Bacterial stains and smear

preparation
Lab 12
Prof.dr.lazem altaie
Stain in microbiology
• To differentiate the microorganisms under the microscope it must be
stained because bacterial cells are the same resolution of the
microscopic light.
• First step for the staining , the bacterial must be smeared and fixed on
a glass slide

• Bacterial smear: small amount of the bacterial culture spread in a

very thin film on the surface of the clean slide
Features for differentiate stained bacteria
• 1- Cellular morphology • 2- Arrangement
 Steps in preparation of bacterial smear
• 1- spreading
• 2- fixation : To prevent the bacteria from washing away during the staining
steps, the smear may be chemically or physically “fixed” to the surface of
the slide by Heat fixing
• 3- staining: Using dyes, in general the dye will be used two mechanisms:
• I- attracted by charge (a cationic dye such as methylene blue or crystal
violet) that bind to bacterial cell with no staining for the surrounding
• II- repelled by charge (an anionic dye such as eosin or India ink) that stain
the surrounding with no stain for bacterial cell
Gram stain
the name from the physician
Hans Christian Gram

• Gram’s staining techniques to aid in the identification of bacteria, beginning

with a preliminary classification into one of two
groups: Gram positive or Gram negative.
• Gram staining involves four steps.
• First cells are stained with crystal violet, for one mint
• followed by the addition of a setting agent for the stain (iodine).for one mint
• Then alcohol is applied, which selectively removes the stain from only the
Gram negative cells. For 30 second
• Finally, a secondary stain, safranin, is added, which counter stains the
decolorized cells pink. F0r one mint
Step
Primary stain(crystal violet)
Mordant (iodine)
Decolorization (ethanol)
Counterstain(safranin)
Procedure
Add several drops of crystal violet
to the smear and allow it to sit for
1 minute. Rinse the slide with
water.
Add several drops of iodine to the
smear and allow it to sit for 1
minute. Rinse the slide with water.
Add drops of
ethanol one at a time until the
runoff is clear. Rinse the slide with
water.
Add several drops of safranin to the
smear and allow it to sit for one
minute. Rinse the slide with water
and blot dry.
Outcome
Both Gram-positive and Gram-
negative cells will be stained purple
by the crystal violet dye.
Iodine “sets” the crystal violet, so
both types of bacteria will remain
purple.
Gram-positive cells resist
decolorization and remain purple.
The dye is released from Gram-
negative cells.
Gram-negative cells will be stained
pink by the safranin. This dye has
no effect on Gram-positive cells,
which remain purple.
 Acid Fast stain
• Some bacteria produce the waxy
substance mycolic acid when they
construct their cell walls. Mycolic
acid acts as a barrier, protecting the
cells from dehydrating, as well as
from phagocytosis by immune
system cells in a host. This waxy
barrier also prevents stains from
penetrating the cell, which is why
the Gram stain does not work with
mycobacteria such
as Mycobacterium, which are
pathogens of humans and animals.
For these bacteria, the acid–
fast staining technique is used.
Procedure of acid fast stain
• Prepare bacterial smear on clean slide
• Allow smear to air dry
• Cover the smear with strong carbol fuchsine stain. Heat the stain until
vapor just begins to rise (i.e. about 60 C). Do not overheat. Allow
the heated stain to remain on the slide for 5 minutes.
• Wash off the stain with clean water.
• Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear
is sufficiently decolorized, i.e. pale pink.
• Wash well with clean water.
• Cover the smear with malachite green or methylene blue stain for 1–2
minutes, using the longer time when the smear is thin.
• Wash off the stain with clean water.
• Examine the smear microscopically, using the 100 X oil immersion
objective.