Outcome 2.5 Staining methods Flashcards
Name the 5 staining methods in this course
Immunoflourescence, electron microscopy, light microscopy, gram stains and acid fast stains
What are gram stains used for
To classify bacteria as gram positive or gram negative and ascertain information about morphology and cell arrangement
What is the difference between gram positive and gram negative bacteria
Gram positive bacteria contain a thick layer of peptidoglycan in their cell wall and retain violet dye.
Gran megative bacteria contain a thin layer of peptidoglycan and retain a pink counterstain
Describe the steps of a gram staining procedure
- Crystal violet is applied, dying all cells purple
- Iodine is applied - forms a CV-I complex
- Alcohol wash performed - CV-I is washed away, except gram postive bacteria retain CV-I
- Apply safranin - the pink counterstain, dyes gram negative bacteria pink and doesnt affect gram positive bacteria
What is an acid-fast stain used for
To differentiate two different gram positive bacilli usually of genus Mycobacterium. It is an important part of TB diagnosis.
Why are acid-fast stains preferred to gram stains when investigating Mycobacterium
Mycobacterium cell walls contain mycolic acid which makes their cell walls resistant to water based stains
Describe the acid-fast stain method
- The primary stain carbofuchsin is added dying the cells red.
- An acid-alcohol wash is perfomed - acid fast bacteria retain the red dye whilst non acid-fast bacteria dont.
- The counterstain methylene blue is added, dyding non acid fast bacteria blue
What is immunofluorescence used to detect
The antigens of a specific pathogen in infected issue by using flourescently-labelled antibodies specific to the pathogen.
Describe the steps of direct immunofluorecsence
- The primary antibody is tagged with a fluorescent label
- The antibodies are incubated with the infected issue allowing the antibody to bind to the surface of the pathogen
- The tissue is washed with buffer to remove unbound antibody
- The tissue section is examined under a fluorescent microscope that excites the label with a wavelength of light that causes the label to emit a longer wavelength of light.
- The location of the pathogen antigen is located
Describe the steps of indirect immunofluorescence
- The antibodies are incubated with the tissue section allowing the antibodies to bind to the antigens of the pathogen.
- The tissue is washed with buffer, removing unbound antibodies.
- A secondary antibody tagged with a fluorescent label and that is against the primary antibody is incubated with the tissue.
- The tissue is washed with buffer, removing any unbound secondary antibody
- The tissue section is examined via fluorescent microscope
- The location of the antibody-antigen complexes can be identified
What are 2 advantages of direct immunofluorescence over indirect
Direct is cheaper and takes less time to be carried out
What is the disadvantage of direct immunofluorescence compared to indirect
Direct is less sensitive - the fluorescent signals cant be amplified and so there is a greater limit of detection
What is electron microscopy?
Using a beam of accelerated electrons instead of a beam of light to magnify a specimen. Transmission electron microscopy can magnify thin sections of of specimens in 2 dimensions, whilst Scanning electron microscopy can magnify specimens coated in gold ions in 3 dimensions
How is light microscopy used
Once specifc stains have been applied to samples, light microscopy can be used to presumptively identify bacteria based on morphology and structural properties
Name and the two ways samples are prepared for light microscopy
Wet mount and fixation (via heat)
Wet mount - specimen is placed on a slide in a liquid drop and covered with a cover slip
Fixation - heat is applied to attach cells to slide. This kills microorgansim whilst affixing them and preserves there structural and morphological properties
