauges.
What is the exposure time required to kill bacterial spores using wet sterilization?
Typically, 15 minutes at 121 °C is sufficient for killing bacterial spores.
What is steam sterilization in the context of autoclaving?
Steam sterilization involves generating pressurized saturated steam to effectively sterilize materials.
What materials should not be used to clean an autoclave chamber?
Avoid using bleach, steel wool, steel brushes, or abrasive materials.
What methods are used to remove air from the autoclave chamber?
Air removal methods include downward displacement, steam pulsing, vacuum pumps, and various atmospheric cycles.
What is a critical step in the autoclave cleaning procedure?
Remove instruments and trays, then use a cleaning packet evenly in the chamber.
Describe the function of the pressure sensor in an autoclave?
The pressure sensor monitors the pressure within the sterilizing chamber to ensure effective sterilization conditions.
What is sterilization?
Sterilization is a process that removes or kills all forms of microbial organisms, including fungi, bacteria, viruses, and spores.
What methods can be used for sterilization?
Methods include heat, chemicals, irradiation, high pressure, filtration, and combinations of these.
What does the additional cleaning cycle help eliminate?
It eliminates excessive dirt in the pipes after a sterilization cycle without cleaning powder.
What is the function of an autoclave?
An autoclave uses high pressure and high temperature steam to eliminate microorganisms and spores.
Who were key historical figures in the development of sterilization techniques?
Notable figures include Hippocrates, Lazzaro Spallanzani, Louis Pasteur, and Charles Chamberland.
How does pressure in an autoclave affect the boiling point of water?
Pressure increases in an autoclave allows water to reach temperatures above 100°C, enabling effective sterilization.
