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Questions and Answers
What is the primary reason culture media cannot be sterilized using chemical agents?
What is the primary reason culture media cannot be sterilized using chemical agents?
What is a common method used to sterilize blood chemically?
What is a common method used to sterilize blood chemically?
Which pore size filter is suitable for retaining bacterial cells?
Which pore size filter is suitable for retaining bacterial cells?
Which of the following liquids can be sterilized using filtration?
Which of the following liquids can be sterilized using filtration?
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What type of filter is commonly made from cellulose acetate or nitrate?
What type of filter is commonly made from cellulose acetate or nitrate?
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What is the correct sequence for using a syringe filter?
What is the correct sequence for using a syringe filter?
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Why is it necessary to autoclave the filter and suction flask before filtration?
Why is it necessary to autoclave the filter and suction flask before filtration?
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Which of the following materials can be part of a syringe filter?
Which of the following materials can be part of a syringe filter?
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What is the primary purpose of sterilization?
What is the primary purpose of sterilization?
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Which method of sterilization generally requires more time?
Which method of sterilization generally requires more time?
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Why is moist heat sterilization more effective than dry heat sterilization?
Why is moist heat sterilization more effective than dry heat sterilization?
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What is a key characteristic of the Bunsen flame during sterilization?
What is a key characteristic of the Bunsen flame during sterilization?
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What occurs to microbial cells when exposed to heat during sterilization?
What occurs to microbial cells when exposed to heat during sterilization?
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What is a characteristic of dry heat sterilization?
What is a characteristic of dry heat sterilization?
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Which of the following is NOT a method of physical sterilization?
Which of the following is NOT a method of physical sterilization?
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What is the role of the upward flow of air around the Bunsen flame?
What is the role of the upward flow of air around the Bunsen flame?
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What is the purpose of using cedar-wood oil in microscopic examination?
What is the purpose of using cedar-wood oil in microscopic examination?
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What is the first step in the microscopic examination process using an oil-immersion lens?
What is the first step in the microscopic examination process using an oil-immersion lens?
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How does the iris diaphragm affect the lighting intensity in a microscope?
How does the iris diaphragm affect the lighting intensity in a microscope?
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Which objective lens is typically used for low power magnification?
Which objective lens is typically used for low power magnification?
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What is the correct adjustment for the iris diaphragm when using a high-power lens?
What is the correct adjustment for the iris diaphragm when using a high-power lens?
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What should be done after placing a drop of cedar oil on a smear during examination?
What should be done after placing a drop of cedar oil on a smear during examination?
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What is the typical magnification range for the oil-immersion lens?
What is the typical magnification range for the oil-immersion lens?
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Which item is NOT necessary for microscopic examination of stained smears?
Which item is NOT necessary for microscopic examination of stained smears?
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What is the first step that should be taken to sterilize the culture medium in a glass bottle?
What is the first step that should be taken to sterilize the culture medium in a glass bottle?
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Which cell arrangement is characteristic of spherical cells (cocci)?
Which cell arrangement is characteristic of spherical cells (cocci)?
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What is the main purpose of preparation of a stained smear in microbiology?
What is the main purpose of preparation of a stained smear in microbiology?
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Which part of the microscope is responsible for focusing the light onto the slide?
Which part of the microscope is responsible for focusing the light onto the slide?
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Which of the following is a characteristic of Actinomycetes?
Which of the following is a characteristic of Actinomycetes?
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What is the function of the coarse adjustment knob on a microscope?
What is the function of the coarse adjustment knob on a microscope?
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Which of the following objective lenses has the highest magnification power?
Which of the following objective lenses has the highest magnification power?
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How does adjusting the iris diaphragm affect light intensity on the microscope slide?
How does adjusting the iris diaphragm affect light intensity on the microscope slide?
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What is the first step in the process of using a sterilized syringe filter?
What is the first step in the process of using a sterilized syringe filter?
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Which method is primarily used to test the sterility of culture media and filtrates?
Which method is primarily used to test the sterility of culture media and filtrates?
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Why is boiling considered insufficient as a sterilization method?
Why is boiling considered insufficient as a sterilization method?
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What indicates efficient sterilization of the culture medium?
What indicates efficient sterilization of the culture medium?
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What must be done after filtering the sample with a syringe filter?
What must be done after filtering the sample with a syringe filter?
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How should the rubber plug of a culture medium bottle be treated for effective sterilization?
How should the rubber plug of a culture medium bottle be treated for effective sterilization?
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What should be done to the distilled water before sterilization using tyndallization?
What should be done to the distilled water before sterilization using tyndallization?
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What is the first action to take when using an autoclave?
What is the first action to take when using an autoclave?
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Study Notes
Sterilization
- Sterilization is the process of killing or removing all microorganisms from a material.
- There are three main methods of sterilization: physical, chemical, and radiation.
Physical Sterilization Methods
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Heat sterilization: This method involves using heat to kill microorganisms.
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Dry heat: This method involves exposing materials to high temperatures in the absence of moisture.
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Direct flaming: This method uses a Bunsen flame to sterilize instruments.
- The Bunsen flame temperature is 1560°c.
- Instruments must be heated to a red heat without altering their chemical and physical properties.
- Hot air: This method uses an oven to sterilize materials at high temperatures.
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Direct flaming: This method uses a Bunsen flame to sterilize instruments.
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Moist heat: This method involves using heat in the presence of moisture.
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Tyndallization: This method uses steam under atmospheric pressure to sterilize materials.
- The process is done by heating the material at 100°c for 30 minutes for three consecutive days.
- This method is suitable for sterilizing thermolabile materials.
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Autoclaving: This method uses steam under pressure to sterilize materials.
- The pressure is higher than atmospheric pressure.
- This method is used for sterilizing culture media, surgical instruments, and other materials that can withstand high temperatures.
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Tyndallization: This method uses steam under atmospheric pressure to sterilize materials.
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Dry heat: This method involves exposing materials to high temperatures in the absence of moisture.
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Filtration: This method involves passing a liquid through a filter with a pore size that is small enough to trap microorganisms.
- Filters with a pore size of 0.45 µm can retain bacteria.
- Filters with a pore size of 0.22 µm can retain bacterial cells and viruses.
- This method is used for sterilizing heat-sensitive liquids, such as carbohydrates, enzymes, hormones, vitamins, toxins, blood, serum, and amino acids.
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Different filter types:
- Membrane filters: These filters are made of cellulose acetate or nitrate.
- Sintered-glass filters: These filters are made of glass fibers.
- Seitz filters: These filters are made of asbestos.
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Filtration unit components:
- Funnel: This is a cone-shaped device used to guide the liquid into the filter.
- Filter holder: This device holds the filter.
- Rubber disk: This acts as a seal.
- Rubber tubing: This connects the suction flask to the vacuum pump.
- Suction flask: This is a flask where the filtrate is collected.
- Vacuum pump: This pump creates a vacuum that helps draw the liquid through the filter.
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Using the filter:
- The filter and suction flask must be sterilized before use.
- After filtration, the filter must be disposed of safely by autoclaving.
Chemical Sterilization Methods
- Chemical sterilization methods involve using chemicals to kill microorganisms. However, direct exposure to chemicals is not recommended when sterilizing culture media because it can inhibit growth of desired microbes.
- Chemical methods for sterilizing materials include:
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Antiseptics: These are chemicals that can kill microorganisms or inhibit their growth, making them suitable for use on living surfaces.
- Examples include 5% phenol solution, 5% cresol solution, 0.25% formaldehyde solution, 0.25% chloroform solution, mercuric chloride, and alcohol.
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Preservatives: These are chemicals used to prevent microbial growth in food and other products.
- Examples include benzoic acid, lactic acid, sorbic acid, acetic acid, and their salts.
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Antiseptics: These are chemicals that can kill microorganisms or inhibit their growth, making them suitable for use on living surfaces.
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Blood sterilization:
- Blood can be sterilized by adding 0.25% formaldehyde solution for 30 minutes and then heating the mixture in a water bath at 57°c to remove the formaldehyde.
Syringe Filters
- Syringe filters are single-use filters that are attached to a syringe.
- They are used to filter small volumes of liquids.
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Using syringe filters:
- The sample is loaded into the syringe.
- The filter is attached to the syringe with a twisting movement.
- The syringe and filter are held vertically over a sterilized bottle or tube.
- The syringe plunger is pressed to push the sample through the filter.
- After filtration, the container with the sterilized filtrate is closed.
- The filter and syringe are autoclaved in a bag for disposal.
Sterility Test
- This test is used to determine the presence or absence of viable microorganisms in culture media and filtrates that require sterility.
- Testing culture media: The medium is incubated at 30-37°c for 24 hours. If no microbial growth occurs, the sterilization is considered effective.
- Testing filtrates: A small volume of filtrate is inoculated into a previously sterilized culture medium. The inoculated medium is incubated at 30-37°c for 24 hours. If no microbial growth occurs, the sterilization is considered effective.
Tyndallization
- Tyndallization is a sterilization method that uses moist heat under atmospheric pressure.
- It involves heating the material at 100°c for 30 minutes for three consecutive days.
- The purpose of the multiple heating cycles is to allow for the germination of bacterial spores into vegetative cells, making them vulnerable to the heat treatment on subsequent days.
Boiling
- Boiling is considered an insufficient sterilization method.
- Although boiling can kill non-spore forming microorganisms, many bacterial spores are resistant to boiling temperatures and survive.
Microscopic Examination
- To examine the morphology of microbial cells microscopically, a stained smear must be prepared.
- Smear preparation: A thin film of microbial cells is spread on a slide for microscopic examination.
Microscope Components
- Mechanical Parts:*
- Base: Supports the microscope.
- Stage: Holds the slide.
- Arm: Connects the base to the body tube.
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Focusing Knobs:
- Coarse adjustment knob: Adjusts the sample into view.
- Fine adjustment knob: Sharpens the focus of the sample.
- Optical Parts:*
- Mirror: Directs light towards the slide through the condenser.
- Condenser: Consists of lenses and an iris diaphragm that controls and concentrates light on the slide.
- Ocular lens: The eyepiece through which the observer views the image.
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Objective lenses: These are attached to the nosepiece and magnify the image.
- Low-power objective lens (8-10x).
- High-power objective lens (40-45x).
- Oil-immersion lens (90-100x).
Controlling Lighting Intensity
- Condenser: Can be racked up to obtain light with high intensity or racked down for low intensity light.
- Iris Diaphragm: Opening the iris diaphragm allows for high intensity light, while closing it produces low intensity light.
Relation Between Magnification and Lighting Intensity
- As microscope magnification increases, the required lighting intensity also needs to increase.
Using Cedar-wood Oil
- Cedar-wood oil is used in microscopy with the oil-immersion lens because it has the same refractive index as glass.
- This eliminates refraction of light as it passes from the glass slide through the oil and into the lens, improving image clarity.
Steps for Using the Oil-Immersion Lens:
- Rack up the condenser.
- Open the iris diaphragm completely.
- Apply a drop of cedar-wood oil onto the stained smear.
- Place the slide on the stage.
- Lower the oil-immersion lens using the course adjustment knob until the oil is flattened.
- Use the coarse adjustment knob with very slow movements to bring the cells into view.
- Adjust focus sharply using the fine adjustment knob.
Microbial Cell Morphology
- Microorganisms are classified based on their cell shapes, including:
- Spherical Cells (cocci): Can be found in various arrangements, including chains, clusters, and pairs.
- Short Rods: Found as single cells or in chains.
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Long Rods:
- Non-sporulated long rods: Found in chains.
- Sporulated long rods: Have spores within their structure.
- Spiral Shape (Spirillum): Curved, helical-shaped bacteria.
- Vibrio Shape: Slightly curved, comma-shaped bacteria.
- Actinomycetes: Long, thin, branched and aseptate hyphae that reproduce by conidia.
- Molds: Large, thick, branched, septate hyphae that reproduce by conidia.
- Yeasts: Large oval cells that can be found as single cells or in groups and reproduce by budding.
Important Tools for Microbiology
- Lab coat: Protects the user from contamination.
- Slides: Glass plates used to prepare samples for microscopic examination.
- Slide covers: Thin pieces of glass that cover the sample on a slide to protect it and improve clarity.
- Wooden or plastic slide box: Used to store stained slides.
- Matches: Used to light the Bunsen burner.
- Small towel: Used to wipe spills and clean the microscope.
- Wooden clip (clothes peg): Used to hold slides in place.
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Description
Test your knowledge on the various methods of sterilization, including physical, chemical, and radiation techniques. Understand the details of heat sterilization methods such as dry heat and moist heat, along with sub-methods like direct flaming and Tyndallization.
