Microscopy - 2.1 - BASIC COMPONENTS OF LIVING SYSTEMS (3) Flashcards
Module 2, Chapter 2, 2.1, Page 11 & 12
In basic light microscopy, where is the sample illuminated?
from below with white light and observed from above (brightfield microscopy)
What happens to the sample after it is illuminated from below?
the whole sample is illuminated at once (wide-field microscopy)
What do the images tend to have?
low contrast as most cells do not absorb a lot of light
What is resolution limited by?
the wavelength and diffraction of light as it passes through the sample
What is diffraction?
it is the bending of light as it passes close to the edge of an object
What is the cytosol?
the liquid found inside of cells
What is often transparent?
the cytosol of cells and other cell structures
What do stains increase?
they increase contrast as different components within a cell take up stains to different degrees
What does the increase in contrast allow?
it allows components to become visible so they can be identified
What do you do in order to prepare a sample for staining?
the sample is first placed on a slide and allowed to air dry
What happens after the sample is allowed to air dry?
it is then heat-fixed by passing through a flame
What happens to the sample after it is heat-fixed?
the specimen will adhere to the microscope slide and will then take up stains
What is crystal violet and methylene blue?
they are postively charged dyes
What is crystal violet and methylene attracted to?
negatively charged materials in cytoplasm leading to staining of cell components
Which dyes are negatively charged?
nigrosin and Congo red
What are nigrosin and Congo red repelled by?
negatively charged cytosol
Where does the nigrosin and Congo red dye stay?
they stay outside cells
What happens when dyes such as nigrosin and Congo red stay outside of the cell?
this leaves the cells unstained, which then stand out against the stained background -> this is a negative stain technique
What can differential staining distinguish?
it can distinguish between two types of organisms that would be hard to identify
What can differential staining also differentiate between?
different organelles of a single organism within a tissue sample
What is the Gram stain technique used for?
it is used to seperate bacteria into two groups
What are the names of the two groups where Gram stain technique is used?
gram-positive bacteria and gram-negative bacteria
Gram stain technique - What happens first in the gram stain technique?
crystal violet is first applied to a bacterial specimen on a slide, then iodine, which fixes the dye
Gram stain technique - What happens after the dye is fixed by iodine?
the slide is then washed with alcohol
Gram stain technique - what happens after the slide is washed with alcohol?
the gram-positive bacteria will keep the crystal violet stain and will appear blue or purple under a microscope
Gram stain technique - what happens to the gram-negative bacteria?
as gram-negative bacteria have thinner cell walls, they will lose the stain
Gram stain technique - what happens after the gram-negative bacteria loses the stain?
they are then strained with safranin dye, which is called a counter stain
Gram stain technique - what colour will the gram-negative bacteria appear after being stained with safranin dye?
red
Gram stain technique - what is the gram-positive bacteria susceptible to?
antibiotic penicillin, which prevents the formation of cell walls
Gram stain technique - as gram-negative bacteria have much thinner cell walls, are they susceptible to penicilin or not?
they are not susceptible to penicillin
Acid fast technique - what is this used for?
to differentiate species of Mycobacterium from other bacteria
Acid fast technique - what is a lipid solvent used for?
to carry carbolfuchsin dye into the cells being studied
Acid fast technique - what happens after the lipid solvent is used?
the cells are then washed with a dilute acid-alcohol solution
Acid fast technique - what happens after the cells are then washed with a dilute acid-alcohol solution?
mycobacterium are not affected by the acid-alcohol and retain the carbolfuchsin stain, which is bright red
Acid fast technique - what happens to the other bacteria?
other bacteria lose the stain and are exposed to a methylene blue stain, which is blue
