Preparation of Specimens

for Microscopic
Examination
1. wet mount preparation – used to observe
living organisms’ actual shape and motility

a.simple wet mount – uses ordinary glass

slide and cover slip

b. hanging drop technique – uses

depression slide and cover slip
2. stained smear preparation – uses stains/dyes;
microorganisms observed are in their dead state-
enables us to:

-make transparent and semi-transparent objects

visible
-observe the size and shape of microorganisms
-detect the presence of various internal and
external structures
-detect physical and chemical reactions
▪ Smear- A thin film of a microbe solution on a
slide,
▪ smear is usually fixed to attach microbes to the
slide and kill microbes

LMBorines
1st sem 2021-2022
 Types of Dyes/Stains:

1. basic dyes – positively-

charged. Chromophore is a
cation
e.g. methylene blue, crystal
violet, safranin

2. acidic dyes- negatively

charged
e.g. congo red, acid fuchsin,
eosin. Chromophore is an
anion
Basic Types of Staining
Procedures

1.Positive staining – dye

actually sticks to the specimen
and gives it color

2.Negative staining – dye

does not stick to the
specimen but settles around
its outer boundary forming a
silhouette
Kinds of Positive
Staining Methods
1. Simple staining –
involves application of a
single stain/dye to a fixed
smear; e.g. methylene blue
staining of bacteria
-cells exhibit more or
less the same color
2. Differential staining – involves application of 2 or
more stains/dyes to a fixed smear;

- stained cells exhibit different colors (e.g.

methylene blue + safranin in staining of cheek cells
(stained pink) and Diplococcus salivarious (stained
blue)
Another example of differential
staining
a. Gram staining – most basic staining procedure
▪ divides bacteria into Gram positive and Gram negative

Gram positive bacteria – appear blue or

violet
e.g. Bacillus, Mycobacterium, Clostridium, Lactobacillus,
Staphylococcus

▪ Gram negative bacteria – appear red or pink

e.g. Escherichia, Pseudomonas, Shigella,
Salmonella
 Gram positive bacteria Gram negative red or
(violet) after staining pink after staining
WHY?
Eg. Bacillus subtilis Eg. Escherichia coli

Gram + Gram -
thick peptidoglycan Thin peptidoglycan
None or thin lipopolysaccharide Thick lipopolysaccharide
With teichoic acid Without teichoic acid
 What happen during each step in
Gram staining Gram + Gram -

1.Ammonium oxalate-crystal violet Violet Violet

(primary stain) - 1 min
Wash slide
2. Gram Iodine (Mordant)- 1 min Violet Violet
Wash slide
3. 95% EthylAlcohol (decolorizer) Violet Colorless
Drain or blot dry-30 secs
4. Safranin – secondary stain-10- 15 secs Violet Pink or red
Wash, air dry, examine under the
microscope Take up:

1o stain 2o stain
& retained
stain
LMBorines
1st sem 2021-2022
b. Acid fast staining
- determines resistance of a stained cell to be decolorized

• by acids.
-acid fast organisms are stained red while non-acid fast ones stained
blue
-used to detect Mycobacterium tuberculosis and Mycobacterium
leprae and Nocardia spp. Stained waxy cell wall is not decolorized by
acid-alcohol

Primary stain- carbol fuchsin

Secondary stain – methylene blue

LMBorines
1st sem 2021-2022
c. Endospore Staining - distinguishes
between endospores and the cells that
they come from, the vegetative cells
- endospores are stained green while the
vegetative cells are stained pink
Stains: malachite green – stains the endospores
Safranin – stains the vegetative cells
- example of endospore-
Bacillus Sporosarcina
3. Special staining - done to emphasize certain cell parts
that are not revealed by conventional staining methods
a. flagella staining – reveals presence of flagella,
their number and arrangement on the cell
b. capsule staining – determines presence of capsule;
mostly negatively stained with India ink

LMBorines
1st sem 2021-2022