Exercise 3C: Microscopic Characteristics
Group 5:
 Quilang, Aleia Sharisse F.
 Quinto, Thea Marie Dolores N.
 Recio, Jose Angelo G.
 Refuerzo, Jirah Angelica M.
 Robles, Andreu Vinzent F.
 Rodriguez, Joanne Claire E.
 Romero, Mavourneen Tracy Chanel V.
 Samonte, Muriel Fay D.
Introduction
All bacteria, both pathogenic and saprophytic, are unicellular organisms that reproduce
by binary fission. Most bacteria are capable of independent metabolic existence and growth, but
species of Chlamydia and Rickettsia are obligately intracellular organisms. Bacterial cells are
extremely small and are most conveniently measured in microns (10-6 m). They range in size
from large cells such as Bacillus anthracis (1.0 to 1.3 µm X 3 to 10 µm) to very small cells such
as Pasteurella tularensis (0.2 X 0.2 to 0.7 µm). Mycoplasmas (atypical pneumonia group) are
even smaller, measuring 0.1 to 0.2 µm in diameter. Bacteria therefore have a surface-to-volume
ratio that is very high: about 100,000.
Bacteria have characteristic shapes. The common microscopic morphologies are cocci
(round or ellipsoidal cells, such as Staphylococcus aureus or Streptococcus, respectively); rods,
such as Bacillus and Clostridium species; long, filamentous branched cells, such
as Actinomyces species; and comma-shaped and spiral cells, such as Vibrio
cholerae and Treponema pallidum, respectively. The arrangement of cells is also typical of
various species or groups of bacteria. Some rods or cocci characteristically grow in chains;
some, such as Staphylococcus aureus, form grapelike clusters of spherical cells; some round
cocci form cubic packets. Bacterial cells of other species grow separately. The microscopic
appearance is therefore valuable in classification and diagnosis. The higher resolving power of
the electron microscope not only magnifies the typical shape of a bacterial cell but also clearly
resolves its prokaryotic organization.
Typical Shapes and Arrangements of Bacterial Cells
Bacteria are microscopic organisms that cannot be seen with unaided eye. They can be seen
even in unstained preparations such as a wet mount or hanging drop preparation but the morphology
is not clear. Bacteria are colorless and when suspended in saline they don’t offer any contrast.
Besides, bacterial motility makes it difficult to observe the morphology clearly. Hence, bacteria have
to be stained to observe them. The dyes often used are toxic chemicals that kill the bacteria. The
process of smearing, fixing and drying often kill the bacteria. This process fixes the bacteria to the
slide and their position on slide remains unaltered.
Objectives
 To know the different staining methods and how to execute them.
 To be able to identify the different characteristics of bacteria.
 To know why it’s important to fix bacteria on the slide.
 To know the unknown bacteria.
Results
We we’re not able to perfectly culture all eight bacteria but we were able to get 5. These are
what we think the identities of those 5 bacteria:
CR_9
A_21
A_1
CR_8
A_22
Unknown
Shape
Arrangement
Color
A_1 (BA)
coccus
staphylococcus
Blue
A_21 (NA)
coccus
staphylococcus
red
A_22 (NA)
coccus
staphylococcus
violet
S_CR8 (BA)
rods
-
colorless
S_CR9 (NA)
coccus
streptococcus
Blue cell wall
Discussion
Bacteria display a wide diversity of shapes and sizes, called morphologies. Bacterial cells are
about one-tenth the size of eukaryotic cells and are typically 0.5–5.0 micrometres in length.
However, a few species — for example,Thiomargarita namibiensis and Epulopiscium fishelsoni —
are up to half a millimetre long and are visible to the unaided eye; E. fishelsoni reaches
0.7 mm. Among the smallest bacteria are members of the genusMycoplasma, which measure only
0.3 micrometres, as small as the largest viruses. Some bacteria may be even smaller, but
these ultramicrobacteria are not well-studied.
Most bacterial species are either spherical, called cocci (sing. coccus, from
Greek kókkos, grain, seed), or rod-shaped, called bacilli (sing. bacillus, from Latin baculus,
stick). Elongation is associated with swimming. Some bacteria, called vibrio, are shaped like
slightly curved rods or comma-shaped; others can be spiral-shaped, called spirilla, or tightly
coiled, called spirochaetes. A small number of species even have tetrahedral or cuboidal
shapes. More recently, bacteria were discovered deep under Earth's crust that grows as
branching filamentous types with a star-shaped cross-section. The large surface area to volume
ratio of this morphology may give these bacteria an advantage in nutrient-poor
environments. This wide variety of shapes is determined by the bacterialcell
wall and cytoskeleton, and is important because it can influence the ability of bacteria to acquire
nutrients, attach to surfaces, swim through liquids and escape predators.
Many bacterial species exist simply as single cells, others associate in characteristic
patterns: Neisseria form diploids (pairs), Streptococcus form chains, and Staphylococcus group
together in "bunch of grapes" clusters. Bacteria can also be elongated to form filaments, for
example the Actinobacteria. Filamentous bacteria are often surrounded by a sheath that
contains many individual cells. Certain types, such as species of the genus Nocardia, even form
complex, branched filaments, similar in appearance to fungal mycelia.
Classification seeks to describe the diversity of bacterial species by naming and
grouping organisms based on similarities. Bacteria can be classified on the basis of cell
structure, cellular metabolism or on differences in cell components such as DNA, fatty acids,
pigments, antigens and quinones. While these schemes allowed the identification and
classification of bacterial strains, it was unclear whether these differences represented variation
between distinct species or between strains of the same species. This uncertainty was due to
the lack of distinctive structures in most bacteria, as well as lateral gene transfer between
unrelated species. Due to lateral gene transfer, some closely related bacteria can have very
different morphologies and metabolisms. To overcome this uncertainty, modern bacterial
classification emphasizes molecular systematics, using genetic techniques such
as guanine cytosine ratio determination, genome-genome hybridization, as well
as sequencing genes that have not undergone extensive lateral gene transfer, such as
the rRNA gene. Classification of bacteria is determined by publication in the International
Journal of Systematic Bacteriology, and Bergey's Manual of Systematic
Bacteriology. The International Committee on Systematic Bacteriology (ICSB) maintains
international rules for the naming of bacteria and taxonomic categories and for the ranking of
them in the International Code of Nomenclature of Bacteria
The term "bacteria" was traditionally applied to all microscopic, single-cell prokaryotes.
However, molecular systematics showed prokaryotic life to consist of two separate domains,
originally called Eubacteria and Archaebacteria, but now called Bacteria and Archaea that
evolved independently from an ancient common ancestor. The archaea and eukaryotes are
more closely related to each other than either is to the bacteria. These two domains, along with
Eukarya, are the basis of the three-domain system, which is currently the most widely used
classification system in microbiology. However, due to the relatively recent introduction of
molecular systematics and a rapid increase in the number of genome sequences that are
available, bacterial classification remains a changing and expanding field. For example, a few
biologists argue that the Archaea and Eukaryotes evolved from Gram-positive bacteria.
Identification of bacteria in the laboratory is particularly relevant in medicine, where the
correct treatment is determined by the bacterial species causing an infection. Consequently, the
need to identify human pathogens was a major impetus for the development of techniques to
identify bacteria.
The Gram stain, developed in 1884 by Hans Christian Gram, characterizes bacteria
based on the structural characteristics of their cell walls. The thick layers of peptidoglycan in the
"Gram-positive" cell wall stain purple, while the thin "Gram-negative" cell wall appears pink. By
combining morphology and Gram-staining, most bacteria can be classified as belonging to one
of four groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci and Gramnegative bacilli). Some organisms are best identified by stains other than the Gram stain,
particularly mycobacteria or Nocardia, which show acid-fastness on Ziehl–Neelsen or similar
stains. Other organisms may need to be identified by their growth in special media, or by other
techniques, such as serology.
Culture techniques are designed to promote the growth and identify particular bacteria,
while restricting the growth of the other bacteria in the sample. Often these techniques are
designed for specific specimens; for example, a sputum sample will be treated to identify
organisms that cause pneumonia, while stool specimens are cultured on selective media to
identify organisms that cause diarrhea, while preventing growth of non-pathogenic bacteria.
Specimens that are normally sterile, such as blood, urine or spinal fluid, are cultured under
conditions designed to grow all possible organisms. Once a pathogenic organism has been
isolated, it can be further characterized by its morphology, growth patterns such as
(aerobic or anaerobic growth, patterns of hemolysis) and staining.
As with bacterial classification, identification of bacteria is increasingly using molecular
methods. Diagnostics using such DNA-based tools, such as polymerase chain reaction, are
increasingly popular due to their specificity and speed, compared to culture-based
methods. These methods also allow the detection and identification of "viable but nonculturable" cells that are metabolically active but non-dividing. However, even using these
improved methods, the total number of bacterial species is not known and cannot even be
estimated with any certainty. Following present classification, there are a little less than 9,300
known species of prokaryotes, which includes bacteria and archaea; but attempts to estimate
the true number of bacterial diversity have ranged from 107 to 109 total species – and even
these diverse estimates may be off by many orders of magnitude.
Answers to Questions
Different Staining Methods
 Direct Stain
Microbial cells are small and transparent. Stains are often used to increase
contrast between the cells and the background, making them easier to see under the
microscope. Since many of the cell components are negatively charged, stains with
positively charged chromophores (the colored ion of the dye) will attach to the cells.
Examples of such stains include methylene blue, crystal violet, and carbol fuchsin.
Steps:
 Make a heat-fixed smear, taking care not to use too much culture if working from an
agar culture.
 Place the slide on a staining rack over a sink or catch basin.
 Add a drop of dye to the smear. You need enough stain to just cover the smear, not
the whole slide.
 Allow the dye to act. (One minute is generally adequate.)
 Gently rinse the dye from the slide with water from a squirt bottle.
 Gently blot the slide and observe under the microscope.

Negative Staining
This is an established method, often used in diagnostic microscopy, for
contrasting a thin specimen with an optically opaque fluid. In this technique, the
background is stained, leaving the actual specimen untouched, and thus visible. This
contrasts with 'positive staining', in which the actual specimen is stained.
Steps:
 Place a very small drop (more than a loop full--less than a free falling drop from the
dropper) of nigrosin near one end of a well-cleaned and flamed slide.
 Remove a small amount of the culture from the slant with an inoculating loop and
disperse it in the drop of stain without spreading the drop.
 Use another clean slide to spread the drop of stain containing the organism using the
following technique.
 Rest one end of the clean slide on the center of the slide with the stain. Tilt the clean
slide toward the drop forming an acute angle and draw that slide toward the drop
until it touches the drop and causes it to spread along the edge of the spreader
slide. Maintaining a small acute angle between the slides, push the spreader slide
toward the clean end of the slide being stained dragging the drop behind the
spreader slide and producing a broad, even, thin smear.
 Allow the smear to dry without heating.
 Focus a thin area under oil immersion and observe the unstained cells surrounded
by the gray stain.

Gram Staining
This is a method of differentiating bacterial species into two large groups (grampositive and gram-negative). The name comes from its inventor, Hans Christian Gram.
Gram staining differentiates bacteria by the chemical and physical properties of their cell
walls by detecting peptidoglycan, which is present in a thick layer in gram-positive
bacteria. In a Gram stain test, gram-positive bacteria retain the crystal violet dye, while a
counterstain (commonly safranin or fuchsin) added after the crystal violet gives all gramnegative bacteria a red or pink coloring. The Gram stain is almost always the first step in
the identification of a bacterial organism. While Gram staining is a valuable diagnostic
tool in both clinical and research settings, not all bacteria can be definitively classified by
this technique. This gives rise to gram-variable and gram-indeterminate groups as well.
The reagents used are Crystal violet, Gram's iodine solution, acetone/ethanol (50:50
v:v), 0.1% basic fuchsin solution
Steps:
1. Prepare a Slide Smear:
A. Transfer a drop of the suspended culture to be examined on a slide with an
inoculation loop. If the culture is to be taken from a Petri dish or a slant culture tube, first
add a drop or a few loopful of water on the slide and aseptically transfer a minute
amount of a colony from the Petri dish. Note that only a very small amount of culture is
needed; a visual detection of the culture on an inoculation loop already indicates that too
much is taken.
If staining a clinical specimen, smear a very thin layer onto the slide, using a wooden
stick. Do not use a cotton swab, if at all possible, as the cotton fibers may appear as
artifacts. The smear should be thin enough to dry completely within a few seconds. Stain
does not penetrate thickly applied specimens, making interpretation very difficult.
B. Spread the culture with an inoculation loop to an even thin film over a circle of 1.5 cm
in diameter, approximately the size of a dime. Thus, a typical slide can simultaneously
accommodate 3 to 4 small smears if more than one culture is to be examined.
C. Air-dry the culture and fix it or over a gentle flame, while moving the slide in a circular
fashion to avoid localized overheating. The applied heat helps the cell adhesion on the
glass slide to make possible the subsequent rinsing of the smear with water without a
significant loss of the culture. Heat can also be applied to facilitate drying the smear.
However, ring patterns can form if heating is not uniform, e.g. taking the slide in and out
of the flame.
2. Gram Staining:
A. Add crystal violet stain over the fixed culture. Let stand for 10 to 60 seconds; for thinly
prepared slides, it is usually acceptable to pour the stain on and off immediately. Pour off
the stain and gently rinse the excess stain with a stream of water from a faucet or a
plastic water bottle. Note that the objective of this step is to wash off the stain, not the
fixed culture.
B. Add the iodine solution on the smear, enough to cover the fixed culture. Let stand for
10 to 60 seconds. Pour off the iodine solution and rinse the slide with running water.
Shake off the excess water from the surface.
C. Add a few drops of decolorizer so the solution trickles down the slide. Rinse it off with
water after 5 seconds. The exact time to stop is when the solvent is no longer colored as
it flows over the slide. Further delay will cause excess decolorization in the gram-positive
cells, and the purpose of staining will be defeated.
D. Counterstain with basic fuchsin solution for 40 to 60 seconds. Wash off the solution
with water. Blot with bibulous paper to remove the excess water. Alternatively, the slide
may shake to remove most of the water and air-dried.

Acid Fast Staining
Mycobacterium and many Nocardia species are called acid-fast because during
an acid-fast staining procedure they retain the primary dye carbol fuchsin despite
decolorization with the powerful solvent acid-alcohol. Nearly all other genera of bacteria
are nonacid-fast. The acid-fast genera have lipoidal mycolic acid in their cell walls. It is
assumed that mycolic acid prevents acid-alcohol from decolorizing protoplasm. The
acid-fast stain is a differential stain.
Ziehl Neelsen Acid-fast stain
ACID-FAST STAIN
Cell Color
Cell Color
Procedure
Reagent
Acid-fast
Bacteria
Nonacid-fast Bacteria
Primary dye
Carbolfuchsin
RED
RED
Decolorizer
Acid-alcohol
RED
COLORLESS
RED
BLUE
Counterstain Methylene blue
Step 1: Smear Preparation
 Add one loopful of sterile water to a microscope slide.
 Make a heavy smear of Mycobacterium smegmatis. Mix thoroughly with your
loop. Then transfer a small amount of Staphylococcus epidermidis to the same
drop of water. You will now have a mixture of M. smegmatis and S. epidermidis.
 Air dry and heat fix.
Step 2:
 Cover the smear with carbolfuchsin dye. Carbolfuchsin a potential carcinogen.
Please wear gloves and work with the stain in the hood.
 Place a piece of paper towel on top of the dye. Be sure the paper towel is
saturated with the dye.
Step 3:
 Dry heat for 2 minutes.
Step 4:
 Cool and rinse with water.
 Decolorize with acid-alcohol for 15-20 seconds.
Step 5:
 Wash the top and bottom of slide with water and clean the slide bottom well.
Step 6:
 Counterstain with Methylene Blue for 30 seconds to 1 minute.
 Wash and blot the slide with bibulous paper.
 Focus 10X - then use oil immersion.

Spore Staining
The primary dye malachite green is a relatively weakly binding dye to the cell wall
and spore wall. In fact, if washed well with water, the dye comes right out of the cell
wall, however not from the spore wall once the dye is locked in. That is why there is no
need for a decolorizer in this stain: it is based on the binding of the malachite green and
the permeability of the spore vs. cell wall. The steaming helps the malachite green to
permeate the spore wall.
Steps:
1. Make a smear of the Bacillus species---air-dry and heat-fix.
2. Put a beaker of water on the hot plate and boil until steam is coming up from the
water. Then turn the hot plate down so that the water is barely boiling.
3. Place the wire stain rack over the beaker which now has steam coming up from the
boiled water.
4. Cut a small piece of paper towel and place it on top of the smear on the slide. The
towel will keep the dye from evaporating too quickly, thereby giving more contact
time between the dye and the bacterial walls.
5. Flood the smear with the primary dye, malachite green, and leave for 5 minutes.
Keep the paper towel moist with the malachite green. DO NOT let the dye dry on the
towel.
6. Remove and discard the small paper towel piece.
7. Wash really WELL with water.
8. Place the smear over the sink and flood the smear with the counterstain dye,
safranin, and leave for 1 minute.

Flagella Staining
Bacterial flagella are fine, threadlike organelles of locomotion. They are slender
(about 10 to 30 nm in diameter) and can only be seen directly using the electron
microscope. In order to observe them with the light microscope, the thickness of the
flagella are increased by coating them with mordants like tannic acid and potassium
alum, and staining them with basic fuchsin (Gray method), pararosaniline (Leifson
method), silver nitrate West method), or crystal violet (Difco's method). Although flagella
staining procedures are difficult to carry out, they often provide information about the
presence and location of flagella, which is of great value in bacterial identification.
Difco's Spot Test Flagella stain employs an alcoholic solution of crystal violet as the
primary stain, and tannic acid and aluminum potassium sulfate as mordants. As the
alcohol evaporates during the staining procedure, the crystal violet forms a precipitate
around the flagella, thereby increasing their apparent size.
Procedure (West):
1. With a wax pencil, mark the left-hand comer of a clean glass slide with the name of
the bacterium.
2. Aseptically transfer the bacterium with an inoculating loop from the turbid liquid at the
bottom of the slant to 3 small drops of distilled water in the center of a clean slide that
has been carefully wiped off with clean lens paper. Gently spread the diluted bacterial
suspension over a 3 cm area using the inoculating needle.
3. Let the slide air dry for 15 minutes.
4. Cover the dry smear with solution A (the mordant) for 4 minutes.
5. Rinse thoroughly with distilled water.
6. Place a piece of paper toweling on the smear and soak it with solution B (the stain).
Heat the slide in a boiling water bath for 5 minutes in an exhaust hood with the fan on.
Add more stain to keep the slide from drying out.
7. Remove the toweling and rinse off excess solution B with distilled water. Flood the
slide with distilled water and allow it to sit for 1 minute while more silver nitrate residue
floats to the surface.
8. Then, rinse gently with water once more and carefully shake excess water off the
slide.
9. Allow the slide to air dry at room temperature.
10. Examine the slide with the oil immersion objective. The best specimens will probably
be seen at the edge of the smear where bacteria are less dense. Record your results in
the report.
Procedure (Difco):
1. Draw a border around the clear portion of a frosted microscope slide with a wax
pencil.
2. Place a drop of distilled water on the slide, approximately I cm from the frosted edge.
3. Gently touch a colony of the culture being tested with an inoculating loop and then
lightly touch the drop of water without touching the slide. Do not mix.
4. Tilt the slide at a slight angle to allow the drop preparation to flow to the opposite end
of the slide.
5. Let the slide air-dry at room temperature. Do not heat-fix.
6. Flood the slide with the contents of the Difco Spot Test Flagella stain ampule.
7. Allow the stain to remain on the slide for approximately 4 minutes. (Note: The staining
time may need to be adjusted from 2 to 8 minutes depending on the age of the culture,
the age of the stain, the temperature, and the depth of staining solution over the culture.)
8. Carefully rinse the stain by adding water from a faucet or wash bottle to the slide while
it remains on the staining rack. Do not tip the slide before this is done.
9. After rinsing, gently tilt the slide to allow excess water to run off and let the slide airdry at room temperature or place on a slide warmer.
10. Examine the slide microscopically with the oil immersion objective. Begin
examination at thinner areas of the preparation and move toward the center. Look for
fields which contain several isolated bacteria, rather than fields which contain clumps of
many bacteria. Bacteria and their flagella should stain purple.

Capsule Staining
Many bacteria have a slimy layer surrounding them, which is usually referred to
as a capsule. The capsule's composition, as well as its thickness, varies with individual
bacterial species. Polysaccharides, polypeptides, and glycoproteins have all been found
in capsules. Often, a pathogenic bacterium with a thick capsule will be more virulent than
a strain with little or no capsule since the capsule protects the bacterium against the
phagocytic activity of the host's phagocytic cells. The capsule stain is of some
importance in clinical microbiology (e.g., in the diagnosis of bacterial pneumonia and the
fungus Cryptococcus neoformans). However, one cannot always determine if a capsule
is present by simple staining procedures, such as using negative staining and India ink.
An unstained area around a bacterial cell may be due to the separation of the cell from
the surrounding stain upon drying. Two convenient procedures for determining the
presence of a capsule are Anthony's capsule staining method and the Graham and
Evans procedure.
Anthony's procedure employs two reagents. The primary stain is crystal violet, which
gives the bacterial cell and its capsular material a dark purple color. Unlike the cell, the
capsule is nonionic and the primary stain cannot adhere. Copper sulfate is the
decolorizing agent. It removes excess primary stain as well as color from the capsule. At
the same time, the copper sulfate acts as a counterstain by being absorbed into the
capsule and turning it a light blue. In this procedure, smears should not be heat-fixed
since shrinkage is likely to occur and create a clear zone around the bacterium, which
can be mistaken for a capsule.
Procedure: Capsule Staining (Anthony):
1. With a wax pencil, label the left-hand comer of a clean glass slide with the name of
the bacterium that will be stained.
2. As shown in figure 15.3, aseptically transfer a loopful of culture with an inoculating
loop to the slide. Allow the slide to air dry. Do not heat-fix!
3. Place the slide on a staining rack. Flood the slide with crystal violet and let stand
for 4 to 7 minutes.
4. Rinse the slide thoroughly with 20% copper sulfate.
5. Blot dry with bibulous paper.
6. Examine under oil immersion (a coverslip is not necessary) and draw the
respective bacteria in the report for exercise 12. Capsules appear as faint blue halos
around dark blue to purple cells.
Modified Capsule Stain (Graham and Evans):
1. Thoroughly clean the slide to be used with Bon Ami and alcohol.
2. Mix two loopfuls of culture with a small amount (1 to 2 drops) of India ink at one
end of the slide.
3. Spread out the drop using a second slide in the same way one prepares a thin
smear.
4. Dry the smear.
5. GENTLY rinse with distilled water.
6. Stain for 1 minute with Gram's crystal violet.
7. Rinse again with water.
8. Stain for 1.5 minutes with safranin stain.
9. Rinse with water and blot dry.
10. If a capsule is present, the pink to red bacteria are surrounded
by a clear zone. The background is blue-black
Importance of Fixing Bacteria on the Slide and How It’s Done
Fixation terminates any ongoing biochemical reactions, and may also increase the
mechanical strength or stability of the treated tissues. The purpose of fixing a slide that is to be
stained is to kill the organism, make it adhere to the slide, and alter the organism so that they
more readily accept stains (dyes).
In order to heat fix a bacterial smear, it is necessary to first let the bacterial sample air
dry. Then either place the slide in the slide holder of a micro incinerator, or pass the dried slide
through the flame of a Bunsen burner 3 or 4 times, smear side facing up. Once the slide is heat
fixed, it can then be stained.
Conclusion
Therefore, I conclude that bacteria have different characteristics (shapes and sizes). The
different staining techniques make it easy to view these characteristics under the microscope
and to differentiate them from one another.
References
 http://www.ncbi.nlm.nih.gov/books/NBK8477/
 http://www.microrao.com/simple_staining.htm
 http://en.wikipedia.org/wiki/Bacteria
 http://en.wikipedia.org/wiki/File:Bacterial_morphology_diagram.svg
 http://www.microeguide.com/lab_skills/direct_stain.asp
 http://en.wikipedia.org/wiki/Negative_stain
 https://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Negative%20Stain%20Proce
dure.html








http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gram2.htm
http://en.wikipedia.org/wiki/Gram_staining
http://www.uwyo.edu/virtual_edge/units/acidfast_stain.html
https://www.google.com.ph/search?q=spore+staining+procedure&newwindow=1&ei=TRBdU9oI8vkkgW-soC4CQ&start=10&sa=N&biw=1366&bih=600#
http://nhjy.hzau.edu.cn/kech/wswx/en/syzd/shiyan/s7.htm
http://nhjy.hzau.edu.cn/kech/wswx/en/syzd/shiyan/s6.htm
http://www.ask.com/question/what-is-the-purpose-of-fixing-a-slide-that-is-to-be-stained
http://www.scienceprofonline.com/microbiology/how-to-prepare-microscope-slide-ofbacteria.html