BACTERIAL STAINING

STAIN:    Stain is a dye used to colour                                                                                  STAINING:   The process of colouring a cell is called staining.                                                    BACTERIAL STAINING:  Bacteria are the transparent and colourless, so staining is required to see or observe or view them, bacterial staining is simple, fast, in expensive and commonly used technique.   We most often use basic stains to examine bacteria. Basic stains, due to their positive charge will bind electrostatically to negatively charged molecules such as many polysaccharides, proteins and nucleic acids. Acid stains bind to positively charged molecules which are much less common, meaning acidic stains are used only for special purposes. Some commonly encountered basic stains are crystal violet, safranin (a red dye) and methylene blue. Basic stains may be used alone (a simple stain) or in combination (differential stain) depending on the experiment involved.                              TYPES OF STAINING:    There are five types of staining:

1.      Simple staining

2.      Differential or gram staining

3.      Selective staining

4.      Wet mount staining

5.      Negative stain technique.

1)      Simple staining: a single dye is used to stain the microbial cell. Example: crystal violet or methylene blue.

2)      Differential or gram staining: two different dyes are used. Example: Gram staining or Acid fast staining.

3)      Selective staining: process of colouring a particular structure. Example: cell wall, flagella, etc

4)      Wet mount staining: used to study microbial motility. 

5)      Negative stain technique: negatively charged dyes are used, which repel the negatively charged cytoplasm leavingthe cel clear unstained.                                                                                           SIMPLE STAINING:                                                                                                                                           A single dye is used to stain the microbial cell. Example: crystal violet or methylene blue.(these are the positively charged dyes)                                                         REQUIREMENT:                                                                                                                               CULTURE: 24 hours oldof E.coli,B.subtilis, and Staph Aureus                                                                    EQUIPMENTS:wire loop, microscope, glass slide, and bunsen burner                                      PROCEDURE:                                                                                                                                             Step 1.           Bacteria from colonies. Clean a glass slide and place a small mark slightly off center using a grease pencil. Using your loop, transfer one small drop of water to the center the slide, being careful to be close to but not overlapping the grease pencil mark. Do not transfer too much water because these drops will have to air dry. Sterilize your loop and touch a single colony and transfer the bacteria to the water droplet on the slide and mix well. DO NOT scoop up a whole colony; you'll have vastly too much bacteria. If while mixing you see an opaque slurry of bacteria on the slide you have too many bacteria for effective staining.                                                                                                                  Step 1. Bacteria from broth. Clean a glass slide and place a small mark slightly off center using a grease pencil. Mix the broth containing the bacteria well because the bacteria may sediment to the bottom of the container. Use a sterile loop and transfer one or two droplets of bacteria to the center of a cleaned glass slide, close to but not overlapping the grease pencil mark.   Step 2. Drying. Allow the bacterial slurry (called the smear) to air dry. You CAN NOT heat the sample nor blow on it to hasten drying time because that could force bacteria into the air leading to contamination and possible infection.                                                                                          Step 3. Heat fixation. Holding the slide by one edge, pass it slowly through a bunsen burner flame. Do not move so slowly that the edge of the slide you're holding heats up to uncomfortable levels. This heat fixation step denatures bacterial proteins causing the cells to stick to the slide while also killing the bacteria making them safe for the following steps.                   Step 4. Staining. Place the stain in a staining rack and cover the smear with the stain of choice. Allow the stain to work for 30 seconds (some stains may have different staining times but this time will work well for simple stains). Remove the stain by rinsing with water from the squeeze bottle and gently blot (do not rub) the stain dry using bibulous paper. The slide is now ready to look at under the microscope. Because the bacteria were heat fixed, it will not be necessary to use a cover slip.               DIFFERENTIAL OR GRAM STAINING

6)      Two different dyes are used. Example: Gram staining or Acid fast staining.                          REQUIREMENT:                                                                                                                CULTURE: 24 hours old of E.coli,B.subtilis, and Staph Aureus                                         EQUIPMENTS: wire loop, microscope, glass slide, and bunsen burner.                                             THE GRAM STAINING:                                                                                                                The Gram stain is classifed as a differential stain because it allows us to distinguish between different types of bacteria. Bacteria can be quickly divided into two distinct morphological and functional groups on the basis of the Gram stain. By this technique, Gram positive bacteria stain purple and gram negative stain red. The bacteria are first stained with crystal violet followed by a brief treament with Gram's iodine. The iodine functions as a mordant to help the crystal violet bind more firmly. The bacteria are then rinsed with ethanol. Gram positive bacteria, which have multiple layers of peptidoglycan, retain the crystal violet while it is quickly rinsed out of Gram negative bacteria because their peptidoglycan is a single layer thick. The bacteria are stained a second time (counter stained) with the dye safranin which will not show up on the already purple Gram positive but will stain the decolorized Gram negative bacteria red.                                                                                Step 1. Preparation. Smear and heat fix the bacteria as described above (steps 1 through 3) for the simple stain.                                                                                                                          Step 2. Primary stain. Cover the smear with crystal violet and incubate for 30 seconds. Rinse the dye off with distilled water (dH2O) from the squeeze bottle.                                                              Step 3. Mordant. Cover the smear with grams iodine. After 20 seconds, rinse the slide with dH2O.                                                                                                                                                          Step 4. Decolorization Rinse the stain with 95% ethanol. This step must be done very carefully. Hold the slide at a 45^o angle over the staining rack and rinse with ethanol one drop at a time. Watch the ethanol as it runs off the slide looking for blue color. Stop dropping ethanol as soon as no more color is releases and rinse the slide immediately with water. A few drops of ethanol too many and the gram positive bacteria will also lose their crystal violet.                                                                                                                                                                     Step 5. Counterstain. Cover the bacteria with safrinin for 30 seconds. Rinse with dH2O and and blot the slide dry with bibulous paper.                                                                                           THE ACID-FAST STAIN                               REQUIREMENT:                                     CULTURE: 24 hours old of E.coli,B.subtilis, and Staph Aureus               EQUIPMENTS: wire loop, microscope, glass slide, and bunsen burner.                       ACID-FAST STAIN

7)       Bacteria with an acid-fast cell wall when stained by the acid-fast procedure, resist decolorization with acid-alcohol and stain red, the color of the initial stain, carbol fuchsin. The genus Mycobacterium and the genus Nocardia are acid-fast. All other bacteria will be decolorized and stain blue, the color of the counterstain methylene blue.    The acid-fast stain is an especially important test for the genus Mycobacterium. Besides the many saprophytic forms of mycobacteria, there are two distinct pathogens in this group: M. tuberculosis, the causative organism of tuberculosis, and M. leprae, the causative agent of leprosy. Mycobacterium tuberculosis (the tubercle bacillus) causes tuberculosis, although atypical species of Mycobacterium may occasionally cause tuberculosis-like infections, especially in the debilitated or immunosuppressed host. Mycobacterium avium-intracellulare complex (MAC), for example, frequently causes systemic infections in people with HIV/AIDS.                                                            PROCEDURE

8)      (Ziehl-Neelsen Method)                                                                                          1. Heat-fix a smear of a bacterium as follows:                                                                                                        a. Using the dropper bottle of distilled water found in your staining rack, place 1/2 a drop of water on a clean slide by touching the dropper to the slide.                                                                        b. Aseptically remove a small amount of the bacterium from the agar surface and mix it with the water. Flame the loop and let it cool.                                                                                                      c. Using the loop, spread the mixture over the entire slide to form a thin film.                                            d. Allow this thin suspension to completely air dry.                                                                                 e. Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4 times to heat-fix.                                                                                                                                                             2. Cover the smear with a piece of blotting paper and flood with carbol fuchsin.                                         3. Steam for 5 minutes by passing the slide through the flame of a gas burner.                                   4. Allow the slide to cool and wash with water.                                                                                  5. Add the acid-alcohol decolorizing slowly dropwise until the dye no longer runs off from the smear.                                                                                                                                                          6. Rinse with water.                                                                                                                                                    7. Counterstain with methylene blue for 1 minute.                                                                                        8. Wash with water, blot dry, and observe using oil immersion microscopy.                               Acid-fast bacteria will appear red; non-acid-fast will appear blue.                                                  WET MOUNT STAINING: The hanging drop and wet mount techniques allow for observation of living organisms. The wet mount tend to dry out quickly under the heat of the microscope light; it is simpler to perform than the wet mount, but it is useful for short-term observation only.  The hanging drop is a more complex technique, but it allows for longer-term obervation and more reliable observation of motility.    These techniques are usually performed without the addition of any stains; therefore, the organisms can be difficult to see. Reduce the illumination on your microscope as much as you can while still allowing yourself enough light to observe the organism.                                                                                                      How To Make A Wet Mount:                                                                                                    1. Gather a thin slice/piece of whatever your specimen is. If your specimen is too thick, then the coverslip will wobble on top of the sample like a see-saw:

                                                                                                                 2. Place ONE drop of water directly over the specimen. If you put too much water over the specimen, then the coverslip will float on top of the water, making it harder to draw the specimens as they float past the field of view!
3. Place the coverslip at a 45 degree angle (approximately), with one edge touching the water drop, and let go.

How To Stain a Slide:          1. Place one drop of Methylene Blue stain on one edge of the coverslip, and the flat edge of a piece of paper towel on the other edge of the coverslip. The paper towel will draw the water out from under the coverslip, and the cohesion of the water (due to those perennial favorites - Hydrogen Bonds) will draw the stain under the coverslip.

2. As soon as the stain has covered the area containing the specimen you are finished. The stain does not need to be under the entire coverslip. If the stain does not cover the area needed, get a new piece of paper towel and add more stain until it does.
3. Be sure to wipe off the excess stain with a paper towel, so you don’t end up staining the objective lenses.
4. You are now ready to place the slide on the microscope stage. Be sure to follow all the instructions on the previous pages as to how to use the microscope.
5. When you have completed your drawings, be sure to wash and dry both the slide and the coverslip and return them to the correct places!
6. All slides must be put away in the proper trays! Students will not leave until all materials have been put way properly.