Download Stains - Dr Magrann

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