[Music] welcome back to microbiology lab my name is Kevin tow cough please make sure to like this video and subscribe to my channel for future videos and notifications in this video we're going to discuss one of the staining procedures from experiment 4 and that is the acid bath stain the acid fast stain is a staining procedure that refers to organisms called acid fast organisms and it can be used to identify those so first of all what is an acid fast organism acid fast organisms are bacteria that have a very wakes like lipid dense cell wall so here's a schematic of an acid fast organism here's their cytoplasmic membrane or plasma membrane and all this stuff out here is outside of the cell in the cell wall and you can see here there's some carbohydrate components here's the peptidoglycan but out here we have a lot of lipids in particular this lipid called mycolic acid and this substance in particular an addition to the phenolic lipids make the cell wall very lipid like and hydrophobic okay and so all this combined will make the organism very resistant to a lot of conditions such as disinfectant dry conditions and then also resistant to stains such as the Gram stain okay the Gram stain arguably is one of the most important staining procedures because it's really the first step in identifying any species of bacteria but if you have an acid fast organism those stains of the Gram stain do not penetrate the cell wall because of these lipids right here such as mycolic acid and so gram stain's therefore are ineffective at identifying acid fast organisms and so because of that they require this special staining technique called the acid fast stain and that's what we're gonna discuss here now just so you know what we're dealing with here's an example up here of an acid fast organism these are bacilli right here and they belong to the species known as Mycobacterium tuberculosis which as the name suggests is the causative agent of the disease tuberculosis in fact one of the genera meaning plural of genus that is acid-fast is the Mycobacterium the other one being nocardia which we'll discuss a little bit more on the next slide but the point is michael bacteria are going to be one of the major two genera that belong to the acid fast classification here's another acid fast organism you can see again it's a bacillus or these are Vasili this is belonging to the species Mycobacterium leprae which you might be able to guess causes leprosy so actually those are two important things to know right off the bat about house acid fast organisms first of all the two genera that both that comprise the acid fast organisms those are Mycobacterium and then also as I mentioned on the previous slide nocardia right and then two major diseases that are caused by acid fast organisms particularly under the genus Mycobacterium are tuberculosis and leprosy right now what about the actual staining procedure what's the theory of this procedure well at least in steps it's very similar to the Gram stain you have a primary stain you have a decolorization step and a counter stain although the stains themselves are different now initially you're gonna heat fix a slide you're gonna you know make a smear heat fix and so forth just like we did with the Gram stain but you're actually going to add a different primary stain and that's car ball fusion now the specifics of the procedure involved you know you we put the paper towels over the slides and then we add the car ball fusion but the important thing here when we're adding the car ball fusion remember we use the Fischer burners and we use heat to drive this stain into the cells okay now I mentioned that the Gram stain is ineffective at staining acid-fast organisms that has to do with these lipid lipids in the cell wall that make it extremely tough and resilient and waxing but still in order to get this primary stain carpal fusion into the acid fast organisms you have to actually apply heat so without the heat this card ball fusion will not actually get into into the cell or at least penetrate through the cell wall so you actually have to apply heat from something like Fischer burner to actually get the car ball to penetrate into here okay and actually that's the critical step of the acid bath stain is the actual heating you can put all the carbo fusion you want on those cells on the slides but if you do not heat them with some heat source such as a Fischer burner and the card ball fusion will not make its way into the cell and therefore it will not stain red in the end okay now in this stain unlike the Gram stain there's no Morden so there's no iodine or anything like that so we proceed straight to decolorization right and when we decolorize we're going to use an acid alcohol very similar to what we used in the Gram stain and so after this step after the decolorization the acid fast organism will still be red while any non acid fast organism will have that red that car buff you should washed out and will be colorless after this step this is a similar pattern to what we saw in the Gram stain after that decolorization step okay and then finally we counter stain with methylene blue and so really both of these cells absorb the methylene blue but the carbon fusion and the acid fast organism is simply going to outweigh the blue and so it'll still appear red and then the non acid fast organisms will take up that methylene blue and since before that they were colorless at non acid fast organisms will appear blue when you look at them under a microscope okay so in a similar way to the Grand Stand it's a colorimetric stain so we're looking at the difference in the two colors to identify what type of organism it is except in this stain we have acid fast organisms that are red and non acid fast organisms that are blue okay let's take a look at this micrograph we'll blow it up here on the next slide so these organisms that are labeled as B right here these are red or kind of a fuchsia I guess you could say thus the name car ball fusion these organisms are going to be your acid fast organisms okay and if you were to actually zoom in on these you can't see it too well you would actually see that these are infected with syl.i which makes sense because pretty much all these organisms that are acid fast are going to be bacilli such as Michael Beck cerium tuberculosis and Mycobacterium leprae and in fact in the lab the species we used was non-pathogenic that was mycobacterium smegmatis that was a bacillus as well right these organisms over here that are blue these are going to be non acid fast and that's because they're blue they do not retain the carbon fusion after the decolorization step okay so now that we've looked through all of this I want to reiterate four major points about the acid bath thing that are gonna be important for you to remember other than the results which is obvious and that is remember that there are two genera that are gonna make up the acid fast organisms primarily and those are going to be Mycobacterium and nocardia also we have a couple diseases that are caused by acid fast organisms we have tuberculosis which is caused by this agent right here and then leprosy which is caused by this agent now in no way do you need to memorize these specific species but you do need to know the two diseases that are caused by these organisms another thing I want to reiterate is that unlike the Gram stain we don't have a positive result in a negative result like gram positive or gram negative we simply refer to this as acid fast if it's red and non acid fast if it's blue okay and then the other thing is to remember the critical step the critical step is the heating that we must do to drive the car ball fusion or the primary stain into the cell so that at the end acid fast organisms will stain red all right so hopefully this made sense to you please make sure to like this video and subscribe to my channel for future videos and notifications thank you very much