Complex staining techniques Flashcards
Auesky method
Spore staining
spore- red color and vegetative part- blue color
Burry-Gins method
Identify capsules (UNSTAINED) (red bacteria surrounding by unstained capsule on dark background)
Ziehl-Nielsen method
Stain acid- fast bacilli
(Acid-stain bacteria: red)
(non acid-fast bacteria: blue)
Romanovsky-Giemsa method
Stain spirochetes, protozoa, rickttsia and chlamydia
Neisser method
Volutin granules detection
Volutin granules: dark blue and bacterial body: pale yellow
Gram-stain method
Differentiate Gram-positive from Gram-negative bacteria .
(Gram +: blue or violet)
(Gram - : red or pink)
What is the difference between simple and complex staining?
Simple staining uses one color for the identification of structures, while Complex staining uses more than one color for the identification of structures in the analyzed microbial.
What is the difference between Gram positive and Gram negative bacteria?
The Gram + bacteria has a thick layer of peptidoglycan and do not present an outer lipid membrane, while the Gram - bacteria has a thin layer of peptidoglycan and presents an outer lipid membrane layer.
-What is the principle of the Gram-staining?
The crystal blue dye basically stain the peptidoglycan layer (Gram +), but because the Gram - bacteria has a thin peptidoglycan layer plus a lipid membrane over it, the crystal blue is easilly washed off from it, when rinsed with alcohol. Therefore, when the saffarine is added what it is really stained is the lipid outer layer of the Gram - bacteria.
That is why:
Gram + = violet
Gram - = red/pink
-HOW is the Gram’s staining technique procedure?
1- A first dye is applied into the slide containing the bacteria-in this stage, it is not differentiated-
(dye 1: Crystal violet).
2- The slide is wahsed with water.
3-Iodine is added (2min) and is washed of with alcohol and then water.
4- A second dye is added (dye 2: Saffarine).
5- The slide is washed, dried and analyzed.
Why is the Ziehl- Neelson staing method is used?
The method is for acid-fast bacteria. That bacteria and some other eukaryotic cells (which have the acid-fast property) have their cell walls impermeable to most stains (including the Gram stain).
Why acid-fast bacteria resist other process of staining?
They do have the peptidoglycan layer, just like any other bacteria. However, outside their cell wall they have a wax coat (mycolic acid), which have the function of protection and resistance to the environment. The Gram-stain, just like other dyes cannot penetrate the wax coat.
How is the procedure for the Ziehl- Neelson staing method?
1- The slides firstly fixed and then dyed with methylene blue.
2- The slide is washed with alcohol and then water.
3- Then the counter dye is applied (Fuchsin- red) and the slide is once again washed.
In the end: The non- acid-fast bacteria pick it up the first dye (blue) but the acid- fast bacteria does’t because of the wax coat. However, once the second dye (red) is added, it stains the wax coat of the acid-fast bacteria.
