Microbiology Portage Learning 3
Define the measurements micrometer and nanometer.
The micrometer is used to measure the size of cells. It is one-millionth of a meter and written as 10^-6m. The
nanometer measures cellular organelles and viruses. It is one-billionth of a meter and written out as 10^-9m.
What are the two critical factors that influence your ability to see an object?
Resolution and contrast. The resolution refers to the distance between two objects at which the objects can still
be seen as separate. The contrast is the difference in light absorbance between two areas (objects).
If you wish to increase the amount of light going into a microscope, what part would you adjust?
The iris diaphragm of the microscope. This part controls the amount of light that passes through the sample and
into the objective lens.
As light passes through a microscope, what is the last piece that light passes before reaching your eyes?
Once the light is through the sample and objective lens, it's directed through the ocular lens (eyepiece) which
then goes to your eyes.
How is the total magnification of an object calculated?
Multiply the power of the objective and the power of the eyepiece.
What is one limitation of fixing your sample?
One limitation of fixing the sample is that it can kill the organism that is on the slide. This prevents the ability to
look at the motility of the sample.
Phase-contrast microscopy provided what benefits to imaging?
Phase contrast allows to the viewing of internal structures of live specimen and observe motility (cilia and flagella)
without staining. With the use of specialized condensers and objects, this microscope amplifies the slight
differences between the cell and the surrounding background.
What is the distinguishing feature of dark field microscopy?
With dark field microscopy, its feature is that the filter inhibits light from going through the organism and instead
light is reflected by the organism.
Unlike brightfield microscope, fluorescence microscopes illuminate samples through what spectrum?
The energy of the incoming light is in the form of the ultraviolet (UV) spectrum.
What is the primary difference between TEM and SEM?
Transmission Electron (TEM) visualizes internal ultras structural details of cells, viruses, and bacteria where the
electron beams pass through the sample.
Scanning Electron (SEM) visualizes surface details and the electron beams reflect off the surface of the
specimen.
Gram staining is based on what basic principle?
The basic principle of Gram staining is that it began with the basic observation that different types of bacteria
react differently to various dyes. Some bacteria will readily take up the specific dye while others will not.
, What is a key determinant in a bacteria being Gram-positive?
Gram-positive bacteria have a thick peptidoglycan layer which allows for this type of bacteria to take up the
staining used. Using dye combinations of crystal violet and iodine, the crystal violet will be retained by the thick
peptidoglycan cell wall and form a stable complex with the addition of iodine effectively trapping the dyes. The
result of this combination is that the cell wall appears purple.
What is the purpose of heat fixing a sample?
Heat fixing is when the sample of the glass slide is passed over a flame until the liquid has been removed from
the sample. The purpose of this allows for the sample to be adhered to the slide prior to going through the
staining process.
What is the primary purpose of a wet mount?
The primary purpose to use a wet mount is for the viewing of live cells for their motility and behavior.
The acid-fast stain is most often used to identify what specific microorganism?
Acid-fast staining is used to identify bacterial stains showing a high degree of resistance to decolorization. This
staining method is used to identify Tuberculosis, Leprosy, and lung and skin infections.
True of False: A nanometer is longer than a micrometer.
False, it is a thousand times smaller then a micrometer
Resolution
Refers to the distance between two objects at which the objects still can be seen as separate. The closer the 2
objects are to each other, the greater the resolution requirement needed to maintain viewing the two objects as
separate
Contrast
The difference in light absorbance between two objects. The lower contrast between an object and its
background, the harder it will be to see that object. The greater the contrast between the two objects, the easier
it will be to visualize.
Magnification
Increase in size of the object
Microscopy
Using light or electrons to magnify objects
Assuming a fixed ocular, identify the part of the microscope you would adjust to enhance the magnification
of a sample.
The objective
What is the total magnification of a sample imaged with a 20x objective and a 15x eyepiece? Show your
math.
20 x 15 = 300x magnification
True or False: A cell that is adherent, flat and unstained is easily identified using bright field microscopy?
False; this type of sample is almost invisible due to the limits of both resolution and contrast
Define the measurements micrometer and nanometer.
The micrometer is used to measure the size of cells. It is one-millionth of a meter and written as 10^-6m. The
nanometer measures cellular organelles and viruses. It is one-billionth of a meter and written out as 10^-9m.
What are the two critical factors that influence your ability to see an object?
Resolution and contrast. The resolution refers to the distance between two objects at which the objects can still
be seen as separate. The contrast is the difference in light absorbance between two areas (objects).
If you wish to increase the amount of light going into a microscope, what part would you adjust?
The iris diaphragm of the microscope. This part controls the amount of light that passes through the sample and
into the objective lens.
As light passes through a microscope, what is the last piece that light passes before reaching your eyes?
Once the light is through the sample and objective lens, it's directed through the ocular lens (eyepiece) which
then goes to your eyes.
How is the total magnification of an object calculated?
Multiply the power of the objective and the power of the eyepiece.
What is one limitation of fixing your sample?
One limitation of fixing the sample is that it can kill the organism that is on the slide. This prevents the ability to
look at the motility of the sample.
Phase-contrast microscopy provided what benefits to imaging?
Phase contrast allows to the viewing of internal structures of live specimen and observe motility (cilia and flagella)
without staining. With the use of specialized condensers and objects, this microscope amplifies the slight
differences between the cell and the surrounding background.
What is the distinguishing feature of dark field microscopy?
With dark field microscopy, its feature is that the filter inhibits light from going through the organism and instead
light is reflected by the organism.
Unlike brightfield microscope, fluorescence microscopes illuminate samples through what spectrum?
The energy of the incoming light is in the form of the ultraviolet (UV) spectrum.
What is the primary difference between TEM and SEM?
Transmission Electron (TEM) visualizes internal ultras structural details of cells, viruses, and bacteria where the
electron beams pass through the sample.
Scanning Electron (SEM) visualizes surface details and the electron beams reflect off the surface of the
specimen.
Gram staining is based on what basic principle?
The basic principle of Gram staining is that it began with the basic observation that different types of bacteria
react differently to various dyes. Some bacteria will readily take up the specific dye while others will not.
, What is a key determinant in a bacteria being Gram-positive?
Gram-positive bacteria have a thick peptidoglycan layer which allows for this type of bacteria to take up the
staining used. Using dye combinations of crystal violet and iodine, the crystal violet will be retained by the thick
peptidoglycan cell wall and form a stable complex with the addition of iodine effectively trapping the dyes. The
result of this combination is that the cell wall appears purple.
What is the purpose of heat fixing a sample?
Heat fixing is when the sample of the glass slide is passed over a flame until the liquid has been removed from
the sample. The purpose of this allows for the sample to be adhered to the slide prior to going through the
staining process.
What is the primary purpose of a wet mount?
The primary purpose to use a wet mount is for the viewing of live cells for their motility and behavior.
The acid-fast stain is most often used to identify what specific microorganism?
Acid-fast staining is used to identify bacterial stains showing a high degree of resistance to decolorization. This
staining method is used to identify Tuberculosis, Leprosy, and lung and skin infections.
True of False: A nanometer is longer than a micrometer.
False, it is a thousand times smaller then a micrometer
Resolution
Refers to the distance between two objects at which the objects still can be seen as separate. The closer the 2
objects are to each other, the greater the resolution requirement needed to maintain viewing the two objects as
separate
Contrast
The difference in light absorbance between two objects. The lower contrast between an object and its
background, the harder it will be to see that object. The greater the contrast between the two objects, the easier
it will be to visualize.
Magnification
Increase in size of the object
Microscopy
Using light or electrons to magnify objects
Assuming a fixed ocular, identify the part of the microscope you would adjust to enhance the magnification
of a sample.
The objective
What is the total magnification of a sample imaged with a 20x objective and a 15x eyepiece? Show your
math.
20 x 15 = 300x magnification
True or False: A cell that is adherent, flat and unstained is easily identified using bright field microscopy?
False; this type of sample is almost invisible due to the limits of both resolution and contrast
