Lecture 2 - Microscopy & Staining Flashcards
what do microscopes do?
they produce magnified images of specimens using a source of illumination and lenses
light microscopes have what type of illumination, lens, magnification, resolution, and is used to view what?
light microscopes have light illumination, glass lenses, 1000x magnification, 200 nm resolution, and is used to view cells.
electron microscopes have what type of illumination, lens, magnification, resolution, and is used to view what?
electron microscopes have electrons as illumination, electromagnetic lenses, 100,000 - 2,000,000X magnification, 0.05 nm resolution (clearer resolution), and are used to view cells, viruses, and molecules.
what does the iris diaphragm lever do?
it adjusts the contrast and controls the amount of light that enters the objective lens.
what does the rheostat do?
it controls the brightness of the light
what does the light source do?
it shines light on the specimen
what does the condenser lens do?
it focuses the light
what is the specimen stage?
where the specimen is
what is the objective lens?
They are near the object/specimen. They provide different magnifications and then go to the ocular lens to get further magnified.
what is the ocular lens (eye piece)?
They are near the eyes. They further magnify the image, usually 10-fold (10x).
what is the magnification of the objective lens?
4X, 10X, 40X, and 100X (which is the maximum)
What are 3 differences between light and electron microscopes?
Light microscopes: has a max magnification of 1000X, has a resolution of 200 nm, and has glass lenses.
Electron microscopes: has a magnification of 100,000 - 2,000,000X, has a resolution of 0.05 nm, and has electromagnetic lenses.
what is the ocular lens magnification?
10X (only one)
what is the formula for total magnification?
Total magnification = Objective lens * Ocular lens
what is the total magnification at 40X?
40X * 10X = 400X; which is 400 micrometers
what is the maximum magnification?
1000X
what total magnification should bacteria always be evaluated at?
1000X total magnification
If you look at an E. coli cell at 1 micrometer, and you magnify it by 40X, what would me its micrometer now?
40 micrometer (??) 400?
at a low resolution, how do two really close objects appear?
two close objects will appear as one blurry object at low resolution
what is resolution defined as?
the minimum distance that 2 separate objects can be distinguished (objects closer than this distance will be blurred together)
AKA
the ability to distinguish two objects that are very close to each other
is resolution limited by the wavelength of light?
yes
do electron microscopes have better resolution?
yes
what increases resolution at 100x objective?
oil immersion
what is refracting of light?
bending; whenever light enters from the glass to the air and vice versa
what is a light microscope resolution?
200 nm
at 200 nm apart, light microscope can distinguish the 2 objects. when they are closer together, is the resolution better or worse?
worse
does oil have a similar refractive index to glass?
yes
How does oil increase resolution at 100X objective?
Oil increases resolution at 100x objective by preventing refraction and loss of light rays.
T/F: Oil immersion prevents refraction (bending) and loss of light
True
what is contrast?
the difference in color intensity between the object and the background
why would be need to increase the contrast?
so that the object doesn’t blend into the background, and therefore the object is easier to see
what are the properties of microbes that make them hard to see in low contrast?
microbes are thin and transparent, so they do not transmit enough light to the objective lens and appear white against a white background
what are two ways to increase the contrast?
-to adjust the iris diaphragm lever
-staining
why might staining not be desirable?
because it kills cells
what are the different types of light microscopes?
- bright-field microscopes
- dark-field microscopes
- fluorescence
what is a bright-field microscope?
it is the most commonly used and it is a compound light microscope
what is a compound microscope?
using 2 or more lenses
what is the contrast like for bright-field microscopes?
it illuminates the field evenly generating a bright background
what is the total magnification of bright-field microscope?
1000X
what is the resolution of bright-field miroscope?
0.2 micrometers
what is the light path of the bright-field microscope?
-light from the bulb is focused on the specimen using the condenser lens
-the specimen absorbs, reflects, and transmits light, some of which enters the objective lens forming a magnified image
-the image is further magnified by the ocular lens
-the field is bright as the light focused on it is transmitted and enters the objective lens
what is the dark-field microscope?
it makes the specimen bright compared to the dark field around it
how does the specimen look bright against the dark field?
a hollow cone of light is focused on the field and the specimen, such that only the light transmitted by the specimen enters the objective lens to form an image, thus the specimen looks bright against a dark field
what specimens are the fluorescence microscope used to see?
it is used to see specimens that are either NATURALLY fluorescent or STAINED with fluorescent dyes
what happens when a UV light is focused on the specimen?
it emits visible light that forms an image, so that the background is completely dark
T/F: the fluorescence microscope is a powerful way to detect specific microbes and subcellular structures while avoiding signals from dust and other nonspecific material
TRUE
what are all the ways we can increase the contrast?
- Use the iris diaphragm lever
- Use staining
- Use a dark-field microscope
- Use fluorescence microscope
how do electron microscopes work?
a beam of electrons is focused on the specimen by electromagnetic lenses to form an image
what is the total magnification of electron magnification?
100,000X
what is the resolution of the electron microscope?
0.3 nanometers
is there special preparation required to view the specimen?
yes
what are the benefits of electron microscopes?
better resolution and magnification
what are the two types of electron microscopes?
transmission and scanning
where are the e-beams transmitted through the transmission electron microscopes (TEM) vs the scanning electron microscopes (SEM)?
-TEM: through the specimen for
-SEM: scans just the specimen surface
how is the specimen in TEM vs SEM?
- TEM: very thinly sliced sections (100 nm)
- SEM: thick (~cm)
what is used to view in TEM vs SEM?
- TEM: internal structures
- SEM: surface details only
is the image 3D or 2D in TEM vs. SEM?
- TEM: 2D
- SEM: 3D
what is an atomic force microscope used to see?
molecules
how does an atomic force microscope work?
it uses a laser probe that scans across a surface looking for bumps/valleys to create a topographic map
is an atomic force microscope better resolution than electron microscope?
YES
are special preparations needed for atomic force microscopes?
No
which type of microscope achieves contrast by shooting ultraviolet light at the specimen?
Fluorescence microscope
what is the purpose of staining?
to increase contrast
what is the wet mount method?
the method of observing living organisms (microbes) in a drop of liquid using a microscope
what are the benefits of wet mount?
to prevent the microbes from moving around and makes it easier to stain
what does staining with dyes do?
increases contrast and immobilizes microbes (makes them stay in place)
what is the basic preparation for staining/What are the 4 basic steps in staining? Write the purpose of each step.
- Smear (to spread the specimen on the glass slide in a thin layer, you could shape it as the size of a dime).
- Air dry (to let the smear sit to dry before proceeding and to avoid aerosols being released before its fully dry)
- Heat fix (to attach the specimen and fix it onto the glass slide by moving it across the flame 4-5 times)
- Stain (to increase contrast and see the specimen more clearly)
what are the different types of stains?
- Simple
- Negative
- Differential (including Gram stain and acid-fast stain)
what are simple stains?
Adding basic dye (that is positively charged +) to stain cells (that are negatively charged -)
what is an example of simple stains?
methylene blue
what are the charges for simple staining?
+ dye is attracted to - cells
what are negative stains?
Using acidic dyes (that are negatived charged -) that cannot penetrate cells (that are negatively charged -) but stain the surrounding background area
what is are the charges for negatively stains?
- negative acidic dyes repel - negaitve cells
what is an example of negative stains?
India ink
what is differential stains? what are examples of them?
-Separating bacteria into 2 different categories (2 different microbes)
-Gram-stain and acid-fast stain
what is Gram stain?
the “go to” stain for unknowns
what are the two categories/bacteria under Gram stains?
Gram-positive or Gram-negative
who came up with Gram stains?
Hans Christian Gram (1853-1938)
what are the 4 steps of Gram stain?
- Use crystal violet stain (which is the primary stain and the first stain). This stains the cell purple. The bacteria are not differential yet, because they are both the same color - purple. (both the Gram-negative and Gram-positive are purple).
- Use iodine staining (mordant stain) - which is a clumper. This keeps the cells purple, and they are still not differential yet. (both the Gram-negative and Gram-positive are purple).
- Use alcohol (which is a decolorizer). This washes away the stain from the Gram-negative cells only. The Gram-positive cells REMAIN PURPLE while the Gram-negative cells become COLORLESS. The alcohol only washes away the staining from 1 type of the cell - which is the Gram-negative. This is the differential step.
- Use Safranin (which is a counterstain). This makes the Gram-positive cells REMAIN PURPLE. This makes the Gram-negative cells APPEAR PINK. We use this Safranin counterstain because it is hard to see the colorless cell.
can the order of the Gram-stain process be changed?
NO, it has to be this specific sequence.
what is the second type of the differential stain?
Acid-fast stain (aka Ziehl-Neelsen stain)
The Gram stain had two categories, Gram-positive and Gram-negative. What are the two categories for Acid-fast stain?
Acid-fast and Non-acid-fast
what things stain pink in the Acid-fast stain?
Mycobacterium spp. (tuberculosis, leprosy, etc.)
what does Mycolic acid do in the acid-fast stain?
the mycolic acid creates a waxy cell wall that doe snot take up the Gram stain
Why don’t we use the Gram stain for mycobacterium?
it doesn’t work because of the mycolic acid
what is the acid-fast stain used for?
used if patient has related symptoms like for TB - coughing up blood, night sweats, weight loss, etc.
what are the 4 steps for acid-fast stains?
- Cells prior to the staining are both transparent.
- After staining the cells with the primary and first stain Carbolfuchsin, the cells become reddish-purple. No steaming is necessary.
- Acid alcohol creates decolorization and removes the stain from the acid-fast negative cells.
- Methylene blue is used to counterstain the acid-fast negative cells.
Lesions on face and lips in the case we learned are the result of what?
of Leprosy, a bacterial infections caused by Mycobacterium leprae.
The lesions contain the bacteria which can only be observed in skin scrapings stained with what method?
with the acid-fast method, which uses heat to allow the stain to penetrate the waxy coating created by the mycolic acid in the cell wall.
which type of stain uses a pink counterstain?
Gram stain
A patient is coughing up bloody sputum. A PPD skin test for tuberculosis comes up positive. You do a staining test to see if the patient has an active infection. Which test do you choose? Explain the procedure, using correct terminology in your answer. If tuberculosis bacteria are present, how would they stain?
I would choose the acid-fast stain for Mycobacterium spp. since tuberculosis is caused by one species in this group. After smearing a sample of the sputum on a slide, I would air dry and heat fix the sample. I would then use the acid-fast stain to stain the sample since the Gram stain will not work. If Mycobacterium spp. are present on the slide, they will be acid-fast and appear pink under the microscope. All other cells will appear blue.
Which of the following aspects of a bacterial cell would you not be able to observe in a stained sample?
Motility
What is the function of the iris diaphragm lever on the microscope?
Give contrast to the specimen
Which type of light microscope is most useful for increasing contrast?
Dark-field microscope
Place the following steps in the correct sequence:1-Staining; 2-Making a smear; 3-Fixing
2-3-1
What is the appearance of Gram-negative bacteria after addition of the decolorizing agent?
Colorless
During the Gram staining procedure, how would Gram-negative cells appear if you forgot to use the counterstain?
Colorless
Which part of the microscope focuses light on the specimen?
Condenser
Which microscope is appropriate to view the SARS-CoV-2 virus?
Electron Microscope
For which bacteria would you use the acid fast staining procedure?
Mycobacterium leprae and Mycobacterium tuberculosis
Define Fimbriae
A short cell surface structure that help in the attachment of bacterial cells.
I noticed there was some confusion about pili vs. fimbriae. Fimbriae are a type of pili that help bacterial cells attach to surfaces.
Define Capsule
A gel-like coating that surrounds some bacterial cells and helps them to attach and resist phagocytosis.
Define Lipopolysaccharides
Lipopolysaccharide (LPS) - a molecule that makes up the outer layer of the outer membrane of Gram-negative bacteria.
