Basic staining techniques in microbiology

1. Basic Staining Techniques in
Microbiology
Mr. Vikas Lalji Gupta
Asst. Professor,
VIVA College,MH-India

2. Staining
• Staining is a technique used to improve the contrast of samples
at microscopic level.
• Stains and dyes are commonly used to study the tissue or cells under
the microscope.
• In the medical fields of histopathology, hematology,
and cytopathology which will be focus on the study
and diagnose disease at a microscopic level.
• Biological staining is also used to mark cells in flow cytometry.
• Staining is not restricted to only biological materials, it can also be
used to study the structure of other materials like lamellar structures
of semi-crystalline polymers.

3. Types of stain/dye
• Acidic – Eosin, Nigrosin, India ink.
• Basic – Malachite green, crystal violet, basic fuschin,
safranin, methylene blue.
• Neutral – Giemsa stain.

4. Prepare a bacterial smear and heat fix it
Add basic dye on the smear for
approximately 1 minute
Rinse the slide gently under running tap
water
Carefully blot dry with Filter paper.
Observe the slide under the microscope, using
proper microscope technique.
Simple/Positive Staining
Simple staining provide a quick and easy way to determine
cell shape, size, and arrangement.

5. Negative Staining
Negative staining is an excellent way to determine an organism’s cellular
morphology. The nigrosine provides a dark background against which the shapes
of the unstained cells are clearly visible.
Place a single drop of Nigrosin on a clean glass slide
Take a loopful of culture suspension and mix it
into the drop of Nigrosin
Place the end of another clean slide at an angle to
the end of the slide containing the organism and
spread the drop out into a film.
Allow the film to air dry.
Observe the slide under the microscope

6. Gram Staining
Prepare a
bacterial
smear and
heat fix it
Flood the
smear with
crystal violet
for 1 minute.
Rinse the slide
gently under
running tap
water
Flood the
smear with
iodine for 1
minute.
Rinse the slide
gently under
running tap
water
Decolorize with
decolorizer
(acetone/alcohol)
for 30-45 seconds
Rinse the slide
gently under
running tap
water
Counterstain
with safranin
for 1 minute
Rinse the slide
gently with
water
Carefully blot
the slide dry
with filter
paper
Observe the
slide under
the
microscope
With proper staining technique…
Gram positive bacteria will stain
purple.
Gram negative bacteria will stain
red/pink.

7. Capsule Staining
Place a single
drop of India ink
on a clean
microscope slide
Take a loopful of
K. pneumoniae
culture suspension
and mix it into the
drop.
Place the end of another
clean slide at an angle to
the end of the slide
containing the organism
and spread the drop out
into a film
Allow the film
to air dry
Saturate the
slide with
crystal violet
for 1 minute
Rinse the slide
gently under
running tap
water
Allow the slide
to air dry
Observe the
slide under the
microscope
The
background
will be dark.
The bacterial
cells will be
stained purple.
The capsule will
appear clear
against the dark
background.

8. Acid-Fast Staining
Prepare a
bacterial
smear
Place a small
piece of filter
paper over the
smear
Add
carbolfuchsin
on the filter
paper
Heat the slide
gently over the
burner for 5
minutes
If carbolfuchsin
dry then add
more
carbolfuchsin
Remove the
filter paper
from the slide
Rinse the slide
gently under
running tap
water
Decolorize the
slide with
acid-alcohol
Rinse the slide
gently under
running tap
water
Counterstain
with methylene
blue for 1
minute
Rinse the slide
gently under
running tap
water
Observe the
slide under the
microscope
Acid-fast cells will stain fuchsia. Non-acid-fast cells will stain blue.

9. Endospore Staining
Prepare a
bacterial
smear of
Bacillus
Place a small
piece of Filter
paper over the
smear
Saturate the
paper with
malachite
green
Heat the slide
gently over the
burner for 10
minutes
If malachite
greendry then
add more
malachite green
Remove the
filter paper
from the slide
Rinse the slide
gently under
running tap
water
Counterstain
with safranin
for 1 minute
Rinse the slide
gently under
running tap
water
Carefully blot
the slide dry
with filter
paper
Observe the
slide under the
microscope
Endospores will stain green. Parent cells will stain red.

10. THANK
YOU