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STAINING
Most bacteria are difficult to see under the bright field microscope. Bacteria are almost colorless (remember, all cells are composed primarily of water) and therefore show little contrast with the broth in which they are suspended. To visualize bacteria, either dyes or stains, or an alternative source of illumination (phase contrast or differential interference contrast) are used. Since staining of bacterial cells is relatively fast, inexpensive, and simple, it is the most commonly used technique to visualize bacterial cells. Staining not only makes bacteria more easily seen, but it allows their morphology (e.g. size and shape) to be visualized more easily. In some cases, specific stains can be used to visualize certain structures (flagella, capsules, endospores, etc) of bacterial cells.
There are several staining methods that are used routinely with bacteria. These methods may be classified as 1) simple (nonspecific) and 2) differential (specific). Simple stains will react with all microbes in an identical fashion. They are useful solely for increasing contrast so that morphology, size and arrangement of organisms can be determined. Differential stains give varying results depending on the organism being treated. These results are often helpful in identifying the microbe.
Stains (dyes) are chemicals containing chromophores, groups that impart color. Their specificity is determined by their chemical structure. Stains are generally salts in which one of the ions is colored. (A salt is a compound composed of a positively charged ion and a negatively charged ion.) For example, the dye methylene blue is actually the salt methylene blue chloride which will dissociate in water as positively charged methylene blue ion which is blue in color and a negatively charged chloride ion which is colorless. Commonly used microbiological stains generally fall into one of two categories - basic stains or acidic stains (although there are a few stains such as Indian Ink which are neutral).
Basic dyes: A basic dye is a stain that is cationic (positively charged) and will therefore react with material that is negatively charged. The cytoplasm of all bacterial cells have a slight negative charge when growing in a medium of near neutral pH and will therefore attract and bind with basic dyes. Some examples of basic dyes are crystal violet, safranin, basic fuchsin and methylene blue.
Acid dyes: Acid dyes have negatively charged chromophores and are repelled by the bacterial surface forming a deposit around the organism. They stain the background and leave the microbe transparent. Nigrosine and congo red are examples of acid dyes.
At first glance, the easiest way to stain bacterial cells would appear to be simply mixing the bacterial suspension with the dye and making a wet mount of this mixture. Unfortunately, if you were to try staining bacterial cells in this manner you would find that there was too much background (unbound dye) to allow for visualization of the cells. Therefore, you need to remove the unbound dye. Simply washing off the dye would result in removal of the cells along with the excess dye. Therefore, you need a mechanism to fix the cells to the slide before staining to allow for removal of excess dye while keeping the cells on the slide. A simple method is that of air drying and heat fixing. The organisms are heat fixed by passing an air-dried smear of the organisms through the flame of a gas burner. The heat coagulates the organisms' proteins causing the bacteria to stick to the slide. Be very careful not to over heat the organisms when fixing them to a slide. This distorts the sample of the organisms.
Preparation of a Bacterial Smear and Heat Fixation (If the culture is taken from solid (agar) medium)
- Clean the slide using alcohol like cleaning material. Gently dry the slide with a lint free cloth.
- Place a very small drop of distilled water on the surface of the slide.
- Aseptically remove a small amount of the culture from the agar surface by inoculation needle and just touch it several times to the drop of water until it just turns cloudy.
- Burn the remaining bacteria off of the loop. (If too much culture is added to the water, you will not see clearly stained individual bacteria).
- Using the loop, spread the suspension over the entire slide to form a thin film.
- Allow this thin suspension to completely air dry.
- Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4 times to heat-fix. (Caution: Too much heat might distort the organism and, in the case of the gram stain, may cause gram-positive organisms to stain gram-negatively. The slide should feel very warm but not too hot to hold)
(If the organism is taken from a broth culture)
- Clean the slide using alcohol like cleaning material.
- Gently dry the slide with a lint free cloth or tissue paper
- Aseptically place 2 or 3 loops of the culture on a clean slide. Do not use water.
- Using the loop, spread the suspension over the entire slide to form a thin film.
- Allow this thin suspension to completely air dry.
- Pass the slide (film-side up) through the flame of the Bunsen burner 3 or 4 times to heat-fix.
Simple (Direct) Staining with Methylene Blue
- A simple stain that stains the bacterial cell is called as direct staining. Many of the bacterial stains are basic chemicals these basic dyes react with negatively charged bacterial components to colour the cell.
- Prepare a heat fixed smear of the culture you wish to examine
- Cover the smear with methylene blue by pouring over the smear Allow the dye to remain on the smear for approximately 1 minute. (Note: staining time is not critical. Somewhere between 30 seconds and 2 minutes should give you an acceptable stain. The longer you leave the dye on, in general, the darker the stain.)
- Wash the excess stain off the slide, pick up the slide by one end and hold it at an angle over the staining tray.
- Using the distilled water wash bottle, gently wash off the excess methylene blue from the slide by directing a gentle stream of water over the surface of the slide.
- Wash off any stain that got on the bottom of the slide as well.
- Blot off excess stain using tissue paper. DO NOT rub the slide, rather place the slide between two sheets of tissue paper and press down gently. Paper will absorb excess dye.
- Examine the slide under the bright field microscope. Record the shape, arrangement, and approximate size of the organisms.
- You should have data on two cultures which you have stained.
Positive (direct) and Negative (Indirect) Stains
When a cell is stained without staining the background, it is called direct or positive staining. Many simple strains are direct or positive stains. However, in some cases, negative straining also used. Here the cells are not stained, but the background is stained. Here, an acidic dye like nigrosin or neutral dye like Indian ink is used. Acidic stain carries a negative charge and repelled by the bacteria, which also carry a negative charge on their surface. Hence they appear transparent upon examination. A stain that stains the background and does not stain the bacteria is called negative stain. It is useful for visualizing capsulated bacteria that are difficult to stain.
Gram Staining
Gram Staining
Gram staining is the single most useful test in the microbiology laboratory given its simplicity and ability to differentiate bacteria into two main groups: gram-positive organisms and gram negative organisms. Hans Christian Joachim Gram first devised the original procedure in the late 19th century, and although modifications have since been made, the basic principles and results remain the same. The staining spectrum includes almost all bacteria with notable exceptions include intracellular pathogens such as Chlamydia and Rickettsia, and those organisms lacking a true cell wall such as Mycobacterium, Mycoplasma, and Ureaplasma.
The staining procedure occurs in four parts: the first step is the addition of the primary stain, crystal violet, this initial step stains the contents of the slide purple. After the rinse, Gram's iodine is added to chemically bond the alkaline dye to the bacterial cell wall, if possible. The third step is the decolorizing step where the slide is exposed to an alcohol wash to take off any unbound crystal violet. The last step is the application of a counterstain. The most common counterstain is safranin, which colors decolorized cells pink. An alternate counterstain is basic fuchsin, which gives the decolorized cells more of a bright pink or fuchsia coloration. The basic fuchsin counterstain works particularly well for anaerobic bacteria, but poorly for aerobic organisms like Bacillus.
Principle :The differential properties of the staining process are attributed to the differences in composition between gram-positive and gram-negative cell walls. Gram-positive cell walls contain a thick layer of peptidoglycan, numerous teichoic acid cross-linkages, and low lipid content. These structural elements make the cell less permeable to organic solvents making it resistant to the decolorizing process. Conversely, the cell walls of gram-negative organisms show higher lipid content and an increased permeability to decolorizer and thus lose the crystal violet dye.
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