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STAIN TECHNIQUES Making a stained slide of your own cheek cells involved several steps. First, the slide is cleaned and dried. Then the flat part of a wooden toothpick is used to scrape the inside of your cheek. The toothpick is then smeared onto the slide and allowed to air dry. If we look at the smear under the microscope at this point, the cells will be very difficult to see because there is little or no contrast: they will be almost clear against a bright background. Therefore, stain is applied. Preparing a smear of bacteria is similar; using a sterile loop, a colony of bacteria is smeared on a clean slide and allowed to dry before staining. The first step in preparing a slide for staining is to “fix” the slide. This is done by passing the slide through a flame a few times. The purpose of this is to attach the cells (or the bacteria) to the slide and kill the microbes. This procedure shrinks the cells and causes the proteins in the cells to become like glue. The slide is then stained so they can easily be seen. You must beware of “artifacts” when you are viewing slides you prepare. They are pieces of dried dye, dust, or other substances that are not part of the specimen. The stain is a dye that is made of a salt with a colored ion (called a chromophore). If the ion has a positive charge it is called a cation; if it has a negative charge it is called an anion. A cation creates a basic dye (pH higher than 7) and an anion creates an acidic dye (pH lower than 7). A basic dye is used to stain the cells that you wish to observe. This is the type of stain that we will mainly be using in lab. An example is the Gram stain. An acidic dye, also called a negative stain, is used when you want to stain the background instead of the cells. An example that we will use in lab is Nigrocin. With negative stains, no heat fixation is necessary; the dye is sticky, so you simply mix the cells in with the dye and spread it out thinly. Since there is no heat fixation, the cells don’t shrink. Therefore, this is the stain technique to use when you want to measure the size of cells. We can measure the cells by using a tiny ruler in the eyepiece of the microscope called an ocular micrometer. TYPES OF STAINS There are different types of basic stains. A simple stain uses only one stain; an example is methylene blue. This is what we will use to stain cheek cells. A differential stain uses several stains; and example is the Gram stain. This is used to stain bacteria. There are also a number of special stains for viewing spores, capsules, or flagella. DIFFERENTIAL STAINS A. GRAM STAIN 1. PRIMARY STAIN: Crystal violet. This is the first stain used. 2. MORDANT: Iodine. The mordant is what allows the primary stain to react chemically with the cell. It forms a complex with crystal violet and peptidoglycan in the cell wall of bacteria. It keeps the crystal violet from being washed out by the alcohol. 3. DECOLORIZER: Alcohol or acetone. This removes the primary stain from some of the cells (decolorizes some of the cells). 4. COUNTERSTAIN: Safranin. This is a red color that stains the cells that became decolorized. The Gram stain is used to distinguish between Gram positive bacteria (will look violet because they are not decolorized) and Gram negative bacteria (will look pink from the safranin because they were decolorized). Since all bacteria are either Gram positive or Gram negative, this stain is the first thing used to determine what type of bacteria is present in the specimen. This helps us figure out what organism we are dealing with. The results are recorded as Gram positive or Gram negative. B. ACID-FAST STAIN The results of this stain are recorded as acid-fast or non acid-fast. An example is the Ziehl-Neelsen stain. Acid-fast bacteria look pink and non acid-fast look blue. 1. 2. 3. 4. PRIMARY STAIN: Carbol fuchsin (purplish-pink color) MORDANT: heat DECOLORIZER: acid alcohol COUNTERSTAIN: Methylene blue This is the stain of choice if one suspects an organism with a cell wall made of mycolic acid, which is a waxy substance that resists Gram stains. The heat in this procedure will melt down the wax in the cell wall to allow the stain to get in. Two organisms that are acid-fast that are pathogens (cause disease) are Mycobacterium and Nocardia. MYCOBACTERIUM 1. Mycobacterium tuberculosis: an air-borne pathogen that causes tuberculosis. 2. Mycobacterium leprae: Causes Hansen’s disease (formerly known as leprosy). NOCARDIA 1. Nocardia asteroides: lives in the soil. When inhaled, it can cause pneumonia, but usually only an opportunistic infection in immunocompromised patents. Opportunistic infections are infections caused by organisms that usually do not cause disease in a person with a healthy immune system, but can affect people with a poorly functioning or suppressed immune system. They need an "opportunity" to infect a person. Immunocompromised patients include the following: 1. Elderly people or infants 2. AIDS or HIV-infection 3. Immunosuppressing agents for organ transplant recipients 4. Chemotherapy for cancer patients 5. Malnutrition 6. Medicines (some antibiotics) 7. Medical procedures (surgeries, especially implanted joint replacements or internal fixation hardware such as screws and plates for broken bones) SPECIAL STAINS A. SPORE OR ENDOSPORE STAIN: When the environment becomes too harsh to survive, some bacteria have the ability to eliminate all their cytoplasm and condense all their essential DNA and organelles into a highly resistant structure called a spore, which is metabolically inactive. When the environment improves, they can re-establish themselves. Only sterilization can kill a spore. Spores are usually only produced by bacillus bacteria that are found in the soil, such as Bacillus (non-pathogenic) and Clostridium (tetanus and botulism) a. b. c. d. PRIMARY STAIN: Malachite green MORDANT: Heat (allows dye to penetrate the spore) DECOLORIZER: Water COUNTERSTAIN: Safranin B. CAPSULE STAIN: Some bacteria have a capsule which resists phagocytosis (being eaten by our white blood cells). An example is Streptococcus pneumoniae. This stain colors the background but the capsule remains clear. This will reveal the presence of a capsule, assisting in the diagnosis. C. FLAGELLA STAIN: Certain bacteria have flagella, which is a whip-like tail used to help them move. The tail is so thin it is not easily seen with ordinary stains. A special stain will reveal this structure. G+ (PURPLE) G – (PINK) ACID FAST SPORE STAIN Crystal violet Crystal violet Carbol Malachite PRIMARY Fuschia green STAIN Iodine Iodine Heat Heat MORDANT ETOH or ETOH or Acid alcohol Water DECOLORIZER acetone: acetone: Cell is purple Cell is clear Safranin: Safranin: Methylene Safranin: COUNTERCell is purple Cell is pink Blue Spores are STAIN green Cell is pink