microbiological Techniques [stm 312] Class three
positive stain
positive stain is a stain procedure in which the cells structures of the organism are stained against a clear background e.g. safranin
Negative stain
Negatne staining is a staining procedure in which the background is stained while the cell and cell structures remain unstained e.g. India - ink.
Simple staining
Simple staining is a staining in which only a single dye is applied on an organism in order to allow someone to distinguish the shape of the bacteria cell.
Differential staining
This staining Procedure is a complex one requiring more than one stain to differentiate cellular component in bacteria.
STAINING
Gram-StainingGram-staining was introduced by a man named "Han christian Gram"
Gram staining procedure
Gram-staining is an example of a differential staining as it is used to differentiate bacterias into two different groups which are; gram positive and gram negative, in this procedure two distinct dye are employed which are; crystal violet (primary dye) and secondary dye (safranin red). The mordant in this process is Lugos iodine solution.
Procedure
1. Make a smear of the organism on a clean glass slide.
2. Heat fix the smear by passing it through the flame 2-3 times.
3. Flood the smear with crystal violet stain and allow it for 30-60 seconds.
4. Wash off excess dye with water.
5. Flood with lugos iodine for 30 seconds.
6. Wash off excess with water.
7. Flood the slide with alcohol or acetone and leave it for five seconds (decolonization).
8. Wash off excess alcohol with water.
9. Counter stain with safranin red and leave it for 30 seconds.
10. Wash off excess dye with water.
11. Air dry and view under the microscope using oil immersion objective lense.
Result: Gram positive organisms remain purple under the microscope, while Gram negative organisms remains pinkish or red under the microscope.