Required Practicals 1 and 2

Question 1

What does PBS stand for?

Answer 1

phosphate-buffered saline

Question 2

Why is the lid loosened on the E.coli broth (1/2 - 3/4 of a turn) ?

Answer 2

In the autoclave, the contents will expand causing a pressure to build and the container to shatter (explosion).

Question 3

Why do we use aseptic technique when innoculating sterile growth media?

Answer 3

To prevent contamination of cultures with other organisms.
To prevent contamination of workers and the environment.

Question 4

When using Gilson pipettes, why must the tips be sterlilsed before use?

Answer 4

To prevent contamination with unknown and unwanted organisms which may cause disease.
To prevent contamination with unknown and unwanted organisms that may effect the outcome of the experiment.

Question 5

How should you label your petri dish for incubation?

Answer 5

Your name
Bench number
Date (to know how long organism has been growing)
Organism name (to identify microbe for safety and ensure correct disposal)
On the base (in case the lid gets detached/mixed up)

Question 6

Why is a well defined lab strain of E.coli used?

Answer 6

This strain has a low disease potential and is safe to be handled in this manner in this laboratory.

Question 7

Why must vessels be open for the minimum amount of time possible?

Answer 7

To prevent any microorganisms other than E.coli entering and contaminating the vessel.

Question 8

How should you work when vessels are open? Why?

Answer 8

Close to a Bunsen burner blue flame because air currents are drawn upwards and microbes away from the vessel.

Question 9

Why must you flame the inoculation loop?

Answer 9

To kill microorganisms.

Question 10

Why must the loop have plenty of time to cool?

Answer 10

So E.coli isn’t killed.

Question 11

You are provided with two 9ml bottles of PBS and a bottle of E.coli which is diluted to concentration 10^-5.
Describe how to make solutions diluted to 10^-6 and 10^-7.

Answer 11

Label bottles 10^-6 and 10^-7.
Using a Gilson P1000 pipette and blue sterile tips, dispense 1ml into 10^-6.
Mix gently with pipette.
Using a fresh blue sterile tip, transfer 1ml of 10^-6 into 10^-7.
Mix gently with pipette.

Question 12

How should you spread out 100 microlitres onto nutrient agar?

Answer 12

Starting at the side of the solution spread 6 times horizontally, then 6 times vertically after rotating the plate, then 6 times anticlockwise and 6 times clockwise.

Question 13

Which agar was used for the spread plate, why?

Answer 13

Nutrient agar - general purpose medium for growing various microorganisms.

Question 14

Which agar was used for the streak plate, why?

Answer 14

MacConkey agar - differentiates between gram-negative and gram-positive bacteria.
Gram-negative bacteria (E.coli) grows well whereas gram-positive bacteria are inhibited from growing.

Different cell wall structure affects how bacteria interact with antibiotics and immune system. Gram-negative = thinner cell wall (but also have lipopolysaccharide outer membrane).

Question 15

Why shouldn’t spread plates be inverted immediately?

Answer 15

To allow the bacterial suspension to adsorb (adhere to surface) onto agar.

Question 16

Why should the agar plate be inverted?

Answer 16

To ensure any condensation doesn’t drip onto the agar surface and run the separation of individual colonies/cause the agar to become too wet.

Question 17

Why should you flame your inoculating loop at an angle?

Answer 17

To flame up the loop and sterlilse it all.

Question 18

Why should you move your inoculating loop up the flame?

Answer 18

To draw the bacteria into the flame.
(Blue flame = hotter).

Question 19

How do you prepare a streak plate?

Answer 19

Flame and cool inoculating loop before dipping into a single E.coli colony (scoop up gently).
Zig-zag over a small area at the side of the plate.
Flame and cool loop.
Draw 5 lines across top of plate.
Flame and cool loop.
Rotate plate and draw 5 more lines.
Flame and cool loop.
Rotate plate and draw 5 lines.
Flame and cool loop.
Draw a zig-zag stroke into the centre of the plate.
Invert agar plate.

Question 20

Why is the final zig-zag into the centre of the plate done?

Answer 20

So separate colonies form.

Question 21

Give the CFU/ml^-1 equation

Answer 21

(counts x dilution factor) / volume plated (ml)

Question 22

Calculate the number of viable bacteria cells per milliliter
when grown on a petri plate using the information given:
91 colonies
10^-5 dilution
100 microlitres plated

Answer 22

(91 x 10^5) / 0.1 = 91, 000, 000

Question 23

What should you consider when evaluating the effectiveness of a streak plate?

Answer 23

Do you have bacterial cultures on agar?
Yes - you cultured viable cells
No - inoculating loop was too hot so killed microorganisms
Do you have heavy confluent growth where first zig-zag was applied?
Do you have discrete single colonies where last zig-zag was applied?

Do you have uniform colony morphology?
Yes - cultured a pure strain (some colonies will appear larger if two have merged)
No - culture is contaminated/genetic variants are growing

Question 24

Before preparing a microscope slide of yeast culture Saccharomyces why should you thoroughly agitate the culture?

Answer 24

To ensure an even suspension of cells.

Question 25

For enumeration which plate is considered? Why?

Answer 25

The plate with 30-300 CFUs because this number is high enough to have statistical accuracy and low enough to avoid mistakes due to overlapping colonies.

Question 26

Why is oil immersion used instead of bright field illumination and phase contrast?

Answer 26

Reduces refraction and increases the amount of light reaching the objective lens.

Refraction = bending of light as it goes through material.

Question 27

Name as many parts of the Zeiss Primo Star microscope as you can.

Answer 27

Power switch
Stage
Coarse focussing knob
Fine focussing knob
Stage
Stage controls
Intraocular lens
Field diaphragm
Condenser knob (focuses edges of diaphragm)
Condenser centering screws
Levers (Ph or magnification) - for condenser diaphragm
Phase slider
Objective lenses

Question 28

What is the magnification of the eyepiece?

Answer 28

10x

Question 29

What decreases as magnification increases?

Answer 29

Depth of focus (may be able to see 3 stacked objects clearly on 100x but not at 400x

Question 30

If there are 3 stacked objects on a slide and you lower the stage (anticlockwise on focussing knobs), what object are you bringing into focus?

Answer 30

The one nearest to the top.