hi my name is dr alice lee and lecture 6 will focus on staining specimens for bright field microscopy one of the types of stains that we will talk about is ground staining which is shown in this particular image here of a gram stain image you can see distinct cell types both round and rod shaped cells and their different colors reddish pink and purple blue now most microbes will appear actually colorless if you were actually going to look at them through a standard light microscope such as an image a here and that's because cells are mostly made of water one way to actually visualize these colorless cells is by staining the specimen such as an image b here this will help improve contrast which is the difference in visual properties that make an object distinguishable from other objects and the background so you'll get a better image staining is what will improve the contrast between the specimen and the background of the slide the dyes that that we rv that we will be using are usually organic compounds which will bind to specific cellular materials the dyes are usually positively charged or cationic and therefore they will bind to negatively charged cell components such as the negatively charged backbone of dna cell proteins and the cell membrane there are three basic types of stains simple stains which use one basic dye and its purpose is to highlight the entire organism so that the cellular shapes and basic structures are visible unlike simple sayings differential stains are going to react differently with different kinds of bacteria and thus can be used to distinguish between them special stains are used to color and isolate a specific part of a microbe such as endospores or flagella at first glance you think the easiest way to stain bacterial cells would simply be to mix a bacterial suspension with the dye and make a wet mount of this particular mixture unfortunately if you were to try staining bacterial cells in this matter you would find that there was way too much background or too much unbound dye in order for you to allow visualization of the cells therefore you really need to remove any of this unbound dye but simply washing off the dye would result in removal of the cells along with the excess dye so you've got to find a way to fix the cells to the slide before staining in order to allow for removal of any of this extra dye while keeping the cells on the slide themselves a simple method is that of air drying the sample and then heat fixing it the organisms are heat fixed by passing an air dried smear of the organisms through a flame of a gas bunsen burner or a slide or putting it on a slide warmer the heat will coagulate the bacterium's proteins causing it to stick to the slide just be careful not to overheat or when you when you're fixing them to the slide this will ultimately distort the sample specimen when you heat fix a slide you want to apply just enough heat to precipitate the proteins which will then allow the cells to stick to the slide but it won't drastically change the shape of the cells or reduce them to charred remains once you've fixed the cells then you can flood the slab the slide with your stain rinse well and pat dry with some blotting paper occasionally a chemical is added to the solution in order to identif to intensify the stain such as a mordant which will help to increase the affinity of the stain for the particular specimen now differential stains will stain one kind of cell but not another this will help to separate them into groups by utilizing different properties of the cell in order to distinguish between them one example of a differential stain is the gram stain it looks at the fundamental differences in the cell wall of bacteria here you can see an image of a sample that has been gram stained you can see a mixture of cell shapes you'll see some rods you'll see some cocci these round ones and vibrios or spirals also they're just called curved cells you also notice the two different colors reddish pink pink which are going to be the indication for negative cell types like this vibrio here and these purple blue ones which will indicate a gram positive cell type like these cocci and rods the differential stain the gram stain is widely used in medical microbiology it will provide the initial characterization and classification of bacteria which is an important step in screening of an infectious bacteria for a patient this will help a doctor to prescribe the right course of treatment gram staining is based on the ability of the bacterial cell wall to retain a certain dye during solvent treatment the gram stain will separate the bacteria into two main groups the gram-positive bacteria will stain purple blue and tend to be sensitive to both penicillins and cephalosporin antibiotics gram-negative cell-type bacteria will seem reddish pink and tend to be more resistant to antibiotics but some types of antibiotics such as chromophilical and streptomycin might work any type of staining is ultimately going to kill cells and distort its features to some degree the acid fat stain is another important differential stain it will bind strongly to bacteria that have a particular waxy lipid material called mycolic acid in their cell walls microbiologists use the stain to identify all bacteria in the genus mycobacterium which includes some important pathogens such as mycobacterium tuberculosis which is the causative agent of tuberculosis and mycobacterium lepreide which causes leprosy this stain is also used to identify some pathogenic strains of nocardia the acid fast stains will retain a reddish pink carbofusion stain because it is much more soluble in the waxy cell wall lipids of these types of bacteria in the acid alcohol mixture while the non-acid fast bacteria lacking these waxy lipid components this red carbofusion dye will be removed and it ultimately leaves the cell colorless whereby a counter stain isn't applied and the cells will then stain blue distinguishing it from the acid fast microbes the image here shows a mixed culture that's been acid fast stained non-acid-fast bacteria will stain bluish purple and the acid fast bacteria will stain reddish pink there are also special stains that are used to color and isolate specific parts of microbes such as endospores or flagella or capsules in this particular image carbofusion dyes are used along bomborden to build up the diameters of the flagella until they become visible underneath the microscope endospores are special resistant dormant structures formed within a cell that protects the bacterium from adverse environmental conditions in this image the endospores are stained green within these pink rod shaped bacteria many microbes also contain a gelatinous covering called the capsule the presence of a capsule is a means of determining an organism's virulence or the degree to which a pathogen can cause disease note these colorless capsules that surround these purple stained rods here's some check your understanding questions and finally the suggested reading is chapter 3 for this particular lecture here's a picture of the gram stain kit that's actually sitting on my desk right now there are four reagent bottles each containing a different solution two are dyes crystal violet and safranin one is a mordant which is iodine that's used here and one is a decolorizer the acetone alcohol mixture