Submitted to: Dr. Tomas J. Fernandez Jr.

Submitted by: Cherry Luz L. Rezaga

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4. Knowledge of the subjected matter (15%)
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7.1. Complete uniform (5)

Total


1

INTRODUCTION
Bacterial cells are usually colorless because cytoplasm, for the most part, is transparent.
Staining the organisms makes them contrast in color with their surroundings, so they are more
readily visible. Certain stains can also be used to identify internal structures of the cell, which
would otherwise be unseen. Further, in order to use the oil immersion objective of the
microscope and thereby obtain the greatest degree of magnification, it is convenient to use
stained preparations rather than wet mounts.
1
Although bacteria do not appear greatly different from their surroundings, they differ
chemically. It is this chemical difference that enables us to distinguish bacteria by staining, the
stain or dye readily reacting with the bacterial cell but not with the background.
2

Preparation of Smear Before Staining (figure 1)

1. Prepare a clean slide. Put a proper label.
2. Heat your loop to sterilize it.
3. For solid media:
Using a sterile inoculating loop, place a 1 or 2 loopfuls of distilled water on the center
of the slide.
Scrape a small amount of the culture off the slant.
Smear on the center of the slide, with the distilled water, the scraped material.
For liquid media:
Make a smear from the broth. You dont have to add water as the bacteria are already
suspended in water.
Use 2 or 3 loopfuls of the culture.
Spread the culture on the center of the slide.
4. Reheat the loop to clean it.
5. Let the smear dry.
- air dry
- heat fix- passing the slide through a flame 2 or 3 times
2











Classification of Stain Based on Function
1
1. Simple Staining Method
- In this method one aniline dye is used to stain the organism to be studied.

2. Differential Staining Method
- Under this type of classification, the staining method employed divides the
microorganism into groups. The Grams Staining Method and Acid-Fast (Ziehl-Neelsen
Method) fall under this category.

3. Selective or Special Staining Method
- Under this category, parts or portion of the cell are stained differently from the rest of
the cell.

4. Indirect Staining Method
- Indirect stains are also relief stains because it is the background which takes up the
stain, not the organism and the organism are only seem by contrast.



Figure 1. Preparation of smear.
3

Examples of Staining
1. Simple Staining Method
1,2

Use only 1 stain.
Use to determine cell morphology, size and arrangement.
Procedure:
a. Make a smear.
b. Staining:
1. Place the slide on a staining rack.
2. Flood the smear with several drops of the dye, allow it to remain for the following
intervals:
- Carbol-fuchsin 15 to 30 seconds
- Methylene Blue 2 to 5 minutes
- Crystal violet 30 to 45 seconds
3. Carefully wash the excess stain off with distilled water from a wash bottle. Let the
water run down the tilted slide.
4. Gently blot the smear with a paper towel or absorbent paper and let it dry.
c. View the prepared slide under the microscope.
Results and reaction:
Reaction / Results Principle Samples of
Bacteria
All bacteria in smear takes stain
and appears in colour of stain
Shape:
Spherical cocci
Rod bacilli
Arrangement :
Cocci in clusters
staphylococci
Cocci in chains
streptococci
Simple stains use basic dyes
which are positively
charged. These positive dyes
interact with the slightly
negatively charged bacterial
cell wall thus lending the color
of the dye to the cell wall.

All types of
bacteria.

2. Differential Staining Method
A. Grams Stain
4

most common technique
the gram stain is valid only when performed on young (less than 24 hours old) cultures of
bacteria
Procedure:
1

a. Make a smear.
b. Gram staining:
Steps Purpose
1. Use a clothespin or slide rack to hold the
slide.

4

2. Cover the smear with crystal violet and
leave for 30 seconds.
Primary stain all bacteria
are stained purple.
3. Wash the slide carefully with distilled water
from a wash bottle.
o do not squirt the water directly
onto the smear

4. Without drying, cover the smear with Grams
iodine for 30 seconds.
Mordant this intensifies
the ionic bond between the
primary stain and the
bacteria.
5. Without washing, decolorize with 95% ethyl
alcohol. Let the alcohol run through the
smear until no large amount of purple wash
out.
o do not over decolorize
Primary stain is washed
out of some bacteria, while
others are unaffected.
6. Immediately wash with distilled water.
7. Add safranin for 30 seconds. Secondary stain or
counterstain stains the
decolorized bacteria red.
8. Wash with distilled water and blot the slide
with a paper towel or absorbent paper. Let
dry.

c. Examine under the microscope.
9. Results:
Reactions /
Result
Principle Samples of Bacteria
Gram-
positive (+)
purple
colored
cell wall are thick and chemically
simple, composed mainly of
protein and cross-linked
mucopeptides
- alcohol causes
dehydration and shrinkage
of the gram+ cell wall
- reducing the loss of
substances such as
crystal violet

Gram positive cocci in
clusters (figure 2a):
Staphylococci
species
Gram positive bacilli (figure
2d):
Clostridium species
Corynebacterium
species
Bacillus anthracis
Gram-
negative (-)
pink
colored
wall is a thin, complex, multi-
layered structure containing
protein, mucopeptides and lipids
- when treated with alcohol,
the lipid dissolves and the
primary stain is wash out
Gram negative cocci in
chains:
- Streptococci
species
Gram negative cocci (figure
2e):
5

- Neisseria species
Gram negative bacilli
(figure 2c):
- Escherichia coli
- Klebsiella
pneumonia




a b
c d
Figure 2. Different observations in Gram's Staining. (a)gram+ cocci in clusters (b)gram+ cocci in chains
(c)gram- bacilli (d)gram+ bacilli (e)gram- cocci.(f)gram stain mixed
e
f
6

B. Acid Fast Stain (Ziehl Neelsen Stain)
1,5

to stain Mycobacterium species especially M.tuberculosis - Contain large amount of fatty
waxes (mycolic acid) within their cell wall resist staining by ordinary methods
Procedure:
a. Make a smear.
b. Staining:
Steps Purpose
1. Flood smear with Carbol Fuchsin
stain.

Carbol Fuchsin is a lipid soluble,
phenolic compound, which is able to
penetrate the cell wall.
2. Cover flooded smear with filter paper
3. Steam for 10 minutes. Add more
Carbol Fuchsin stain as needed.

4. Cool slide.
5. Rinse with distilled water.
6. Flood slide with acid alcohol (leave 15
seconds). The acid alcohol contains
3% HCl and 95% ethanol or 20% H2
SO4.
The waxy cell wall then prevents the
stain from being removed by the acid
alcohol (decolorizer) once it has
penetrated the cell wall. The acid
alcohol decolorizer will remove the
stain from all other cells.
7. Tilt slide 45 degrees over the sink and
add acid alcohol drop wise (drop by
drop) until the red color stops
streaming from the smear.

8. Rinse with distilled water
9. Add Loefflers Methylene Blue stain.
Leave Loefflers Blue stain on smear
for 1 minute.
Counter stain - This stain adds blue
color to non-acid fast cells.
10. Rinse slide. Blot dry.

c. Allow to dry and focus under microscope.
Results:
Reactions /
Result
Principle Bacteria
Acid fast
Bacteria
Primary stain binds cell wall mycolic
acids
Intense decolonization does not
release primary stain from the cell wall
of Acid Fast Bacteria
Color of AFB-based on primary stain
Counterstain provides contrasting
background
Pink bacilli Acid fast
bacteria/bacilli (figure 3)
*E.g. M.tuberculosis
long slender bacilli
*M. leprae short thick
bacilli
Non Acid fast Blue colored bacteria
Non-acid fast
*E.g. Epithelial cells,
pus cells, other bacteria

7


Figure 3. (A)Non Acid-fast bacteria (B) Acid-fast bacteria

3. Selective or Special Staining Method
A. Capsule Staining
1,6

A capsule is composed of mucoid substance that covers the cell membranes of some
bacteria.
Majority are too thin to be detected by the usual staining procedures.
Procedure:
a. Prepare a smear.
b. Staining.
Steps Purpose
1. Stain the smear with a 1%
aqueous solution of Crystal
Violet.
Primary stain - gives the bacterial cell and its
capsular material a dark purple color.
2. Allow to act for 2 minutes.
3. Wash off the stain with a 20%
solution of copper sulfate,
drain, blot and air dry.
Decolorizing agent - It removes excess
primary stain as well as color from the
capsule.
- Acts as a counterstain by being
absorbed into the capsule and
turning it a light blue.

c. View under the microscope.
Result:
Result / observation Principle Bacterial Samples
The capsule is revealed
as a clear halo between
the colored background
and the stained cell.
The bacterial cells and
the background will be
stained by the crystal
violet while the capsule
are unstained.
Encapsulated Bacillus sp.
(figure 4)


8


Figure 4. Encapsulated Bacillus sp.

B. Spore Staining
1,7

Spore not considered as a part of reproduction, yet play a part in the continuity of the life
of an organism.
Procedure:
a. Make smear.
b. Staining:
1. Malachite green- 2 min- heat stain till steam rises -2 min - wash.
2. Counterstain with safranin 1 min- wash.
3. Dry the slide.
c. Examine under the microscope
Results:
Result/Reaction Principle Sample
Spores are
stained green,
while vegetative
cells are stained
red.

Spores have a durable outer coating
that is composed of the protein
keratin.
This keratin coat resists staining so in
order to stain a spore the primary
stain, malchite green, must be heated
to drive the stain into the spores.
Vegatative cells are then decolorized
with water and 0.5% safranin is used
to counterstain.
Spore forming bacteria
(figure 5):
Eg., Clostridium
species.
Bacillus species
Eg. B. anthracis

9


Figure 5. Spore stain.

C. Stains for Granules
1,8,9

To demonstrate polar granules of Corynebacterium diphtheria.
Take up the stain of methylene blue but appears bluish black hence granules called
metachromatic granules.
Procedure:
a. Smear preparation.
b. Staining.
1. Flood the smear 10 30 seconds with solution prepared as follows:
- Methylene blue 0.1 gm
- Absolute alcohol 20.0 mL
- Glacial acitic acid 5.0 mL
- Distilled water 100.0 mL
2. Wash in water.
3. Counterstain with Bismarck brown or dilute safranin for a few second.
4. Blot, dry.
c. Examine.
Results:
Result/Reaction Principle
A row of irregularly shaped circles
(metachromatic granules) appear close
together because they are contained
within one bacterium. The cell wall (outer
border) of the bacterium is not readily
seen. (figure 6)
When stained with methylene blue,
metachromatic granules take up the
dye more intensely than the rest of
the rod-shaped bacterium.

10


Figure 6. Metachromatic granules stain black and vegetative cells yellow.

D. Stain for Flagella
1,8,9

this stain increases the thickness of flagella thus easy to see under light microscope
Procedure:
a. Smear.
b. Staining:
1. Add about 1 ml. of the Flagella Stain solution (one dropper full) to the smear
and allow to stain for 10-15 minutes. The solution should not be allowed to flow
outside the waxed area.
2. Flood off the stain by adding tap water to the slide while it remains on the
rack. Do not tip the slide before this is done.
3. Drain and flood the slide with carbol fuchsin for one minute. Rinse by
flooding. Drain and air dry. Do not blot.
c. Examine.
Result:
Result/ Reaction Principle Sample Bacteria
Flagella can easily be
observed (figure 7).
Because bacterial flagella are very
thin and fragile a special stain
(flagella stain) is prepared that
contains a mordant. This mordant
allows piling of the stain on the
flagella, increasing the thickness
until they become visible.
Escherichia coli
Salmonella
typhimurium
Caulobacter
crescentus
Vibrio alginolyticus

11


Figure 7. Flagellated bacteium.


4. Indirect (Negative) Staining Method
To determine morphology, arrangement and size of bacteria, that may be affected by heat
fixing
11
.
Procedure:
a. Smear.
b. Staining
10,11
:
1. Place a small drop of the negative stain (India ink) near the end of the slide.
2. Transfer one loop=full of the bacterial sample to the india ink and mix the two
together.
3. Hold a clean slide at about a 20
o
angle to the first slide. Touch the edge of the
clean slide to the bacteria/stain mixture so that the mixture spreads across the
edge.
4. Spread the suspension across the surface of the slide by drawing the clean slide
away from the mixture. Essentially, you will be using the clean slide to push the
mixture across the surface of the slide. When you have finished spreading the
slide, place the "clean" slide in a jar of disinfectant.
5. Air dry the slide. Do not heat fix.
c. Examine under the bright field microscope.

Principle
11
The negative stain uses the dye nigrosin, which is an acidic dye. By giving up
a proton (as an acid) the chromophore of the dye becomes negatively
charged. Because the cell wall is also negatively charged only the background
around the cells will become stained, leaving the cells unstained (figure 8 and
9).

12





References:
1. Microbiology Laboratory Manual, CVM VSU
2. http://www2.hendrix.edu/biology/CellWeb/Techniques/microstain.html
3. http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/bacterial_smears_
and_stains.html
4. en.wikipedia.org/wiki/Staining
5. www.sciencebuddies.org/science-fair-projects/project_ideas/microbio_stainingbacteria.pdf
6. www.slideshare.net/doctorrao/bacterial-staining-methods
7. www.personal.psu.edu/faculty/k/h/khb4/enve301/301labs/Lab2_Simple_staining.html
8. http://faculty.lacitycollege.edu/hicksdr/The%20bacteria%20Lab%20page.htm
9. http://www.cliffsnotes.com/sciences/biology/microbiology/microscopy/staining-
techniques.html
10. http://www.personal.psu.edu/faculty/k/h/khb4/enve301/301labs/Lab2_Simple_staining.ht
ml
11. https://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Negative%20stain%2
0results.html


Figure 8. Negatively Stained Bacillus: (A) Vegetative
Cell (B) Endospore.
Figure 9. Negatively Stained Cocci.