Transcript Gram staining

Staining
Bacteria are microscopic organism, they tend to be
transparent even when they are magnified.
The simplest way to make bacteria visible to human eyes
is stain them with a visible dye.
The best bacterial stains are aniline dyes. These dyes
react in either:
an acidic , basic, or neutral manner.
Acidic dyes are anions(negative charged)
Basic dyes are cations (+ve charged)
Bacteria are slightly negatively charged so stain very well
with basic dyes.
Stains and Staining
Basic dye stains bacteria
Acidic dye stains background
We stain bacteria to study there :
A) Morphology and Arrangement :
• Coccus: pairs, chains, clusters
• Bacillus:
• spiral
Bacterial arrangement and Morphology
B)Differentiated bacteria to groups according to
there biochemical composition of cell wall
C) Study structures of bacteria (capsule,
flagella)
Stains are classified into:
• Simple stain (methylene blue, crystal violet)
• Differential stain(Gram's stain ,acid fast stain).
• Special stain (capsule stain, flagella stain).
Preparation of smear
• Clean the slide
• Place a loopfull of water in the centre of the slide
• Mix a small amount of bacteria using a sterile loop
(heating it at Benzen' burner) with the water and
spread it out.
• Allow the slide to air dry
• Heat-fix the smear by passing the slide through the
Benzen' burner three times.
Simple Stains
•Prepare the smear
•Flood the slide with methylene blue for 3 mins
•Wash the slide with tap water genetly, drain off excess water
then let the slide dry in air or by using filter paper
•Exam it microscopically
•The bacteria will appear blue cells.
Differential Stains
Gram Staining
Basic classification of bacteria is based on the cell
wall structure.
Gram staining is a differential staining technique
that provides an easy differentiation of bacteria into
one of two groups:
Gram positive and Gram negative.
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The characteristic compound found in all true bacterial cell walls
is peptidoglycan. The amount of PPG is among one of the
differences between the GP and GN cell walls.
Gram-positive cell walls
• Thick peptidoglycan
(90% peptidoglycan)
• Teichoic acids
• Not many
polysaccharides
• In acid-fast cells,
contains mycolic acid
Gram-negative cell walls
• Thin peptidoglycan
(5-10% peptidoglycan)
• No teichoic acids
• Outer membrane has
lipids, polysaccharides
• No acid- fast cells
(mycolic acid)
The process includes the use of:
a primary stain (crystal violet)
a mordant (helper) iodine solution,
a decolorizer (95% ethanol),
a counterstain (safranin).
The Gram stain
1. prepare a smear
2. Flood slide with crystal violet and let
stain for 1 minute.
3. Drain off crystal violet and rinse off
with distilled water; flood slide
with Gram's iodine for 1min,rinse off
iodine with distilled water.
4. Remove excess water from slide and
blot, so that alcohol used for decolorization is
not diluted. Hold the slide on an angle and
drop 95% ethyl alcohol for 10 seconds and
wash off with tap water.
5. flood the slide with safranin and let stain
for 2-3 minutes.
6. Rinse with distilled water and blot dry
with filter paper.
Gram
positive
Gram
negative
Mechanism of action
• the PPG is found in layers in GP CW and the stain molecules are
trapped within these layers, when they form the complex with the
mordant Iodine molecules.
Since the GN CWs lack much PPG the amount of stain captured in
those CWs is much lesser.
• When the cells are treated with the decolorizer – the ethanol – this
causes denaturation of the proteins in the outer membrane of the
GN CWs resulting in gaping holes in these CWs that lead to the
removal of the crystal violet-iodine complexes easily, leaving these
cells unstained.
• The counterstain -safranin- thus is used to make these cells visible.
There are 4 conditions to be followed for a valid Gram
staining :
• Young cultures - must be young within 18-24hrs old
(older cultures lose their Gram staining properties
due to changes in the CWs as the cells get older)
•Thin smear- thicker or uneven smears will result in uneven
staining and decolorization
•Fresh reagents - of proper strength
•Control cultures - for a known GP bacterium and GN culture
(S.aureus & E.coli)
History
Hans Christian Gram (born in 1853) studied botany in
Denmark
In 1883, he graduated from medical school, he settled
in Berlin.
In 1884, while examining lung tissue from patients
who had died of pneumonia, Gram had discovered
that certain stains were taken up and retained by
bacterial cells.Certain bacteria (pneumococci) retained
the color (gram-positive), while other species became
bleached or de-colorized by the alcohol (gramnegative). His initial work with this staining process
was performed on Streptococcus pneumoniae and
Klebsiella pneumoniae.
Gram did not use a counter stain in his
procedure.
the German pathologist Carl Weigert
(1845-1904, added a final step of
staining with safranin.
In 1891, Gram became a lecturer in
pharmacology
In 1900 he resigned his Chair in Pharmacology
to become Professor of Medicine.
The king of Denmark awarded him in
1912, and 1924.
He retired in 1923 and died in 1938.
Special Stains
Capsule stain
Many bacteria, including both gram-positive
and gram-negative, may be surrounded by
an outer polysaccharide-containing layer
termed the glycocalyx
When the composition of this layer is tightly bound
and remains attached to cells, it is referred to as
a capsule.
The capsule or glycocalyx is a gelatinous outer layer
that is secreted by the microbe and remains stuck
to it.
Capsules may be polysaccharide, glycoproteins or
polypeptides depending on the organism.
The capsule functions:
• It protects the cell from DRYING.
• source of NUTRITION. in times of need.
• It helps the cells stick or attach to things because of
its sticky (adhesive) nature.
• It may be TOXIC or inhibitory to a host's defense
system and so aid in the disease process.
Capsule staining methods depend upon revealing the presence
of the capsule indirectly.
There are 3 procedure to stain capsule
1. India Ink Method
2. Anthony’s method
3. Hiss’s method
Anthony’s capsule stain
Anthony’s capsule stain procedure
1.
2.
Prepare a smear from a 12- to 18-hour culture
Allow the smear to air dry. DO NOT HEAT FIX (to avoid destroying or distorting the capsule or
causing shrinkage).
3.
Cover the slide with 1% crystal violet for 2 minutes
4.
Rinse gently with a 20% solution of copper sulfate.
5.
Air dry the slide. DO NOT BLOT. (Blotting will remove the un-heat-fixed bacteria from the slide
and/or cause disruption of the capsule.)
6.
Examine the slide under an oil immersion lens
the bacterial cells and the background will be stained by the crystal violet while the unstained capsule
will appear transparent.
Mechanism of action
•
crystal violet is used as the primary stain, interacting with the
protein material in the culture broth
• 20% copper sulfate serves as the mordant stabilize the capsule
structure.
.
• the bacterial cells and the background will be stained by the crystal
violet while the unstained capsule will appear white.
Encapsulated Bacillus anthracis
using india ink method
Nonencapsulated Bacillus megaterium stained
using Anthony's capsule stain.
Encapsulated Streptococcus
lactis stained using Anthony's
capsule stain.
India ink staining
Staining procedure
1. Place a loopfull of India ink on the side of a clean
slide.
2. mix a loopful of liquid culture with the ink.
3. Place a clean cover slip over the preparation
avoiding air bubbles.
4. Examine microscopically.
demonstrate the capsule which is seen as an
unstained halo around the organisms distributed
in a black background.
Principle: India ink is not absorbed by the cells
or capsules of organisms.
When encapsulated forms are mixed -with India
ink on a glass slide, the dark background of ink
particles clearly outlines the colorless capsule
surrounding the more dense appearing,
centrally situated cell.
• The flagella stain allows observation of bacterial flagella
under the light microscope. Bacterial flagella are normally
too thin to be seen under such conditions. The flagella
stains employs a mordant to coat the flagella with stain
until they are thick enough to be seen.
• Many bacteria are motile; some accomplish this motility by
means of flagella. Flagella can vary by number and
location. Some bacteria only have one flagella; this is called
monotrichous. In most monotrichous bacteria, the flagella
is at the end of the cell; this placement is called polar. Some
bacteria have a flagella at either end of the cell; this
arrangement is called amphitrichous. Many bacteria have
multiple flagella; these may all be located in a tuft at one
end of the cell, in which case the arrangement is
lophotrichous, or they may be all over the cell, in which
case the arrangement is peritrichous.
• The flagella stain allows observation of bacterial flagella under the light
microscope. Bacterial flagella are normally too thin to be seen under such
conditions.
• The flagella stains employs a mordant to coat the flagella with stain until
they are thick enough to be seen.
• Many bacteria are motile; some accomplish this motility by means of
flagella.
• Flagella can vary by number and location.
• Some bacteria only have one flagella at the end of the cell this is called
monotrichous.
•
Some bacteria have a flagella at either end of the cell; this arrangement is
called amphitrichous.
• Many bacteria have multiple flagella; these may all be located in a tuft at
one end of the cell, in which case the arrangement is lophotrichous,
• or they may be all over the cell, in which case the arrangement is
peritrichous.
Special Stains
• Flagella stain
Differential Stains
Acid-fast stain
– Used to detect Mycobacterium species
Special Stains
• Spore stain (Schaeffer-Fulton)
Bacillus subtilis
Microscopy
• Measurement
– Microorganisms are very small
– Use metric system
– Metre (m) : standard unit
– Micrometre (m) = 1 x10-6 m
– Nanometre (nm) = 1 x10-9 m
– Angstrom (Å) = 1 x10-10 m