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. PRIMARY STAIN: Carbol fuchsin (purplish-pink color)
  2. MORDANT: heat
  3. DECOLORIZER: acid alcohol
  4. 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 cancause 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

  1. 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)
  1. PRIMARY STAIN: Malachite green
  2. MORDANT: Heat (allows dye to penetrate the spore)
  3. DECOLORIZER: Water
  4. COUNTERSTAIN: Safranin
  1. 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.
  1. 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
PRIMARY STAIN / Crystal violet / Crystal violet / Carbol Fuschia / Malachite green
MORDANT / Iodine / Iodine / Heat / Heat
DECOLORIZER / ETOH or acetone:
Cell is purple / ETOH or acetone:
Cell is clear / Acid alcohol / Water
COUNTER-STAIN / Safranin:
Cell is purple / Safranin:
Cell is pink / Methylene Blue / Safranin:
Spores are green
Cell is pink

SIMPLE STAIN (Methylene Blue)Of Cheek Cells

Place one drop of methylene blue on the slide.

Obtain bacteria by scraping the inside of your cheek with the FLAT end of the toothpick.

Place the toothpick with bacteria directly into the stain and spread in circles until the stain is spread out very thin with no gaps in the circle of stain.

When the slide is completely air dried (if wet, the water will spatter, spreading bacteria), heat fix the slide by passing it through a flame three times. The purpose of heat fixing is to make the cells stick to the slide so they don’t rinse off when we apply the stain.

Place the slide on a rack over the sink and cover the smeared area with methylene blue.

Allow to sit for only one minute. Tilt the slide over the sink and use a water bottle to gently rinse the stain off; make sure the stream of water is on the upper edge of the slide where there is no bacteria. Otherwise, you might rinse the bacteria right off the slide.

Press the slide in bilbulous paper several times and measure some bacteria.

SIMPLE STAIN (Methylene Blue) OF BACTERIAL COLONY

Take one of your clean slides and mark the underneath surface, in the center of the slide, with a circle the size of a dime. Sterilize a loop and allow the air to cool it. Place several drops of water from the water bottle onto the loop until you get a loop full of water. Tap the loop onto the center of the circle on the slide.

Obtain the colony sample with the inoculating needle and smear the bacteria into the water drop in progressively wider circles. Air dry completely to avoid heat-fixing a wet slide that will spatter bacteria (aerosols). The next step is to heat fix the slide. Why do we do this? Two reasons: to kill the bacteria so there is less danger, and to stick the bacteria onto the slide.

Place one drop of methylene blue onto the slide for 1 minute, rinse gently with water bottle, blot dry with Bibulous paper, and observe under 1000x.

NEGATIVE STAIN (Nigrosin)

What makes a stain a negative stain? It is acidic with its negative anion on the chromophore (color portion of the dye). That means the color portion of the dye has a negative charge. Bacteria cells also have a negative charge, so the bacteria cells repel the chromophore (dye). Therefore, the cell will have no color. Just the background is stained.

Negative Stain Prep

With NO gloves on, clean several slides, rinse, then squirt alcohol on the slides and dry with a paper towel and set them aside on a clean paper towel. Place one SMALL drop of Nigrosin to the edge of a slide and set aside.

We will be using aseptic technique, so we need the Bunsen burner, gloves, and lab coat.

MOUTH BACTERIA

Clean four slides, rinse, and dry.

Place one drop of Nigosin on the edge of a slide.

Scrape the pointed end of a toothpick between your teeth to pick up some plaque.

Wipe the plaque from the toothpick to the edge of the slide.

Mix the plaque with the stain using the toothpick in a tap-and-mix motion.

Throw the toothpick in the biohazard bag.

Take a clean slide (not a coverslip) and place it at a 45 degree angle facing TOWARD the stain drop. Slide it into the drop and wait five seconds so the capillary action draws the stain up and down the edge of the slide.

Maintaining the angle, quickly draw the top slide across the bottom slide to smear the drop. Leave the slide out to air dry.

The bacteria cells will look clear against a gray background.

You do not need to heat fix this slide, and don’t use a coverslip.

Whenever you look at a slide with oil immersion, you can change the objectives back to one of the lower two lenses, but do not ever use the hi-dry lens on a slide that already has oil on it, or it will get oil on the wrong lens.

TAKING PURE COLONY WITH A NEEDLE

Place on drop of Nigrosen in the center of a new slide. Take the sample with an inoculating needle instead of a loop. A loop picks up too much of the colony and it will be hard to see the individual bacteria cells. An inoculation needle picks up a much smaller amount of sample, so it will be easier to see.

Sterilize the needle from the tip to the end of the wire where it attaches to the handle by passing it through the tip of the inner blue cone of the flame. Cool it by dipping the wire tip into the agar in a clear area of the plate. What will happen if we touch a hot needle to a colony? It will be destroyed and change the normal morphology (size, shape, and arrangement of cells). Remember, when you lift the lid of the plate, hold it above the plate as a shield from air contaminants. Touch the inoculating needle to one colony and replace the lid.

Touch the inoculating needle tip into the Nigrosin drop and mix well by stirring in circles with the tip of the needle, but do not enlarge the drop much. Place a spreading slide at 45° to the drop, contact the drop for 5 seconds and spread the stain across the slide. Sterilize the needle again, and because it has sticky Nigrosin on it, rinse in water and wipe with a towel before you return it or use it again. This will remove the Nigrosin that the flame does not remove.

There is no need to heat-fix Nigrosin because the stain makes the bacteria stick to the slide. Just let it air dry. When your slides are all prepared, you can clean up your area and remove your gloves. When viewing the Nigrosin slide, observe under 1000x and oil. Observe the colony morphology (color, margin, elevation) of the pure culture you took a sample from. Also observe what you see under the microscope, including shape of cells (bacilli, cocci, etc) and arrangement (staphylo, tetrads, strepto, etc).

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