00:01
What could be a possible reason that you stain a colostrum, a discephal, a gram -positive pothilus and if you observe mostly pink rashes? so this is a gram -positive, so it's supposed to to stain purple, but now it become pink and red.
00:22
So what could be the possible error? so after we through all the options, the most likely reason is going to be too much decolorization i want to talk a little bit about the process of gram stain.
00:46
You start out obviously with a heat fix.
00:49
So the bacteria will be fixed on a slide and they glue on top of the slide.
00:52
Then you treat the sample with crystal violet.
00:57
The crystal violet is going to stain the cell wall of the bacteria.
01:05
If the cell wall has peptidoglycan in gram -positive material, it will be stained purple.
01:15
At this moment, with excessive dye, the gram -negative will also show purple.
01:20
The next step, you will have iodine.
01:23
Iodine forms a complex with the crystal violet, so it fixes the crystal violet.
01:30
Make sure that they form a complex that they cannot be removed by the decolorizing reagent.
01:35
After that, you have the decolorizing agent, usually alcohol.
01:44
This step differentiates the gram -positive negative.
01:47
Gram -positive one because the iodide and crystal violet will retain those structures and show purple.
01:55
Because the crystal violet plus iodide form this unremovable complex shield purple.
02:07
But the gram -negative does not have the peptidoglycan.
02:11
The crystal violet cannot be retained.
02:14
So the decolorizing agent is going to remove the dye.
02:30
Colorless.
02:33
Dye.
02:34
Remove.
02:35
Nothing to retain it to the cell because it has different cell wall structure and then the next step you will actually restain that with saffroning which is a red dye now because gram positive is already purple so it's going to remain purple for the entire time but the gram negative it's clear and now with with saffroning treatment, it's red, so it will show a red -pinkish color at the end...