Podcast
Questions and Answers
Why is understanding how various kinds of microscopes work crucial in microbiology?
Why is understanding how various kinds of microscopes work crucial in microbiology?
- It reduces the cost of maintaining microscopes.
- It helps in adjusting the colors in the image to match the actual colors of the microorganisms.
- It allows for the manipulation of light to produce highly detailed images of microbes for research and clinical applications. (correct)
- It simplifies the process of sterilizing microscope lenses.
What benefit do different staining techniques provide when viewing microscopic specimens?
What benefit do different staining techniques provide when viewing microscopic specimens?
- They alter the size of the microorganisms to make them easier to view.
- They manipulate color and contrast to help identify species and diagnose diseases. (correct)
- They increase the speed at which microorganisms move under the microscope.
- They prevent the microorganisms from reproducing during observation.
How does darkfield microscopy enhance the visualization of specimens compared to brightfield microscopy?
How does darkfield microscopy enhance the visualization of specimens compared to brightfield microscopy?
- Darkfield microscopy renders a brighter image on a darker background, increasing contrast. (correct)
- Darkfield microscopy allows for the observation of live specimens without staining.
- Darkfield microscopy uses higher magnification to see smaller structures.
- Darkfield microscopy uses different stains that are not visible under brightfield.
In the context of microscopy, what does the manipulation of light properties primarily allow scientists to achieve?
In the context of microscopy, what does the manipulation of light properties primarily allow scientists to achieve?
Which of the following is the MOST direct application of microscopy in clinical settings?
Which of the following is the MOST direct application of microscopy in clinical settings?
What is the significance of understanding the properties of light in the context of microbiology?
What is the significance of understanding the properties of light in the context of microbiology?
If a researcher needs to observe the motility of a bacterium without altering its natural state, which microscopic technique would be most appropriate?
If a researcher needs to observe the motility of a bacterium without altering its natural state, which microscopic technique would be most appropriate?
Why is it important to manipulate size when examining microbial cells through a microscope?
Why is it important to manipulate size when examining microbial cells through a microscope?
Aside from species identification and disease diagnosis, what other application benefits from the detailed images produced by advanced microscopic technologies?
Aside from species identification and disease diagnosis, what other application benefits from the detailed images produced by advanced microscopic technologies?
A lab technician is having trouble differentiating a bacterial sample using standard brightfield microscopy. What adjustment to the microscope could MOST likely improve the clarity and resolution of the image?
A lab technician is having trouble differentiating a bacterial sample using standard brightfield microscopy. What adjustment to the microscope could MOST likely improve the clarity and resolution of the image?
If a researcher requires a microscope with both high magnification and high resolution, which adjustment would MOST directly contribute to achieving this?
If a researcher requires a microscope with both high magnification and high resolution, which adjustment would MOST directly contribute to achieving this?
In microscopy, what is the relationship between the wavelength of light used and the resolution achievable?
In microscopy, what is the relationship between the wavelength of light used and the resolution achievable?
When light passes through a lens, refraction occurs. What property of the lens MOST influences the degree of refraction?
When light passes through a lens, refraction occurs. What property of the lens MOST influences the degree of refraction?
A scientist observes a specimen under a microscope and notices a high degree of diffraction. What can they infer about the specimen and the light interacting with it?
A scientist observes a specimen under a microscope and notices a high degree of diffraction. What can they infer about the specimen and the light interacting with it?
How does fluorescence microscopy enhance the visualization of specific cellular structures?
How does fluorescence microscopy enhance the visualization of specific cellular structures?
If a light wave transitions from air into glass, and the refractive index of glass is significantly higher than air, what change will occur to the light wave's direction?
If a light wave transitions from air into glass, and the refractive index of glass is significantly higher than air, what change will occur to the light wave's direction?
Why is enhancing contrast important when observing microorganisms under a microscope?
Why is enhancing contrast important when observing microorganisms under a microscope?
How does the numerical aperture of a microscope lens affect its performance?
How does the numerical aperture of a microscope lens affect its performance?
Consider a scenario where a researcher is using a prism to separate white light into its constituent colors. Why does this dispersion occur?
Consider a scenario where a researcher is using a prism to separate white light into its constituent colors. Why does this dispersion occur?
What is the fundamental difference between magnification and resolution in microscopy?
What is the fundamental difference between magnification and resolution in microscopy?
Girolamo Fracastoro's hypothesis about disease transmission was limited by what factor?
Girolamo Fracastoro's hypothesis about disease transmission was limited by what factor?
How did Antonie van Leeuwenhoek's work significantly advance the field of microbiology?
How did Antonie van Leeuwenhoek's work significantly advance the field of microbiology?
What distinguishes a simple microscope from a compound microscope?
What distinguishes a simple microscope from a compound microscope?
Why were Robert Hooke's observations of cork cells significant?
Why were Robert Hooke's observations of cork cells significant?
Aside from advancements in microscopy, what other contribution did Galileo Galilei make to science?
Aside from advancements in microscopy, what other contribution did Galileo Galilei make to science?
Why is there uncertainty surrounding the exact origins and inventors of the earliest microscopes?
Why is there uncertainty surrounding the exact origins and inventors of the earliest microscopes?
What was it about van Leeuwenhoek's reports to the Royal Society that initially sparked skepticism?
What was it about van Leeuwenhoek's reports to the Royal Society that initially sparked skepticism?
Robert Hooke's Micrographia played what role in the scientific revolution?
Robert Hooke's Micrographia played what role in the scientific revolution?
How did Reinier de Graaf contribute to Antonie van Leeuwenhoek's recognition in the scientific community?
How did Reinier de Graaf contribute to Antonie van Leeuwenhoek's recognition in the scientific community?
What implication can be drawn about the impact of early microscopes, despite their limitations?
What implication can be drawn about the impact of early microscopes, despite their limitations?
What is the primary function of the condenser lens in a brightfield microscope?
What is the primary function of the condenser lens in a brightfield microscope?
Why is it important to clean oil from immersion lenses after using a microscope?
Why is it important to clean oil from immersion lenses after using a microscope?
What characteristic of light is manipulated in phase-contrast microscopy to enhance the contrast of a specimen?
What characteristic of light is manipulated in phase-contrast microscopy to enhance the contrast of a specimen?
In indirect immunofluorescence assays (IFA), what is the role of the secondary antibody?
In indirect immunofluorescence assays (IFA), what is the role of the secondary antibody?
Which type of microscopy is MOST suited for examining thick, live specimens like biofilms without fixing or staining?
Which type of microscopy is MOST suited for examining thick, live specimens like biofilms without fixing or staining?
A researcher observes a bacterial specimen using a 10x ocular lens and a 40x objective lens on a brightfield microscope. What is the total magnification of the specimen?
A researcher observes a bacterial specimen using a 10x ocular lens and a 40x objective lens on a brightfield microscope. What is the total magnification of the specimen?
What limits the effective sensitivity of fluorescence microscopy when viewing thick specimens?
What limits the effective sensitivity of fluorescence microscopy when viewing thick specimens?
In brightfield microscopy, the use of stains on a specimen primarily serves to:
In brightfield microscopy, the use of stains on a specimen primarily serves to:
What is the function of the opaque disk in darkfield microscopy?
What is the function of the opaque disk in darkfield microscopy?
Which of the following best explains why oil immersion lenses improve resolution?
Which of the following best explains why oil immersion lenses improve resolution?
A researcher is investigating a new bacterial species and wants to observe its natural color and motility. Which preparation method would be MOST appropriate?
A researcher is investigating a new bacterial species and wants to observe its natural color and motility. Which preparation method would be MOST appropriate?
A clinical microbiologist observes a bacterial sample after Gram staining and notices a mix of purple and pink cells. What is the MOST likely explanation for this observation?
A clinical microbiologist observes a bacterial sample after Gram staining and notices a mix of purple and pink cells. What is the MOST likely explanation for this observation?
A researcher is examining a sample of lung tissue from a patient suspected of having tuberculosis. Which staining technique would be MOST appropriate to identify the causative agent?
A researcher is examining a sample of lung tissue from a patient suspected of having tuberculosis. Which staining technique would be MOST appropriate to identify the causative agent?
Why is heat used in the Ziehl-Neelsen method of acid-fast staining, but not in the Kinyoun method?
Why is heat used in the Ziehl-Neelsen method of acid-fast staining, but not in the Kinyoun method?
A microbiologist suspects a bacterial species possesses a capsule, but it is not clearly visible using simple staining. Which staining method would BEST reveal the presence of a capsule?
A microbiologist suspects a bacterial species possesses a capsule, but it is not clearly visible using simple staining. Which staining method would BEST reveal the presence of a capsule?
A researcher is trying to identify Clostridium difficile in a stool sample. Which staining technique would be MOST useful in the rapid presumptive identification of this organism?
A researcher is trying to identify Clostridium difficile in a stool sample. Which staining technique would be MOST useful in the rapid presumptive identification of this organism?
Why is it important to use a mordant in flagella staining?
Why is it important to use a mordant in flagella staining?
In preparing samples for transmission electron microscopy (TEM), what is the purpose of dehydrating the cells through a series of ethanol solutions?
In preparing samples for transmission electron microscopy (TEM), what is the purpose of dehydrating the cells through a series of ethanol solutions?
What is the MAIN reason for sputter-coating specimens with a metal, such as palladium, when preparing them for scanning electron microscopy (SEM)?
What is the MAIN reason for sputter-coating specimens with a metal, such as palladium, when preparing them for scanning electron microscopy (SEM)?
How does differential staining improve the identification of microorganisms compared to simple staining?
How does differential staining improve the identification of microorganisms compared to simple staining?
How do differences in refractive indices between two materials affect light passing from one to the other?
How do differences in refractive indices between two materials affect light passing from one to the other?
Which adjustment would MOST improve resolution when using a microscope?
Which adjustment would MOST improve resolution when using a microscope?
In microscopy, what is the function of lenses?
In microscopy, what is the function of lenses?
What is the primary difference between a simple and a compound microscope regarding their lenses?
What is the primary difference between a simple and a compound microscope regarding their lenses?
What is the role of fixation in sample preparation for microscopy?
What is the role of fixation in sample preparation for microscopy?
Why is it necessary to cut thin sections of samples for Transmission Electron Microscopy (TEM)?
Why is it necessary to cut thin sections of samples for Transmission Electron Microscopy (TEM)?
Consider a light wave moving from air to glass. If the refractive index of glass is higher than that of air, what happens to the light wave?
Consider a light wave moving from air to glass. If the refractive index of glass is higher than that of air, what happens to the light wave?
How does increasing the numerical aperture of a microscope lens improve its performance?
How does increasing the numerical aperture of a microscope lens improve its performance?
In fluorescence microscopy, what is the purpose of fluorochromes?
In fluorescence microscopy, what is the purpose of fluorochromes?
When using a prism to separate white light into its constituent colors, what property of light is being demonstrated?
When using a prism to separate white light into its constituent colors, what property of light is being demonstrated?
Which of the following has the highest energy?
Which of the following has the highest energy?
You place a specimen under the microscope and notice that parts of the specimen begin to emit light immediately. These materials can be described as _____________.
You place a specimen under the microscope and notice that parts of the specimen begin to emit light immediately. These materials can be described as _____________.
Who was the first to describe "cells" in dead cork tissue?
Who was the first to describe "cells" in dead cork tissue?
Who is the probable inventor of the compound microscope?
Who is the probable inventor of the compound microscope?
Which type of microscope is especially useful for viewing thick structures such as biofilms?
Which type of microscope is especially useful for viewing thick structures such as biofilms?
Which type of microscope would be the best choice for viewing very small surface structures of a cell?
Which type of microscope would be the best choice for viewing very small surface structures of a cell?
What type of microscope uses an annular stop?
What type of microscope uses an annular stop?
What type of microscope uses a cone of light so that light only hits the specimen indirectly, producing a light image on a dark background?
What type of microscope uses a cone of light so that light only hits the specimen indirectly, producing a light image on a dark background?
What mordant is used in Gram staining?
What mordant is used in Gram staining?
What is one difference between specimen preparation for a transmission electron microscope (TEM) and preparation for a scanning electron microscope (SEM)?
What is one difference between specimen preparation for a transmission electron microscope (TEM) and preparation for a scanning electron microscope (SEM)?
When you see light bend as it moves from air into water, you are observing _________.
When you see light bend as it moves from air into water, you are observing _________.
A microscope that uses multiple lenses is called a _________ microscope.
A microscope that uses multiple lenses is called a _________ microscope.
Chromophores that absorb and then emit light are called __________.
Chromophores that absorb and then emit light are called __________.
In a(n) _______ microscope, a probe located just above the specimen moves up and down in response to forces between the atoms and the tip of the probe.
In a(n) _______ microscope, a probe located just above the specimen moves up and down in response to forces between the atoms and the tip of the probe.
What is the total magnification of a specimen that is being viewed with a standard ocular lens and a 40⨯ objective lens?
What is the total magnification of a specimen that is being viewed with a standard ocular lens and a 40⨯ objective lens?
Ziehl-Neelsen staining, a type of _______ staining, is diagnostic for Mycobacterium tuberculosis.
Ziehl-Neelsen staining, a type of _______ staining, is diagnostic for Mycobacterium tuberculosis.
The _______ is used to differentiate bacterial cells based on the components of their cell walls.
The _______ is used to differentiate bacterial cells based on the components of their cell walls.
Explain how a prism separates white light into different colors.
Explain how a prism separates white light into different colors.
Why is Antonie van Leeuwenhoek's work much better known than that of Zaccharias Janssen?
Why is Antonie van Leeuwenhoek's work much better known than that of Zaccharias Janssen?
Why did the cork cells observed by Robert Hooke appear to be empty, as opposed to being full of other structures?
Why did the cork cells observed by Robert Hooke appear to be empty, as opposed to being full of other structures?
What is the function of the condenser in a brightfield microscope?
What is the function of the condenser in a brightfield microscope?
Label each component of the brightfield microscope.
Label each component of the brightfield microscope.
How could you identify whether a particular bacterial sample contained specimens with mycolic acid-rich cell walls?
How could you identify whether a particular bacterial sample contained specimens with mycolic acid-rich cell walls?
In which of the following has the lowest energy?
In which of the following has the lowest energy?
When focusing a light microscope, why is it best to adjust the focus using the coarse focusing knob before using the fine focusing knob?
When focusing a light microscope, why is it best to adjust the focus using the coarse focusing knob before using the fine focusing knob?
You need to identify structures within a cell using a microscope. However, the image appears very blurry even though you have a high magnification. What are some things that you could try to improve the resolution of the image? Describe the most basic factors that affect resolution when you first put the slide onto the stage; then consider more specific factors that could affect resolution for 40⨯ and 100⨯ lenses.
You need to identify structures within a cell using a microscope. However, the image appears very blurry even though you have a high magnification. What are some things that you could try to improve the resolution of the image? Describe the most basic factors that affect resolution when you first put the slide onto the stage; then consider more specific factors that could affect resolution for 40⨯ and 100⨯ lenses.
You use the Gram staining procedure to stain an L-form bacterium (a bacterium that lacks a cell wall). What color will the bacterium be after the staining procedure is finished?
You use the Gram staining procedure to stain an L-form bacterium (a bacterium that lacks a cell wall). What color will the bacterium be after the staining procedure is finished?
Protozoa have which of these characteristics (choose all that apply):
Protozoa have which of these characteristics (choose all that apply):
Cyanobacteria are the only Prokaryotes to:
Cyanobacteria are the only Prokaryotes to:
Which of the following are characteristics of Gram-Positive Bacteria?
Which of the following are characteristics of Gram-Positive Bacteria?
One potential bacterial benefit of horizontal gene transfer could be:
One potential bacterial benefit of horizontal gene transfer could be:
Where do Extremophiles thrive?
Where do Extremophiles thrive?
Where can Methanogens thrive?
Where can Methanogens thrive?
What do we know about Archaea?
What do we know about Archaea?
What is a key difference between eukaryotic cells genetic makeup and prokaryotic genetic makeup?
What is a key difference between eukaryotic cells genetic makeup and prokaryotic genetic makeup?
What is a key difference between eukaryotes and prokaryotes?
What is a key difference between eukaryotes and prokaryotes?
What are some of the characteristics of microorganisms?
What are some of the characteristics of microorganisms?
What does HLM stand for?
What does HLM stand for?
These colors represent:
These colors represent:
This allows for what to occur?
This allows for what to occur?
How are the #D portions of this object able to communicate with the cells living on the surface?
How are the #D portions of this object able to communicate with the cells living on the surface?
What is being discussed in this second video?
What is being discussed in this second video?
What is the difference between these two images?
What is the difference between these two images?
What are some of the benefits of this new technology (mark all that apply)
What are some of the benefits of this new technology (mark all that apply)
What is a downfall with conventional passive guidewire?
What is a downfall with conventional passive guidewire?
This device is controlled by:
This device is controlled by:
Engineers at what University developed this Robo-thread?
Engineers at what University developed this Robo-thread?
Flashcards
Microscopy
Microscopy
Microscopes manipulate light to visualize and magnify images of microbial cells, aiding in species identification and disease diagnosis.
Brightfield Microscopy
Brightfield Microscopy
Brightfield microscopy renders a darker image on a lighter background, useful for observing stained cells.
Darkfield Microscopy
Darkfield Microscopy
Darkfield microscopy increases contrast, rendering a brighter image on a darker background. Good for viewing live, unstained specimens.
Wavelength
Wavelength
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Amplitude
Amplitude
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Frequency
Frequency
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Reflection
Reflection
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Absorbance
Absorbance
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Transmission
Transmission
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Diffraction
Diffraction
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Refraction
Refraction
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Refractive Index
Refractive Index
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Magnification
Magnification
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Resolution
Resolution
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Contrast
Contrast
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Girolamo Fracastoro
Girolamo Fracastoro
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Fomes
Fomes
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Antonie van Leeuwenhoek
Antonie van Leeuwenhoek
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Simple Microscope
Simple Microscope
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Compound Microscope
Compound Microscope
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Robert Hooke
Robert Hooke
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Hans & Zaccharias Janssen
Hans & Zaccharias Janssen
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Light Microscopes
Light Microscopes
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Total Magnification
Total Magnification
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Coarse Focusing Knob
Coarse Focusing Knob
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Fine Focusing Knob
Fine Focusing Knob
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Condenser Lens
Condenser Lens
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Oil Immersion Lens
Oil Immersion Lens
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Phase-Contrast Microscopes
Phase-Contrast Microscopes
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Differential Interference Contrast (DIC) Microscopes
Differential Interference Contrast (DIC) Microscopes
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Fixation
Fixation
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Wet Mount
Wet Mount
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Positive Stain
Positive Stain
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Negative Stain
Negative Stain
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Simple Staining
Simple Staining
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Differential Staining
Differential Staining
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Gram Staining
Gram Staining
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Crystal Violet
Crystal Violet
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Mordant
Mordant
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Acid-Fast Staining
Acid-Fast Staining
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Reflection (Light)
Reflection (Light)
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Absorption (Light)
Absorption (Light)
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Transmission (Light)
Transmission (Light)
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Refraction (Light)
Refraction (Light)
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Lens
Lens
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Dispersion (Light)
Dispersion (Light)
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Numerical Aperture
Numerical Aperture
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Study Notes
Introduction
- Most cells and microorganisms lack color and contrast in their natural state, making it difficult to observe cellular structures without artificial treatment.
- Staining techniques are used to identify specific microbes, cellular structures, DNA sequences, or infection indicators in tissue samples.
Preparing Specimens for Light Microscopy
- Two basic preparation types for light microscopes are wet mounts and fixed specimens.
Wet Mounts
- The specimen is placed on a slide in a liquid drop.
- Liquid specimens can be deposited directly, while solid specimens require a liquid drop.
- Stains are often added to enhance contrast.
- A coverslip is placed on top before examination.
Fixed Specimens
- Fixation attaches cells to a slide, typically by heating (heat fixing) or chemical treatment.
- Fixation kills microorganisms, halts movement and metabolism, and preserves cellular components.
- Heat fixing involves spreading a thin specimen layer (smear) on the slide and briefly heating it.
- Chemical fixatives like acetic acid, ethanol, methanol, formaldehyde (formalin), and glutaraldehyde denature proteins, stop reactions, and stabilize cell structures.
Staining
- Staining is typically applied to color features before observation under a light microscope.
- Stains contain salts with a positive and a negative ion.
- The chromophore (colored ion) can be positive or negative, while the uncolored ion is the counterion.
- Basic dyes have a positively charged chromophore, acidic dyes have a negatively charged chromophore.
- Positive stains are absorbed by the cells or organisms, adding color for contrast.
- Negative stains are absorbed by the background, outlining the organisms.
Types of Dyes
- Basic dyes with positive chromophores stick to negatively charged cell walls and act as positive stains.
- Examples of basic dyes include basic fuchsin, crystal violet, malachite green, methylene blue, and safranin.
- Acidic dyes with negatively charged chromophores are repelled by negatively charged cell walls and act as negative stains.
- Examples of acidic dyes include acid fuchsin, eosin, and rose bengal.
Simple vs. Differential Staining
- Simple staining uses one dye to emphasize particular structures, making all organisms appear the same color.
- Differential staining uses multiple stains to distinguish organisms based on their interactions, resulting in different colors.
- Differential staining techniques include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining.
Gram Staining
- A differential staining procedure developed by Hans Christian Gram in 1884.
- Distinguishes bacteria with different cell wall types.
Gram Staining Steps
- Crystal violet (primary stain) colors all cells purple.
- Gram’s iodine (mordant) stabilizes stains and dyes.
- Mordant acts like a trapping agent, complexes with the crystal violet.
- Decolorizing agent (ethanol or acetone/ethanol) washes dye out of cells with thin peptidoglycan layers, making them colorless.
- Safranin (counterstain) stains decolorized cells pink.
- Gram-positive cells appear purple, while gram-negative cells appear red.
- Older bacterial cells with damaged cell walls may appear gram-negative, so fresh cultures are best.
- Errors like leaving the decolorizer on too long can affect results.
- Gram staining helps classify bacterial pathogens and their properties.
- Gram-negative bacteria tend to be more resistant to certain antibiotics.
Clinical Focus: Gram Staining
- Gram staining is commonly used as an early step in identifying pathogenic bacteria.
- Purple, grape-like clusters of spherical cells under a brightfield microscope indicate gram-positive bacteria.
Acid-Fast Stains
- A differential staining technique used to differentiate gram-positive cells with and without waxy mycolic acids in their cell walls.
- Two methods: Ziehl-Neelsen and Kinyoun techniques.
- Both use carbolfuchsin as the primary stain.
- Acid-fast cells retain carbolfuchsin after decolorizing, while non-acid-fast cells are counterstained blue with methylene blue.
- The Ziehl-Neelsen method uses heat to infuse carbolfuchsin, while the Kinyoun method does not.
- Important diagnostic tools for diseases caused by acid-fast bacteria (AFB).
Micro Connections: Using Microscopy to Diagnose Tuberculosis
- Mycobacterium tuberculosis, which causes tuberculosis, can be detected based on presence of acid-fast bacilli.
- A smear is prepared from the patient’s sputum and stained using the Ziehl-Neelsen technique.
- If acid-fast bacteria are confirmed, they are generally cultured to make a positive identification.
- Immunofluorescence uses fluorochrome-labeled antibodies to bind to M. tuberculosis.
Capsule Staining
- Capsules are protective outer structures related to microbial virulence.
- Capsules do not absorb basic dyes, so negative staining is used.
- The dye stains the background, leaving capsules as halos around the cells.
- Specimens do not need to be heat-fixed prior to negative staining.
- India ink or nigrosin can be added to the specimen.
- Positive and negative staining can be combined: positive stain colors the cell, negative stain colors the background, leaving a halo around each cell.
Endospore Staining
- Endospores are structures within bacterial cells that allow them to survive harsh conditions.
- Gram staining alone cannot visualize endospores, which appear clear.
- Endospore staining uses two stains to differentiate endospores from the rest of the cell.
- The Schaeffer-Fulton method uses heat to push malachite green (primary stain) into the endospore.
- Washing with water decolorizes the cell, but the endospore retains the green stain.
- The cell is counterstained pink with safranin.
- Green endospores appear within pink vegetative cells or separately, if no endospores are present, only pink vegetative cells are visible.
- Endospore-staining identifies Bacillus, Clostridium, and Clostridioides.
Flagella Staining
- Flagella are tail-like structures used for locomotion by some bacteria, archaea, and eukaryotes.
- Flagella are too thin to be seen without specialized staining.
- Flagella staining thickens flagella by applying mordant (tannic acid), which coats the flagella.
- The specimen is stained with pararosaniline or basic fuchsin.
- The location and number of flagella classify and identify bacteria.
Preparing Specimens for Electron Microscopy
- Samples for TEM must have very thin sections.
- Cells are embedded in plastic resin, dehydrated through ethanol solutions, and thin sections are cut using an ultramicrotome.
- Samples are fixed to copper wire or carbon-fiber grids and stained with uranyl acetate or osmium tetroxide.
- Samples for SEM are dehydrated using an ethanol series and critical point drying with liquid carbon dioxide.
- After drying, the specimens are sputter-coated with metal to prevent charging by the SEM’s electron beam.
Properties of Light
- Light waves interacting with materials may be reflected, absorbed, or transmitted, depending on the properties of the material.
- Light waves can interact with each other (interference) or be distorted by interactions with small objects or openings (diffraction).
- Refraction occurs when light waves change speed and direction as they pass from one medium to another.
- Differences in the refraction indices of two materials determine the magnitude of directional changes when light passes from one to the other.
- A lens is a medium with a curved surface that refracts and focuses light to produce an image.
- Visible light is part of the electromagnetic spectrum.
- Light waves of different frequencies and wavelengths are distinguished as colors by the human eye.
- A prism can separate the colors of white light (dispersion) because different frequencies of light have different refractive indices for a given material.
- Fluorescent dyes and phosphorescent materials can effectively transform nonvisible electromagnetic radiation into visible light.
- The power of a microscope can be described in terms of its magnification and resolution.
- Resolution can be increased by shortening wavelength, increasing the numerical aperture of the lens, or using stains that enhance contrast.
Peering Into the Invisible World
- Antonie van Leeuwenhoek is credited with the first observation of microbes, including protists and bacteria, with simple microscopes that he made.
- Robert Hooke was the first to describe what we now call cells.
- Simple microscopes have a single lens, while compound microscopes have multiple lenses.
Instruments of Microscopy
- Numerous types of microscopes use various technologies to generate micrographs.
- Most are useful for a particular type of specimen or application.
- Light microscopy uses lenses to focus light on a specimen to produce an image.
- Commonly used light microscopes include brightfield, darkfield, phase-contrast, differential interference contrast, fluorescence, confocal, and two-photon microscopes.
- Electron microscopy focuses electrons on the specimen using magnets, producing much greater magnification than light microscopy.
- The transmission electron microscope (TEM) and scanning electron microscope (SEM) are two common forms.
- Scanning probe microscopy produces images of even greater magnification by measuring feedback from sharp probes that interact with the specimen.
- Probe microscopes include the scanning tunneling microscope (STM) and the atomic force microscope (AFM).
Staining Microscopic Specimens
- Samples must be properly prepared for microscopy, this may involve staining, fixation, and/or cutting thin sections.
- A variety of staining techniques can be used with light microscopy, including Gram staining, acid-fast staining, capsule staining, endospore staining, and flagella staining.
- Samples for TEM require very thin sections, whereas samples for SEM require sputter-coating.
- Preparation for fluorescence microscopy is similar to that for light microscopy, except that fluorochromes are used.
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