Exercise 1: Endospore, Flagella, and Motility Flashcards
(40 cards)
Flagella: Hangin Drop Technique
What temperature should bacterial cultures be incubated at for flagella observation?
5°C below the organism’s optimum temperature (e.g., ~25°C for mesophiles).
Flagella: Hanging drop technique
Why avoid using broth in preparing suspensions for the hanging drop technique?
Broth components may interfere with staining and motility observation.
Flagella: Hanging drop technique
What is the purpose of adding water to the corners of the cover slip in the hanging drop method?
To hold the depression slide in place and prevent the drop from spreading.
Flagella: Hanging drop technique
What magnification should be used to observe motility in the hanging drop method?
High power objective (HPO), typically 40x or 100x with oil immersion (OIO).
Flagella: Motility Band
A visible diffused growth away from the inoculation point, indicating motility.
motility band
Flagella: Motility Band
How is bacterial suspension added to motility medium in the second method? (2)
By dropping 10–20 µL of suspension into the tube or by stabbing with a needle.
Flagella: Motility Band
How long should motility medium tubes be incubated? (Include temp.)
2 days at 25°C (or 5°C below optimum growth temperature).
Flagella Staining
They act as mordants to thicken flagella and make them visible under the microscope. (2)
Solution A
–> Tannic acid (10% aq sol’n.) 18 mL
–> FeCl3.6H2O (Iron (III) Chloride Hexahydrate (6% aq sol’n) 6 mL
Solution B
–> solution A 3.5 mL
–> basic fuschin (0.5% in ethyl alcohol) 0.5 mL
–> HCl, concentrated 0.5 mL
–> Formalin 2 mL
Flagella Staining
What stain is used in Modified Bailey’s method for flagella visualization?
Ziehl’s carbol fuchsin
Flagella Staining
What is the final step before viewing a flagella stain under the microscope?
Air-dry the slide and examine under oil immersion objective (OIO).
Flagella Staining
How to prepare Ziehl’s carbol fuchsin?
solution A:
* basic fuchsin (90% dye content) 0.3 g
* ethyl alcohol (95%) 10.0 mL
solution B:
* phenol 5.0 g
* distilled water 95.0 mL
Mix solutions A and B.
Flagella: Hanging Drop Technique
What type of motility does E. coli use in a homogeneous environment?
Run-and-tumble motility
Flagella
How would you describe E. coli’s movement in a uniform environment?
Undirected and random
Flagella
What triggers a biased movement in E. coli?
The presence of a chemical gradient (e.g., attractants).
Flagella
The ability to sense and move toward or away from chemical stimuli by adjusting motility.
Chemotaxis
Flagella
How does E. coli respond to an attractant gradient?
It prolongs runs toward the attractant and reduces tumbles.
Flagella
What is the effect of chemotaxis on E. coli movement?
It leads to directed movement toward favorable environments.
Flagella: Hanging drop technique
1) _______: directional movement (usually by flagella)
2) _______: random vibration (not true motility)
1) True motility
2) Brownian movement
Why incubate bacterial cultures 5°C below optimum when studying flagella?
To slow down growth, allowing better observation of flagella formation and function.
Flagella: Motility Band
What is the function of tryptone in motility medium?
Nutrient source
Flagella: Motility Band
What does glycerol provide in the medium?
Carbon source
Flagella: Motility Band
Why is bacto agar used in small amounts?
Semi-solid support
Flagella: Motility Band
Why is EDTA included in the motility medium?
Metal ion chelator
It binds to divalent metal ions like Mg²⁺ and Ca²⁺, which are essential cofactors for many enzymes and can also stabilize cell wall and membrane structures. By chelating these ions, EDTA:
What structures do some bacteria form to survive harsh environments?
Endospores
