MICROBIOLOGY LAB 1 and 2 – Staining Modified kinyoun stain phenol

USTMED ’07 Sec C – AsM; Photos provided by JV.N. & A.M.

USTNotesGroup2009
Updated 2006 - A. Abad 3. Decolorizer – a chemical to remove the primary stain
- Ex.
Stain – coloring of organisms with a dye that emphasizes Gram Stain 95% C2H2OH
certain structures (acetone alcohol)
Smear – a thin film of material containing the microorganisms Acid fast stain Acid alcohol
spread over the surface of the slide
Fixation – a procedure done before staining a smear to: 4. Secondary stain – a chemical which imparts a
1. attach the microorganisms to the slide contrasting color to the primary stain
2. kill the microorganisms - Ex.
3. preserve the various parts of microbes in their Gram stain Safranin
natural state with only minimal distortion Acid fast Methylene blue,
malachite green
3 Methods of Fixation Spore safranin
1. Air drying stain
2. Passing over flame of Bunsen burner
3. cover slide with methyl alcohol for 1 minute
I. Gram Stain
- if not fixed, stain may wash the microbes Purpose Reagents Gram + Gram -
Primary Crystal violet Violet or Violet or
Stains – salts composed of a positive and a negative ion stain purple purple
Chemical Grams iodine Dark violet Dark violet
1. basic dyes (chromatophore) – positive ion
Mordant or purple or purple
2. acidic dyes – negative ion Decolorizer Acetone Violet or Colorless
Alcohol purple
Bacteria – slightly negative charge; pH 7 Secondary Safranin Violet or Red
stain purple
Add (+) ion [basic dye] to charge bacteria = attract End Result Violet Red
Ex. Crystal violet, methylene blue, safranin
Add (-) ion [acidic dye] to charge bacteria = repel
Ex. Eosin, nigrosin, acid fuchsin Gram stain of Micrococcus
species. Microscopically,
Negative stain – prepare colorless bacteria against a colored micrococci are larger than
background staphylococci and appear in
tetrads rather than
Importance: grapelike clusters.
1. valuable in the observation of overall shapes,
sizes and capsules
2. distortion of cell size and shape are minimized
a. heat fixing is not necessary
b. cells do not pick up the stain Urethral discharge with PMN
and intracellular gram
Types of stain negative diplococci
1. Simple stain suggestive of Neisseria
- Purpose – cellular shape and structure is made gonorrhoeae.
visible
- Ex. Methylene blue, carbol fuchsin, crystal violet,
safranin
2. Differential stain
- Distinguishes two groups of organisms or different
parts of a bacterial cell
- Ex. Principle:
a. Gram stain 1. When applied to both gram-positive and gram negative cells,
b. Acid fast stain crystal violet and then iodine readily enter the cells.
i. Ziehl Neelsen stain 2. Inside the cells, the crystal violet and iodine combine to
ii. Modified Kinyoun stain fome CV-1 which is larger than the crystal violet molecule
c. Spore stain that entered the cells.

i. Schaefer Fulton method 3. In Gram positive cells, crystal violet-iodine complex is

trapped and therefore, retains the color.
ii. Wirtz Conklin method
4. In Gram negative cells, the alcohol disrupts the
Reagents in Differential stain lipopolysaccharides and the CV-I complex is washed out
1. Primary Stain – imparts color to all cells through the thin peptidoglycan layer.
- Ex. 5. Gram negative cells are colorless until counterstained with
Gram stain Crystal violet safranin after which they are pink.
Acid fast stain Carbol fuchsin
Clinical Significance
Spore stain Malachite green
- Provides valuable information for the treatment of disease
Gram Penicillin
2. Mordant – a chemical added to primary stain to:
+ Cephalosporin
a. Intensify
Gram More resistant because the antibiotics do not
b. Increase affinity
- penetrate the lipopolysaccharides
c. To coat a structure to make it thicker

- Types of mordant II. Acid Fast Stain

o Physical mordant – steaming Purpose Reagents Gram + Gram -
o Ex. Primary Carbol Red Red
stain fuchsin
Ziehl Neelsen stain
Mordant Steam or Red Red
Schaefer Fulton method
phenol
Wirtz Conklin method
Decolorizer Acid alcohol Red Colorless
Secondary Methylene Red Blue or green
o Chemical mordant
stain blue or
o Ex.
malachite
Gram stain Grams iodine green
Flagellar stain Tannic acid
 Mordant Steam Green Green
Rinse Tap water Green Colorless
Secondary safranin green red
stain

Modified Kinyoun acid-fast Spore stain of Bacillus cereus.

stain. Nocardia species The arrows are pointed at green
appear acid-fast when spores in a pink vegetative cell.
stained with a modified
Kinyoun stain using 2% H2SO4
as the decolorizing agent.
This feature helps to
distinguish this
mircroorganism from other
actinomycetes. Gram Stain
Prinicple Purpose Reagent Spores Vegetative cell
1. Acid fast organisms retain the red color because the Primary stain crystal Violet or Violet or purple
carbol fuchsin is more soluble in the cell wall lipids than violet purple
in the acid alcohol. Mordant Grams Dark violet Dark violet or
2. Non-acid fast organisms – cell walls lack the lipid iodine purple
components; carbol fuchsin is rapidly removed during Decolorizer 95% Colorless Violet or purple
decolorization C2H2OH
Secondary Safranin colorless Violet or purple
Mycolic acid – a cell wall component responsible for the
stain red
acid fastness of the organism
Gram stain of Bacillus cereus.
Acid fast bacilli – organisms which are hard to stain but
The arrow is pointed at a
once stained, they are hard to decolorize
spore, which is clear inside
the gram-positive vegetative
Ex. Genus Mycobacteria, Genus Nocardia
cell.
Clinical significance
- the finding of acid fast bacilli in a sputum is a
presumptive evidence that the organisms is
Mycobacterium tuberculosis.
D. Metachromatic granules
- Ex. Neisser stain, Loefflers methylene blue stain
III. Special stains or selective stains – stains used to color
and isolate specific parts of microorganisms such as
Loefflers methylene blue stain.
endospores, flagella, capsules and metachromatic granules
Corynebacterium diptheriae
Demonstrate metachromatic
A. Negative staining for capsules
granules when stained with
1. India ink technique
Loeffler methylene blue stain or
- mix the bacteria in a solution containing a fine
Neisser stain. The stain is best
colloidal suspension of colored particles.
performed on colonies grown on
- then stain with a simple stain, safranin.
a Loeffler agar slant.
- Result – Halos surrounding each stained bacterial
Metachromatic deposits are
cell.
reddish purple in Loeffler
methylene blue stain.
Capsule stain. The cell is the purple
rod in the center of the clear area.
- fin -
The purple color is from the basic
stain, crystal violet.
Demo slides…..
Vibrio spp. (curved bacilli)
The clear area is the capsule, and the
background is colored by the negative,
acidic stain (India ink).

B. Flagella stain – a tedious and delicate staining

procedure using a mordant and the stain carbol
fuchsin to build up the diameters of the flagella
Gram + stain
until they become visible.

Vibrio cholerae.

Monotrichous flagella – one

polar flagellum

Streptococcus lactis (cocci in chains)

Proteus.

Peritrichous flagella – flagella

over the entire bacterial cell

C. Endospore (spore) staining – both selective and

differential
- Ex. Schaefer Fulton method, Wirtz Conklin method
Sarcinae lutea (cocci in tetrads)
Purpose Reagent Spores Vegetative cell
Primary Malachite Green Green
stain green
 structure itself. The exact mechanism of the staining reaction is
not fully understood, however, this does not detract from its
usefulness.

Congo red staining The Gram staining method

1. A small sample of a bacterial culture is
removed from a culture. In this example it
is being taken from a broth culture of the
pure microbe but it could be removed
from a culture on solid medium or from
material containing bacteria eg faeces or
soil.

India Ink 2. The bacterial suspension is smeared

unto a clean glass slide. If the bacteria
have been removed from a culture or a
solid media or it is from a soil or feces
sample It will have to be mixed with a
drop of bacteria-free saline solution
3. The bacterial smear is then dried slowly
at first and the, when dry, heated for a
few seconds to the point when the glass
slide is too hot to handle. This fixes ie
kills the bacteria making the slide safe to
Malachite green staining
handle. Care must be taken not to
overheat which will char the cells.

4. Once cool, the slide is transferred to a

support over a sink and flooded with a
stain called Gentian Violet (a dye
consisting of a methyl derivative of
pararosaniline). The stain is left on the
slide for about 1 minute. This stains all
the bacteria on the slide a dark purple
S. aureus colour. Note, this stain will not penetrate
the waxy cell walls of some bacteria eg
mycobacteria.
5. The Gentian Violet is gently washed off
the slide with running water

6.The bacterial smear is then treated with

Bacillus subtillis (gram positive bacilli in chains) Gram’s solution which consists of 1 part
iodine, 2 parts potassium iodide, and 300
parts water. This iodine solution reacts
with the Gentian Violet turning it a very
dark shade of blue. It also causes it to be
retained by certain types of bacteria in a
way which is not really understood.
7.After about 30 seconds the slide is
gently rinsed with ethyl alcohol which
causes the dye-iodine complex to be
washed out of some bacteria but not
Streptococcus lactis (cocci in chains) others. This is called decolourisation.

If we now look at the smear down a microscope, the bacteria

which had retained the Gentian Violet-iodine complex will
appear blue-black. These are called Gram-positive.
However wi would not be able to see those which had lost
the dye-iodine complex which are called Gram-negative. The
final step in the gram stain method is, therefore, to stain the
Gram-negative cells so they can be seen.
8. This is achieved by treating the smear
The Gram Stain Introduction with a compound which stains the Gram-
negative cells a colour which contrasts
Gram’s Stain is a widely used method of staining bacteria as markedly with the blue-black colour of the
an aid to their identification. It was originally devised by gram-posiitve cells. The stain common
Hans Christian Joachim Gram, a Danish doctor. used for this is either eosin or fuchsin,
both of which are red. These are called
Gram’s stain differentiates between two major cell wall counterstains. Bacteria in the smear
types. Bacterial species with walls containing small amounts which are Gram-positive are unaffected by
of peptidoglycan and, characteristically, lipopolysaccharide, the counterstain.
are Gram-negative whereas bacteria with walls containing 9. The counterstain is left on the smear
relatively large amounts of peptidoglycan and no for about 30-60 seconds and then gently
lipopolysaccharide are Gram-positive. rinsed away with running water.

It’s a mystery

Although it may seem strange, the reason why bacteria with

these two major types of bacteria cell walls react differently
with Gram’s stain appears to be unconnected with the wall
10. After the counterstain has been rinsed
off, the slide is placed between some Staphylococcus aureus.
absorbent paper and the excess water Gram stain of culture
gently blotted off. Care must be taken showing characteristic
not to rub the slide with the blotting irregular clusters of gram-
paper because this would remove the positive cocci. There are no
adhering bacteria. spores or capsules.
11. The slide is gently warmed to dry off
any residual moisture and then a drop of
oil immersion oil is placed on the stained
bacterial smear. This helps transmit light Bacillus subtilis
through the specimen directly to the high- Stain used: Gram stain
powered microscope lens. Results: Gram positive Bacilli
12. The slide is the placed on a in chain (violet rods in chain)
microscope stage and the oil-immersion Aerobic Sporeformer Bacilli
lens lowered into the immersion oil. High- Note: Spores elliptical and
powered lenses are required because centrally located
bacteria are very small SPORES – unstained
VEGETATIVE PORTION – violet

Kinyoun’s acid fast stain. Mycobacteria are readily stained with

carbol fuchsin, which binds the cycolic acid in their lipid-rich cell
walls. This stain cannot be removed (decolorized) with acid
alcohol and, therefore, the microorganisms are referred to as
acid-fast. There are two common acid-fast stains, Ziehl-Neelsen
(ZN), and Kinyoun’s. The difference is that the ZN stain requires
heat during the staining process because the phenol
concentration used is less than in the Kinyoun’s method. The
decolorizer, acid alcohol, and the counterstain, methylene blue,
are the same for both methods. When stained, acid-fast bacilli
stain red and the background is blue.

Clinical specimen :
Urethral
discharge
Staining used:
Gram staining
Results:
Gram neg
cocci in pairs
Intracellularly
located Kinyoun’s Acid Fast Staining (Cold Method)
Most possible microorg : i. Fix smear by passing slide over the flame for 3 times
Neisseria
gonorrhea ii. Cover the smear with enough Kinyoun’s Carbol Fuchsin stain
(initial stain) for 2-3 minutes.
Pseudomonas iii. Rinse with tap water
aeruginosa
Stain used:
Gram Staining iv. flood the smear with 3% acid alcohol (decolorizer) until the
Results: smear appears colorless or until only a suggestion of pink
Gram Negative bacilli in remains (30-45 secs).
singly/random (red
slender rods in singly or v. rinse with tap water
random)
vi. cover the smear with methylene blue (counter stain) for 20-
30 seconds)
Streptococcus pyogenes
Stain used: vii. rinse with tap water
Gram Staining
Results: viii. air dry or blot with filter paper then examine the slide
Gram Positive under oil immersion lens.
Cocci in chains (violet
round/spherical cocci in Manner of Reporting/Recording for AFB Smear Results (2001)
chains) IUATLD System
No AFB in at least 100 fields 0 or Negative
1-9 AFB in 100 Fields + n (report the actual AFB
Escherichia coli count)
Stain used: Gram stain Suggest repeat collection
Gram reaction: 10-99 AFB per 100 field +1
Gram 1-10 AFB/field in at least 50 +2
negative (red) fields
Morphology: >10 AFB/field in at least 20 +3
Coccobacilli fields
arragned singly or
random Mycobacterium tuberculosis
Note: Stain used:
E. coli is a Kinyoun’s Acid fast stain
Gram Negative Bacilli Clinical specimen used:
but it appears as a short Sputum
plump bacilli so it is Results:
called coccobacilli (see Positive for acid fast
arrow) bacilli. acid fast stain of direct
smear to show acid fast bacilli
 staining deep red (arrow
A) and non-acid fast bacilli
cells staining blue with the
counter stain methylene
blue (arrow B)

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Jayveeh Navarro
Amelia Mendoza Gram (-) Bacilli Gram (+) Cocci
Auds Martinez
audrey_cl@yahoo.com
Gram stain of Clostridium
paraputrificum (x1250).
This microorganism displays
terminal, swollen spores
and the gram-variable
staining typical of
Clostridium species. Spores Loeffier methlene blue stain Sarcina lutea
may not take up the stain, Corynebacterium diphtheriae Gram positive tetrad
so they may appear as clear metachromatic granules
areas. (terminal spore)
Sputum smear stained with
Gram’s stain shows
neutrophil amorphous
debris, and filamentous,
beaded, branched gram-
positive bacilli (oil
immersion)
Gram stain of pus from an
empyema cavity showing Sprillum (spiral bacterium) Urethral smear report
long and short chains of
Gram-positive streptococci
and large numbers of pus
cells (stained red). X3,900

large boxcar shapped Gram

(+) bacilli

Vibria comma (curved bacilli)

Gram stain Nocardia spp.

(x1250). Nocardia species
are branching, beaded,
filamentous gram-positive
bacilli, approximately 1 um
in diameter. They can also
appear as coccoid or
coccobacillary forms. Care
should be taken when
examining the slides
because of the faint
staining properties of these
microorganisms and other
actinomycetes.
Flagella stain, Proteus sp.
Lab 2 addition
India Ink Method
(Staining of Capsule)
Indirect or negative or relief staining
1. To a small loopful of water on a clean glass slide, ad a minute
amount of growth from the agar culture by using a sterile
inoculating needle. (Broth culture maybe used directly.)
2. Mix well, then add a small drop of INDIA INK and immediately
cover with a thin coverslip allowing the fluid to spread as a thin
film beneath the coverslip.
3. Examine under the high power objective and then with the oil
immersion lens. Regulate the light with the sunstage condenser
of the Iris diaphragm.

Klebsiella pneumoniae
Capsule stain
Sputum stained with
Gram’s stain shows many
neutrophil amorphous
debris, and coryneform
gram-positive bacilli (oil
immersion)

Congo Red Method

(Staining of Capsule)
Demo Slides Indirect or negative or relief staining
Diplococcus pneumonia 1. Using a sterile inoculating needle fish out a small amount of
colony of Klebsiella pneumoniae and mix it with a drop of CONGO
RED stain and spread 2 cm. Diameter.
2. Air dry. Do not heat.
3. Cover the smear with 1% ACID ALCOHOL until the smear
turns blue.
4. AIR DRY and examine under Oil Immersion lens.

Malchite Green Method

(Staining of Spores)

1. Prepare smear from bacillus subtilis

2. Flood the slide with 5% AQUEOUS SOLUTION OF MALACHITE
GREEN and heat to gentle steaming for 2 or 3 minutes. Avoid
overheating and drying.
3. Pour off excess stain and rinse thoroughly with tap water.
4. Flood the smear with SAFRANIN for 30 seconds
5. Rinse again with tap water, blot dry and examin under Oil
immersion objective.

SPORE: Green
Vegatative Portion: Red

Spore stain of Bacillus cereus. The arrows are pointed at

green spores in a pink vegetative cell.

Kinyoun’s acid fast stain (x1500). Mycobacteria are readily

stained with carbol fuchsin, which binds the mycolic acid in
their lipid-rich cell walls. This stain cannot be removed
(decolorized) with acid alcohol and, therefore, the
microorganisms are referred to as acid-fast. There are two
common acid-fast strains, Ziehl-Neelsen (ZN), and Kinyoun’s.
The difference is that the ZN stain requires heat during the
staining process because the phenol concentration used is
less than in the Kinyoun’s method. The decolorizer, acid
alcohol, and the counterstain, methylene blue, are the same
for both methods. When stained, acid-fast bacilli stain red
and the background is blue, as shown here.

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