Download 2c Types of Stains

STAIN TECHNIQUES
Proper order for staining: Place organism on slide, air dry, heat fix, stain
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 of a negative stain that we will use in lab is Nigrosin (or
India ink). 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. It is also good for seeing the shapes of the organisms.
We can measure the cells by using a tiny ruler in the eyepiece of the microscope called an
ocular micrometer.
TYPES OF STAINS
1) A SIMPLE STAIN uses only one stain; an example is methylene blue. This is
what we can use to stain cheek cells. We used it to stain bacteria last time.
2) A DIFFERENTIAL STAIN uses several stains; examples are the Gram stain
and the Acid-Fast stain. These are used to find out what category of bacteria are
present.
3) A NEGATIVE STAIN stains the background instead of the bacteria.
4) There are also a number of SPECIAL STAINS for viewing endospores, capsules,
or flagella.
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SIMPLE STAIN (Methylene Blue) Of Cheek Cells or bacteria
Obtain bacteria by scraping the inside of your cheek with the FLAT end of the
toothpick. Place the toothpick with bacteria directly into a dime-sized circle on the
center of the slide. 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 over the gray bucket on the white tray and cover the smeared area with
methylene blue. Allow to sit for only one minute. Tilt the slide over the bucket 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 are no bacteria. Otherwise, you might rinse the
bacteria right off the slide.
Press the slide in bibulous paper several times and examine under microscope. If
there are no bacteria, you did not heat fix it long enough.
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.
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 (like cement) with crystal violet and
peptidoglycan in the cell wall of bacteria. It keeps the crystal violet from being washed
out by the alcohol in Gram positive organisms (because the PG layer is thick in G+).
3. DECOLORIZER: Alcohol or acetone. This removes the primary stain
(decolorizes) from Gram negative cells.
4. COUNTERSTAIN: Safranin. This is a red color that stains the cells that became
decolorized (Gram negative).
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 almost 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. A few organisms are
neither. Instead, they are acid-fast, so this stain is the next to do.
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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 (pink color)
MORDANT: heat
DECOLORIZER: acid alcohol
COUNTERSTAIN: Methylene blue
If a bacterium is positive for the acid-fast stain, this indicates it has mycolic acid in the
cell wall. Mycolic acid 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 carbol fuchsin 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. Patients on immunosuppressing agents (eg. organ transplant recipients,
autoimmune diseases, etc)
4. Cancer patients on chemotherapy
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, artificial heart
valve, etc)
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NEGATIVE STAIN (Nigrosin or India Ink)
If an ion has a negative charge it is called an ANION. A negative stain is one that
contains anions on the chromophore (color portion of the dye). That means the color
portion of the dye has a negative charge (it will also be acidic). 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. The purpose of using a negative stain is to
determine the size and shape of an organism, and its arrangements (staphylo, strepto,
diplo, etc).
There are three types of negative stains:
a) Nigrosin
b) India ink
c) Congo red
Negative Stain Prep
Clean two 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 use another slide at 45 degrees, and smear the drop (see photo in lab book).
We will be using aseptic technique, so we need gloves and lab coat.
MOUTH BACTERIA
Clean two 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.
There is no need to heat-fix Nigrosin because the stain makes the bacteria stick to the
slide. Just let it air dry. When viewing the Nigrosin slide, observe under 1000x and oil.
Observe shape of cells (bacilli, cocci, or spirals) and arrangement (staphylo, tetrads,
strepto, diplo).
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Above is a drawing of bacteria with
a negative stain. The most practical
use for a negative stain is to
determine cell size and morphology
(shape) because there is no need to
heat-fix the slide. Heat-fixing
causes the cells to shrink.
This is an actual photo of a
negative stain. See the
spirochete in the center (looks
like a wavy noodle)? This
organism is frequently found
between the teeth.
SPECIAL STAINS
A. 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 an
endospore, which is metabolically inactive. It is resistant to chemicals and heat. It
is not the same thing as a spore, which is the result of sexual conjugation in
bacteria. When the environment improves, they can re-establish themselves. Only
sterilization can kill an endospore. Endospores are only produced by Gram
positive rods that are found in the soil, such as Bacillus (non-pathogenic) and
Clostridium (tetanus and botulism). Spores will be green and the bacteria is pink.
a.
b.
c.
d.
PRIMARY STAIN: Malachite green
MORDANT: Heat (allows dye to penetrate the endospore)
DECOLORIZER: Water
COUNTERSTAIN: Safranin
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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. Thus, it is a type
of negative stain. It is also a simple stain since it uses only one stain. 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.
A.
B.
C.
D.
Monotrichous
Lophotrichous
Amphitrichous
Peritrichous
GRAM STAIN
This is the most common type of stain. It is the first step in identifying an organism. It
does not identify the organism, but it is the first step. It is one of the differential types of
stain. The reaction of stains is different because of differences in the chemistry of the
cell wall.
Unlike Gram positive bacteria, the cell wall of a gram negative organism has many lipids
in the cell wall:
LPS – lipopolysaccharide
LP – lipoproteins
PL – phospholipids
GRAM POSITIVE
Purple
Thick peptidoglycan
Very little lipid
GRAM NEGATIVE
pink
thin peptidoglycan
Lots of lipid (in the outer membrane)
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Gram Stain Procedure
First, prepare the sample on a slide, air dry and heat fix.
1. PRIMARY STAIN (the stain used first)
a. Crystal Violet
b. Leave on for 1 minute
c. Rinse gently with distilled water
2. MORDANT (a mordant is something that causes the primary stain to form a
complex with it to chemically react with the cell).
a. Gram’s Iodine (not regular iodine)
b. Leave on for 1 minute
c. Rinse gently with distilled water.
d. The iodine forms a complex with crystal violet which reacts with the
peptidoglycan in the cell wall.
e. Without iodine, all of the stain would wash away.
3. DECOLORIZER
a. 95% Ethyl alcohol (ETOH)
b. As an alternative, you can use ETOH mixed with acetone.
c. This is the most critical step because the alcohol must be left on for just
the right amount of time.
d. Drip three drops of alcohol onto the slide then quickly rinse gently with
water from the water bottle.
4. COUNTERSTAIN
a. Safranin
b. Leave on for 1 minute
c. Rinse gently with distilled water
d. Blot dry.
What are some reasons why you might get a Gram positive culture show up with both
purple and pink cells that are all the same size?
1. The culture is too old (more than 24 hours). Some of the cells have died, and the
peptidoglycan breaks apart, so it appears Gram negative.
2. The decolorizer was left on for too long.
3. The sample smear was too thick and the stain did not get through to all the cells.
Suppose you look at a Gram stain of staphylococcus and you see staphylo, diplo, singles,
and tetrads on your slide. They are all purple and they are all the same size. Is the culture
contaminated? No, it is probably still pure because the cells are all the same size. The
clusters just broke apart during the preparation, or you are seeing a new cell in the
process of dividing into a cluster. If you see both purple and pink cells of different
shapes, that is not a pure culture. If the culture is supposed to be pure, it became
contaminated.
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Get three clean slides and prepare one slide of the following cultures that are less than 24
hours old:
1. E. coli (Gram neg rod)
2. B. Megaterium (Gram + rod)
3. Staphylococcus aureus (Gram + cocci)
Problems:
When you do a Gram stain:
What would happen if you forget to use Gram’s iodine? (G+ cells would be pink)
Over-decolorize? (G+ cells would be pink)
Use no alcohol? (G neg cells would be purple)
Use an old culture? (Gram negative cells might be purple, Gram + cells might be pink
or purple and pink because some PG has broken down)
COMPARISONS OF STAINS
PRIMARY
STAIN
MORDANT
DECOLORIZER
COUNTERSTAIN
G+
(PURPLE)
Crystal violet
Iodine
ETOH or
acetone:
Cell is purple
Safranin:
Cell is purple
G – (PINK)
ACID
FAST
Crystal violet Carbol
Fuschia
Iodine
Heat
ETOH or
Acid alcohol
acetone:
Cell is clear
Safranin:
Methylene
Cell is pink
Blue
ENDOSPORE
STAIN
Malachite green
Heat
Water
Safranin:
Endospores are
green
Cell is pink
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