Staining and Microscopy
Objectives
▪ Preparation of Smear
▪ Fixation of Smear
▪ Staining of Smear
▪ Observation
Background
▪ There are many different ways to stain bacteria so that they can
be more easily visualized under the microscope. Some stains
can also be used to identify and classify bacteria
▪ The Gram stain is a differential stain that allows you to classify
bacteria as either Gram-positive or Gram-negative
The staining technique consists of four steps
• A primary stain (crystal violet). All bacteria are stained purple
by this basic dye
• A mordant (Gram’s iodine). The iodine combines with the
crystal violet in the cell to form a violet-iodine complex
• A decolorizer (ethyl-alcohol). The primary stain is washed
out of some bacteria, while others are unaffected
• A secondary or counterstain (safranin). This basic dye
stains bacteria that have been decolorized red
Smear Formation
• Before bacteria can be stained, a smear of bacteria must be
made on a slide and heat fixed
• A smear is made by spreading a bacterial suspension on a
clean slide and letting it air dry
• The dry smear is heated on a hot plate or passed through a
flame several times to heat fix it
Heat fixing denatures bacterial enzymes, preventing them
from digesting cell parts, which causes the cell to break, a
process called autolysis
The heat also enhances the adherence of bacterial cells to
the slide
THE GRAM STAIN
Prepare a bacterial smear
– Place 10 μl of sterile water in the center of a clean glass slide
– Remove lid from culture plate with free hand. Do not place
lid on bench. Hold lid face down just above culture plate to
help prevent contaminations from falling on plate or lid.
– Use a sterile inoculating loop to scrape up a visible cell mass
from a colony. Do not gouge agar. Replace culture lid.
– Mix bacteria with water drop on slide. Spread the water-
bacteria mixture over an area of about 1 inch square
• Allow the smear to air dry.
• Hold the slides with forceps and heat fix the smears by
heating on the hot plate for several minutes
Staining
–Cover the smear with a few drops of crystal
violet (1) and leave for 1 minute.
–Turn the slide over so the smear is facing
down. Wash the slide carefully over the top
with distilled water from a wash bottle or the
sink until no large amounts of color wash off.
–
–Cover the smear with Gram’s iodine (2) and
leave for 1 minute.
–Without washing, decolorize with 95% ethyl
alcohol (Decolorizer (3)). Wash the slide
carefully over the top with distilled water from a
wash bottle or the sink until no large amounts
of color wash off.
–Immediately rinse with distilled water.
• Cover the smear with safranin (4) and leave for 1
minute
– Gently rinse with distilled water.
– Blot dry with a paper towel or absorbent paper. Do not
rub slide, as the bacterial smear can be rubbed off.
• Observe under the microscope.
• Record your observations from the microscope.
• Is it Gram-positive or Gram-negative?
• What shape are the bacteria?
• How are the cells arranged relative to one another?
Bacillus
Bacillus
Gram Positive Cocci
Gram Negative Cocci
Spirillium
List of Advantages of Gram Staining
It gives quick results when examining infections
In most cases, Gram stains are performed on biopsy or
bodily fluids when infection is suspected, and they yield
results much more quickly than other methods, such as
culturing
It is simple and cost-effective
This general stain is observed as the simplest, least
expensive and most useful among the quick methods that
used extensively in microbiology for the preliminary
differentiation, identification and classification of
microbiological organisms
It helps with determining appropriate treatments
for infection
▪ In some cases, it would not be clear whether an infection is
caused by bacteria, fungi or viruses, and these kinds of
infection would be treated in different ways
▪ Now, a Gram stain will let physicians determine whether a
certain type of bacteria is causing the infection and what type of
microorganism is present
It is basically a key procedure in identifying
bacteria
This method is a very important step in the preliminary
characterization and classification of bacteria, and it is a
key procedure in identifying bacteria based upon staining
characteristics, which enables the bacteria to be examined
with the use of a light microscope
Disadvantages
It has significant limitations when used for
environmental microbiology
The Gram stain is definitely not an infallible
technique for identification, diagnosis or phylogeny,
and it faces extreme limitations when used in
environmental microbiology
References
http://medicine.wustle.edu/ysp
THANKS
