Mcb101 Exer 8 Postlab by Rizon, Kristina
-
Upload
kriztina-rizon -
Category
Documents
-
view
111 -
download
8
description
Transcript of 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
you can get this at:www.scribd.com/krizon