Exercise 8 Bacterial Motility and Flagella

EXERCISE 8 STUDY OF BACTERIAL MOTILITY

AND FLAGELLA 1 EXERCISE 8 MCB101 UV-3L

FLAGELLA • most common organelle which is responsible

for bacterial locomotion

• semi-rigid and helical

• made up of protein called flagellin

• rotates like a propeller depending on direction of motion

2 EXERCISE 8 MCB101 UV-3L

FLAGELLA Ultrastructure:

1. Filament – with rotary motors which are capable of turning clockwise or counterclockwise

2. Hook – a sharp bend which allows the flagella to point away from the cell


FLAGELLA Ultrastructure:

3. Basal body – with hollow cores where flagellar components pass through during assembly and synthesis.


FACTORS AFFECTING FLAGELLAR SYNTHESIS 1. Temperature – 5˚C lower than optimum

temperature for growth

2. pH - should be close to neutral (pH tolerated by most bacteria)

3. Presence of metallic ions – would block flagellin assembly by binding with its amino end.


4. Oxygen – accounts for active motility of aerobes and facultative anaerobes

5. Nutrients – high nutrient content reduces motility; no need to move when there is abundant nutrients.

- Glucose inhibits flagellar synthesis because it chelates poorly with metal ions at normal pH.


3 Methods used to Evaluate Bacterial Motility 1. Hanging Drop Technique

2. Observation of Motility Band

3. Flagella Staining using Bailey Method


1. Hanging Drop Technique

Purpose: Determine bacterial motility using live, unstained cells suspended in water or broth.

True Motility is different from Brownian Motion!


Motile Pseudomonas (Mag:1000x) 9 EXERCISE 8 MCB101 UV-3L

True Motility (√ result) - Unique and directional movement of

individual cells from one point to another.

Brownian Motion - aimless undirected movement

- oscillating and quivering motion


Brownian Motion


Advantage: The Hanging Drop Technique preserves

bacterial cell shape and arrangement. It also allows for longer observation since the drying-out process would occur slowly.

Disadvantage: Too risky to use with highly pathogenic

organisms.


2. Observation of Motility Band

Sulfide-Indole Motility Medium Purpose: Semi-solid medium used for the

identification of members of the family Enterobacteriaceae by detecting indole formation, sulfide production and motility.


Principle: Stabbing

Motile bacteria will migrate away from the stab line by means of their flagella causing turbidity to the medium. Non-motile organisms grow just along the stab line leaving the surrounding medium clear.


Dropping

Motile bacteria will move down from the surface of the medium and disperse throughout the media. Non-motile bacteria will clump on the surface, leaving the rest of the medium clear.


Components: • Tryptone – provides amino acids needed by

growing bacteri a

• Glycerol – accounts for hydration in cells for prolonged observation; slows down metabolism

• EDTA (Ethylenediaminetetraacetic Acid)

- chelating agent; prevents clumping of cells


• Phosphate Buffer – resist drastic pH changes

• Bacto Agar – solidifying agent


Reading the SIM Media: Stabbing

*Visible stab line, cloudy - √ motile

*No distinct stab line, cloudy - √ motile

*Visible stab line, clear media – Non-motile

Dropping

*Visible growth on surface, cloudy - √ motile

*Barely visible growth on surface, cloudy - √ motile

*Visible growth on surface, clear media – Non-motile


1 2 3 1 - Dropping√ motile 2 - Stabbing √ motile 3 - Uninoculated Control


1 2

1 - √ motile 2 - √ motile


3. Flagella Staining Using Bailey Method Purpose: To determine presence/absence and

arrangement of flagella on bacterial cells. Principle: The mordant would allow for higher

affinity of the dye to the flagella. The stain molecules will pile on the flagella, increasing its thickness, therefore, making it easier viewed under the microscope.


Components: *Tannic Acid – binds to glycoproteins

regardless of overall charge; fixating agent

*Basic Fuchsin – colors the cell and the flagella RED

* FeCl36H2O - binds with Tannic Acid to form mordant

*Formalin – fixating agent for Basic Fuchsin


*Ziehl’s Carbol Fuchsin

- Phenol + Basic Fuchsin

- commonly used for staining Mycobacteria

- high affinity for mycolic acids




ANSWERS TO

STUDY QUESTIONS


WHY DO YOU HAVE TO INCUBATE CULTURES FOR THE STUDY OF FLAGELLA AT

TEMPERATURES 5˚C LOWER THAN OPTIMUM FOR GROWTH?

*At the optimum temperature, or at temperatures above optimum, the cell might prioritize synthesis of other cellular structures. This is because the said temperature range would cause damage to flagellin synthesis. . .


. . . Also, the RNA messenger for flagellin synthesis is inactivated or not being

synthesized at this range. At 5˚C below optimum, proteins needed for the

synthesis of flagellin could be manufactured by the cell, therefore promoting production of flagella.


WHAT IS THE FUNCTION OF EDTA IN THE MOTILITY MEDIUM?

*EDTA is a chelating agent which prevents clumping of cells. Molecules of EDTA would form multiple bonds with metal ions. These

metal ions bind to flagellin, hindering flagellar synthesis. The metal ions bind to EDTA

instead of flagellin and thus, flagellar synthesis would proceed.


WHY ARE SMEARS FOR FLAGELLA NOT FIXED BY HEAT?

*Heat would coagulate and denature flagellin and could also cause the

detachment of flagella from the cell body. With this, proper observations

could not be made.


REFERENCES: http://student.ccbcmd.edu/~gkaiser/puzzles/Un

it1/Answers_flagella.html

• Lodish H, Berk A, Zipursky SL, et al. 2000. Molecular Cell Biology. 4th edition. New York: W. H. Freeman.

• http://homepages.wmich.edu/~rossbach/bios312/LabProcedures/FlagellaStainProcedure.html


http://student.ccbcmd.edu/~gkaiser/puzzles/Unit1/Answers_flagella.html

http://student.ccbcmd.edu/~gkaiser/puzzles/Unit1/Answers_flagella.html

http://www.whfreeman.com/

http://homepages.wmich.edu/~rossbach/bios312/LabProcedures/FlagellaStainProcedure.html




• http://www.microbelibrary.org/library/

• DIFCO Manual of Microbiological Culture Media 11th Edition


http://www.microbelibrary.org/library/


Date post:	08-Oct-2014
Category:	Documents
Upload:	jonah-feliza-liz-mora
View:	475 times
Download:	1 times

Exercise 8 Bacterial Motility and Flagella

Documents