EXERCISE 8

STUDY OF BACTERIAL MOTILITY AND FLAGELLA EXERCISE 8 MCB101 UV-3L

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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

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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

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

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FACTORS AFFECTING FLAGELLAR SYNTHESIS 1. Temperature 5C 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.
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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.
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3 Methods used to Evaluate Bacterial Motility 1. Hanging Drop Technique 2. Observation of Motility Band 3. Flagella Staining using Bailey Method

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1. Hanging Drop Technique Purpose: Determine bacterial motility using live, unstained cells suspended in water or broth.
True Motility is different from Brownian Motion!

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Motile Pseudomonas (Mag:1000x) 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

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Brownian Motion

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Advantage: The Hanging Drop Technique preserves bacterial cell shape and arrangement. It also allows for longer observation since the dryingout process would occur slowly.

Disadvantage: Too risky to use with highly pathogenic organisms.

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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.

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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.

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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.

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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

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 Phosphate Buffer resist drastic pH changes Bacto Agar solidifying agent

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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

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

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1 - motile 2 - motile

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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.
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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

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*Ziehls Carbol Fuchsin - Phenol + Basic Fuchsin - commonly used for staining Mycobacteria - high affinity for mycolic acids

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ANSWERS TO STUDY QUESTIONS

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WHY DO YOU HAVE TO INCUBATE CULTURES FOR THE STUDY OF FLAGELLA AT TEMPERATURES 5C 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. . .

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. . . Also, the RNA messenger for flagellin synthesis is inactivated or not being synthesized at this range. At 5C below optimum, proteins needed for the synthesis of flagellin could be manufactured by the cell, therefore promoting production of flagella.
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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.

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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.
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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/bios 312/LabProcedures/FlagellaStainProcedure.ht ml
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 http://www.microbelibrary.org/library/ DIFCO Manual of Microbiological Culture Media 11th Edition

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