hello in this video we'll talk about the principles behind acid fast staining acid fast staining is a differential staining method used in microbiology the stain is used to identify acid-fast organisms such as that of indigenous mycobacterium in this staining the acid first bacteria would appear reddish whereas the non-acid-fast bacteria would appear blue in this video it would be totally clear what is the basis of this differential coloring but let's understand what are the acid fast organisms acid fast organisms are characterized by the presence of wax-like nearly impermeable cell wall they have very high level of mycolic acid large amount of fatty acids waxes and complex lipid which make their cell wall literally impermeable to all the substances so their cell wall is so much resistant that they don't take up any other stains such as gram stain and acid first organisms are highly resistant to disinfectants or dry conditions and we need to do this kind of acid for staining to identify mycobacterium tuberculosis and that's why this is really important from a clinical perspective as well so let's hit the virtual lab and try to do a acid first training first of all let's get introduced with the reagents we need carbolfuse gene as the primary stain then methylene blue as the counter stain there would be acid alcohol which would be the decolorizing agent there would be a distilled water for washing purposes and some other reagents like pipette forceps all of these things would be very common to any microbiol lab so first step involves smearing the sample on a slide here the patient derived sample would be smeared on a particular slide and we are expecting that this particular sample might have mycobacterium tuberculosis in that at the end of this video we would understand whether it has it or not so the primary stain would involve carbol fusion carpal fusion is actually a particular stain which has phenol in it and it can really penetrate even this impermeable mycolic acid containing cell wall so they would penetrate and get inside the cell wall carbon fusing actually has basic fusion ethanol then phenol i mean the melt heat metal melted crystals of phenol and also distilled water so all the bacteria at this particular step would be colored red with the carbol fusion be it acid fast or be it non-acid fast and this incubation would happen for let's say 30 seconds after this step there is a heat fixation step which would fix the stain even further and it's kind of important such that these bacteria should adhere to the slide and should not wash away when we are adding other reagents now in the step three we would be using a decolorization agent after applying this decolorization agent the bacteria which are non-acid fast cannot retain this carbol fusion stain this decolorization agent is very strong and this decolorizing agent contains ethanol and hydrochloric acid so it would wipe out all the staining from the non-acid-first organisms remember the acid-first bacteria has very rigid impermeable cell wall so actually this decolorizing agent cannot wipe out the stain from these acid fast bacteria so by the end of this step the acid first bacteria should retain their red staining whereas the non acid fast bacteria would fail to retain their carbol fusion stain now in the last step we would be doing counter staining with a counter stain popularly used known as methylene blue it's a blue colored counter stain so obviously the bacteria which has failed to retain carbol 15 now would take the counter stain methylene blue remember this acid-first bacteria has very impermeable cell wall so they would not allow methylene glue to stem them so acid-first bacteria would retain their red coloration due to carbol fusion and the non-acid fast bacteria would ultimately take the countest in methylene blue and thereby they would be they would be visualized as blue cells so there are two methods used in the microbiology lab one is xyle nielsen method and another is kinyon method both these methods are useful for microbiologists and you can take a look at the reagents really quickly but i'm not going to get into details of these exact protocols now as per summary we have used a primary stain carbol fusion which is highly permeable it can even penetrate the almost impermeable cell walls of acid fast bacterium now after that we have used a strong decolorizing agent such as acid alcohol which would wipe out all the stain from non-acid fast bacteria and we have counter stain with methylene blue where the non acid fast bacteria took a blue stain and the acid first bacteria has retained their red curbal fusion stain this video tells us what is the basis of acid fasting and how it is really important for medical microbiologist you can get all the notes and flashcards in my facebook page the link is provided in the description you can click on the link and don't forget to like that page as well as usual don't forget to like 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