Lecture 1

Question 1

Give some examples of where bacteria can be found

Answer 1

Air
Water
Soil
Deep subsurface rocks
Acidic, alkaline, very hot & cold and salty environments
Internal & external surfaces of the body

Question 2

What are the basic steps in bacterial identification?

Answer 2

Using light microscopy:

1. Size and shape
2. Cell arrangement
3. Motility (flagella)
4. Spores- one per cell
5. Capsules

Question 3

Describe the different size and shapes of bacteria and which genus each one relates to.

Answer 3

1. Coccus e.g. Staphylococcus aureus, Streptococcus pneumoniae. Size: 0.5-1.0microns in diameter
2. Rod (also vibrios) e.g. Escherichia coli, Bacillus cereus, Vibrio cholerae. Size: < 8.0 x 0.5 microns
3. Spiral- rigid helix e.g. Spirillum spp. Found in water cooling towers, stagnant water
4. Spirochetes- flexible, helical & very long e.g. Treponema pallidum (syphilis); Borrellia burgdorferi (Lyme’s disease)
5. Square-flat, rectangular, straight edges e.g. genus Arcula (box) Haloquadratum walsbyi. Isolated from brine colected from the shore of the Red Sea. Only grows in 20% (3.3M) salt solution. Gram -ve.
6. Star shaped- genus Stella. Isolated from garden compost, House manure in Moscow region. Gram -ve non-motile
7. Absolutely huge- Epulopiscium fishelsoni (0.6mm long x 75um wide) Lives in gut of surgeon fish. Gram +ve symbiant. Thiomargarita namibiensis bacteria (0.75mm long)

Question 4

What is the Gram stain, and what is the difference between Gram +ve and Gram -ve?

Answer 4

• Developed in 1884 by Christian Gram
• Differentiates bacteria on the basis of cell wall structure into Gram +ve and Gram -ve.
• Gram +ve is purple, and has a thick layer of peptidoglycan (20nm).
• Pencillin can work on Gram +ve but not Gram -ve
• the cell wall of Gram +ve is split into 13/14 layers of peptidoglycan & teichoic acid, and a cytoplasmic membrane.
• Gram -ve is pink/red and has a thin layer of peptidoglycan (3nm). The cell membrane of the Gram -ve bacteria is composed of an outer membrane, peptidoglycan (5-8nm) and a cytoplasmic membrane.

Question 5

How do you prepare a Gram stain?

Answer 5

1. Prepare the cells on slide
2. Stain with crystal violet
3. Stain with iodine (CVI complex forms)
4. Acetone (dissolves outer membrane)
5. Counter stain with safranin
6. Gram -ves colourless–> pink/red
7. Gram +ves purple –> purple

Question 6

Why does the Gram stain differentiate between Gram +ve and Gram -ve cells?

Answer 6

crystal violet> iodine > safranin

• Dependent on cell wall chemistry
• The CVI complex is trapped in Gram +ve bacteria. Acetone causes dehydration of peptidocylcan sealing the CVI complex into the cell.
• In Gram -ve bacteria acetone dissolves the outer membrane, and eventually permeates the inner membrane, releasing the CVI complex

Question 7

What are the biological functions of peptidogylcan?

Answer 7

1. Rigid structure of wall- gives cell shape
2. Protects from osmotic lysis
3. Withstands a high internal osmotic pressure:
   Gram +ve -high internal pressure (thick walls)
   Gram -ve -low internal pressure (thinner walls)

Question 8

What is different about pleomorphic cells?

Answer 8

• of Mycoplasma pneumoniae
• have no peptidogycan and are surrounded by a membrane.
• lost ability to make mRNA

Question 9

What percentage of the Gram +ve cell wall is peptidoglycan?

Answer 9

90%