Microbiology Quiz: Electron Microscopy
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Electron MicroscopyQuestion 1 of 20

A researcher has a purified suspension of bacteriophages and needs to quickly assess their basic morphology (e.g., head and tail structure) using TEM, without resorting to the lengthy process of embedding and sectioning. Which of the following is the most appropriate and rapid preparation technique?

Negative staining
Freeze-fracture replication
Critical-point drying
Immunogold labeling
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Microbiology Quiz

Microbiology Quiz: Electron Microscopy

Practice Electron Microscopy in Microbiology with focused quiz questions that help you check what you know, review explanations, and build confidence with test-style prompts.

What this quiz covers

This quiz focuses on Electron Microscopy, giving you a quick way to practice the rules, question types, and explanations that matter most for Microbiology.

How to use this quiz

Try each quiz question before looking at the correct answer. Use the explanations to review missed ideas, then come back to similar questions until the pattern feels familiar.

All questions

Question 1

A researcher has a purified suspension of bacteriophages and needs to quickly assess their basic morphology (e.g., head and tail structure) using TEM, without resorting to the lengthy process of embedding and sectioning. Which of the following is the most appropriate and rapid preparation technique?

  1. Negative staining (correct answer)
  2. Freeze-fracture replication
  3. Critical-point drying
  4. Immunogold labeling
Explanation: Negative staining is a simple and rapid technique for visualizing small particulate specimens like viruses or protein complexes. The sample is mixed with a solution of a heavy metal salt (e.g., uranyl acetate), which is allowed to dry on the grid. The stain pools around the particles and penetrates surface crevices, creating a dark background against which the unstained, electron-lucent particle is clearly outlined. Freeze-fracture (B) is for viewing internal membrane faces. Critical-point drying (C) is for SEM. Immunogold labeling (D) is for localizing specific antigens, not for general morphology.

Question 2

A researcher has a purified suspension of bacteriophages and needs to quickly assess their basic morphology (e.g., head and tail structure) using TEM, without resorting to the lengthy process of embedding and sectioning. Which of the following is the most appropriate and rapid preparation technique?

  1. Negative staining (correct answer)
  2. Freeze-fracture replication
  3. Critical-point drying
  4. Immunogold labeling
Explanation: Negative staining is a simple and rapid technique for visualizing small particulate specimens like viruses or protein complexes. The sample is mixed with a solution of a heavy metal salt (e.g., uranyl acetate), which is allowed to dry on the grid. The stain pools around the particles and penetrates surface crevices, creating a dark background against which the unstained, electron-lucent particle is clearly outlined. Freeze-fracture (B) is for viewing internal membrane faces. Critical-point drying (C) is for SEM. Immunogold labeling (D) is for localizing specific antigens, not for general morphology.

Question 3

A large, dynamic, multi-protein complex has proven impossible to crystallize for X-ray diffraction studies. Which alternative technique is most suitable for determining a near-atomic resolution 3D structure of this complex, and why?

  1. Single-particle cryo-electron microscopy, because it analyzes thousands of images of individual, frozen complexes, bypassing the need for crystals. (correct answer)
  2. Scanning electron microscopy, because it can directly image the three-dimensional surface of the complex.
  3. Electron tomography, because it can reconstruct the complex by tilting the specimen.
  4. Atomic force microscopy, because it can physically probe the atomic contours of the complex.
Explanation: When you encounter questions about determining protein structure, especially for large, dynamic complexes that resist crystallization, you need to consider which techniques can achieve atomic-level resolution without requiring ordered crystal lattices. Single-particle cryo-electron microscopy (cryo-EM) is the ideal solution here because it captures thousands of images of individual protein complexes frozen in vitreous ice, preserving their native conformations. Advanced computational algorithms then align and average these images to reconstruct a high-resolution 3D structure, often reaching near-atomic resolution (2-4 Å). This technique has revolutionized structural biology precisely because it bypasses crystallization requirements while maintaining exceptional detail. Option B (scanning electron microscopy) only provides surface topology information at much lower resolution and cannot reveal internal atomic arrangements. Option C (electron tomography) reconstructs 3D structures by tilting specimens, but it typically achieves only moderate resolution (20-50 Å) - insufficient for atomic detail. Option D (atomic force microscopy) can probe surface contours at high resolution, but only examines surfaces, not complete 3D atomic structures, and requires the protein to be immobilized on a substrate. The key distinction is that cryo-EM combines the preservation advantages of flash-freezing with sophisticated image processing to overcome the traditional crystallization bottleneck while still achieving the atomic-level detail needed for understanding protein function. Remember: when you see "large, dynamic, impossible to crystallize" paired with "near-atomic resolution," cryo-EM is almost always your answer. It's specifically designed to solve this exact structural biology challenge.

Question 4

A researcher is examining a TEM image at 50,000x and can just resolve two adjacent protein subunits. If the magnification is increased to 200,000x on the screen, the image of the subunits becomes larger but remains a single blurred object. This phenomenon demonstrates that:

  1. the accelerating voltage of the microscope is set too low.
  2. the useful magnification has been exceeded due to the microscope's resolution limit. (correct answer)
  3. the specimen has been damaged by the intensity of the electron beam.
  4. the heavy metal stain has precipitated, obscuring fine details.
Explanation: This is a classic example of 'empty magnification.' The resolving power (resolution) of a microscope is its ability to distinguish two closely spaced points. Once the magnification is increased beyond the point where the finest details resolvable by the instrument are already visible, further magnification simply enlarges the blur. The useful magnification has been exceeded because it is limited by the actual resolution achieved. While a low accelerating voltage (A) could limit resolution, the core concept being illustrated is the relationship between resolution and useful magnification. Beam damage (C) or stain precipitation (D) would present different visual artifacts.

Question 5

In immunoelectron microscopy, a secondary antibody is often used to detect the primary antibody bound to its target antigen. For the location to be visible in the TEM, this secondary antibody must have a specific modification, which is:

  1. conjugation to an enzyme like horseradish peroxidase.
  2. labeling with a fluorophore like fluorescein isothiocyanate (FITC).
  3. attachment to an electron-dense particle such as colloidal gold. (correct answer)
  4. cross-linking with glutaraldehyde to increase its molecular weight.
Explanation: Electrons in a TEM are scattered by atoms with high atomic numbers. To make an antibody visible, it must be linked to something highly electron-dense. Colloidal gold particles are ideal because they are extremely dense, inert, and can be manufactured in uniform sizes. Horseradish peroxidase (A) is used for colorimetric detection in light microscopy or blot assays. Fluorophores (B) are used in fluorescence microscopy. Glutaraldehyde (D) is a fixative and is not used to label antibodies for visualization.

Question 6

A TEM image of a bacterium prepared by ultramicrotomy displays a series of fine, parallel lines running in the direction of the knife travel. This artifact is known as 'knife marks' and is caused by an issue in which preparation step?

  1. Fixation
  2. Staining
  3. Sectioning (correct answer)
  4. Embedding
Explanation: Knife marks are a classic artifact of the sectioning step. They are caused by imperfections in the edge of the glass or diamond knife used in the ultramicrotome, such as nicks, debris, or a dull edge, which then drag across the block face, creating scratches in the resin section. Staining artifacts (B) typically appear as precipitates or crystals. Fixation (A) or embedding (D) errors lead to poor preservation or polymerization, causing issues like holes or chatter (vibrations during cutting), not fine parallel scratches.

Question 7

A researcher attempts to use immunogold labeling on thin sections to find the subcellular location of a specific enzyme. The tissue was fixed with 2.5% glutaraldehyde, post-fixed with 1% osmium tetroxide, and embedded in epoxy resin. Despite using a high-titer primary antibody, no gold particles are detected bound to the sections. What is the most likely reason for the labeling failure?

  1. The osmium tetroxide post-fixation is electron-dense and completely obscured the gold particles.
  2. The glutaraldehyde fixation aggressively cross-linked proteins, likely destroying the antigen's epitope. (correct answer)
  3. The gold particles, being heavy, were dislodged and washed away during the staining steps.
  4. The epoxy resin is hydrophobic and repelled the aqueous antibody solution from the section.
Explanation: Glutaraldehyde is a powerful cross-linking fixative that is excellent for preserving ultrastructure. However, this extensive cross-linking can alter the three-dimensional conformation of proteins, including the specific epitope recognized by the primary antibody. This 'masking' of the epitope is a common cause of failure in immunolabeling on conventionally processed samples. While osmium tetroxide (A) is electron-dense, it would not completely hide the distinct, spherical gold particles. Gold particles bind very tightly via the antibody-antigen interaction and are not easily washed away (C). While resin hydrophobicity (D) can be a factor, epitope masking by the fixative is a more fundamental and frequent problem.

Question 8

An SEM image of a bacterial biofilm on a catheter surface shows some regions with excellent detail, while others are excessively bright, distorted, and lack clear focus. This phenomenon, known as 'charging,' is most likely due to a failure in which preparatory step?

  1. Primary fixation with glutaraldehyde
  2. Sputter coating with a conductive metal (correct answer)
  3. Critical-point drying
  4. Dehydration with acetone
Explanation: Charging artifacts in SEM occur when the electron beam strikes a non-conductive surface, causing electrons to accumulate. This accumulated negative charge deflects the incoming primary electron beam, leading to bright, distorted patches in the image. To prevent this, biological specimens are coated with a thin layer of a conductive metal like gold or palladium via sputter coating. Insufficient or uneven coating is the direct cause of charging. Failures in fixation (A), drying (C), or dehydration (D) would cause structural artifacts (decomposition, collapse, shrinkage) but not the specific electronic artifact of charging.

Question 9

A researcher is investigating a novel virus that buds from the plasma membrane of infected host cells. To best visualize the three-dimensional morphology of the viral particles as they emerge from the cell surface, which microscopy technique is most appropriate?

  1. Transmission electron microscopy (TEM) of ultrathin sections
  2. Scanning electron microscopy (SEM) (correct answer)
  3. Cryo-electron tomography
  4. Phase-contrast light microscopy
Explanation: Scanning electron microscopy (SEM) is the ideal technique for visualizing surface topography. It scans the surface of a specimen with a focused beam of electrons to produce a three-dimensional image, which is exactly what is needed to observe viruses budding from a cell surface. TEM of thin sections (A) would show internal cross-sections of the cell and budding viruses but not the overall 3D surface morphology. Cryo-electron tomography (C) can provide high-resolution 3D reconstructions but is typically used for isolated macromolecules or subcellular structures, and SEM is more direct for surface topography of whole cells. Phase-contrast microscopy (D) lacks the necessary resolution to visualize individual virus particles.

Question 10

A student's protocol for observing the surface pili on Escherichia coli includes the following steps: (1) chemical fixation, (2) dehydration in ethanol, (3) embedding in epoxy resin, (4) ultrathin sectioning, and (5) viewing with a scanning electron microscope. Why is this protocol fundamentally flawed for the stated objective?

  1. Chemical fixation will destroy the delicate pili structures before they can be imaged.
  2. The ethanol dehydration step will cause the bacteria to shrink and lyse.
  3. An SEM cannot achieve the resolution necessary to visualize individual pili.
  4. Embedding in resin and sectioning are for TEM internal views and will obscure the cell surface. (correct answer)
Explanation: When approaching questions about microscopy techniques, you need to match the preparation method to the type of microscope and the specific structures you want to observe. Surface features require different protocols than internal structures. The correct answer is D because this protocol is designed for transmission electron microscopy (TEM) of internal cellular structures, not for scanning electron microscopy (SEM) of surface features. Embedding specimens in epoxy resin and cutting ultrathin sections are hallmarks of TEM sample preparation, where you need to see through the specimen. However, pili are external appendages on the bacterial surface that need to be viewed from the outside. When you embed and section the bacteria, you're essentially cutting through them and destroying the very surface structures you want to observe. SEM requires specimens to be dried, coated with metal, and viewed whole to see surface topography. Let's examine why the other options are incorrect: A is wrong because chemical fixation actually preserves cellular structures, including pili, by cross-linking proteins and preventing degradation. B is incorrect because ethanol dehydration is a standard step in electron microscopy preparation and, when done properly through graded alcohols, doesn't cause significant cellular damage. C is false because SEM can absolutely resolve individual pili, which are typically 2-8 nanometers in diameter—well within SEM's resolution capabilities. Remember this key distinction: TEM uses thin sections to see internal structures, while SEM examines whole specimens to visualize surface features. Match your sample preparation to your microscopy goal and the structures you're targeting.

Question 11

What is the principal advantage of using cryo-electron microscopy (cryo-EM) to determine the structure of a large protein complex compared to traditional TEM of a negatively stained or sectioned sample?

  1. Cryo-EM achieves higher levels of magnification than are possible with conventional TEM instruments.
  2. It allows for the visualization of macromolecules in a fully hydrated, near-native conformation. (correct answer)
  3. The sample preparation for cryo-EM is significantly faster and less technically demanding.
  4. Cryo-EM does not require the use of a high vacuum in the microscope column.
Explanation: The main advantage of cryo-EM is its ability to preserve the specimen in a vitrified (non-crystalline), hydrated state. This avoids the potentially damaging and artifact-inducing procedures of chemical fixation, dehydration, and staining used in conventional TEM. Consequently, the structure observed is much closer to its native state in solution. Magnification levels (A) are comparable between cryo-EM and conventional TEM. Cryo-EM sample preparation (C) is technically very demanding. Cryo-EM absolutely requires a high vacuum (D) to prevent the frozen sample from sublimating and to allow the electron beam to travel.

Question 12

A TEM micrograph of a thin-sectioned bacterium reveals several small, dark, granular structures scattered throughout the cytoplasm and a particularly large, highly electron-dense (very dark) spherical body. Which of the following correctly identifies these structures?

  1. Small structures are ribosomes; large body is a polyphosphate granule. (correct answer)
  2. Small structures are plasmids; large body is the nucleoid.
  3. Small structures are vesicles; large body is a ribosome.
  4. Small structures are polyphosphate granules; large body is the nucleoid.
Explanation: When interpreting TEM micrographs of bacterial cells, you need to recognize structures based on their size, electron density, and distribution patterns. Electron-dense (dark) structures contain materials that scatter electrons strongly, while less dense areas appear lighter. The small, dark granular structures scattered throughout the cytoplasm are ribosomes. Bacterial ribosomes are numerous (thousands per cell), roughly 20nm in diameter, and appear as small dark dots because their RNA and protein components are electron-dense. The particularly large, highly electron-dense spherical body is most likely a polyphosphate granule - an inclusion body that stores phosphate and appears very dark due to its high mineral content. Looking at the wrong answers: Option B incorrectly identifies small structures as plasmids, but plasmids are DNA molecules that wouldn't appear as distinct granular structures in thin sections, and the nucleoid (bacterial chromosome region) appears as a lighter, less electron-dense area, not dark. Option C suggests the small structures are vesicles, but bacterial vesicles are membrane-bound and would appear as light circles with dark borders, plus no ribosome could be "particularly large" since they're uniformly small. Option D reverses the identification - polyphosphate granules vary in size but the nucleoid characteristically appears light, not dark, because DNA is less electron-dense than the surrounding cytoplasm. Remember this pattern: in bacterial TEM images, numerous small dark dots = ribosomes, and the nucleoid appears relatively light while inclusion bodies (like polyphosphate granules) appear very dark.

Question 13

A scientist aims to improve the theoretical resolution limit of a transmission electron microscope to better resolve the fine structure of a bacterial ribosome. Which of the following adjustments would most directly achieve this goal?

  1. Increasing the current of the tungsten filament in the electron gun
  2. Decreasing the thickness of the specimen section from 70 nm to 30 nm
  3. Increasing the accelerating voltage of the electron beam from 100 kV to 200 kV (correct answer)
  4. Improving the vacuum level within the microscope column from 10⁻⁵ Pa to 10⁻⁷ Pa
Explanation: The theoretical resolution of an electron microscope is fundamentally limited by the de Broglie wavelength of the electrons, which is inversely proportional to their velocity. Increasing the accelerating voltage imparts more kinetic energy to the electrons, increasing their velocity and thereby shortening their wavelength. A shorter wavelength allows for better resolution. Increasing filament current (A) increases beam intensity (brightness) but not resolution. Decreasing specimen thickness (B) improves practical resolution by reducing scattering, but does not change the theoretical limit set by the wavelength. Improving the vacuum (D) is necessary for operation but does not alter the fundamental physics of the electron wavelength.

Question 14

Upon examining a TEM micrograph of a yeast cell, a researcher observes that the cytoplasm appears shrunken and the mitochondria and nucleus are distorted with large, clear spaces separating them from the surrounding cytoplasm. Which step in the sample preparation protocol was most likely performed inadequately?

  1. Staining with uranyl acetate and lead citrate
  2. Fixation with glutaraldehyde
  3. Dehydration with a graded ethanol series (correct answer)
  4. Sectioning with an ultramicrotome
Explanation: The observed artifacts—cellular shrinkage and distortion—are classic signs of improper dehydration. If water is not completely removed by the ethanol series, the remaining water will rapidly sublimate in the high vacuum of the microscope, causing the delicate cellular structures to collapse. Inadequate staining (A) would result in poor contrast, not shrinkage. Poor fixation (B) would lead to poor preservation of ultrastructure (e.g., membrane breaks, extracted cytoplasm) but the described artifact is most characteristic of dehydration failure. Improper sectioning (D) causes artifacts like knife marks (scratches) or chatter (variations in thickness), not general shrinkage.

Question 15

What is the principal advantage of using cryo-electron microscopy (cryo-EM) to determine the structure of a large protein complex compared to traditional TEM of a negatively stained or sectioned sample?

  1. Cryo-EM achieves higher levels of magnification than are possible with conventional TEM instruments.
  2. It allows for the visualization of macromolecules in a fully hydrated, near-native conformation. (correct answer)
  3. The sample preparation for cryo-EM is significantly faster and less technically demanding.
  4. Cryo-EM does not require the use of a high vacuum in the microscope column.
Explanation: The main advantage of cryo-EM is its ability to preserve the specimen in a vitrified (non-crystalline), hydrated state. This avoids the potentially damaging and artifact-inducing procedures of chemical fixation, dehydration, and staining used in conventional TEM. Consequently, the structure observed is much closer to its native state in solution. Magnification levels (A) are comparable between cryo-EM and conventional TEM. Cryo-EM sample preparation (C) is technically very demanding. Cryo-EM absolutely requires a high vacuum (D) to prevent the frozen sample from sublimating and to allow the electron beam to travel.

Question 16

A TEM image of a bacterium prepared by ultramicrotomy displays a series of fine, parallel lines running in the direction of the knife travel. This artifact is known as 'knife marks' and is caused by an issue in which preparation step?

  1. Fixation
  2. Staining
  3. Sectioning (correct answer)
  4. Embedding
Explanation: Knife marks are a classic artifact of the sectioning step. They are caused by imperfections in the edge of the glass or diamond knife used in the ultramicrotome, such as nicks, debris, or a dull edge, which then drag across the block face, creating scratches in the resin section. Staining artifacts (B) typically appear as precipitates or crystals. Fixation (A) or embedding (D) errors lead to poor preservation or polymerization, causing issues like holes or chatter (vibrations during cutting), not fine parallel scratches.

Question 17

A TEM micrograph of a thin-sectioned bacterium reveals several small, dark, granular structures scattered throughout the cytoplasm and a particularly large, highly electron-dense (very dark) spherical body. Which of the following correctly identifies these structures?

  1. Small structures are ribosomes; large body is a polyphosphate granule. (correct answer)
  2. Small structures are plasmids; large body is the nucleoid.
  3. Small structures are vesicles; large body is a ribosome.
  4. Small structures are polyphosphate granules; large body is the nucleoid.
Explanation: When interpreting TEM micrographs of bacterial cells, you need to recognize structures based on their size, electron density, and distribution patterns. Electron-dense (dark) structures contain materials that scatter electrons strongly, while less dense areas appear lighter. The small, dark granular structures scattered throughout the cytoplasm are ribosomes. Bacterial ribosomes are numerous (thousands per cell), roughly 20nm in diameter, and appear as small dark dots because their RNA and protein components are electron-dense. The particularly large, highly electron-dense spherical body is most likely a polyphosphate granule - an inclusion body that stores phosphate and appears very dark due to its high mineral content. Looking at the wrong answers: Option B incorrectly identifies small structures as plasmids, but plasmids are DNA molecules that wouldn't appear as distinct granular structures in thin sections, and the nucleoid (bacterial chromosome region) appears as a lighter, less electron-dense area, not dark. Option C suggests the small structures are vesicles, but bacterial vesicles are membrane-bound and would appear as light circles with dark borders, plus no ribosome could be "particularly large" since they're uniformly small. Option D reverses the identification - polyphosphate granules vary in size but the nucleoid characteristically appears light, not dark, because DNA is less electron-dense than the surrounding cytoplasm. Remember this pattern: in bacterial TEM images, numerous small dark dots = ribosomes, and the nucleoid appears relatively light while inclusion bodies (like polyphosphate granules) appear very dark.

Question 18

A research team wants to produce a detailed 3D reconstruction of the internal mitochondrial network (the 'chondriome') within an intact eukaryotic cell. Which electron microscopy approach would be most suitable for this specific objective?

  1. High-resolution scanning electron microscopy (SEM) of the intact cell.
  2. Transmission electron microscopy (TEM) of a single ultrathin section.
  3. Freeze-fracture TEM to visualize the mitochondrial membranes.
  4. Electron tomography using a series of tilted TEM images from a thick section. (correct answer)
Explanation: Electron tomography is specifically designed for 3D reconstruction of internal cellular structures. It involves taking multiple TEM images of a relatively thick (200-500 nm) section as it is tilted through a range of angles. These 2D projection images are then computationally combined to reconstruct the 3D volume. SEM (A) only shows surface features. A single thin section (B) provides only a 2D slice of the network. Freeze-fracture (C) reveals the surfaces of internal membranes but does not show the overall 3D organization of the entire network within the cell.

Question 19

A student's protocol for observing the surface pili on Escherichia coli includes the following steps: (1) chemical fixation, (2) dehydration in ethanol, (3) embedding in epoxy resin, (4) ultrathin sectioning, and (5) viewing with a scanning electron microscope. Why is this protocol fundamentally flawed for the stated objective?

  1. Chemical fixation will destroy the delicate pili structures before they can be imaged.
  2. The ethanol dehydration step will cause the bacteria to shrink and lyse.
  3. An SEM cannot achieve the resolution necessary to visualize individual pili.
  4. Embedding in resin and sectioning are for TEM internal views and will obscure the cell surface. (correct answer)
Explanation: When approaching questions about microscopy techniques, you need to match the preparation method to the type of microscope and the specific structures you want to observe. Surface features require different protocols than internal structures. The correct answer is D because this protocol is designed for transmission electron microscopy (TEM) of internal cellular structures, not for scanning electron microscopy (SEM) of surface features. Embedding specimens in epoxy resin and cutting ultrathin sections are hallmarks of TEM sample preparation, where you need to see through the specimen. However, pili are external appendages on the bacterial surface that need to be viewed from the outside. When you embed and section the bacteria, you're essentially cutting through them and destroying the very surface structures you want to observe. SEM requires specimens to be dried, coated with metal, and viewed whole to see surface topography. Let's examine why the other options are incorrect: A is wrong because chemical fixation actually preserves cellular structures, including pili, by cross-linking proteins and preventing degradation. B is incorrect because ethanol dehydration is a standard step in electron microscopy preparation and, when done properly through graded alcohols, doesn't cause significant cellular damage. C is false because SEM can absolutely resolve individual pili, which are typically 2-8 nanometers in diameter—well within SEM's resolution capabilities. Remember this key distinction: TEM uses thin sections to see internal structures, while SEM examines whole specimens to visualize surface features. Match your sample preparation to your microscopy goal and the structures you're targeting.

Question 20

A researcher attempts to use immunogold labeling on thin sections to find the subcellular location of a specific enzyme. The tissue was fixed with 2.5% glutaraldehyde, post-fixed with 1% osmium tetroxide, and embedded in epoxy resin. Despite using a high-titer primary antibody, no gold particles are detected bound to the sections. What is the most likely reason for the labeling failure?

  1. The osmium tetroxide post-fixation is electron-dense and completely obscured the gold particles.
  2. The glutaraldehyde fixation aggressively cross-linked proteins, likely destroying the antigen's epitope. (correct answer)
  3. The gold particles, being heavy, were dislodged and washed away during the staining steps.
  4. The epoxy resin is hydrophobic and repelled the aqueous antibody solution from the section.
Explanation: Glutaraldehyde is a powerful cross-linking fixative that is excellent for preserving ultrastructure. However, this extensive cross-linking can alter the three-dimensional conformation of proteins, including the specific epitope recognized by the primary antibody. This 'masking' of the epitope is a common cause of failure in immunolabeling on conventionally processed samples. While osmium tetroxide (A) is electron-dense, it would not completely hide the distinct, spherical gold particles. Gold particles bind very tightly via the antibody-antigen interaction and are not easily washed away (C). While resin hydrophobicity (D) can be a factor, epitope masking by the fixative is a more fundamental and frequent problem.