2. IMPORTANCE OF IDENTIFICATION
• Determining the clinical significance of particular pathogen.
• Guiding physician care of the patients.
• Determining the laboratory testing for detection of antibacterial
resistance is warranted.
• Determining the type of antibacterial therapy that is
appropriate.
• Determining the whether infectious organisms are risk for others
patients in the hospital,the public and other laboratory workers.
• Because the clinical samples will most likely contain many
microorganisms, both normal flora and pathogens, it is important
to isolate the pathogen in a pure culture using various types of
selective and differential media.
• And without accurate identification it is impossible to determine
how many species exist in a given area.
3. IDENTIFICATION METHODS
• Traditional method/phenotypic method
• Microscopic morphology and staining characteristics.
• Macroscopic ( colony) morphology.
• Environmental requirement for growth.
• Resistance or susceptibility to antibacterials agents.
• nutritional requirement and metabolic capabilities.
• culturing and simple biochemical tests
• Immunochemical method/serological methods
• Genotypic method/molecular method
4. STAINING TECHNIQUES
4
As bacteria consist of clear protoplasmic matter,
differing but slightly in refractive index from the
medium in which they are growing, it is difficult with
the ordinary microscope, except when special
methods of illumination are used, to set them in the
unstained condition.
Staining, therefore, is of primary importance
for the recognition of bacteria.
Staining may be simple staining and differential
staining.
5. DYES
5
ACIDIC: Acidic dyes, in their ionized form, have a negative
charge and bind to positively charged cell structures.
Ex: Eosin, Rose Bengal and Acid fuchsin possess groups such as
carboxyls (-COOH) and phenolic hydroxyls (-OH), malachite
green, nigrosin, Indian ink.
BASIC: Positively charged basic radicals combines with negatively
charged particles in cytoplasm and gives color.
Ex: Haematoxillin, methylene blue, crystal violet, gention violet.
NEUTRAL: Both positively and negatively charged imparts
different colors to different components.
Ex: Geimsa’s stain, Leishman’s stain, Wright’s stain.
6. SIMPLE STAINING
6
• These show not only the presence of organisms but also the nature of the
cellular content in exudates .
• Simple stains provide a quick and easy way to determine cell shape, size, and
arrangement.
• Perform a bacterial smear, saturate the smear with basic dye for approximately 1
minute. You may use crystal violet, safranin, or methylene blue.
• Rinse the slide gently with water.
• Carefully blot dry with bibulous paper.
• Observe the slide under the microscope, using proper microscope technique.
• A single stain is used.
• Examples are Loeffler’s methylene blue, polychrome methylene blue,
dilute carbol fuchsin.
7. POSITIVE STAINING
7
POSITIVE STAINING: - where the actual cells are themselves colored and appear in a
clear background
(a) Simple staining: A stain which provides color contrast but gives same color to all
bacteria and cells.
Ex: Loeffler’s methylene blue, Polychrome methylene blue, Diluted carbol fuchsin.
(b) Differential Staining: A stain which imparts different colors to different bacteria is
called differential stain(which contains more than one stain). Ex: Gram’s stain , Acid fast
staining, Special stains.
8. BACTERIAL SMEAR PREPARATION:
Smear - is a distribution of bacterial cells on a slide for the purpose of viewing
them under the microscope.
Method: -
Aseptically a small sample of the culture is spread over a slide surface. -This is then
allowed to air dry.
The next step is heat fixation to help the cells adhere to the slide surface.
The smear is now ready for staining.
SMEAR FIXATION:
1) Heat fixation
a) Pass air-dried smears through a flame two or three times. Do not overheat.
b) Allow slide to cool before staining.
2) Methanol fixation
a) Place air-dried smears in a coplin jar with methanol for one minute. Alternatively,
flood smear with methanol for 1 minute.
b) Drain slides and allow to dry before staining.
9.
10. SIMPLE STAINING
LOEFFLER’S METHYLENE BLUE: It is generally the most useful, it shows the
characteristic morphology of polymorphs, lymphocytes and other cells more clearly than do
stronger stains such as the Gram stain or dilute carbol fuchsin.
POLYCHROME METHYLENE BLUE:
This is made by allowing Loeffler’s methylene blue to ‘ripen’ slowly.
The slow oxidation of the methylene blue forms a violet compound that gives the stain its
polychrome properties.
The ripening takes 12 months or more to complete, or it may be ripened quickly by the
addition of 1.0% potassium carbonate (K2co3) to the stain. It is also employed in
McFadyean’s reaction.
Incontrast to the blue staining of most structures by the methylene blue, the violet component
stains acidic cell structures red-purple , e.g. the acid capsular material of the anthrax bacillus
in the McFadyean reaction.
DILUTE CARBOL FUCHSIN
Made by diluting Ziehl-Neelsen’s stain with 10-20 times its volume of water. Stain for 10-25
seconds and wash well with water.
Over-staining must be avoided, as this is an intense stain, and prolonged application colours
the cell protoplasm in addition to nuclei and bacteria.
11. SIMPLE STAINING REQUIREMENTS
Loefflers Methylene blue
Dil. Carbol Fuchsin
Distilled Water
Compound Microscope
Cedar Wood oil
Fixed smear
PROCEDURE
Make a thin smear on a slide.
Heat fixes the smear by passing the slide 2-3 times gently over the Bunsen
flame with the smear side up
Pour Loeffler’s methylene blue over the smear and allow it to stand for 3
minutes.
Wash the stained smear with water and air dry it.
Observe the smear first under low power (10x) objective, and then under oil
immersion (100x) objective.
Observe the presence of organisms and also the cellular content of sample.
12.
13. NEGATIVE STAINING
13
• Negative staining procedure helps to study the cell shape, cell breakage, refractable
inclusion bodies and spores, where the cells remain clear (uncolored) and the
background is colored to create a contrast to aid in the better visualization of the
image.
• It is useful for those bacteria which are difficult to stain.
• Very slender bacteria like spirochaetes that are not detectable by simple staining
methods can be viewed by negative staining.
(a) Indian ink
(b) Nigrosin .
(c) Principle: Negative staining requires the use of acidic stains such as Indian ink
and nigrosin. The acidic stain with its negatively charged chromogen will not
penetrate the cell because of the negative charges on the surface of the bacteria. So
unstained cells are diffferentiable against the dark background. Since heat fixation
is not required, the cells are not subjected to the de-staining effect of chemicals and
heat, their natural size, shape and arrangement can be seen by this method. It is
possible to observe bacteria that are difficult to stain.
14. 14
• Procedure:
• A loopful of undiluted Indian ink was
placed on one end of clean slide.
• A loopful of inoculum was transferred
into the drop of stain.
• By using a second dirt-free slide
with smooth edge over the
suspension, it was spread
uniformly along the edge.
• The suspension was spread to the end of
the slide so as to form a uniform smear.
• The slide was then air dried and
observed under oil immersion
objective.
• Colorless bacteria are seen against a dark
background.
15. DIFFERENTIAL STAINING
15
• Differential Staining is a staining process which uses more than
one chemical stain. Using multiple stains can better differentiate
between different microorganisms or structures/cellular
components of a single organism.
• Differential staining is used to detect abnormalities in the
proportion of different white blood cells in the blood.
• One commonly recognizable use of differential staining is
the Gram stain.
• Acid-fast Stains are also differential stains.
16. INTRODUCTION
Gram staining is a method of differentiating bacterial species into two large Groups
(Gram-positive and Gram-negative).
The Gram staining is almost always the first step in the identification of
bacteria.
It 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 method is named after its inventor, the Danish scientist Hans Christian
Gram (1853–1938), who developed the technique while working with Carl
Friedländer in the morgue of the city hospital in Berlin in 1884.
In 1884, while examining lung tissue from patients who had died of pneumonia,
Gram had discovered that certain stains were preferentially taken up and retained
by bacterial cells.
17. Gram did not use a counter stain in his procedure. It was a few years later, that
the German Pathologist Carl Weigert(1845-1904) from Frankfurt, added a final
step of staining with Safranin.
Gram himself never used the red counterstaining in order to visualize the gram
negative bacteria.
The difference between the two types of bacteria is that the Gram positive have
thicker and denser peptido-glycan layers in their cell walls, which makes them
less permeable to the COUNTER stain than those of the Gram negative
bacteria.
The iodine has a critical role in enhancing this difference.
It seems to bind temporarily to the peptidoglycan and make it even less
permeable to the dye.
18. Applying a primary stain (Crystal Violet) to a heat-fixed
smear of a bacterial culture.
The addition of Grams Iodine, which binds to crystal violet and traps it in the
cell.
Decolourization with Alcohol or Acetone, and
Counter staining with Safranin
Principle
Violet dye and the iodine combine to form an insoluble, dark purple compound in
the bacterial protoplasm and cell wall.
This compound is dissociable in the decolorizer, which dissolves and removes its
two components from the cell.
But the removal is much slower from Gram-positive than from the Gram-negative
bacteria, so that by correct timing the former stay dark purple whilst the latter
become colorless.
FOUR BASIC STEPS OF GRAM’S STAINING
19. Bacteria All Bacteria will be stained Purple
Stain will be fixed due to formation of
complex of Crystal Violet & Iodine
Crystal Violet
Stained with
Grams Iodine
solution
Add
Decolourizer
(Alcohol orAcetone)
Cells will be
decolourized
while some
cells will retain
the stain
Staining with Safranin(Counter Stain)
Cells that retains the colour of Primary Stain are Gram positive.
Cells that do not retains the colour of Primary Stain and takes up the colour of
Counter Stain are Gram Negative.
20.
21.
22. Prepare a heat fixed smear of the bacterial culture.
Cover the smear with the Crystal Violet for 1 min.
Add Grams Iodine, which washes the crystal violet stain.
Rinse the slide in running water and add decolourizer (Alcohol).
Again rinse the slide and cover the smear with the Safranin for 1 min.
Wash off the safranin with water, air dry the slide and Observe under oil immersion
lens.
Gram positive cocci inchains Gram negativebacilli
23.
24. ACID – FAST STAINING
24
• This is also known as Ziehl – Neelsen staining.
• This method is a modification of Ehrlich’s (1882) original method for the differential
staining of tubercle bacilli and other acid-fast bacilli with aniline-gentian violet
followed by strong nitric acid.
• Stain used consists of basic fuchsin, with phenol added.
PAUL ERHLICH
FIRST IDENTIFIED Mycobacterium tuberculosis
25. Principle
25
• Acid-fast staining is another commonly used, differential staining technique that
can be an important diagnostic tool.
• An acid-fast stain is able to differentiate two types of gram-positive cells: those
that have waxy mycolic acids in their cell walls, and those that do not.
• Two different methods for acid-fast staining are the Ziehl-Neelsen technique and
the Kinyoun technique.
• Both use carbol fuchsin as the primary stain.
• The waxy, acid-fast cells retain the carbol fuchsin even after a decolorizing agent
(an acid-alcohol solution) is applied.
• A secondary counter stain, methylene blue, is then applied, which renders non–
acid-fast cells blue.
• The fundamental difference between the two carbol fuchsin-based methods is
whether heat is used during the primary staining process.
• The Ziehl-Neelsen method uses heat to infuse the carbol fuchsin into the acid-fast
cells, whereas the Kinyoun method does not use heat.
26. • Both techniques are important diagnostic tools because a number of specific
diseases are caused by acid-fast bacteria (AFB).
• If AFB are present in a tissue sample, their red or pink color can be seen clearly
against the blue background of the surrounding tissue cells
• It is most commonly used to identify M.tuberculosis and M.leprae, the pathogen
responsible for tuberculosis and leprosy, respectively.
• These bacteria have cell walls containing lipids constructed from mycolic acids, a
group of branched chain hydroxyl fatty acids, which prevent dyes readily binding to
the cells.
• However, M.tuberculosis and M.leprae can be stained by procedures such as the
Zeihl-Neelson method which uses heat and phenol to derive basic fuchsin into cells.
• Once basic fuchsin has penetrated, M.tuberculosis and M.leprae are not easily
decolorized by acidified alcohol (acid-alcohol) and thus are said to be acid-fast.
• Non acid-fast bacteria are decolorized by acid-alcohol and thus are stained blue by
methylene blue counterstain.
26
27. Procerdure
27
•Prepare bacterial smear on clean and grease free slide, using sterile technique.
Allow smear to air dry and then heat fix.
•Alcohol-fixation/Heat-fixation of untreated sputum will not kill M. tuberculosis whereas
alcohol-fixation is bactericidal.
•Cover the smear with carbol fuchsin stain.
•Heat the stain until vapour just begins to rise (i.e. about 60◦C). Do not overheat. Allow
the heated stain to remain on the slide for 5 minutes.
•Heating the stain: Great care must be taken when heating the carbol fuchsin especially if
staining is carried out over a tray or other container in which highly flammable chemicals have
collected from previous staining. Only a small flame should be applied under the slides using an
ignited swab previously dampened with a few drops of acid alcohol or 70% v/v ethanol or
methanol.
• Do not use a large ethanol soaked swab because this is a fire risk.
•Wash off the stain with clean water.
28. Procerdure
28
•Note: When the tap water is not clean, wash the smear with filtered water or clean
boiled rainwater.
•Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear is
sufficiently decolorized, i.e. pale pink.
•Caution: Acid alcohol is flammable, therefore use it with care well away from an
open flame.
•Wash well with clean water.
•Cover the smear with malachite green/ Methylene Blue stain for 1–2 minutes, using
the longer time when the smear is thin.
•Wash off the stain with clean water.
•Wipe the back of the slide clean, and place it in a draining rack for the smear to air-
dry (do not blot dry).
•Examine the smear microscopically, using the 100 X oil immersion objective.
32. VOLUTIN-GRANULE STAINING
32
• Volutin granules are a type of cytoplasmic inclusion bodies found in many bacteria
as well as in some fungi, algae, protozoa.
• These granules are composed mainly of polyphosphate, RNA and protein.
• These granules are found most prominent in old cultures before starvation occurs.
• The method of volutin granule staining is known as ALBERT- LAYBOURN
METHOD.
Volutin
granules
33. 33
PRINCIPLE
Albert’s stain contains cationic dyes like toludine blue and malachite
green.
Due to the highly acidic nature of the granules, they can be selectively
stained by acidified basic dyes.
The toludine blue preferentially stain volutin granules while malachite green
stains the cytoplasm.
Later due to application of Albert’s iodine, the dye molecule are fixed by
precipitation.
Well developed granules of volutin (polyphosphate) may be seen in unstained
wet preparations as round refractile bodies within the bacterial cytoplasm
34. 34
• PROCEDURE
• A thin uniform smear of culture was made. It was air dried and heat fixed.
• Lower the slide with Albert’s stain A (consists of Toluidine blue, malachite green, glacial
acetic acid, and ethyl alcohol) and allowed to react for 3-5 min.
• The slide was then washed under running tap water.
• Flood the slide with Albert’s Iodine and allowed to react about 1 min.
• Slide was then washed and blot dried.
• The slide was observed under oil immersion objective of a microscope.
OBSERVATION
35. SPORE STAINING
35
• The morphology of bacterial endospores is best observed in
unstained wet films under the phase contrast microscope, where
they appear as large, refractile, oval or spherical bodies within a
bacterial mother cells or else from the bacteria.
• If spore-bearing organisms are stained with ordinary dyes , or by
Gram’s stain , the body of the bacillus is deeply colored , whereas
the spore is unstained and appears as clear area in the organism.
• This is the way in which spores are most commonly observed.
• If desired , however , it is possible by vigorous staining procedures
to introduce dye into the substance of the spore.
• When thus stained , the spores tends to retain the dye after treatment
with decolorizing agents, and in this respect behaves similarly to the
tubercle bacillus, but is more weakly acid-fast.
36. Principle
36
• The spores are thick walled structures and very resistant to physical and
chemical agents.
• To differentiate between vegetative cells and endospores.
• The spores have a capacity to survive for long periods even in unfavourable
environmental conditions.
• The heat resistance by spores is due to the high content calcium-
dipicolinic acid.
• The spores are differentially stained using special procedures that help dye to
penetrate the spore wall.
• An aqueous primary stain, malachite green is applied and steamed to enhance
the penetration of the impermeable spore coat.
• Once stained the endospore does not readily decolorize even with the application
of decolorizer and they appear, but the cytoplasm of the cell takes the color of
safranine and appears red.
• A modified Ziehl-Neelsen stain in which weak, 0.25% sulphuric acid is used
as decolorizer, yields red spores in blue-stained bacteria. Lipid granules also
stain red, appearing like small spherical spores.
38. Procedure
38
Films are dried and fixed with minimal flaming.
1. Place the slide over a beaker of boiling water , resting it on the rim
with the bacterial film uppermost.
2. When , within several seconds , large droplets have condensed on
the underside of the slide , flood it with 5% aqueous solution of
malachite green and leave to act for 1 min while the water
continues to boil.
3. Wash in cold water.
4. Treat with 0.5% safranine or 0.05% basic fuchsin for 30 seconds.
5. Wash and dry.
This method colors the spores green and the vegetative bacilli red.
Lipid granules are unstained.
39. Observation
39
Positive: Clostridium perfringens, C. botulinum, C. tetani, Bacillus anthracis, Bacillus
cereus, Desulfotomaculum spp, Sporolactobacillus spp, Sporosarcina spp, etc.
Negative: E. coli, Salmonella spp, etc.
40. CAPSULE STAINING
40
•A bacterial capsule can define as the mucilaginous coating that surrounds the cell wall of
bacteria. The capsule also refers as glycocalyx as it is composed of glycoproteins.
•A cytoplasm of bacteria partially forms a capsule which then goes to the cell wall and surrounds
it as a mucous or slime covering.
•The capsule protects a cell from desiccation because of the mucous content. A mucus layer also
protects a cell from phagocytosis.
•The capsule also acts as a virulence factor and responsible for the pathogenicity of many
microorganisms like Streptococcus pneumoniae, Escherichia coli, Neisseria meningitis etc.
•On the majority, a capsule consists of polysaccharides but besides this, also contains
polypeptides or glycoproteins.
•The formation of a capsule is a process which is controlled genetically. Capsules can be easily
visible under the light microscope, by the use of differential capsule stain.
•Bacteria having capsule will refer as “Capsulated bacteria”, and those who lack a capsule will
refer as “Non-capsulated bacteria”.
•Capsule are of two types, namely micro and macro capsule. Microcapsule has a size less than
0.2µ whereas Macrocapsule has a size more than 0.2µ.
41. CAPSULE STAINING
41
•The capsule is non-ionic in nature, i.e. it will neither stain by acidic nor by basic
stains.
•The basic dye will stain the negative bacterial cell, and an acidic stain will stain the
positive background.
•Thus, to stain a capsule, we need a special capsule stain that will focus on the
capsule.
•A capsule can be easily destroyed by a heat treatment that’s why a step of heat fixing
is skipped while performing capsule staining.
• In addition to this, a step of washing or rinsing is also avoided because it can
dislodge the capsule from the bacterial cell.
•To enhance the size of the capsule or to increase its visibility, we can also add a drop
of serum. The addition of serum provides a more unobstructed view of a capsule
under a light microscope.
Principle
The principle of capsule staining is based on staining of background with an acidic
stain and staining of bacterial cell with a basic stain.
As a capsule is non-ionic, it will not stain by either of the two dyes.
Thus a capsule staining creates a contrast by staining a bacterial cell and its
background in between which, capsule appears as a colourless halo.
42. • Methods of Capsule Staining
• There are different methods for capsule staining, among which the
most common methods are:
• India ink method
• Anthony’s method
• Maneval’s method
• Hiss method
43. • India ink Method
• India ink method uses two types of stain, i.e. a basic stain (Crystal violet) and an
acidic stain (India ink). Crystal violet being positive stain will stain the negatively
charged bacterial cell. India ink being negative stain will stain the positively charged
background. After staining:
• The Background appears darker or black.
• A Bacterial cell appears violet.
• The capsule appears as a clear halo.
• Procedure
• India ink method involves the following steps:
• Take a clean, sterilized or grease free slide.
• Add a drop of crystal violet to the centre of the glass slide.
• Prepare a smear, by taking an inoculum from the bacterial culture and mix it with a
drop of crystal violet.
44. • Procedure
• Then, allow the smear to air dry(Do
not heat fix, as it can cause cell
shrinkage and distortion of the
bacterial capsule).
• Flood a smear with the India ink for
30 seconds and remove the extra
stain by tilting a glass slide.
• Add oil immersion to the stained
area and observe it under the
microscope having a 100X
objective.
• India ink method is a type
of negative staining method, which
stains both the bacterial cell and its
background but not a capsule. As a
result, a capsule appears as a bright
halo between the violet bacterial cell
and a darker background.
45. • Anthony’s Method
• This method makes the use of two reagents, namely crystal violet as primary
stain and 20% of CuSO4 solution as a decolouring agent and counterstain.
• Crystal violet will stain the bacterial cell and background. The CuSO4 solution
will stain the non-ionic capsule. After staining:
• A Bacterial cell appears violet.
• The background appears light violet.
• The capsule appears as a faint blue halo
• Procedure
• Anthony’s method involves the following steps:
• Take a clean, sterilized or grease free slide.
• Add a drop of crystal violet to the centre of the glass slide.
• Prepare a smear, by taking an inoculum from the bacterial culture and mix it
with a drop of crystal violet.
• Then, allow the smear to air dry (Do not heat fix, as it can cause cell shrinkage
and distortion of the bacteria).
46. • Procedure
• Flood a smear with 20% of
CuSO4 solution for at least
30 seconds and remove the
extra stain by tilting a glass
slide.
• Add oil immersion to the
stained area and observe it
under the microscope
having a 100X objective.
• Anthony’s method is a type
of positive staining
method that stains the
capsule along with the
bacterial cell but not stains
the background. As a
result, a capsule appears as
faint blue halo between the
violet bacterial cell and
purple background.
47. • Maneval’s Method
• This method makes the use of two stains, i.e. acidic stain (Congo red) and a
special stain (Maneval’s stain). The composition of Maneval’s dye includes:
• 10% Ferric chloride, which acts as a “Mordant”.
• 5% phenol, which increases the penetration of the stain between the smear.
• Acid fuschin, stains the bacterial cell being a basic dye.
• Acetic acid, which decreases the pH of the smear to the acidic side.
• Congo red appears red (at neutral pH) and appears blue in colour (at acidic pH).
After staining:
• A Bacterial cell appears bright red-pink.
• The background appears dark blue colour.
• The capsule appears as a clear halo.
• Procedure
• Maneval’s method involves the following steps:
• Take a clean, sterilized or grease free slide.
• Add a drop of 1% Congo red to the centre of the glass slide.
• Prepare a smear, by taking an inoculum from the bacterial culture and mix it with
a drop of Congo red.
48. • Procedure
• Then, allow the smear to air dry
(Do not heat fix, as it can cause
cell shrinkage and distortion of the
bacteria).
• Flood a smear with Maneval’s
stain for at least 1 minute and
remove the excess stain by tilting a
glass slide.
• Add oil immersion to the stained
area and observe it under the
microscope having a 100X
objective.
• Maneval’s method is also a type
of negative staining method,
which stains the bacterial cell and
its background but not a capsule.
As a result, a capsule appears as a
clear halo between the pink
bacterial cell and blue background.
49. • Hiss Method
• This method makes the use of two reagents, namely crystal violet and copper
sulphate solution. After staining:
• A Bacterial cell appears dark violet.
• The background appears brighter in colour.
• The capsule appears as a light violet colour.
• Procedure
• Hiss method involves the following steps:
• Take a clean, sterilized or grease free slide.
• Add a drop of crystal violet to the centre of the glass slide.
• Prepare a smear, by taking an inoculum from the bacterial culture and mix it
with a drop of crystal violet.
• Then, allow the smear to air dry (Do not heat fix, as it can cause cell shrinkage
and distortion of the bacteria).
• Flood a smear with copper sulphate solution and remove the excess stain by
tilting a glass slide.
50. • Procedure
• Add oil immersion to the
stained area and observe it
under the microscope having a
100X objective.
•
Hiss method is also a type
of positive staining
method that stains the capsule
and bacterial cell with a
brighter background. As a
result, a capsule appears as a
light violet colour between a
dark violet coloured bacterial
cell and colourless
background.
51. FLAGELLAR STAINING
51
Flagellar staining provides taxonomically valuable information about the presence
and distribution pattern of flagella on prokaryotic cells. Bacterial and archaeal
flagella are fine, threadlike organelles of locomotion that are so slender (about 10
to 30 nm in diameter) they can only seen directly using electron microscope. To
observe bacterial flagella with the light microscope, their thickness is increased by
coating them with mordants such as tannic acid and potassium alum, and then
staining with pararosalineor basic fuchsin.
52. Procedure
52
• Grow bacteria for 16 – 24 hrs on a non-inhibitory medium ,
e.g. tryptic soy agar or blood agar.
• Touch a loopful of water onto the edge of a colony and let motile bacteria
swim into it.
• Then transfer the loopful into a loopful of water on a slide to get a faintly turbid
suspension and cover it with a cover-slip.
• The bacterial suspension is thus prepared with a minimum of agitation , which
would detach the flagella.
• After 5-10 min , when many bacteria have attached to the surfaces of the slide and
cover-slip , apply two drops of Ryu’s stain to the edge of the cover-slip and leave
the stain to diffuse into the film.
• Examine with the microscope after standing 5-15 min at ambient
temperature.
