Bacteriology: 2. Practical SessionBacteriology: 2. Practical Session

1. Motility Test1. Motility Test

2. Gram Staining

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Flagellum Flagellum -- FlagellaFlagella

• 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

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In many bacteria, flagella are responsible for

motility in liquid :

• Loose correlation between cell shape and the

presence of flagella:

o almost all spirilla,

o half of all rod-shaped bacteria, and

o only a few of the cocci are motile by flagella

(in fact, most cocci are non-motile)

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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), flagella

cannot be seen in the light microscope unless a

special stain is applied 4

Relevance: Example Relevance: Example Escherichia coliEscherichia coli

• When E coli is 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 flagellar rotation, and when the

flagella turn clockwise, they pull against the

bacterial cell (tumbling)

•• UropathogenicUropathogenic E coli E coli swim upstream to reach the swim upstream to reach the

bladder !bladder !5

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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Gram StainingGram 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 20th century)

• The Gram stain is almost always the first step

in the identification of a bacterial organism

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Gram stainingGram 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:

o Gram-positivepositive bacteria retain the crystal-violet o Gram-positivepositive bacteria retain the crystal-violet dye,

o while a counterstain (commonly safranin or fuchsin) added after the crystal violet

o gives all Gram-negativenegative bacteria a red or pink coloring (= decolorize)

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Four basic steps of the Gram stainFour 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)

� in between: rinsing gently with water !13

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� The decolorization step is criticalcritical and must be

timed correctly; the crystal violet stain is

removed from both gram-positive and negative

cells if the decolorizing agent is left on too long

(a matter of seconds)

• Counterstain, which is usually positively charged

safranin or basic fuchsin, is applied last to give

decolorized Gram-negative bacteria a pink or red

color

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