Microscopy And Fluorescence Flashcards
What is the stain Called that light microscopy uses
Gram stain
What is gram stain dependant on
The outer membrane of gram negative bacteria
What stain is used for TB testing
Acid fast stain
Explain the steps of gram staining on light microscopy
1- both stained purple with crystal violet
2- iodine is used to set the violet into cell wall
3- short term decolorisation with alcohol causes gram -ve to become colourless
4- Safranin stain is added to turn gram -ve pink
What instead of gram staining and light microscope is used to detect bacteria
PCR
What is the biggest advantage of electron microscopy to light
They have a higher resolution due to electron shorter wavelengths
Why do samples in electron microscopes have to be fixed and dehydrated
They are placed into a vacuum which kills cells
What did the electron microscope help to find for the bacterial structure
It is split into 2 by the septum
What are the issues with electron microscopy
Needs a thin sample to see high resolution
Forms artefacts
Not live - only dead cells used
What is another word for Scanning electron microscopy to see 3D structures
Cryoelectron microscopy and tomography
How are cells kept live in cryoelectron and tomography
They are frozen with ethene
What is better about cryoelectron tomography than electron microscopy
Doesn’t need fixation or thin sectioning
Produced a 3D imagine
What is an issue with cryoelectron tomography
Poor contrast
What is the most recent microscopy technique used
Fluorescent microscopy and digital imagining
What does fluorescent microscopy and digital imaging help do that no other does
It shows compartmentalisation in cell components through dyes
What 2 ways are cell components dyed in fluorescent microscopy
GFP
Immunofluorescence
What is the filter and mirror called which helps only blue light wavelength pass in fluorescent microscopy which helps visualise the dye
Emission filter
Dichroic mirror
What is better in fluorescent microscopy which uses light compared to light microscopy
Much better resolution
What is immunofluorescence and name 2 types
Detection through marked antibodies
Direct or indirect
What is direct immunofluorescence
Monoclonal antibodies are attached to fluorescent molecules which bind to the target
What is indirect immunofluorescence and why is it time efficient
Primary antibodies will attach with no fluorescent molecule
Secondary antibodies attach to them with a fluorescent dye which is easier to attach than to a primary antibody
What is bad about immunofluorescence
Cells need to be fixed to allow antibodies to work (no live imaging)
What is GFP tagging
Gene for a green fluorescent protein added onto gene of interest to track its expression over time
How is GFP switched on
When the gene of interest is expressed in transcription mrna GFP also expressed
