Aim: Examination of microorganisms by staining techniques. 2a.

To perform Gram staining of the

given bacterial strain
Principle: Gram staining, a differential staining technique was developed by Dr. Hans Christian
Gram in 1884. It is a very useful stain for identifying and classifying bacteria into two major groups:
Gram-positive and Gram-negative. Gram-negative bacterial cell wall is thin, complex, multilayered
structure and contains relatively a high lipid contents, in addition to protein and mucopeptides. The
higher amount of lipid is readily dissolved by alcohol, resulting in the formation of large pores in the
cell wall which do not close appreciably on dehydration of cell-wall proteins, thus facilitating the
leakage of crystal violet-iodine complex (CV-I) and resulting in the decolorization of the bacterium
which later takes the counter stain and appears red. In contrast, the gram-positive cell walls are thick
and chemically simple, composed mainly of protein and cross-linked mucopeptides. When treated
with alcohol, it causes dehydration and closure of cell wall pores, thereby not allowing the loss of
(CV-I) complex and cell remains purple.
Procedure:
Take a fresh clean slide and mark the smear area on the underside of the slide with a marking
pencil.
Put a looopful of sterile distilled water on a clean slide.
Aseptically transfer a small amount of bacterial growth or colony into the sterile distilled water
droplet with the inoculating loop.
Spread the bacterial suspension thinly to form a smear
Firstly allow the smear to air dry.
Heat fix the smear by passing it rapidly through the tip of the blue portion of the Bunsen burner
flame for four to five times.
Cover the smear with crystal violet for 30 seconds.
Wash slide with DW for a few seconds,using wash bottle.
Cover smear with gram’s iodine solution for 60 seconds.
Wash off the iodine solution.
Add ethyl alcohol for few seconds (10-20 seconds) until no more colour flows from the smear.
Wash the slides with distilled water and drain.
 Apply (counter –stain) safranin to smears for 30 seconds.
Wash with DW and blot dry with absorbent paper.
Examine the slides microscopially under oil-immersion objective.

Observations
Those bacteria that appear purple are referred to as Gram-positive and those that appear pink are
Gram-negative.

2b. To perform Negative staining for the given bacterial strain

Principle: In a negative staining technique, a simple stain is used that does not stain the bacteria but
stains the background. Acidic stains like (nigrosin or India ink) carry a negative charge on their
surface and bacterial cells also carry negative charge and thus they do not take up the stain and
appear transparent and unstained upon examination. Negative staining is advantageous for two
reasons 1. Cells appear less distorted because no heat fixing is done, 2. Capsulated bacteria that are
difficult to stain can be observed by this technique.
Procedure:
Place one drop of nigrosin at one end of a clean glass slide.
Transfer a loopful of the inoculum from the broth culture in the drop of stain and mix gently with
the loop.
Take another clean slide, place it against the drop of suspended organism at an angle of 30 0 and
allow the droplet to spread across the edge of the top slide.
Allow the smear to air dry.
Examine the preparation under oil-immersion objective.

Observations
Spherical cells occurring singly as well as in clusters appear colourless (transparent) against a blue
background.
2c. To perform Capsule staining by negative staining technique.
Principle: Some bacterial cells are surrounded by a mucilaginous substance forming a viscous coat
around the cell. This structure is referred to as a capsule. In negative staining the background but not
the bacteria is stained by the use of an acidic stain which carries a negative charge on its surface and
is repelled by the bacteria that too carry a negative charge on their surface. Capsules are clearly
visible in the light microscope when negative stains and special capsule stains are employed.
Procedure:
 Place one drop of nigrosin at one end of a clean glass slide.
Transfer a loopful of the inoculum from the broth culture in the drop of stain and mix gently with
the loop.
Take another clean slide, place it against the drop of suspended organism at an angle of 30 0 and
allow the droplet to spread across the edge of the top slide.
Allow the smear to air dry. Do not heat dry.
Examine the preparation under oil-immersion objective.

Observations
Spherical cells occurring singly as well as in clusters appear colourless (transparent) against a blue
background.
2d. To perform endospore staining by Schaeffer- Fulton method.
Principle: Some bacteria are capable of changing into dormant structures that are metabolically
inactive and do not grow or reproduce. Since these structures are formed inside the cells, they are
called endospores. A German botanist Fertinand Cohn discovered the existence of endospore in the
bacteria these are remarkably resistant to heat, radiation, chemicals and other agents that are typically
lethal to the organism. The heat resistant of spores has been linked to their high content of calcium
and dipicolenic acid. During sporulation, vegetative cells gives rise to a new, intracellular structure
termed as endospore, that is surrounded by impermeable layers are called spore coats. An endospore
develops in a characteristic position with in a cell that is central, subterminal or terminal.
Endospores are extremely resistant due to their thick wall, the spore coat. The spore coat does not
stain easily. Malachite green, however, penetrates the spore coat of endospore after considerable
heating. Once stained, the endospore does not decolourizes easily hence appears green even after
washing. In contrast, the counter stains entering the endospore but stains rest of the cell content that
appears red.
Procedure:
Take a fresh clean slide and mark the smear area on the underside of the slide with a marking
Pencil.
Put a looopful of sterile distilled water on a clean slide.
Make a smear of the bacterial culture on a clean slide.
Air dry & heat fix the smear.
Flood the smears with malachite green.
Heat the slides to steaming and steam for 5 minutes, adding more stain to the smear from time to
time.
 Wash the slides under slowly running tap water.
Counterstain with safranin for 30 seconds.
Wash smear with DW.
Blot dry slides with blotting paper.

Observations
Endospores stain green and the vegetative cells stain red...
2e. Staining of Fungi using Lactophenol cotton blue.
Principle: Lactophenol cotton blue is a stain commonly used for microscopic preparations of fungi.
It stains the fungal cytoplasm and provides a light blue background, against which the walls of
hyphae can readily be seen. It contains four constituents: phenol, which serves as a fungicide; lactic
acid, which acts as a clearing agent; cotton blue, which stains of the cytoplasm of the fungus; and
glycerine, which gives a semi-permanent preparation.
Procedure:
Place a drop of lactophenol cotton blue on a clean slide.
Transfer a small tuft off the fungus, preferably with spores and spore bearing structures, into the
drop, using a flamed, cooled needle.
Gently tease the material using the two needles.
Mix gently the stain with the mold structures.
Observe the slide under low and high power objectives of a microscope.

Observations:
Observe the types of conidia, conidiophores, hyphae and their arrangement.