Techniques

Question 1

What is the difference between true motility and Brownian motion?

Answer 1

Brownian motion

• is caused by water molecules colliding with the organism and moving it around in an irregular jerky pattern.
• Organisms will appear to vibrate in place.

True motility

• cells will exhibit independent movement in some consistent direction over greater distances.

Question 2

(6 pts) You are given a culture of K. pneumoniae and asked make a 1:15 dilution to make a total volume of 40 mLs of culture. What volumes of culture and broth will you use?

_____________ K. pneumoniae culture

_____________ broth

Answer 2

2. 5 mL K. pneumoniae culture
3. 5 mL broth

Question 3

You gram stain a broth culture of E. coli that has been growing for 36 hours. As you will learn soon, by now the E. coli has been fermenting for a while, and produced a number of small organic acids. Give TWO reasons that this culture may give an erroneous or variable Gram stain result.

Reason 1:

Reason 2:

Answer 3

1. Reason 1:
   
   • Cells have grown for too long and are at the death phase. At the death phase, there would probably be a lack a nutrients and maybe a lower that optimal pH. Autolysis occurs and this may cause erroneous results.
2. Reason 2:
   
   1. Production of acidic components due to fermentation. The acids may react with the positive CV dye and give a false positive for gram (+) bacteria since the dye is binding to something other than the peptidoglycan wall.

Question 4

What happens during the ethanol wash in gram staining?

Answer 4

• Dehydration of the peptidoglycan wall and removal of the outer membrane in gram (-) bacteria.
• CV that is bound to the outer membrane in gram (-) is thus lost.
• Since the peptidoglycan wall is dehydrated it “tightens” and the prior use of a mordant makes a large CV-Iodine complex that cannot “leave” through the tightened peptidoglycan layer.