Last Lecture On Microscope And Cultures Flashcards
State three tests that provide specific info unique to a certain microbe
Specimen collection occurs when Microbiologists begin by sampling the object of their interest.
Inoculation: then the sample is put into a container of medium that will support the growth. The medium may be solid or liquid and held in tubes,flasks,eggs and plates. The delivery tool is usually a needle,a loop,a syringe,
Incubation- the sample or inoculated media is placed in a controlled environment (incubator) to promote growth. During the hours or days of this process, a culture develops as the visible growth of the microbes in the container of the medium.
Isolation:some inoculation techniques can separate microbes to create isolated colonies that each contain a single type of microbes. This is important for identifying the specific type of species of microbes in the sample and it paves the way for making pure cultures.
Inspection:cultures are observed for the microscopic appearance of growth characteristics. Cultures are examined under microscope for basic details such as cell type and shape. This may be enhanced through staining and use of special microscopes
Information gathering:additional tests for microbial function and characteristics may include inoculations into specialized media that determine biochemical traits,immunological testing and genetic typing. Such tests provide specific info unique to a certain microbe. More include biochemical tests,immunological tests,drug sensitivity,DNA analysis
Identification: one of the goals of these procedures is to attach a name or identity to the microbe using information gathered from inspections and investigations. Identification is accomplished through the use of keys,charts and computer programs that analyze the data and arrive at final conclusions
What’s the difference between sub cultures and pure cultures under isolation
Subculture and pure culture are both important concepts in microbiology, but they serve different purposes:
- Definition: Subculture refers to the process of transferring microorganisms from one culture medium to another to maintain or propagate the culture. This technique is used to extend the life of a culture by providing fresh nutrients or to isolate specific colonies.
- Purpose: It’s typically used to keep a culture growing, to separate different species from a mixed culture, or to maintain a particular strain.
- Process: A small sample from a growing culture is transferred to a new medium under sterile conditions.
- Application: Often used in laboratories to maintain bacterial or fungal cultures over long periods.
- Definition: A pure culture consists of a single species of microorganism growing in a medium, without any contamination from other species. This is achieved by isolating one type of microorganism from a mixture.
- Purpose: It’s essential for studying the characteristics of a particular microorganism, such as its morphology, biochemical activities, or antibiotic sensitivity.
- Process: Pure cultures are often obtained using techniques like streaking, dilution, or selective media.
- Application: Used in research, diagnostic labs, and industrial processes to study or utilize a specific microorganism.
In summary, subculture is a method to propagate or maintain a culture, while pure culture refers to a culture that contains only one type of microorganism, free from contamination by others.
State two key characteristics of a reliable microscope
Key characteristics of a reliable microscope are:
• Magnification - ability to enlarge objects
• Resolving power - ability to show detail
What shines light up through the aperture and reflects light into the same space? It makes it easier to see the object on the slide?
Where do you look into the microscope to see the image of the object you intend to study?
Which part holds the two or three objective lenses, and rotates around in a circle, allowing you to choose which objective lens you want to use?
What holds the microscope in place?
What allows the light to come up from behind the object you’re observing so that it’s easier to see?
What’s the long, skinny tube that holds the eyepiece above the microscope, and allows light to travel up from the objective lenses to the eyepiece?
What are attached to the nosepiece and vary in size?
Function: you would use this to change the amount of light that comes through the aperture?
things that magnify the object your viewing. you can adjust the power of this thing by switching it for another one?
https://microscopy4kids.org/compound-microscope-parts-function/
- Location: At the top of the microscope.
- Function: These are the lenses you look through to view the specimen. They typically magnify the image 10x. In a binocular microscope, there are two ocular lenses to allow viewing with both eyes, which provides depth perception and reduces eye strain.
- Location: Mounted on the revolving nosepiece, just above the stage.
- Function: These are the primary lenses that magnify the specimen. Most microscopes have multiple objective lenses (e.g., 4x, 10x, 40x, 100x) to provide different levels of magnification.
- Location: Holds the objective lenses and is located above the stage.
- Function: Allows you to rotate between different objective lenses to change the magnification.
- Location: The flat platform below the objective lenses.
- Function: The stage holds the slide with the specimen. It often includes stage clips to secure the slide in place.
- Location: On the stage.
- Function: These clips hold the slide in a fixed position on the stage during observation.
- Location: Below the stage.
- Function: Focuses light from the illuminator onto the specimen, which enhances clarity and contrast.
- Location: Beneath the condenser.
- Function: Controls the amount of light reaching the specimen, allowing for adjustments in contrast and resolution.
- Location: On the side of the microscope.
- Function: Moves the stage up and down to bring the specimen into general focus. It’s used primarily with lower magnification objectives.
- Location: Usually located on the side of the microscope, near the coarse adjustment knob.
- Function: Provides precise focusing after the coarse adjustment has been made, especially important at higher magnifications.
A mirror placed below the aperture can reflect light upwards, while a lamp positioned above the aperture can shine light through it. This combination allows the light to be directed into the same space, making it easier to see the object on the slide.
- Location: At the base of the microscope.
- Function: Provides the necessary light to view the specimen. It may be an LED, halogen, or a mirror that reflects external light.
- Location: Within or near the light source at the base.
- Function: Filters the light to improve the quality of illumination, reducing glare and enhancing contrast or adjusting the color of the light.
- Location: The curved structure connecting the base to the body of the microscope.
- Function: Provides support and is used for carrying the microscope.
- Location: The bottom part of the microscope.
- Function: Provides stability and support to the microscope, ensuring it remains steady during use.
Aperture: allows the light to come up from behind the object you’re observing so that it’s easier to see. The aperture refers to the opening in a camera lens that controls the amount of light that enters the camera. By adjusting the size of the aperture, more or less light can pass through, affecting the brightness and clarity of the image. In the context of the question, a larger aperture would allow more light to come up from behind the object being observed, making it easier to see.
The aperture and condenser are essential parts of a microscope that control light for optimal image quality:
- Aperture: An adjustable opening in the condenser system that controls the amount of light and contrast in the image. allows the light to come up from behind the object you’re observing so that it’s easier to see
- Condenser: A lens system below the stage that focuses light onto the specimen for uniform illumination.
The aperture primarily adjusts light intensity and contrast, while the condenser ensures the specimen is well-lit for clear, detailed imaging.
Yes, that’s a good way to summarize it! Here’s a more detailed breakdown:
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Mirror or Illuminator:
- The mirror (in older microscopes) or the illuminator (in modern microscopes) provides the source of light.
- If a mirror is used, it reflects light from an external source (like a lamp or sunlight) up into the condenser.
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Condenser:
- The condenser collects and focuses the light from the mirror or illuminator and directs it up through the specimen on the slide.
- It ensures that the light is concentrated and properly aligned for optimal illumination of the specimen.
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Aperture Diaphragm:
- The aperture diaphragm, which is part of the condenser system, controls the amount of light that passes through the specimen.
- By adjusting the aperture diaphragm, you can control the contrast and depth of field in the resulting image. This helps in optimizing the clarity of what you see through the eyepiece.
- The mirror or illuminator brings light through the condenser, which focuses and directs it through the specimen.
- The aperture diaphragm determines the amount of light and controls its contrast and depth, affecting the clarity and quality of the image seen through the eyepiece.
So the mirror brings the light through the condenser and the aperture determines the amount of light that comes through to the eyepiece?
What’s the long, skinny tube that holds the eyepiece above the microscope, and allows light to travel up from the objective lenses to the eyepiece? Body tube
. The legs. The legs of the microscope hold it in place. They provide stability and support, preventing the microscope from moving or tipping over while in use. The legs are designed to be sturdy and durable, ensuring that the microscope remains steady and secure during observations and experiments.
What are attached to the nosepiece and vary in size?
Correct answer:
Objective lenses
Function: you would use this to change the amount of light that comes through the aperture.
Ans: The diaphragm is a part of a camera that controls the size of the aperture, which in turn determines the amount of light that passes through the lens. By adjusting the diaphragm, the photographer can change the size of the aperture opening, allowing more or less light to reach the camera sensor or film. This adjustment is crucial in controlling the exposure of the image and achieving the desired level of brightness or darkness in the photograph.
things that magnify the object your viewing. you can adjust the power of this thing by switching it for another one.?
Objective lens. Objective lenses are a type of optical component that are used to magnify the object being viewed. By switching the objective lens for another one, the power of the magnification can be adjusted. These lenses are commonly used in microscopes and telescopes to enhance the clarity and detail of the image.
The condenser is a component in a microscope that is located beneath the stage and is responsible for focusing and directing light onto the specimen. It shines through the aperture. It contains an aperture (an adjustable opening) that controls the amount of light passing through to illuminate the specimen. By adjusting the condenser’s aperture, you can control the intensity and focus of the light, which is crucial for obtaining clear and well-illuminated microscopic images.
The mirror reflects light unto the aperture
I understand how these parts of a microscope can be confusing since they all relate to light and its control. Here’s a breakdown of the functions of the aperture, condenser, iris diaphragm, and illuminator in a compound light microscope:
- Illuminator:• Function: The illuminator is the light source of the microscope. It is usually located at the base of the microscope.
• Purpose: It provides the light that is directed through the condenser to illuminate the specimen.
• Key Point: Without the illuminator, there would be no light passing through the specimen, making it impossible to see the specimen clearly. - Condenser:• Function: The condenser is located beneath the stage and above the illuminator. It focuses the light from the illuminator onto the specimen.
• Purpose: Concentrates and directs the light from the illuminator into a cone that passes through the specimen, enhancing contrast and resolution.
• Key Point: The condenser helps to sharpen the image by focusing light on the specimen. The height of the condenser can often be adjusted to control how focused the light beam is. - Iris Diaphragm:• Function: The iris diaphragm is part of the condenser system. It is a ring with an adjustable opening (like a camera aperture) that controls the amount of light reaching the specimen.
• Purpose: By adjusting the diaphragm, you can control the intensity and contrast of the light. It helps to improve image clarity and contrast by adjusting the size of the light cone that reaches the specimen.
• Key Point: The iris diaphragm does not focus light but rather adjusts the width of the light beam, affecting brightness and contrast. - Aperture:• Function: The aperture refers to the hole in the stage through which light passes from the condenser and illuminator to the specimen.
• Purpose: It allows the light from the condenser to pass through the specimen to the objective lens.
• Key Point: The aperture is not an adjustable part itself but is simply the opening in the stage. However, the size of the aperture indirectly affects the amount of light and clarity of the image depending on the size of the light cone controlled by the iris diaphragm.
Summary of Differences:
• Illuminator: The light source of the microscope. • Condenser: Focuses the light from the illuminator onto the specimen. • Iris Diaphragm: Controls the amount of light and adjusts contrast by changing the diameter of the light beam. • Aperture: The opening in the stage that allows light to pass through the specimen.
The names X-axis knob and Y-axis knob are often used to describe the movement directions of the slide, but they are not the formal names. The actual names for the knobs on a microscope’s mechanical stage are:
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Stage Control Knobs (sometimes called Stage Adjustment Knobs):
- One knob moves the slide left and right (horizontal movement).
- The other knob moves the slide forward and backward (vertical movement).
These controls are typically positioned together, and while some may refer to them by their movement axes (X and Y), the general term is mechanical stage control knobs.
So X knob or stage knob moves the slide left or right in a horizontal plane and Y knob moves the slide forward or backwards in a vertical plane.
Up and down is the coarse and fine adjustment
Magnification in the microscope so in two phases. State them
How is the real image formed andhow is the virtual image formed
What is total magnification and how is it calculated
Which lens is responsible for the magnification of the image of the specimen?
4x,10x,40x and 100x magnification is provided wht which lens?
In light microscopy, magnification occurs in two main phases, as you’ve correctly outlined:
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Objective Lens Forms the Magnified Real Image:
- When light passes through the specimen, the objective lens (which is closer to the specimen) collects the light and magnifies the image of the specimen.
- This magnified image is called the real image because it can, in theory, be projected onto a surface (like a piece of paper) if the eyepiece is removed.
- The magnification provided by the objective lens varies depending on which lens is being used (e.g., 4x, 10x, 40x, 100x).
Image formed here is inverted or upside down compared to the actual position of the organism
And located in focal plane of objective lens
### Phase 2: Ocular Lens Magnification
- The Real Image is Projected to the Ocular Lens:
- The ocular lens (eyepiece) receives the real image formed by the objective lens.
- The ocular lens further magnifies this real image. The magnification provided by the ocular lens is typically 10x, but it can vary in some microscopes.
- This additional magnification creates the virtual image, which is what the viewer sees when looking through the eyepiece.
- The image is called “virtual” because it cannot be projected onto a surface; it only exists in the viewer’s perception.
The image formed here is upright and larger than the real image.
### Summary of Magnification Process
- Total Magnification: The total magnification of the microscope is the product of the magnification of the objective lens and the magnification of the ocular lens.
- For example, if you are using a 40x objective lens with a 10x ocular lens, the total magnification would be 400x (40x objective * 10x ocular).
This two-phase magnification process is fundamental to how light microscopes function, allowing for detailed examination of microscopic structures by progressively enlarging the image.
Total magnification of the final image is a productofthe separate magnifying powers of the two lenses
. objective power x ocular power =total m a gn i f i c a t i o n
Between air and oil, which has a lower refractive index and how does it make oil useful in high magnification microscopy
In microscopy, especially when using high magnification objectives like the 100x oil immersion lens, immersion oil plays a crucial role in improving image clarity. Here’s how it works in relation to air, the objective lens, and the slide:
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Refractive Index Matching:
- Air vs. Oil: Air has a lower refractive index (about 1.0) compared to glass (around 1.5) and immersion oil (also around 1.5). When light passes from the glass slide (where the specimen is placed) into the air and then into the objective lens, the difference in refractive index causes light to bend (refract) and scatter, leading to a loss of resolution.
- Oil’s Role: Immersion oil, placed between the slide and the objective lens, has a refractive index similar to that of glass. This matching minimizes the bending of light rays as they move from the slide to the objective lens, allowing more light to enter the lens directly and increasing the resolution and brightness of the image.
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Enhancing Image Quality:
- Increased Resolution: By reducing the scattering of light, oil immersion helps to capture more light from the specimen. This results in a sharper, clearer image with greater detail, especially at high magnifications (100x or higher).
- Better Light Transmission: Immersion oil reduces light loss due to refraction, allowing more light to pass through the specimen and into the objective lens, which enhances the image brightness and contrast.
- Without Oil: When using high-power objectives without oil, the difference in refractive index between air and glass can lead to significant light refraction and scattering, resulting in a blurred or less detailed image.
- With Oil: Immersion oil bridges the gap between the slide and the objective lens, minimizing light refraction, improving resolution, and providing a much clearer and detailed view of the specimen.
Thus, the use of oil is essential in high-magnification microscopy to maintain image quality by optimizing light transmission and reducing distortions caused by the refractive index differences between air and glass.
Under variations on the optical microscope, the two main techniques used are bright field and dark field microscopy.
Which of them is used for live and preserved stained specimen?
Which is used for unstained specimen?
Why would you stain the specimen in the first place?
Staining increases the contrast of the specimen true or false
If you decide not to stain the specimen, Why would it appear bright?
What is an optical microscope
• Bright-field- mostwidelyused;specimen is darker than surrounding field;used for live and preserved stained specimens. The specimen is stained because specimen on a regular are transparent so if you view them in bright field, you won’t see the specimen. So you stain them so that they will look darker compared to the rest of the field. Staining them increases contrast so yoj can see more structures in the microorganism
Dark-field - brightly illuminated specimens surrounded by dark field; used for live and unstained specimens.
Here’s why the specimen can appear bright without staining:
How Dark Field Microscopy Works:
1. Light Pathway: In dark field microscopy, a special condenser is used to direct light so that it only enters the objective lens if it is scattered by the specimen. Most of the direct light is blocked by the condenser, so it doesn’t enter the objective lens, creating a dark background.
2. Scattering of Light: When light hits the specimen, some of it is scattered in different directions. This scattered light is what enters the objective lens and is used to form the image. Because only scattered light reaches the objective, the specimen itself appears bright against a dark background.
3. No Need for Staining: Unlike bright field microscopy, where contrast is achieved through staining, dark field microscopy enhances contrast by using scattered light. This technique is particularly useful for viewing live, unstained, and delicate specimens like bacteria, flagella, or thin biological structures, which might be invisible or lack contrast in bright field microscopy.
The different techniques described are all variations of the optical microscope, which uses visible light and lenses to magnify images of small objects. Here’s how each variation fits into the broader category of optical microscopy:
- Optical Basis: This is the standard form of optical microscopy where light passes directly through the specimen.
- Variation: It is the most basic type of optical microscopy, using bright light and straightforward optics to view specimens.
- Optical Basis: Still relies on visible light, but modifies the path of light using a special condenser that creates a dark background by blocking direct light and only allowing scattered light from the specimen to be observed.
- Variation: This technique enhances contrast by illuminating the specimen at an angle, making it appear bright against a dark background, without altering the fundamental optical principles.
- Optical Basis: Uses light but introduces phase-shifting elements to exploit the different refractive indices of various parts of the specimen, which are invisible in bright-field microscopy.
- Variation: Converts phase shifts (which are invisible) into variations in light intensity (which are visible), allowing for the observation of transparent, unstained specimens like living cells.
- Optical Basis: Uses visible or ultraviolet light to excite fluorescent molecules within the specimen.
- Variation: Enhances the optical microscope by using fluorescent dyes or proteins that emit light at a different wavelength when excited by a specific light source, allowing for the visualization of specific components within cells.
- Optical Basis: Uses laser light (still within the visible spectrum) and advanced optics to create high-resolution, three-dimensional images.
- Variation: Uses a pinhole to eliminate out-of-focus light, enhancing the sharpness and resolution of the optical microscope’s images, particularly in three dimensions.
- Important Note: Electron microscopy (TEM and SEM) does not use visible light and is therefore not a variation of the optical microscope. Instead, it uses a beam of electrons for imaging, which is beyond the scope of optical microscopy.
- Use of Light: All the variations (except electron microscopy) use light in some form—whether it’s direct, scattered, refracted, or emitted from fluorescent molecules—to create images.
- Lenses and Magnification: They all employ lenses to magnify the image, which is the core principle of optical microscopy.
- Enhancing Capabilities: Each technique modifies how light interacts with the specimen or how it is captured by the microscope, offering enhanced capabilities for observing different types of specimens or details that may not be visible using standard bright-field microscopy.
These variations allow optical microscopes to be versatile tools, adapted for various scientific and clinical applications by modifying how light is used to observe and record the specimen.
How does the electron microscope work
Electron waves are how many times shorter than waves of visible light
Why do electron microscopes have tremendous power to resolve minute structures?
What is the range of magnification in electron microscope
Electron Microscopy
• Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds
• Electron waves are 100,000 times shorter than the waves of visible light
• Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength or depends on wavelength.
Resolving Power and Wavelength: Resolving power is the ability of a microscope to distinguish between two closely spaced objects. The shorter the wavelength of the light or radiation used in imaging, the higher the resolving power, meaning finer details can be distinguished. In electron microscopy, electrons are used instead of light, and electrons have much shorter wavelengths than visible light.
• Magnification between 5,000X and 1,000,000X
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State the two types of electron microscopes and how they are used
Which of the two have a higher resolution ?
Why does it have a higher resolution?
Which of the electron microscopes is used to study the internal ultrastructure of cells, viruses, and materials at the molecular or atomic level?
Which of the electron microscopes scans the surface with electrons to produce detailed images of surface morphology with a 3D appearance?
Which of the electron microscopes bombards the surface of a whole metal coated specimen with electrons evil scanning back and forth over it?
Transmission electron microscopes (TEM) - transmit electrons through the specimen.
Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts.
Has a higher resolution than SEM cuz it transmits electrons through an ultra thin(slide said thick but cha gpt says thin) specimen which reveals details of the specimen. TEM is used to study the internal ultrastructure of cells, viruses, and materials at the molecular or atomic level.
TEM: Transmits electrons through the specimen to reveal internal structures at extremely high resolution.
• SEM: Scans the surface with electrons to produce detailed images of surface morphology with a 3D appearance.
Scanning electron microscopes (SEM) - provide detailed three-dimensional view.
SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.
- Description: TEMs transmit a beam of electrons through an ultra-thin specimen.
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Image Formation:
- Darker Areas: These represent thicker, denser parts of the specimen that scatter or absorb more electrons, resulting in reduced transmission and a darker appearance in the image.
- Lighter Areas: Indicate more transparent, less dense regions where more electrons pass through, resulting in a lighter appearance in the image.
- Application: Used to observe the internal ultrastructure of cells, viruses, and complex molecules at very high resolution.
- Description: SEMs provide a detailed three-dimensional view of the surface of a specimen.
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Image Formation:
- SEMs work by bombarding the surface of a metal-coated specimen with electrons.
- The electron beam scans back and forth over the specimen’s surface, interacting with the atoms and producing signals that are collected to form a 3D image.
- Application: Ideal for studying the surface topology and morphology of various materials, including biological tissues, metals, and other solid surfaces, providing high-resolution images of the surface details.
State three ways specimen are prepared for optical microscopes
Which way is used to view non living organisms
Which way is used to examine characteristics of live cells such as size, shape,arrangement and motility?
Which way is used to examine cell division or responses in stimuli?
Which way is used if the sample requires hydration to maintain its natural state?
Which way is used to study fluid dynamics?
Which way is made by drying and heating a film of the specimen?
Which way has a smear that is stained using dyes to permit visualization of cells or cell structures in details?
Which is used jn. Histology?
Which is used for specimen that don’t need hydration?
Which are quicker to prepare and which are used when you want to store the specimen for a longer period?
Which is used to observe motility and 3D movement in live specimen?
Which is used when you need a clearer view of microorganisms without squashing them?
2 main ways from the sldies were wet and hanging mounts and then fixed mounts. Then I added dry mounts.
Specimen Preparation for Optical Microscopes
• Wet mounts and hanging drop mounts - allow examination of characteristics of live cells: size, motility, shape, and arrangement. Used to also examine stuff like cell division in microorganisms or responses to stimuli. When Staining Might Be Used:
• Stains can be added to a wet mount if needed, but this typically involves vital stains (like methylene blue or iodine) that are gentle enough not to kill the cells immediately. However, even with vital stains, some cellular activity can be lost or altered.
Key Point:
• Unstained Wet Mounts: Used primarily to view living cells. • Stained Wet Mounts: Rare and generally used with non-toxic stains if cell visualization needs enhancement without significantly compromising viability.
• Fixed mounts are made by drying and heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts and structures in detail.
Fixed mounts involve staining and are used in histology
Dry mounts: for viewing non living things and simple viewing Dry mounts work well for specimens that do not require hydration, such as plant material, fibers, or mineral samples.
Dry and wet mounts are quicker to prepare that fixed mounts but fixed mounts have a longer storage period
In essence, both wet and fixed mounts allow visualization of cell shape, size, and arrangement, but they cater to different purposes:
• Wet mounts show these features in live, unstained cells. • Fixed mounts enhance and preserve these features with added structural detail, often aided by stains.
Wet and hanging drop mounts are temporary and best for short-term viewing, while fixed mounts are permanent.
Guidelines for Choosing the Right Mount
1. Use a Wet Mount If: • You need to observe live cells or organisms. • The sample requires hydration to maintain its natural state. • You are studying motility, behavior, or fluid dynamics. 2. Use a Hanging Drop Mount If: • You are observing motility and 3D movement in a live specimen. • You need a clearer view of microorganisms without squashing them. • You want to study undisturbed behaviors in a suspended drop. 3. Use a Dry Mount If: • The specimen is non-living and doesn’t need hydration. • You need a quick and simple observation without special preparation. • The sample is solid, dry, or powdery, such as crystals, hair, or pollen. 4. Use a Fixed Mount If: • You need detailed visualization of cell structures, morphology, or tissue samples. • Staining is required to enhance contrast and highlight specific features. • The specimen needs to be preserved for long-term studies.
Hanging drop mount:
Requires a depression slide, which may not be as readily available as a standard flat slide.
Is Preferred when a more detailed and accurate observation of motility is required, such as distinguishing true motility from Brownian motion (random movement caused by water molecules).
Choosing Between Wet Mount and Hanging Drop:
• Use a Wet Mount when you need a quick, simple preparation for general motility observation. • Use a Hanging Drop Mount when accurate motility observation is critical, especially for distinguishing true motility from passive movement or when observing motility over a longer period.
Read from chat gpt how hanging drop mounts are prepared
How are dyes used?
What is a basic dye?(Is it positive or negatively charged?)
What is an acidic dye?(is it positively or negatively charged?)
What is positive staining?
What is negative staining?
Between neg and pos stains, which stain the organism and which stain the background?
What are simple stains?
What are differential stains?
Between positive and negative staining, which can be used to stain spirochetes?
What are structural stains ?
Acid fast bacteria stain what color?
Importance of endospore stains
Dyes are used to create contrast by imparting color
• Basic dyes - cationic(positively charged. Don’t confuse it with cathode which is negatively charged and anode which is positively charged), positively charged chromophore(colour bearing ion). Examples are methylene blue,crystal violet and safranin. Hematoxylin is a neutral dye that can act like a basic dye in that it stains nucleus blue but itself is not a basic dye.
• Positive staining - surfaces of microbes are negatively charged and attract basic dyes.
Positive Staining: Involves the use of a dye or stain that binds specifically to the target structures in a specimen, causing them to appear darker or more colored compared to the background. The stain itself is often positively charged, which attracts it to negatively charged cellular components.
Example is the methylene blue staining the nucleus which contains acidic components which are negatively charged.
Acidic dyes - anionic, negatively charged chromophore. Examples are eosin and nigrosin.
• Negative staining - microbe repels dye, the dye stains the background.
H Principle: In negative staining, the dye does not penetrate the microbial cells or structures. Instead, it stains the background while the cells remain relatively clear.
• Mechanism:
• Microbial Surface: Microbes often have a negative charge on their surfaces due to acidic components in their cell walls or membranes.
• Dye Interaction: Since the microbial surfaces repel the negatively charged acidic dye, the dye does not adhere to the cells but spreads around them. The dye or stain used is typically negatively charged, which repels from the negatively charged cellular components of the specimen, thus leaving them unstained and appearing as clear or lighter areas against a dark background.
• Appearance: As a result, the cells appear as clear shapes against a darkly stained background when observed under a microscope.
Applications:
• Negative Staining: This technique is useful for observing cell morphology, size, and arrangement without staining the cells themselves. It is particularly helpful for visualizing cells with delicate structures or those that do not hold stains well. So you can use it to stain spirochetes cuz of this.
Simple stains - one dye is used; reveals shape, size, and arrangement. Examples are methylene blue, crystal violet,safranin,basic fuschin,malachite green(for staining spores)
Differential stains - use a primary stain and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain, and endospore stain)
Structural stains - reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains
Endospore staining is a type of structural stain used to visualize bacterial endospores, which are highly resistant structures formed by certain bacteria to withstand extreme conditions.
- Primary Stain: Malachite Green is used to stain the endospores.
- Procedure: The sample is heated to allow the dye to penetrate the endospores. After staining, the slide is washed, and the endospores retain the green color.
- Counterstain: Safranin or another counterstain is used to stain the vegetative cells, which will appear red or pink against the green endospores.
- Example of Endospore Staining Method: Schaeffer-Fulton Method.
Structural stains are designed to highlight specific cellular structures or components.
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Examples:
- Capsule Stain: Uses a combination of acidic and basic dyes to visualize bacterial capsules, which are often colorless or transparent against a stained background.
- Flagella Stain: Uses special dyes and mordants to make bacterial flagella visible.
- Endospore Stain: As described above, used to visualize bacterial endospores.
Differential stains are used to differentiate between different types of cells or cellular components based on their staining properties.
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Examples:
- Gram Stain: Differentiates bacteria into Gram-positive (purple) and Gram-negative (pink) based on cell wall structure.
- Acid-Fast Stain: Differentiates acid-fast bacteria (such as Mycobacterium tuberculosis) from non-acid-fast bacteria. Ziehl-Neelsen Stain is a common method where acid-fast bacteria retain the red color of carbol fuchsin, while non-acid-fast bacteria take up the blue color of methylene blue.
- Endospore Stain: Differentiates endospores from vegetative cells as described above.
- Endospore Stains: Specifically target endospores.
- Structural Stains: Highlight specific cellular structures.
- Differential Stains: Separate organisms or structures based on their differential staining properties.
State the 6 Is of culturing media
Which of the Is is concerned with :
1. introduction of a sample into a container of media to produce a culture of observable growth?
2.Provides conditions that allow growth
3. observe and identify the characteristics of the colonies.
4. separating one species from another to obtain pure cultures. This is done by techniques such as streak plating or serial dilution.
5.which is used to Examine the colonies’ morphology (color, shape, size), growth pattern, and other visible traits under a microscope or with the naked eye
6. obtain detailed data about the microorganisms.
7. Perform additional tests and assays (e.g., Gram staining, biochemical tests, molecular techniques) to gather information about the microorganisms’ biochemical properties, genetic makeup, and physiological characteristics. Includes collecting data on colony morphology, performing biochemical assays, and assessing environmental conditions.
8. Uses detailed methods like microscopic examination, biochemical testing using biochemical assays such as carbohydrate fermentation profile, molecular techniques such as PCR and sequencing , Antibiotic Sensitivity Testing and serological tests to conclusively determine the exact species of the bacterium.
9. helps in understanding the general characteristics of the bacterium,
10. precisely determines its species or type based on that gathered information.
The 6 l’s of Culturing Microbes
Inoculation - introduction of a sample into a
container of media to produce a culture of observable growth
Incubation - under conditions that allow growth
Isolation - separating one species from another to obtain pure cultures. This is done by techniques such as streak plating or serial dilution.
Inspection-To observe and identify the characteristics of the colonies.
• Procedure: Examine the colonies’ morphology (color, shape, size), growth pattern, and other visible traits under a microscope or with the naked eye
Information gathering-To obtain detailed data about the microorganisms.
• Procedure: Perform additional tests and assays (e.g., Gram staining, biochemical tests, molecular techniques) to gather information about the microorganisms’ biochemical properties, genetic makeup, and physiological characteristics. Includes collecting data on colony morphology, performing biochemical assays, and assessing environmental conditions.
Identification-Uses detailed methods like microscopic examination, biochemical testing using biochemical assays such as carbohydrate fermentation profile, molecular techniques such as PCR and sequencing , Antibiotic Sensitivity Testing and serological tests to conclusively determine the exact species of the bacterium.
In this case, information gathering helps in understanding the general characteristics of the bacterium, while identification precisely determines its species or type based on that gathered information.
Microscopy in Inspection: Used to observe general features (general characteristics of the sample, such as size, shape, arrangement, and staining properties. ) and conditions of the microorganism using bright field,dark field ,phase contrast microscopy.
• Microscopy in Identification: Used to perform detailed, specific analysis to determine the exact species or type of microorganism.using staining and fluorescence microscopy
Key Points:
• Inspection Staining: Typically uses simpler, less specific stains to get an overview of the microorganism.uses basic stains and aciidc stains • Identification Staining: Involves more complex staining methods to provide detailed and specific information for accurate identification. Uses counter stains and differential stains which are more complex
Summary: Both phases utilize staining, but the techniques and goals differ. Inspection focuses on general morphology, while identification uses specialized stains to confirm specific characteristics of the microorganism.
What is Isolation
Explain the microscopic and macroscopic view of isolation
A colony consists of how many species?
If an individual bacterial cell is separated from other cells and has space on a nutrient surface, it will grow into a mound of cells. What is the name of this mound of cells ?
How does separation of a mixture of cells occur in inoculation?
What ends the microscopic view?
If an individual bacterial cell is separated from other cells and has space on a nutrient surface, it will grov into a mound of cells— a colony. A colony consists one species.
When there’s a Mixture of cells in sample,
Parent cells are Separated or there is separation of the mixture of cells by spreading or dilution on agar medium.
Incubation occurs and
There is subsequent Growth which increases the number of cells.
This ends the microscopic view or cellular level.
Microbes become visible as isolated colonies containing millions of cells. This is the Macroscopic view where the colony is seen or the colony level is seen
The image you uploaded showcases different examples of microbial staining techniques. Here’s a breakdown of the different staining techniques displayed:
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Simple and Negative Stains
- Example: Methylene blue stain of Corynebacterium.
- Purpose: These stains are used to observe the general shape and size of cells.
- Application: The simple stain colors the cells, while the negative stain colors the background, leaving the cells clear.
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Differential Stains
- Example: Gram stain.
- Purpose: This stain differentiates bacteria into two groups based on cell wall composition: Gram-positive (purple) and Gram-negative (red).
- Application: It is crucial for the identification and classification of bacteria.
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Structural Stains
- Example: India ink capsule stain of Cryptococcus neoformans.
- Purpose: Special stains highlight specific structures within or on cells, such as capsules, spores, or flagella.
- Application: The India ink capsule stain is used to identify encapsulated bacteria or fungi.
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Negative Stain
- Example: Negative stain of Treponema bacteria made with nigrosin.
- Purpose: Negative staining is used to visualize organisms that are difficult to stain directly, such as spirochetes.
- Application: Provides contrast to visualize delicate and thin structures like flagella.
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Acid-Fast Stain
- Example: Acid-fast stain.
- Purpose: Used to differentiate acid-fast bacteria (red cells) from non-acid-fast bacteria (blue cells).
- Application: Commonly used for identifying Mycobacterium species.
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Flagellar Stain
- Example: Flagellar stain of Proteus vulgaris.
- Purpose: This stain highlights the presence of flagella, which are thin and delicate structures used for bacterial motility.
- Application: Important in studying the motility and classification of bacteria.
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Spore Stain
- Example: Spore stain, showing spores (green) and vegetative cells (red).
- Purpose: Identifies the presence of spores, which are resistant structures formed by some bacteria.
- Application: Used to identify spore-forming bacteria, such as Bacillus and Clostridium.
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Fluorescent Stain
- Example: Fluorescent stain of bacterial chromosome (green) and cell membrane (red).
- Purpose: Uses fluorescent dyes to highlight specific cellular components under a fluorescence microscope.
- Application: Useful in genetic studies and for identifying specific bacterial species.
These stains provide crucial insights into the structure, composition, and function of microbial cells, aiding in their identification and understanding of their biology.
State three isolation techniques
Why is the inoculating loop re-sterilized?
-Streak plate technique-Note: This method works best if the spreading tool (usually an inoculating loop) Is resterized (Bamad) ater each of steps 1-3, Loop containing sample
The inoculating loop is sterilized each time it streaks an amount of bacteria and it puts it in the plate. Re-sterilization: After each streak, the inoculating loop is re-sterilized by passing it through a flame until it is red-hot to kill any residual microorganisms before proceeding to the next streak. This ensures that fewer microorganisms are transferred with each step, aiding in the isolation of single colonies.
By the time you finish the last streak, individual cells are spread far enough apart to grow into separate colonies. These distinct colonies can then be used to grow pure cultures.
5. Importance of Sterilization: Flaming the loop ensures that only a reduced, controlled number of microorganisms are transferred during each step, which is critical for achieving isolation.
Why Use the Streak Plate Technique?
• It’s used when you need isolated colonies for identification, further culture, or analysis. • Effective for studying mixed cultures or assessing contamination.
-pour plate technique or loop dilution technique or pour plate or serial dilution technique
-spread plate technique
The three methods—streak plate, pour plate, and spread plate—are all used to isolate and grow microorganisms, but they differ in their specific applications and techniques. Here’s a detailed explanation to help you understand when to use each method and the differences between them:
- Streak Plate Technique:• Purpose: Best for isolating pure colonies from a mixed sample.
• When to Use:
• When you need to separate different microorganisms in a mixed culture (e.g., isolating bacteria from a clinical sample).
• When you want to study colony morphology (shape, color, size).
• How it Works: The sample is streaked in sections, diluting the sample across the plate to achieve isolated colonies.
• Key Feature: Creates distinct colonies on the surface of the agar. - Pour Plate Technique:• Purpose: Ideal for counting colonies and isolating microorganisms within the agar medium.
• When to Use:
• When you need to quantify microorganisms in a sample (e.g., measuring bacterial load in a food sample).
• When working with microorganisms that might grow better inside the agar rather than on the surface.
• How it Works: The sample is diluted, mixed with molten agar, and poured into a dish. Microorganisms grow throughout the medium, not just on the surface.
• Key Feature: Colonies grow both inside and on the surface of the agar, providing a 3D distribution. Effective for isolating anaerobic or microaerophilic microorganisms since some colonies develop within the agar.
• Provides an accurate count of viable microorganisms.
5. Disadvantages:
• Labor-intensive compared to some other methods.
• Heat-sensitive microorganisms might be affected by the warm molten agar. - Spread Plate Technique:• Purpose: Used to evenly distribute microorganisms on the surface of the agar for isolation and counting.
• When to Use:
• When you want to count the number of colonies on the agar surface (e.g., water or food testing).
• Suitable for samples that have been diluted properly to avoid overcrowded growth.
• How it Works: A small volume of diluted sample is spread across the agar surface using a sterile spreader.
• Key Feature: Colonies only grow on the surface of the agar, making counting and isolation straightforward.
How to Choose:
• Streak Plate: Choose when isolating individual colonies from a mixed culture is the main goal. • Pour Plate: Choose when quantifying microorganisms is important, and when the ability to grow throughout the medium could enhance colony growth. • Spread Plate: Choose when you need a clear, even distribution of colonies on the agar surface for counting.
Practical Examples:
• Streak Plate: Used in clinical labs to isolate bacterial colonies from patient samples. • Pour Plate: Used in food microbiology to count bacteria in contaminated food samples. • Spread Plate: Used in environmental testing to quantify bacteria in water samples.
The spread plate technique differs from the streak plate technique in several ways, particularly in how the inoculum is spread and the resulting colony formation. Here’s a comparison between the two:
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Purpose:
- Used to evenly distribute a liquid sample over the surface of an agar plate to isolate colonies for counting or further analysis.
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Procedure:
- A small, measured volume of a diluted liquid sample is pipetted onto the surface of a pre-poured solid agar plate.
- A sterile spreading tool, such as a glass or metal rod, is used to spread the sample evenly across the surface of the agar.
- The plate is then incubated to allow the colonies to grow.
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Colony Formation:
- Colonies grow on the surface of the agar, making it easier to count and differentiate between colony types.
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When to Use:
- Useful when you need to quantify the number of viable microorganisms in a liquid sample.
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Purpose:
- Primarily used to isolate pure colonies from a mixed sample.
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Procedure:
- A loopful of the sample is streaked across the surface of the agar plate in a series of steps, usually in three or four streak patterns.
- After each streak, the loop is sterilized, ensuring fewer organisms are transferred with each step, eventually leading to isolated colonies.
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Colony Formation:
- Colonies form along the streak lines, often in areas of decreasing concentration.
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When to Use:
- Ideal for obtaining isolated colonies from a mixed culture, often used to purify a specific microorganism.
- Distribution: The spread plate technique distributes the inoculum evenly across the entire agar surface, while the streak plate technique distributes the inoculum in diminishing concentrations through repeated streaking.
- Purpose: Spread plates are better for quantification and even growth, whereas streak plates are better for isolating individual colonies.
- Tools: Spread plates use a spreader, while streak plates use an inoculating loop.
