5.1 Staining Methods Flashcards
What is the significance of staining in microbiology?
Colour used to highlight certain features
Making the characteristics features more visible
What are the types of Stains used?
Usually salts comprised of ions
Coloured ion = Chromophore (+/- charged)
Basic stain (+ charge)
- Methylene blue
- Malachite green
- Gentian violent
- Carbol fuchsin
- Safranin
Attracted to negatively charged cell wall
Acidic stain
- Nigrosin
- Acid fuchsin
Repelled by cell wall
What are the categories of Staining Techniques?
1) Simple (Only one stain)
2) Differential
3) Negative
4) Microbial structures, Eg. Endospores/ Capsules/ Filaments
What do you do before staining?
Bacterial Smear must first be prepared and fixed
1) Bacteria obtained from plate/broth culture
2) Spread bacteria to form a thin layer on microscope slide; must be able to allow light to pass through to be examined under the microscope, allows for a better view of the arrangement of cellular structures
3) Allow smear to dry
4) Fix smear to slide with heat/ chemical such as alcohol to prevent accidental removal of bacteria from washing; Heat > Alcohol; Alcohol is used to study capsules
5) Proceed to stain
What is Simple Staining?
Only one stain is added such as:
Methylene blue
Gentian violet
Carbol fuchsin
A quick method to demonstrate shape, size and arrangement of cells, but there is some cell distortion
Cell distortion due to heat
What is Differential Staining?
Bacteria react differently to various stains and may be differentiated by using a combination of stains
Examples:
Gram staining — Gram-positive VS negative
Acid-fast staining — Acid-fast VS non-acid-fast
What is Gram Staining?
1) Primary Stain: Basic para-rosaniline stain (Eg. Gentian violet)
2) Add iodine (Mordant) to form a complex w the stain (GV-I)
- Both GV and I penetrate cell wall and form a complex within the cell
3) Wash with alcohol/ acetone (Decolourising agent)
- Gram positive — Resist decolourisation retains GV-I, Stained purple
- Gram negative — Decolourised cannot retain GV-I, Colourless
4) Add counterstain (Eg. Safranin-contrasting colour); Must contrast Primary stain
- Gram positive — Remains purple
- Gram negative — Stained red
Why does gram positive bacteria retain GV-I complex but gram negative bacteria does not?
Gram-positive:
Highly cross-linked and thick peptidoglycan entraps GV-I complex
During alcohol washing, cell wall becomes dehydrated, porosity of the cell wall decreases allowing for better entrapment of GV-I
Gram-negative:
Thin peptidoglycan cell wall preventing strong binding of GV-I
During alcohol washing, outer membrane becomes more porous allowing GV-I to be washed out
What are the examples of the Gram bacteria?
Gram-positive:
Staphylococcus aureus
Bacillus subtilis
Gram-negative:
Escherichia coli
Pseudomonas aeruginosa
What is Acid-fast staining?
Not alot of acid-fast bacteria
Staining method aids in the identification of Mycobacterium tuberculosis
How is acid-fast staining done?
1) Primary stain: Strong stain (Eg. Strong carbol fuchsin)
2) Heat (Mycolic acid makes acid-fast bacteria cell wall waxy and impermeable and therefore require heat to allow penetration of stain into the cell)
3) Wash with Acid/ acid Alcohol (Decolourising agent)
- Acid-fast — Resist decolourisation, Stained red;
- Non-acid-fast — Decolourised, Colourless
4) Add Counterstain (Eg. Methylene blue - Contrasting colour)
- Acid-fast — Remain red
- Non-acid-fast — Stained blue
What is Negative Staining?
No fixing and staining of cells
Method:
1) Mix a loopful of bacterial culture with a drop of Indian ink/ Nigrosin on a microscope slide
2) Spread to form a thin film on the slide
3) Allow film to dry
4) Examine with an oil immersion lens
Advantage:
No distortion of cells — No fixing cause no washing
Cells are visible against a dark background (Nigrosin/ Indian ink)
Cells are still colourless
