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Mcb101 Exer 8 Postlab by Rizon, Kristina

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MCB 101 F-1L Exer 8 postlab (1st sem, AY '11-'12)
33
Prepared by : Kristina P. Rizon MCB 101 F1L dy of Bacterial Motil And Flagella Exer 8
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Page 1: 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

Page 2: Mcb101 Exer 8 Postlab by Rizon, Kristina

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

Page 3: Mcb101 Exer 8 Postlab by Rizon, Kristina

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

Page 4: Mcb101 Exer 8 Postlab by Rizon, Kristina

Factors affecting Flagellar Synthesis

Oxygen needed for active movement of

aerobes and facultative anaerobes Nutrients

high nutrient reduces motility glucose inhibits flagellar synthesis

Page 5: Mcb101 Exer 8 Postlab by Rizon, Kristina

Bacterial Motility wasevaluated using 3 methods

Page 6: Mcb101 Exer 8 Postlab by Rizon, Kristina

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:

Page 7: Mcb101 Exer 8 Postlab by Rizon, Kristina

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:

Page 8: Mcb101 Exer 8 Postlab by Rizon, Kristina

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

Page 9: Mcb101 Exer 8 Postlab by Rizon, Kristina

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

Page 10: Mcb101 Exer 8 Postlab by Rizon, Kristina

but sometimes, we cannotreally see the actual cells moving

because they are transparent SO….

Page 11: Mcb101 Exer 8 Postlab by Rizon, Kristina

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:

Page 12: Mcb101 Exer 8 Postlab by Rizon, Kristina

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:

Page 13: Mcb101 Exer 8 Postlab by Rizon, Kristina

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:

Page 14: Mcb101 Exer 8 Postlab by Rizon, Kristina

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:

Page 15: Mcb101 Exer 8 Postlab by Rizon, Kristina

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:

Page 16: Mcb101 Exer 8 Postlab by Rizon, Kristina

Disadvantage: takes longer to provide results than

the wet mount or hanging drop technique

Use of Sulfide Indole Medium2nd method:

Page 17: Mcb101 Exer 8 Postlab by Rizon, Kristina

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

Page 18: Mcb101 Exer 8 Postlab by Rizon, Kristina

+ -

Page 19: Mcb101 Exer 8 Postlab by Rizon, Kristina

Positive Results SIM (by stabbing)

bacteria grows around the stab line and radiate in all directions

will be able to penetrate the medium

Page 20: Mcb101 Exer 8 Postlab by Rizon, Kristina

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

SIM doesn’t work…

Page 21: Mcb101 Exer 8 Postlab by Rizon, Kristina

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:

Page 22: Mcb101 Exer 8 Postlab by Rizon, Kristina

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:

Page 23: Mcb101 Exer 8 Postlab by Rizon, Kristina

Dyes & reagents used: FeCl36H20

acts w/ tannic acid to produce a mordant

Formalin fixating agent for Basic Fuchsin

The Bailey Method3rd method:

Page 24: Mcb101 Exer 8 Postlab by Rizon, Kristina

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:

Page 25: Mcb101 Exer 8 Postlab by Rizon, Kristina

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:

Page 26: Mcb101 Exer 8 Postlab by Rizon, Kristina

Proteus vulgarisflagella & cells appear red,

background ppts finer & less conspicuous

Page 27: Mcb101 Exer 8 Postlab by Rizon, Kristina

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.

Page 28: Mcb101 Exer 8 Postlab by Rizon, Kristina

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

Page 29: Mcb101 Exer 8 Postlab by Rizon, Kristina

What is the function of EDTA in the motility medium?

Answers to Questions:

It is a chelating agent that prevents cell clumping.

Page 30: Mcb101 Exer 8 Postlab by Rizon, Kristina

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

Page 31: Mcb101 Exer 8 Postlab by Rizon, Kristina

Why are smears for flagella staining not fixed by heat?

Answers to Questions:

It can damage the bacterial flagella.

Page 32: Mcb101 Exer 8 Postlab by Rizon, Kristina

The Principle behind it.

heat denatures proteins

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

Page 33: Mcb101 Exer 8 Postlab by Rizon, Kristina

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