Mcb101 Exer 8 Postlab by Rizon, Kristina

Prepared by : Kristina P. Rizon MCB 101 F1L

1st sem, A.Y. 2011-2012

Study of Bacterial MotilityAnd Flagella

Exer 8

long, thread-like appendage Provide motility Composed of proteinic

subunits flagellin Rotation is driven by a proton

motive force (flow of protons, ie: H+ ions)

FLAGELLA

Factors affecting Flagellar Synthesis

Temperature 5 C lower than opt. temp for growth

pH pH opt. for growth of flagella

Presence of Ions metallic ions (Fe3+) binds w/ amino

end of flagellin, blocking its assembly

Factors affecting Flagellar Synthesis

Oxygen needed for active movement of

aerobes and facultative anaerobes Nutrients

high nutrient reduces motility glucose inhibits flagellar synthesis

Bacterial Motility wasevaluated using 3 methods

Advantages: simple method to test motility preserves cell shape & arrangement allows for longer observation of

living organisms. doesn’t involve vigorous physical &

chemical treatment

Hanging Drop Technique1st method:

Disadvantages: far too risky to use with highly

pathogenic organisms may not be able to differentiate if

organism exhibits brownian movement only or true motility

Hanging Drop Technique1st method:

Possible Results Brownian Movement

aimless, undirected movement caused by bombardment of molecules

oscillating & quivering movements True Motility (+ result)

cell swims and progresses from one point to another

brought about by organelles of locomotion

Possible Results Non-motile

does not move at all/stationarymoves a little forward & back but will

not change its position in relation to neighboring cells

but sometimes, we cannotreally see the actual cells moving

because they are transparent SO….

a semi-solid agar/medium that detects indole formation, sulfide production & motility

contains casein peptone which contains amino acids including tryptophan

Use of Sulfide Indole Medium2nd method:

Requirements: must be semi-solid to permit

bacteria to move away from line of inoculation

must be able to support growth should have low concentration of

metabolizable substrates

Use of Sulfide Indole Medium2nd method:

Components: Tryptone

amino acid source for growing bacteria Glycerol

inc. hydration w/in cells for prolonged observation; to slow down metabolic activity

Use of Sulfide Indole Medium2nd method:

Components: EDTA (ethylenediaminetetracetic acid)

chelating agent that prevents cell clumping Phosphate buffer

helps maintain constant pH bacto agar

solidifying agent, reduces extraneous matter & salts

Use of Sulfide Indole Medium2nd method:

Advantages: semi-solid nature makes motility

possible useful for det. motility of pathogenic

bacteria culture of any age can be used for

inoculation as long as pure & viable

Use of Sulfide Indole Medium2nd method:

Disadvantage: takes longer to provide results than

the wet mount or hanging drop technique

Use of Sulfide Indole Medium2nd method:

Positive Results SIM (by dropping)

blackening may be observed if bacteria can reduce S to ferrous sulfide

turbidity (indicates growth) away from point of inoculation

+ -

Positive Results SIM (by stabbing)

bacteria grows around the stab line and radiate in all directions

will be able to penetrate the medium

but then again, there are reallyinstances when both HDT & using

SIM doesn’t work…

recommended for bacteria on w/c flagella are difficult to stain e.g. slime prod’n, fine flagella, flagella already lost

has been found to give the most uniformly satisfactory results in experienced hands

The Bailey Method3rd method:

Dyes & reagents used: Tannic acid

binds to glycoproteins regardless of their charge, fixating agent

Basic Fuchsin mixture of rosaniline, pararosaniline,

new fuchsine & magenta II colors the cell & flagella red

The Bailey Method3rd method:

Dyes & reagents used: FeCl36H20

acts w/ tannic acid to produce a mordant

Formalin fixating agent for Basic Fuchsin

The Bailey Method3rd method:

Dyes & reagents used: Concentrated HCl

functions for hydrolysis prevent tannin-iron reaction that can

color the cell with dark gray instead of red

The Bailey Method3rd method:

Dyes & reagents used: Ziehl’s Carbol Fuchsin

mixture of phenol and basic fuchsin commonly used in staining mycobacteria has affinity for mycolic acids found in cell

walls Carbol fuchsin used as topical antiseptic

The Bailey Method3rd method:

Proteus vulgarisflagella & cells appear red,

background ppts finer & less conspicuous

Why do you have to incubate cultures for the study of flagella at temperatures 5 C lower than optimum growth?

Answers to Questions:

This allows the formationof flagella.

The Principle behind it.

due to a lesion in the flagellin-synthesizing process

temperature has a dramatic effect on flagellation.

active messenger RNA for flagellin is not being synthesized at a temperature above the optimal growth temperature

What is the function of EDTA in the motility medium?

Answers to Questions:

It is a chelating agent that prevents cell clumping.

The Principle behind it. EDTA is a substance whose molecules

can form several bonds to a single metal ion

after being bound by EDTA, metal ions remain in solution but exhibit diminished reactivity

thus, the metallic ions will not anymore bind with the flagellin, resulting to flagellar synthesis

Why are smears for flagella staining not fixed by heat?

Answers to Questions:

It can damage the bacterial flagella.

The Principle behind it.

heat denatures proteins

this may cause the detachment of flagella from it’s basal body

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