The Gram Stain Procedure

The Gram Stain Procedure. This protocol is taken from "Benson's Microbiological Applications: Laboratory Manual in General Microbiology" Eleventh Edition.
Some protocols vary depending on the source. Use the one best suited for your laboratory.
The gram stain follows this procedure:
1. Prep your sample on your slide
2. Heat fix the slide
3. Flood with Crystal Violet for 20 seconds
4. Wash for 2 seconds
5. Flood with Gram's Iodine for 1 minute
6. Decolorize with Grams Alcohol for 10-20 seconds
7. Wash for 2 seconds
8. Flood with Safranin for 1 minute
9.Wash for 2 seconds
10. Blot the slide dry
Gram-Positive bacteria have a thick peptidoglycan layer on their cell membrane which retains the crystal violet-iodine complex and stains the bacteria purple.
Gram-Negative Bacteria have a thin peptidoglycan layer on their cell membrane which does not retains the crystal violet-iodine complex and is instead stained by the Safranin. This will leave the cells stained pink.

[@verah1849]

This my exam today I'm happy to see that I did it well as required. Thanks for posting.

[@ginosanchez7]

can i play this music during my lab practical tmrw??

[@anahita-bn6cy]

Lmao😁

[@subhashkasota7542]

😂

[@GonzaloCayoDigital]

A lot of thanks, very good video, please DO MORE VIDEOS!!!

[@abhishekmajumdar4564]

But we don't use blot paper to dry it, we air dry the slide

[@sci-inspi]

That works too. Some lab techniques differ lab to lab.

[@yashiraghav3408]

Learned and enjoyed watching 😀

[@Jmpaz2010]

Very interesting video. Why aren't the bacteria washed off during any of these steps? Does the "heat fix" keep them in place? Thanks!

[@sci-inspi]

Correct, the heat fix step sticks them to the slide.

[@Jmpaz2010]

@@sci-inspi Thanks for the quick reply.

[@aaronrichardtalley]

@@sci-inspi Why isnt the crystal violet washed away with the water. it hasnt been mixed with the iodine yet to form the complex that's insoluble in water.

[@DeShark88]

@@aaronrichardtalley The water cannot penetrate the cell since it is a polar solvent. Therefore it only washes away crystal violet that hasn't penetrated the cell. This is also the reason that alcohol (a nonpolar solvent) is used for the decolorisation step and not water. In fact, even the alcohol can only penetrate those cells that do not have a thick peptidoglycan layer. In that case, the crystal violet is not washed away, and that is how/why this Gram stain procedure works.

[@naveedahmad8407]

very helpful video thanx...and next time explain mechanism of the Gram staining...good luck

[@DeShark88]

See 2:05 for the mechanism.

[@ellacalle]

What magnification should be used when observing a gram stain? 1000x or 1500x

[@sci-inspi]

Both will work, although 1500X will only look bigger, but not clearer. The limit of light resolution through a light microscope is about 1000X magnification.

[@theshatub962]

Good job useful video 👏🏽👏🏽

[@Jrodrig1138]

Thank you!!!

[@laurenodom7779]

forgot the wax ring and water droplet

[@vidhiwanjari1704]

Very interesting video sir but sir I have one confusion that the smear will be prepare by the process of heat fix?

[@nicolekrantz2172]

Very cool!

[@chinemeogougwu1065]

Wow Thank you 😊 I love it

[@kamalarani5478]

Short n simple... Good

[@lil_weasel219]

Why aren't Gram+ stained by safranin as well?

[@sci-inspi]

Gram + bacteria have a thick peptidoglycan layer that binds to the crystal violet-iodine complex that doesn't wash off with grams alcohol. so Safranin can't stain it since the CV-I complex is already there. Gram - bacteria don't retain the CV-I complex after washing with grams alcohol. so it is stain with safranin.

[@icediverfull]

Thanks you, very nice Video

[@rawyhjamal5215]

A lot of thanks

[@genanhamideh3097]

don't you have to put acetone for less than a second? or does it matter?

[@sci-inspi]

This protocol says 10-20 seconds. I have gram stains that come out fine following this protocol.

[@kamalarani5478]

No need

[@DeShark88]

There are different decolourisers used in Gram staining. Acetone can bleach the dye somewhat, but alcohol will not. So, it depends what decolouriser is used.

[@indianstars2405]

Can i use cover clip

[@sci-inspi]

Yes, a coverslip can be used.

[@dhananjayasingh2838]

Gram + ve bacteria is coccus and Gram -ve bacteria is rod shaped👍

[@timetravelerboutique8009]

Gram positive stains purple, and gram negative stains pink. Both include cocci and rods. Anthrax, for example, is a Gram positive rod.

[@SaadSaad-mb4py]

Fantastic

[@sappuprajapati2345]

Very nice

[@brienYT]

How long do I heat fix the slide for?

[@DeShark88]

About as long as was shown in the video. It's usually only two slow passes over the flame.

[@arunpratapsingh3145]

Awesome ☺️

[@Nova-ke7pk]

Thank u

[@nursepotassium]

Staphylococcus 💜

[@wondimw320]

good

[@xaazoozaa856]

my exam is tommorw

[@jai.m1934]

Better than danish grams

[@NarenManoj7]

👍

[@SAX-cl6lk]

nice but we didnt need so much jimi hendrix in thebackround

[@qmac9966]

I beg to differ