Download Procedure:

Bacterial stains
Most bacteria are difficult to see under the bright field microscope.
Bacteria are almost colorless and therefore show little contrast with the
broth in which they are suspended. To visualize bacteria, either dyes or
stains, are used. Since staining of bacterial cells is relatively fast,
inexpensive, and simple.
 Staining microorganisms enables one to:
1. see greater contrast between the organism and the
background.
2. differentiate various morphological types (by shape,
arrangement, gram reaction, etc).
3. observe certain structures (flagella, capsules, endospores,
etc.).
Stains (dyes): are chemicals containing chromophores, groups that impart
color.
There are several staining methods that are used routinely with bacteria.
These methods may be classified as:
1. Simple stain (nonspecific):
a) Positive (basic stain).
b) Negative (acidic stain)
2. Differential (specific).
a) Gram stain.
b) Acid-fast stain.
3. Special stain
a) Capsular stain
b) Endospore stain
c) Flagellar stain
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Simple Stain:
The simple stain consists of one dye. The dye adheres to the cell wall and
colors the cell making it easier to see.
a) Positive (basic stain): are cationic; exhibits a positive
charge. its bind to negatively charged cell structures like
nucleic
acids.
Methylene
blue,
crystal
violet
and
Carbolfuchsin are common basic stains. Dye binds to
the specimen.
b) Negative (acidic stain): are anionic; exhibits a negative
charge. its bind to positively charged cell structures like
proteins. Eosin , India ink and nigrosin are common acidic
stains. Dye does not bind to the specimen, but
rather around the specimen.
Positive (direct)stain procedure:
1. Prepare a heat fixed smear of the culture you wish to examine
2. Cover the smear with methylene blue (1 minute)
3. Wash the excess stain off the slide by distilled water
4. Blot off excess stain using bibulous paper. DO NOT rub the slide,
rather place the slide between two sheets of bibulous paper and
press down gently. Paper will absorb excess dye.
5. Examine the slide under the bright field microscope.
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Negative (Indirect) Stains:
Because the cell wall is also negatively charged only the background
around the cells will become stained, leaving the cells unstained.
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Negative stain procedure:
1. Place a small drop (one-two loopfuls) of the negative stain (India
ink) near the end of the slide.
2. Transfer one loop=full of the bacterial sample to the india ink and
mix the two together.
3. Hold a clean slide at about a 20o angle to the first slide. Touch the
edge of the clean slide to the bacteria/stain mixture so that the
mixture spreads across the edge.
4. Spread the suspension across the surface of the slide by drawing
the clean slide away from the mixture.
5. Air dry the slide. DO NOT HEAT FIX.
Differential Stains :
uses two or more dyes to differentiate between cells or structures;
A.Gram stain
Principle: Gram-positive cells have a thick peptidoglycan cell wall that
is able to retain the crystal violet-iodine complex that occurs during
staining, while Gram-negative cells have only a thin layer of
peptidoglycan. Thus Gram-positive cells do not decolorize with ethanol,
and Gram-negative cells do decolorize. This allows the Gram-negative
cells to accept the counter stain safranin. Gram-positive cells will appear
blue to purple, while Gram-negative cells will appear pink to red.
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Procedure:
• Prepare a Gram stain
1) Using an inoculating loop prepare smears of the bacteria by spreading
a thin suspension of each bacteria on a glass slide
2) Heat-fix the smears by passing them through the Bunsen burner flame
a 3 times.
3) Place the slides on the staining rack.
4) Cover the smears with the primary dye, crystal violet, and let it stand
for 30 seconds.
5) Rinse the slides with water.
6) Apply the mordant, Gram’s Iodine, by covering the smears and let it
stand for 1 minute.
7) Rinse the slides with water.
8) Decolorize with ethanol for 15-30 seconds. Add the ethanol slowly
until the crystal violet no longer floats up from the slide. Be careful not
to decolorize for too long.
9) Rinse the slides with water.
10) Cover the smears with the counter stain, safranin, and let stand for 1
minute.
11) Rinse the slides with water.
12) Blot dry using the bibulous paper.
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Procedure
Reagent
Cell color
Gram Positive
Gram Negative
Primary stain
Crystal Violet
PURPLE
PURPLE
Mordant
Iodine
PURPLE
PURPLE
Decolorizer
Alcohol
PURPLE
COLORLESS
Counter stain
Safranin
PURPLE
RED
B. Acid-fast Stain (Ziehl Neelsen):
Mycobacterium and many Nocardia species are called acid-fast because
during an acid-fast staining procedure they retain the primary dye carbol
fuchsin despite decolorization with the powerful solvent acid-alcohol
(95% ethanol with 3% HCl). Nearly all other genera of bacteria are
nonacid-fast. The acid-fast genera have the waxy hydroxy-lipid called
mycolic acid in their cell walls. It is assumed that mycolic acid prevents
acid-alcohol from decolorizing protoplasm.
pink to red acid-fast cells and blue non-acid-fast cells; distinguishes the
genera Mycobacterium and Nocardia from other bacteria; differential
stain.
A : acid fast bacteria
B : non acid fast bacteria
Ziehl Neelsen Acid-fast stain
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Procedure
Reagent
Cell color
Primary dye
Carbolfuchsin
Acid-fast Bacteria Acid-fast Bacteria
RED
RED
Decolorizer
Acid-alcohol
RED
COLORLESS
Counter stain
Methylene blue
RED
BLUE
Procedure:
1. Add one loopful of sterile water to a microscope slide.
2. Make a heavy smear of tested bacteria.
3. Air dry and heat fix well.
4. Cover the smear with carbolfuchsin dye. Place a piece of paper
towel on top of the dye. Be sure the paper towel is saturated with
the dye.
5. Place the slide on the rack over dry heat for 2 minutes.
6. Cool and rinse with water.
7. Decolorize by placing a drop of acid alcohol on the slide and
allowing it to sit for 15 seconds.
8. Wash the top and bottom of slide with water and clean the slide
bottom well.
9. Counterstain with Methylene Blue for 30 seconds to 1 minute.
10.Wash and blot the slide with bibulous paper.
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Special stains:
A. Endospore stain:
Principle: Spores have a durable outer coating that is composed of the
protein keratin. This keratin coat resists staining so in order to stain a
spore the primary stain, malchite green, must be heated to drive the stain
into the spores. Vegatative cells are then decolorized with water and
0.5% safranin is used to counterstain. Thus endospores are stained green,
while vegetative cells are stained red.
 (e.g. Bacillus spp., Clostridium spp.)
Procedure:
1. Malachite green is the primary stain .which is placed on blotting
paper over the smear gently heating over a warm water bath to
penetrate the spore coat.
2. The bacteria are decolorized with water. leaves the endospores
green as the stain is driven into the endospore . The malachite
green is washed out of the vegetative cells with the water.
3. It is then counterstained with safranin.
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B. Capsular stain:
Capsules are structures composed of carbohydrate or glycoprotein that
lay outside of an organism's cell wall and thus are in direct contact with
the environment. Many bacteria produce capsules under the right
conditions.
Bacterial capsules are non-ionic, so neither acidic nor basic stains will
adhere to their surfaces.
 In this stain we use acidic and basic dyes:
Acidic dye as India Ink and Nigrosen use to stain the background of
the slide but basic dye as methylene blue and crystal violet use to stain
the cell
results in background being dark; cells are unstained or stained with
simple stain; this reveals bacterial capsules- Klebsiella pneumonia.
Procedures
1. Use an inoculating needle to suspend the organism in a drop of
India Ink at one end of the slide.
2. Place the short end of a clean microscope slide into the suspension
and spread the mixture across the slide to form a thin layer.
3. Allow to air dry. Do not heat fix.
4. Cover the smear with methylene blue for 2-3 minutes.
5. Rinse gently with water and allow to air dry.
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Capsules appear as clear zones (halos) around the organism.
Examples:
Bacteria with capsules: Streptococcus pneumoniae, Klebsiella
pneumoniae, Pseudomonas spp. .
C. Flagellar stain:
Purpose: To determine the presence/absence and location of flagella
on various microorganisms
Principle: Because bacterial flagella are very thin and fragile a
special stain (flagella stain) is prepared that contains a mordant. This
mordant allows piling of the stain on the flagella, increasing the
thickness until they become visible. Various arrangements of flagella
are seen on different cells.
Preparation of smear :
1. Handling the suspension carefully remove a large loopful of the
suspension and place it at one end of the rectangular area.
2. Tilt the slide to permit the suspension to run down to the other
end of the slide. If the drop fails to run, add another drop.
3. Air dry the film. Do not heat.
4. Place the slide on a horizontal staining rack.
Staining procedure :
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1. Add about 1 ml. of the *Flagella Stain solution (one
dropper full) to the smear and allow to stain for 10-15
minutes.
2. Flood off the stain by adding tap water to the slide while
it remains on the rack.
3. Drain and flood the slide with Methylene blue for one
minute. Rinse by flooding. Drain and air dry. Do not
blot.
*Flagella Stain solution:
Nacl 1%
Tannic acid 3%
Basic fuchsine 1.25%
Solvent in ethyl alcohol 95%
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