2. pracical. motility, gram staining

MicroBiology . Practicals on motility and gram Staining .

1. Bacteriology: 2. Practical Session Bacteriology: 2. Practical Session
1.
Motility Test
1.
Motility Test
2.Gram Staining
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2. Flagellum Flagellum --Flagella Flagella
•Flagella give bacteria the ability to move
about
actively
•Made up of a class of linear proteins: flagellins
•Attached to the cell through a complex of
proteins (
hook
and basal
body)
proteins (
hook
and basal
body)
•Cause the cell to move by their rotation
, which is
powered by the proton motive force
2

3. In many bacteria, flagella are responsible for
motility in liquid
:
•Loose correlation between cell shape
and the
presence of flagella:
oalmost all spirilla,
ohalf of all rod-shaped
bacteria, and
oonly a few of the cocciare motile by flagella
(in fact, most cocciare non-motile)
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4. Flagella can be thought of as little semi-rigid
propellers
that are free at one end and attached to
a cell at the other
Due to their small diameter, (about 20 nm), flagell a
cannot be seen in the light microscope
unless a
special stain is applied
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5. Relevance: Example Relevance: Example Escherichia coli Escherichia coli
•When E coliis swimming through a solution, the
flagella turn counter-clockwise and push
the
microbe through solution. This behavior is termed
smooth swimming;
•
It
is possible for
E. coli
to also
reverse
the
•
It
is possible for
E. coli
to also
reverse
the
direction of flagellarrotation, and when the
flagella turn clockwise, they pull
against the
bacterial cell (tumbling)
••Uropathogenic UropathogenicE coli E coli swim upstream to reach the swim upstream to reach the
bladder ! bladder !
5

6. E coli
: the
direction of
flagellar
rotation determines
E coli
: the
direction of
flagellar
rotation determines
straight swimming or tumbling motion;
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10. Gram Staining Gram Staining
•Gram staining, also called Gram's method, is a
method of differentiating bacterial species
into two large groups (Gram + and Gram -)
•
Inventor: the Danish microbiologist Hans
-
•
Inventor: the Danish microbiologist Hans
-
Christian Gram (begin of 20
th
century)
•The Gram stain is almost always the first step
in the identification of a bacterial organism
10

11. Gram staining Gram staining
differentiates bacteria by the chemical and
physical properties of their cell walls
by
1detecting peptidoglycan, which is present in a
thicklayer in gram-positive bacteria:
o
Gram
-
positive positive
bacteria
retain
the crystal
-
violet
o
Gram
-
positive positive
bacteria
retain
the crystal
-
violet
dye,
owhile a counterstain
(commonly safraninor
fuchsin) added after the crystal violet
ogives all Gram-negative negative
bacteria a red or pink
coloring (= decolorize)
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13. Four basic steps of the Gram stain Four basic steps of the Gram stain
1. Applying a primary stain (crystal violet)
2. Addition of iodine, which binds to crystal
violet and traps it in the cell (mordant)
3. Rapid
decolorization
with
ethanol
or
3. Rapid
decolorization
with
ethanol
or
acetone
4. Counterstaining with safranin(carbol
fuchsin)
1in between: rinsing gently with water !
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15. 1Thedecolorization
stepis
critical critical
andmustbe
timedcorrectly;thecrystalvioletstainis
removedfromboth
gram-positiveandnegative
cellsifthedecolorizingagentisleftontoolong
(amatterofseconds)
•Counterstain,whichisusuallypositively
charged
safraninorbasicfuchsin,isappliedlasttogive
decolorizedGram-negativebacteriaapinkorred
color
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