Week 2, Bacteria and fungi, form and function

Question 1

Where might you find bacteria that form a glycocalyx?

Answer 1

→ On surfaces. The capsules help them stick to surfaces.

Question 2

List some properties of lactic acid bacteria (LAB)

Answer 2

→ Aerotolerant anaerobes
→ Gram positive
→ Usually non-motile
→ Non-endospore forming
→ Many produce glycocalyx
→ Produce lactic acid as a major or sole produce of fermentative metabolism
→ Complex nutritional requirements

Question 3

How does the gram stain work?

Answer 3

→ Gram positive bacteria have thick cell walls that have a high peptidoglycan content (~90%) and small pores, meaning that they retain the crystal violet and iodine complex when they are subsequently exposed to alcohol, which shrinks their pores further.
→ Gram negative bacteria have a thin peptidoglycan layer with large pores, which allows the crystal violet-iodine complex to be rinsed out during the decolourisation step (rinsing with alcohol).

Question 4

What shape are lactobacillus?

Answer 4

Rod-shaped, short and bent to long and slender.

Question 5

Other than fermentation products, how are lactic acid bacteria distinguished?

Answer 5

→ Morphology

Question 6

T/F: Bacteria with flagella move by moving their flagella back and forth like a shark swims through the water.

Answer 6

False. The basal body (the base) of the flagella rotates like a propeller.

Question 7

Describe the gram staining process

Answer 7

1. Cells from a fresh, pure culture (< 24 hrs old) are transferred (smeared on) to a glass slide and allowed to dry.
2. Culture slides are then gently heated to fix the bacteria in place.
3. STAIN with CRYSTAL VIOLET for 30-60 seconds
4. Rinse the slide gently with RO water
5. STAIN with IODINE (a mordant (fixes the stain) for at least 1 minute
6. Rinse the slide gently with RO water
7. Rinse with alcohol until it just begins to run clear
8. Rinse with RO water
9. COUNTERSTAIN with SAFRANIN for 30-60 secs
10. Rinse with RO water
11. Blot dry
12. Examine
    * ** Gram positive bacteria will appear purple and gram negative bacteria will appear red.

Question 8

Explain how you would estimate the number of viable bacterial cells in a culture.

Answer 8

1. Perform a dilution series of the culture
2. 1. Put 1ml of original culture into 9ml of sterile distilled water and vortex (= 10⁻¹ dilution factor)
3. 2. Put 1ml of the 10⁻¹ dilution into another 9ml of distilled water and vortex (= 10⁻² dilution factor)
4. 3. Repeat step 1.2 until a 10⁻⁵ dilution factor is achieved
5. Use the spread-plate technique to spread 0.1 ml (100 µl) of each dilution, starting from the 10⁻⁵ dilution (down to 10⁻²), on to agar plates
   NOTE: The dilution factor becomes one order of magnitude higher; e.g. the 10⁻⁵ dilution becomes 10⁻⁶ once its plated on agar.
   NOTE: The same pipette tip can be used so long as you go from the most diluted culture to the least diluted, but the glass spreader should be sterilised between each plate.
6. Incubate plates (inverted) for 1 week at 25 °C
7. Count the (separate) colonies on each plate that contains between 30 and 300 colonies. Don’t count colonies outside of this range.
8. Calculate the number of CFU by multiplying the number of colonies on the plate by the (positive) dilution factor; e.g. 54 colonies observed * 10⁴ dilution factor = 540,000 CFU/ml = 5.4*10⁵ CFU/ml. Make sure to provide the answer using scientific notation.

Question 9

Where might you find bacteria that produce endospores?

Answer 9

→ In an environment where there its lots of competition; e.g. soil.

Question 10

Where might you find bacteria with flagella?

Answer 10

→ Where there’s free water/moisture

Question 11

What is the main difference between gram positive and gram negative bacteria?

Answer 11

GRAM POSITIVE:
→ Cell wall consists of ~90% peptidoglycan
→ Simple (membrane, then peptidoglycan)
→ Retain the violet dye

GRAM NEGATIVE
→ Cell wall consists of ~10% peptidoglycan
→ Has outer membrane
→ More complex (membrane, periplasmic space, peptidoglycan, periplasmic space, outer membrane)
→ Don’t retain the violet dye
→ Can be stained another colour (usually red)

Question 12

List some MICROscopic morphological features that should be recorded when studying bacteria.

Answer 12

→ Size
→ Shape
→ Arrangement
→ Motility
→ Number and position of flagella
→ Presence or absence of endospores

Question 13

Where might you find bacteria that form gas vesicles?

Answer 13

→ In water bodies where buoyancy is beneficial.

Question 14

MICROscopic morphology should be studied on…

Answer 14

Pure cultures

Question 15

Why must cultures be less than 24 hours old if they are going to be gram stained?

Answer 15

The old gram positive cells may lose their ability to retain the violet dye.

Question 16

What are the purposes of a glycocalyx (bacterial capsule) and slime?

Answer 16

→ Attachment to surfaces
→ Biofilm formation
→ Resistance (to being engulfed, desiccation, etc)
→ Pathegonicity

Question 17

What is another method (other that gram-staining) for grouping bacteria, and why could it be preferential over categorising by gram-stain?

Answer 17

Based on the number of membranes.
→ Monoderm = plasma membrane online
→ Diderm = plasma membrane + outer membrane
Some bacteria that stain gram positive are actually diderms, and some bacteria that stain gram negative are actually monoderms.

Question 18

What are bacterial endospores?

Answer 18

→ Non-sexual survival structures (not spores) formed inside a parent cell
→ Occur in bacteria within the Firmicutes phylum
→ Have a thick cell wall to protect them
→ Very resistant/tough/long-living
→ Basically a bacteria’s ultimate survival strategy

Question 19

There are two groups of lactic acid bacteria (LAB): homofermentative and heterofermentative. How are they distinguished?

Answer 19

By their fermentation products, particularly gas production.
→ Homofermentative LAB produce only lactic acid and ATP
→ Heterofermentative LAB produce lactic acid, CO2, and other products.

Question 20

Describe what fimbriae and pili are and what they’re used for.

Answer 20

→ Rigid cylindrical protein filaments
→ Shorter and more numerous than flagella
Used for:
→ Attachment
→ Sex
→ Pathegenocity
→ Twitching motility
→ Barrier to toxic substrates

Question 21

List some MACROscopic morphological features that should be recorded when studying bacteria.

Answer 21

→ Colour
→ Form
→ Edge
→ Elevation
→ Surface (smooth or rough)
→ Opacity (clear or opaque)