Gram Stain Procedure

[jameeljo]

I wish you showed us how the drying by heat is performed!!!

[alm7benc0m]

this is site good in lab

laboratory-testing . org

please like

[jameeljo]

@alm7benc0m HOw can I like something that I haven't tried?

[alm7benc0m]

@jameeljo

(:

[jameeljo]

@alm7benc0m :)

[alm7benc0m]

@alm7benc0m

من وين ؟

[Rojjergeil]

It is a good video to teach the students. its a fact that the things we have seen, become more rememberable n so this video also playing a same role.........

Rajesh M.S.

[geezeweezebabypls]

Bloody procedure

[renesisrx8]

I think I keep washing off the bacterial smear every time I do it. I can't see anything under the microscope. I heat fixed the bacteria on the slide but as soon as I rinse off the crystal violet, the slide looks like it's been washed clean. The smear isn't visible anymore. I get to try it again tomorrow so I will try to leave the crystal violet on a bit longer and maybe add 2 or more loopfuls of bacteria to the slide.

[kingofweightloss]

Everyone complaining about dumping it down the sink, staining kills bacteria!

[eyebal]

@kingofweightloss

Ok, but what about the endotoxins produced by the gram negative bacteria?

OHMYGOSH

[23not24]

You dry it with bibulous paper after all staining and rinsing not over heat.

[Huffdev]

what is nthe best stain

[unnecessaryremarks]

i hope that wasnt a prepared smear of an organism. biohazard contamination? and its DEFINITELY not safe for those chemicals to be dumped in the sink

[nuntyfunt]

one last pest on the streets.. and it wont be shiting in your nabers gatden eather.. if you couldent aford the bill to put the cat down,, you should of got a lend of a dog to sort things out for ya. ha ha

[meercachase]

thank u! i have practical exam tomorrow! and this video explains better than my lecture notes :D

[oOStarsAndStripesOo]

is it safe for all those chemicals to go down the sink??

[OmarChannel]

@oOStarsAndStripesOo Safe or not, I do it in my lab all the time even though my teacher tells us not too; But yah make sure you don't do it at home.

[HarryPotterTrio91]

Thanks so much. I'm learning this out of a text book right now and it helps to see how it's actually done!

:)

[gaiafrances]

shouldnt have done it in the sink . u r going to create super bug in the future . and u didnt put on ur gloves ! its important ya .

[datzfast]

never heat fix with a candle you will splatter living infectious bacteria everywhere dont believe me? do it over a petri dish and amaze your friends. i assume you all are college students. you can air dry your slides under a biological safty cabinet or use a comercial slide dryer under the hood also.

[datzfast]

i gram stain for 23 years, tilt the slide at an angle, spray pure acetone to decolorize until crystal violet stops being removed. about 4-10 seconds use your eyes, rinse with good tap water.

[ElNinoLalibre]

Can someone answer this!! .. what are the differences in gram positive and gram negative emitting bacteria. could u outline the whole scenario

[spacewolf5462]

@ElNinoLalibre, I'm only 5 months too late, but I'll tell you the difference anyway

Gram Positive bacteria have a thick wall of peptidoglycan. When you add the mordant (grams iodine), you crystalize the crystal violet and basically get it stuck in the cells so that it won't wash out.

Gram negative bacteria have a tiny amount of peptidoglycan, so the alcohol wash actually breaks holes in the cell wall. This means the crystal violet will leak out of the cell. All thats left is the counterstain. 

[Wannabepro1]

Why is it necessary to Decolorize for just a few seconds? not a minute (just like the other substances rinsed)?

[beverlyhillschic]

thanks for posting, I had exam and did not "see" my bacteria after staining, my question is, why is your bacteria so pronounce? it looks like alot more than a loopful, when my heat fix smear is dried, i don't see anything, any pointers, I have to repeat it today and I need to pass, is it ok to add 2 loopful instead of one? thanks.

[datzfast]

@beverlyhillschic you must heat fix the bacteria to the slide befor you start staining or they will wash away with the staining steps.

[Archzenom]

Nice! This will surely turn as a blaster of my previous lessons in third year CLS class. Thanks.

[Kimarey]

Didn't it stain your hands when you didn't wear gloves? It takes forever for all the stains to come off if you don't wear gloves. Well not forever but yeah.

[Rodsan555]

maybe if you use gloves next time it would be better for your health!

[AstroBoyLives]

i thought you had to heat-fixate the slide first. if so, can you use a candle? and for how long should it be held over the flame? also, i thought you were supposed to use bibulous paper.  is there a substitute you can use? would a soft tissue work?

[messengercrow]

yes it needs to be heat fixed first. just move the slide over a candle (bacteria face up). don't burn the bacteria or you cannot see anything under microscope after staining.

[AstroBoyLives]

thanks! very helpful.

[tigerstripes379]

In fact. Proper technique requires that you blot dry the slide with bibulous paper. What would happen if you made a mistake a didn't rinse off all of the alcohol from the slide?

[tigerstripes379]

Actually it is necessary to wait 60 seconds because it allows the CVI complex to form better. If you don't do that you will get a false negative and that's not good. If you leave it on too long you will get a false positive and that's not good either.

[Erysipelothrix14]

is it really required to dry heat fix after applying safranin what we do is just air dry or blot dry

[Animechiongster]

i dont think drying over heat is required at the last step.

[rgiegoldt]

you don't have to wait the whole 60 seconds....as long as the crystal violet, iodine and safranin all get the same amount of time it'll be fine. I leave each step on for approx. 15 sec's without problems.

[TheSkripter]

LOL i hoped he waited 60 sec for real in the video XD

[TAKYEELEUNG]

purPle = Positive

piNk = Negative

[graveey]

oh ye sweet cheers, have always had trouble remembering, silly me :)

[D0UKHI]

heat fixation

Cristal violet 60s hen wash

morden with iodine then wash

critical step is dcolarizing with methanol 95% 3s

then wash

or it will become both red like this video

damaging the LPS

[D0UKHI]

it must heat fixation first then gram stain procedure come second !

and use methanol 95% or acetone 50% with Hcl 50%

red G-

blue G+

[yamani0007]

tnx alot cego and welcome AION here ^.^

[TheChaMpMvp]

Im Matthew Cusack and i do this with my mum at home for fun. Instead of partying all night i gram stain with her.

She is a saint

[i0nical]

No, that's the proper way to Gram stain. I don't know if I would dry over heat though. It might destroy the lipid membranes of your sample.

[adrastea99]

Correct me if I'm wrong, but this looks like some screwed up version of the acid fast stain

[D0UKHI]

aid fast use carbon fusion with heat for 5 min

then cool and decolarizing with alcohol+acid

then methel blue

blue non acid fast

red acid fast

[CEast]

wear gloves

[acrane74]

how much trouble did you get into for the sink? CV stains pretty good, did it come out?

[goldenboiaeroul]

tnx

[whyharish]

Good, work,but flush slowly rather than flushing fast, because some times it will erodes the smear. good work,

[tuddyfruity4]

my husband and I do gram staining at home for fun

[somubond27]

lol... nice answer.. :P

[2SXC2H8]

@tuddyfruity4 lol, you must have an exciting life

[dragenov]

thanx reallly thanx for this vid

[hustladoggy]

thanks from chile by the video...

[76ivonneastrid]

Well superman, i love the lack of gloves. And where is the link to the video that shows us how you get rid of THE MRSA that happily live in your room? I also need your zip code... to stay away from there! Youdont work in a restaurant do you? At a doughnut shop? Maybe at the food processing plant that spreaded the salmonella and by which tomatoes and chillies were held responsible? are the one they fired?

[kadgek]

Im with you there... also, decolourizer should be left on no more than 10 secs... otherwise you will have false results.

I work in a food lab and we would never do a gram stain like this. Also these chemicals are hazardous and should not be flushed down the sink!

You can blot dry the stain very gently... I wouldnt hold it over a flame!

[whyharish]

yeah,hazardous,but drying will easily catches to your hand or some where,better procedure is flushing,no need to worry about this method

[whyharish]

well,know before you comment,once the heat fixed there is no problem, so try to observe and then comment.

video pretty good .

[ryoko79]

in class we blot dry

[kadgek]

Yup!  Carry on blotting... that is the correct thing to do.

[izdagrimeyone]

thats easy... its the heat fix i have trouble with how long do u keep it with out ruining the slide

[BeeSoe1]

just a quick pass over the flame would do

[d0ughboy07]

can you decolorize to the point where the slide becomes clear? i figured that if i do that, i may get a false gram - result

[evlunknwn]

you are suppose to decolorize with the alcohol -before- you wash with DI H20 to ensure that the decolorizing process ceases before adding the safranin.

[safranin66]

Crystal violet is 2 g of 90% crystal violet dissolved in 20 ml of 95% ethyl alcohol.

Gram's iodine is 1 g of iodine, 2 g of potassium iodide, dissolved in 300 ml of distilled water.

Decolorizer is 50% ethyl alcohol, 50% acetone

-- i use these materials for my gram staining protocol

[safranin66]

Crystal violet is 2 g of 90% crystal violet dissolved in 20 ml of 95% ethyl alcohol.

Gram's iodine is 1 g of iodine, 2 g of potassium iodide, dissolved in 300 ml of distilled water.

Decolorizer is 50% ethyl alcohol, 50% acetone

- i use this materials for my gram staining protocol

[safranin66]

yah.. i agree.. use alcohol + acetone.. not HCL..

Also, you should use "distilled water".. not

jus water only...

[esanez]

Very helpful

[Sergionewyork]

gloves? hahahaa !

[ImSpartacus811]

Wow, this looks complicated, but very useful. I'm doing a science fair project (10th grade bio) involving identifying bacteria, and I'm struggling to find a good idiot's guide for bacteria testing methods and culturing. Anybody know any good guides or websites?

As for the video, I'm sorry, I don' have enough experience to judge it right or wrong.

[fuckenweak]

grow mushrooms

[meddy1979]

Jesus almighty, what have you done to your sink, LOL! Agree on alcohol/acetone mix;). Actor001 doesn't have a life:).

[cegodsey]

Actually, it came out easily using decolorizer. Gave me a bit of a scare at first!

[actor001]

for gram we dnt use alcohol+HCl thats for zihel stain, if you use alcohol+HCl the peptidglican of the gram- will give you a false gram+ so, we just use alcohol+cetone. please check it.. because remember that.. HCl change the structural configuration of crystal violet and denaturalize the pepids on the superf..

thnks im a BFQ

[actor001]

sorry but you have a little mistake..

you need to decolorate in about 15-30 seconds with alcohol-cetone complex in (50-50) or (20-80) and then wash it..

other thing.. you need to dry it in room temp..or a blower but never use a oven

carbol fuc.. is the AARB thats for mycobacteriums and other mycobacteria type

ta taaa

[cegodsey]

actor001 - we use alchohol-HCL and do it very quickly - just a few seconds. And then we either let it air dry, use a paper towel to pat it gently, or reheat the slide over a flame (gently). Everybody does it a little differently, so don't look at it as a mistake - just a different method.

[nadiaw00t]

@cegodsey I agree you 15-30 secs ethanol is too long - you'll get poor visibility

[datzfast]

@cegodsey your mixing up the procedure for the kinyons cold stain for the zeil-nelson hot stain and the gram stain. good luck with that.

[akmnawab110]

that's a gram positive because the glue/purple dye was retained after the addition of the 95% alcohol-Acetone

[cegodsey]

I used spit. Is spit normally gram positive?

[akmnawab110]

no....your going to die from the spit gram postitive disease.......

[cegodsey]

Aacckkk!

[Cal0yski]

yeah... Normal flora of spit.... may be some cocci's in ur spit, dude. hehehe. You wont die, ofcourse

[maccomplex]

Carbol Fuchsin can also be used for the final differential stain

[Mindymed1]

I really needed to study this. Thanx Dude.