this is one of the best representations of a gram staining procedure I have every seen. Simple and straight forward with all the precautions built it. Well done.
Interesting! Never had to do this before, kinda wish I did. Only disappointment was at the end because I was kind of expecting to see what that slide looked like under the microscope.
Staining is to improve the contrast within a cell to show the organelles in greater clarity and detail. The cytosol is transparent there is little if any contrast. Using positively charged dye it attracts to the cytosol within the cell and you can distinguish between the different organelles. Using a negatively charged dye it repels the outside of the cell and stays outside the cell and distinguishes the cell from the background. The positive dye could be Methylene blue and the negative dye could be Congo red. Differential staining is used to differentiate between two different organelles or organisms. Gram stain technique is used to show gam positive bacteria and gram negative bacteria whilst the acid fast technique is used to show mycobacterium form other bacteria present.
Thank you for making this video! My lab partner hogs all of the lab activities and doesn't care about other people, so I could not learn it well sadly. There is a lab exam on Gram staining, so I am glad that you made this video so I can learn how to do it!
So gram staining is to differentiate between gram positive and gram negative bacteria and acid fast technique is to differentiate mycobacterium from other bacteria
Nice video! I wish I had seen this *before* doing Gram Stain for the first time as a high school student. Instead, I had to rely on an ambiguously worded worksheet for instructions. I ended up washing my slide completely clean of all cells... That was spectacular, I suppose. ah ha ha :)
I have my practical exams tomorrow And today I am watching 😂😂 So thankful to you for explaining each and every step perfectly Hope I will do well tomorrow 😂😘❤️😂
we wash the slide with a bit of detergent before use to make it grease free which helps alot in spreading the water droplet and giving a smear of bigger surface area.........idk if it's right but it's a mandatory step in our university and it has become a part of staining procedure
The video was nice, but I have a question, u marked a circle with wax pencil , and made a smear on the same side???? As I'm a new student, my proff taught me to mark the slide , invert it n prepare a smear on inverted slide.... Why y did so?????😶
at very fast if you add drop of water to bacterial colony, the cells may swell and lyse due to osmotic gradient. better use a drop of isotonic normal saline
i understand this video is 10 years old, but i just want to be sure. is the gram decolouriser Ethanol solution? because i'm sure that's what we used for our gram staining methods to rinse the stain, but maybe i am just mixing up the wrong reagents
How come when I apply the alcohol most of my specimens come out red, even something like gram positive bacillus subtilis comes out half red half blue and sometimes completely red. Professor said that could happen if I'm applying too much alcohol but I do it as instructed, until it runs clear. Could it be that my school has such old specimens that all the peptidoglycan is long gone and alcohol just washes everything away?
I can answer this now that I've finished the semester, by the end I was able to do perfect Gram stains. The reason it could sometimes comes out mixed color or the blue color washes off completely is mostly due to uneven alcohol coverage. You have to place the drop of alcohol all the way at the top of the slide and let it slide down on it's own, also don't follow the directions "until it runs clear" once you see that it's light blue it's fine to stop. It's just a matter of getting the amount and the coverage right. By the way, got an A+ in this class, due in part to videos such as these. Many thanks.
Thanks for explaining the technique. I don't understand the main ideia, though. Was that Gram positive or Gram negative? I've read the Gram positive retains violet and Gram negative doesn't. And that Gram - are pathogenic and gram + have more glucose. What does one thing have to do with another? How come having less glucose means increased pathogenicity? And why is it called positive/negative if the gram technique includes all stains, not the violet one? When I first saw the name I assumed the nagative would be transparent, but it is red. Can someone on the field please show me the light? thanks!!
Bacteria that take up the primary stain is called gram positive and those that take up the counterstain is gram negative. Gram positive bacteria have more of the peptidoglycan layer and no outer lipid layer whereas gram negative bacteria have less of the peptidoglycan layer and more lipid layer. The basic principle is that the crystal violet iodine complex is taken up by the bacterial cell wall of gram positive bacteria and on adding the decolorizer (ethanol) it dehydrates and closes the pore ,effectively locking in the primary stain. On the other hand Gram negative bacteria, on addition of decolorizer has its lipid layer solubilized and loses the primary stain, then on addition of the counter stain, Gram negative picks it up while Gram positive already is saturated with the primary stain.
@@digitalditz3474 Thank you so much for the kind and clear explanation. So the fact that Gram negative are more patogenic has nothing to do with the fact that their wall is thinner? If I add ethanol for less time than required will the gram - look like a gram +? On the same matter, if I leave ethanol for too long will the gram + look like gram -? Or once the pore is dehydrated it will be forever violet? Thank you a lot!
@@oliviapereira364Gram negative is pathogenic not exactly because of their thin peptidoglycan layer but rather the presence of an outer lipid membrane structure confers greater resistance to antibiotics. Also it's more reactive meaning that in the host, the outer membrane having virulence factors like toxins and secretory molecules cause the immune response. I think if you add the ethanol for too long, the gram positive will still stay the same( might change the overall morphology and cause issues during microscopy) but if you add too less , you can expect some experimental error where some take the counter stain and some don't.
You're welcome! If there are basic biology and life-science laboratory techniques you'd like to learn more about, email the Bio-Rad Explorer education program at explorer@bio-rad.com
The wax circle doesn't really interfere with the smear or staining. Although unnecessary, it is pretty commonly used and can be useful for students who are new to laboratory techniques and microscopy (eg. to divide several samples on a single slide and/or to align the field of view in the microscope). Some slides even come with premade circles or grid sections. The most likely issue would be contamination of the slide, which can be avoided using proper aseptic technique. Some students also draw small circles leading to thick smear suspensions, which can interfere with dye penetration and cellular arrangement visualisation, but that's more of an issue with smear technique than it an issue with the wax circle itself.
Last week on my lab exams i did the slide heat fixation perfectly. The mistake I always used to do before was that I used pass the slide through the flame for more than once which eventually incinerated the bacteria.
You can try methanol fixation instead of heat fixing the bacteria. works fine for us. place the air dried slide in 95% methanol for 1 mins. and then proceed for gram staining as usual.
I would have emphasized the decolorizer is alcohol, and kills the crap out of everything so get it off within 3 seconds or less. So many students in my class didn't do that and kept getting bad results.
Good afternoon ma'am, Please tell 1.After culturing in test tube, at which temperature we have to keep it for preservation and upto how many days we can preserve it 2. How identification and characterization of bacteria can be done 3. If I have a single bacterial strain- and I have to culture it. Then how I know that the strain I used is the same, I want to culture. Please tell the method for it
![Blox Fruits Dragon Rework Update [Full Stream]](http://i.ytimg.com/vi/EqDAp8udhm0/mqdefault.jpg)
We can learn this with a sentence
Come In And Stain
C- Crystal violet
I - iodine
A- alcohol ( ethanol)
S- safranin
Pooja Pareek yo thank you!! Have my micro practical tomorrow. Im a Pharm d student in Pakistan 😊😊
Thank you for helping me remember this:)
Pooja di thx,I have exams tomorrow it will help me
@@nocmemer4686 all the best for your exam dear😊...and no need of thanks dear I'll be great full if I can help you 😊😊
So sweet of you😇
this is one of the best representations of a gram staining procedure I have every seen. Simple and straight forward with all the precautions built it. Well done.
Interesting! Never had to do this before, kinda wish I did. Only disappointment was at the end because I was kind of expecting to see what that slide looked like under the microscope.
Yeah...Me too disappointed......
Here you go ---> ruclips.net/video/egpZ0BdJQE8/видео.html
@@ProfRoofs THANK YOU SO MUCHHH
Staining is to improve the contrast within a cell to show the organelles in greater clarity and detail. The cytosol is transparent there is little if any contrast. Using positively charged dye it attracts to the cytosol within the cell and you can distinguish between the different organelles. Using a negatively charged dye it repels the outside of the cell and stays outside the cell and distinguishes the cell from the background. The positive dye could be Methylene blue and the negative dye could be Congo red. Differential staining is used to differentiate between two different organelles or organisms. Gram stain technique is used to show gam positive bacteria and gram negative bacteria whilst the acid fast technique is used to show mycobacterium form other bacteria present.
I have microbiology exam next week. This video is so helpful for students of medical schools like me. Thank you for making it👏
I have a gram stain lab tomorrow and this was really helpful!!
Thank you for making this video!
My lab partner hogs all of the lab activities and doesn't care about other people, so I could not learn it well sadly.
There is a lab exam on Gram staining, so I am glad that you made this video so I can learn how to do it!
nooo i wanna see the results :'(
😭
Me too
Thank you for saving my time, not to see the video. You lost but you saved many.
thank you for this. i hope i'll do well on my practicals tomorrow 😨
ᄏᄏ아라 So, how did it go?
I have praticals tomorrow as well
i have practical tomorrow too
same lol
Mine too
So gram staining is to differentiate between gram positive and gram negative bacteria and acid fast technique is to differentiate mycobacterium from other bacteria
Kathryn Russell yup!
Acid fast different iate Mycobacterium tuberculosis. And m. leprae
Thank you for this!
you saved me from the embarrassment of asking my lab partner who likes to gatekeep information to himself
Ugh I hate lab practicals. These RUclips vids are a godsend!
This is great demonstration of an important concept and very concise. Weldone
Where is the observation?
Nice video!
I wish I had seen this *before* doing Gram Stain for the first time as a high school student. Instead, I had to rely on an ambiguously worded worksheet for instructions. I ended up washing my slide completely clean of all cells... That was spectacular, I suppose. ah ha ha :)
Joules Kin same bit somehow my e. Coli cane out gram-positive which was correct!
Thank u for the information
I did it yesterday in my internship. I'm so happy.
Rinsing in a beaker seems a lot more controlled than using spray bottles as they seem to do in the lab I'm taking classes in.
I have my practical exams tomorrow
And today I am watching 😂😂
So thankful to you for explaining each and every step perfectly
Hope I will do well tomorrow 😂😘❤️😂
armyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy
@@A9poj ARMY 💜💜💜💜
@@A9pojstupid armyyy
we wash the slide with a bit of detergent before use to make it grease free which helps alot in spreading the water droplet and giving a smear of bigger surface area.........idk if it's right but it's a mandatory step in our university and it has become a part of staining procedure
quick and straight to the point explanation
Bestest video short and sweet 😊 staining of gram negetive bacteria 😌
Ok, this is so different from how my lab TA did it but makes so much more sense.
The video was nice, but I have a question, u marked a circle with wax pencil , and made a smear on the same side????
As I'm a new student, my proff taught me to mark the slide , invert it n prepare a smear on inverted slide....
Why y did so?????😶
Awesome. Passionate to perform this on lab.
Full prepared before 1 hours exam❤😁
What are the other counter stains other than safranin can be used ?
thankyou, its all cleared. having exam of Gram staining tomorrow 🙌🏼
loved it except for the smear. it was a little too cloudy in my opinion.
i think it may have been agar residue
Thanks I've understood the technique properly thanks to this video.
Shouldn’t the alcohol be continuously added, drop by drop on the smear for 30-45 seconds?
at very fast if you add drop of water to bacterial colony, the cells may swell and lyse due to osmotic gradient. better use a drop of isotonic normal saline
i understand this video is 10 years old, but i just want to be sure. is the gram decolouriser Ethanol solution? because i'm sure that's what we used for our gram staining methods to rinse the stain, but maybe i am just mixing up the wrong reagents
Am kindly asking why do you heat fix
My histology exam after one day! This vdu was so informative thanks ✨
I have a gram stain skill exam in a few days…can’t wait lol
What's the benefit of using a wax bencil ?
How come when I apply the alcohol most of my specimens come out red, even something like gram positive bacillus subtilis comes out half red half blue and sometimes completely red. Professor said that could happen if I'm applying too much alcohol but I do it as instructed, until it runs clear. Could it be that my school has such old specimens that all the peptidoglycan is long gone and alcohol just washes everything away?
I can answer this now that I've finished the semester, by the end I was able to do perfect Gram stains. The reason it could sometimes comes out mixed color or the blue color washes off completely is mostly due to uneven alcohol coverage. You have to place the drop of alcohol all the way at the top of the slide and let it slide down on it's own, also don't follow the directions "until it runs clear" once you see that it's light blue it's fine to stop. It's just a matter of getting the amount and the coverage right. By the way, got an A+ in this class, due in part to videos such as these. Many thanks.
Oh! This is the greater Graim Strainig procedure I've ever seen. It's so interesting.
Can anybody tell me what kind of iodine that used in this video?
how long does it take to perform and analyze a gram stain ?
Like 15-20 mins?
Very clear explanation keep it up sir
Can you post all your lab experiments...lol I learned so much through this THANKS!!1
If it is time i will post it dr
wish me luk on my practical :)
What happens if you do not heat fix the bacteria? is it detrimental to the experiment?
If you what 24 hours to heat stain what happens?
elizabeth owairu hi
the bacteria will still be alive i guess !? ..
Bacteria will be washed off during staining without heat fixation
Thank you helped me for my bacteriology subj
Thanks for explaining the technique. I don't understand the main ideia, though. Was that Gram positive or Gram negative? I've read the Gram positive retains violet and Gram negative doesn't. And that Gram - are pathogenic and gram + have more glucose. What does one thing have to do with another? How come having less glucose means increased pathogenicity? And why is it called positive/negative if the gram technique includes all stains, not the violet one? When I first saw the name I assumed the nagative would be transparent, but it is red. Can someone on the field please show me the light? thanks!!
Bacteria that take up the primary stain is called gram positive and those that take up the counterstain is gram negative. Gram positive bacteria have more of the peptidoglycan layer and no outer lipid layer whereas gram negative bacteria have less of the peptidoglycan layer and more lipid layer. The basic principle is that the crystal violet iodine complex is taken up by the bacterial cell wall of gram positive bacteria and on adding the decolorizer (ethanol) it dehydrates and closes the pore ,effectively locking in the primary stain. On the other hand Gram negative bacteria, on addition of decolorizer has its lipid layer solubilized and loses the primary stain, then on addition of the counter stain, Gram negative picks it up while Gram positive already is saturated with the primary stain.
@@digitalditz3474 Thank you so much for the kind and clear explanation. So the fact that Gram negative are more patogenic has nothing to do with the fact that their wall is thinner? If I add ethanol for less time than required will the gram - look like a gram +? On the same matter, if I leave ethanol for too long will the gram + look like gram -? Or once the pore is dehydrated it will be forever violet? Thank you a lot!
@@oliviapereira364Gram negative is pathogenic not exactly because of their thin peptidoglycan layer but rather the presence of an outer lipid membrane structure confers greater resistance to antibiotics. Also it's more reactive meaning that in the host, the outer membrane having virulence factors like toxins and secretory molecules cause the immune response. I think if you add the ethanol for too long, the gram positive will still stay the same( might change the overall morphology and cause issues during microscopy) but if you add too less , you can expect some experimental error where some take the counter stain and some don't.
@@digitalditz3474 Thank you so much! I finally get the idea
@@digitalditz3474 why we are rinsing it with water after every step ?
Interesting and helpful episodes.
If the stain of crystal violet came inti my hand how do i take it off
That was so interesting I am not a science student but it was like I can understand everything by watching this video
what if u accidentally swipe the bacteria from the wax pencil circle?
Thank you for making this video!
very useful video.tommorw my practical xam.
This is amazing, so interesting, I like it!
Can I use blot paper to dry it or let it air dry?
you can do either but blotting will be quicker. if youre doing acid fast, let it air dry
Very clear and informative tutorial.
good
Why we are heating the steel (thin rod)
Am i the only one Who had Done It Right and Met The Blueish purple Bacteria and Red pink ones 😍
Hit The Like if you Got that !!
I'm here because one of my character is a scientist and I wanted to describe part of the procedure for Gram straining!
What is the percentage to alcohol used
Post more helpful videos on equipments and related solutions which are used and how can they be used
Great video although you could've shown what the samples looked like after all of those steps.
haha I was waiting for what it looked like under the microscope
How did you get bacteria???
Better than my professor explanation
Amazing video
Thank you so much for this video. ❤
Thank you so much, I learnt a lot from this video!
Thank you so much ,I learnt alot from this video!
How come our lab manual said crystal violet stays for 30 sec?
mine says for 1 minute
this is interesting to watch goodjob 🎉
Video maker- Today, we are out of microscopes but we were in a hurry to make this video
Wow so amazing your explain 😍
where is the bacteria on the slide?
Jeez man, did you flame the loop enough?
hey I wanted to see the microscopic results
what is used to fix the bacterial???????????????????????????????????????????????
Heat from the bunsen burner or flame
PLEASE WHAT IS THIS GRAM STAINING USED FOR I ACCIDENTALLY STUMBLED INTO IT.
Used to differentiate between two types of bacteria.
nic and straight technique
I think its Gram negative
Thank you for this useful video
You're welcome! If there are basic biology and life-science laboratory techniques you'd like to learn more about, email the Bio-Rad Explorer education program at explorer@bio-rad.com
Thank you . Is very interesting
Great video 🫶🏻
Excellent demonstration, but there is no point and even undesirable to draw a circle on the slide using a wax pen.
The wax circle doesn't really interfere with the smear or staining. Although unnecessary, it is pretty commonly used and can be useful for students who are new to laboratory techniques and microscopy (eg. to divide several samples on a single slide and/or to align the field of view in the microscope). Some slides even come with premade circles or grid sections. The most likely issue would be contamination of the slide, which can be avoided using proper aseptic technique. Some students also draw small circles leading to thick smear suspensions, which can interfere with dye penetration and cellular arrangement visualisation, but that's more of an issue with smear technique than it an issue with the wax circle itself.
thanks that was SO helpful 💖👏👩🔬
Its wonderful understand the Subject after this video saved me
Thanks very much
i'm just a random person who thought that gram-negative means something about the weight
thank you for education!
Thanks a lot, would really be helpful in my defense
Excellent video. Just one addition, Safranine being a weak stain should be kept for 2 mins
From my current work experience, one minute is sufficient.
over staining may turn gram positive to negative
@@khgnew763 No, its not that way, Gram positives will not take up counter stain
thank you for this..i hope i'll write the same in my exam👍👍
Good job
simple and easy, thank you.😊😊😊
I always end up incinerating the bacteria while heat fixing. Is there any way to avoid that?
Last week on my lab exams i did the slide heat fixation perfectly. The mistake I always used to do before was that I used pass the slide through the flame for more than once which eventually incinerated the bacteria.
You can try methanol fixation instead of heat fixing the bacteria. works fine for us. place the air dried slide in 95% methanol for 1 mins. and then proceed for gram staining as usual.
How can you tell when the bacteria has been incinerated??
Good job 👍
after microscope what will happen
tank you for this good presentations.i got it good experiance.
I would have emphasized the decolorizer is alcohol, and kills the crap out of everything so get it off within 3 seconds or less. So many students in my class didn't do that and kept getting bad results.
Thanks 😊 that was so helpful ☺
Good afternoon ma'am,
Please tell
1.After culturing in test tube, at which temperature we have to keep it for preservation and upto how many days we can preserve it
2. How identification and characterization of bacteria can be done
3. If I have a single bacterial strain- and I have to culture it. Then how I know that the strain I used is the same, I want to culture. Please tell the method for it
Good questions. Waiting for reply.
I think it’s Gram -ve
Careful ! Never wear gloves near the flame
Thank you so much 🙏