Quiz 1

Question 1

What is Aseptic Technique? and why does it matter ?

Answer 1

• Aseptic technique to obtain and maintain pure cultures
• It matters because it reduces contamination and helps us to only work with the organisms we actually want to work with

Question 2

Pure culture

Answer 2

Contains only 1 species of microorganisms

Question 3

Mixed culture

Answer 3

Contains more than 1 species of microorganisms

Question 4

Contamination

Answer 4

Introduction of undesirable microorganisms into the culture

Question 5

What is the purpose of flaming loops and lips of tubes

Answer 5

To reduce contamination

Question 6

What are main techniques needed to properly transfer cultures from a tube?

Answer 6

Aseptic Technique

Question 7

Sterilization

Answer 7

Destroying/removal of all living organisms

Question 8

What types of sterilization are there? What methods are used to clean laboratory
glassware discarded microbial cultures, and disposable plasticware encountered in a
laboratory environment?

Answer 8

• Types of sterilization - Direct Flame, Dry heat, Autoclaving, Filtration, Chemicals
• Autoclaving is used to clean laboratory glassware and disposable plasticware

Question 9

What is autoclaving and how does it work?

Answer 9

• Autoclaving is sterilizing using heat and pressure to that material is sterilized.

Question 10

How does a light microscope work?

Answer 10

Light Microscope use photons of visible light to form images

Question 11

What is the purpose of immersion oil?

Answer 11

Immersion oil can increase refractive index; loss of light due to refraction is eliminated.

Question 12

What is the appropriate method for focusing a microscope?

Answer 12

• using a 10x objective, slowly bring the stage up with the course knob
• when sample is visible, switch to the 40x objective and continue focusing
• before switching to the 100x objective, add a drop of oil on the slide
  use the fine adjustment knob when working with the 100x objective

Question 13

What is heat fixing and why is it used?

Answer 13

Heat Fixing kills cells prior to staining; This makes it so the dye can penetrate and fixing the cells to the slide

Question 14

Why are bacteria able to be stained?

Answer 14

Microbial Cells have a Negative charge on their surface (Surface charge) and positively charged (Basic) dye is attracted to it and binds

Question 15

What is the purpose of staining bacteria with simple and differential stains?

Answer 15

Simple Stain - Colors the cell so that you can see if cell is straight, curved, rod, or sphere and are paired in pairs or clusters

Differential Stain - Differentiate between different microorganisms

Question 16

What is a negative stain?

Answer 16

appear pink in color; use acidic dyes; do not have a cell wall

Question 17

What morphologies can microbes have?

Answer 17

the shape of the microbes can be rods, cocci, spirals, or a combination; the size of the microbes can also vary from .2 micrometers in diameter 100 micrometers.

Question 18

Why and how are bacterial smears prepared?

Answer 18

Smears help to fix microbes to the cell for tests such as gram staining. This is done by:
1. ensuring you are using a clean slide
2. label the slide (the underside)
3. inoculate the slide
4. let it sit on hot plate until liquid is no longer present
5. heat fix the cell by passing it through the flame
6. repeat this 3x if it is from a broth; Do not repeat if it is from a slant

Question 19

What is media and why are there different kinds?

Answer 19

-It is used as a nutrient and mineral source for the bacteria to grow;

• Varying types of media are used depending on the organism being tested and what types of nutrients they need for growth and survival

Question 20

What are fastidious organisms?

Answer 20

Organisms that require more growth factors

Question 21

What is Trypticase Soy Broth/Agar?

Answer 21

A complex medium that is great for growing a variety of microbes used in microbiology Lab

Question 22

What is complex media vs. defined media?

Answer 22

Complex Media - rich in type and concentration of nutrients available

Defined - composed of pure cultures in measured concentrations

Question 23

How is solid media prepared and what makes it solid?

Answer 23

composed of galactose and galacturonic acid; agar swells in cold water but as it gets close to boiling it becomes transparent (sol). once the agar is allowed to cool back down to room temp the gel becomes firmer and more opaque

Question 24

What reagents are used in Gram staining? How do they interact with the cell?

Answer 24

crystal violet - (primary stain),
gram’s iodine - (mordant),
95%Ethanol - (decolorizer),
Safranin/ fuchsin - (counter stain)

How do they interact with the cell? -

gram-positive microbes have a thicker cell wall. This hold the crystal violet color.

Gram-negative have a thinner cell wall with an extra lipid bilayer. This does not hold the purple as well.

The ethanol will decolorize this and when restrained it will appear pink in color

Question 25

What is the purpose of Gram staining? What does it tell you about the cell?

Answer 25

Purpose of Gram Staining - helps to identify prokaryotic cells

What does it tell you about the cell? - gram staining can help determine if the cell is gram-negative or positive as well as the organism’s shape. both of which can be used in determining what type of cells are seen

Question 26

How do we inoculate agar deeps?

Answer 26

1 - Heat the needle and the lop of the test tube with the bacteria your using

2. take your test tube with the deep and make sure to flame the tip of the tube
3. Stab the deep test tube with your needle with the bacteria
4. heat the lip of the test tube with the deep and close it, heat the needle to kill the bacteria
5. turn off the heat

Question 27

How are agar slants inoculated?

Answer 27

A needle/loop can be used to inoculate an agar slant by gentely spreading the bacteria onto the slant.

Question 28

What are the purposes of using agar deeps, using agar slants?

Answer 28

Agar Deep - Used to identify traits of the organism

Agar Slants - Most often used for growth and storage of pure cultures

Question 29

Which streaking method do we employ?

Answer 29

The most common streaking method used in labs is the T streak

Question 30

What is a primary streak vs. a secondary streak?

Answer 30

Primary streak - is to get individual colonies

Secondary streak - is to obtain a pure culture

Question 31

What is the purpose behind separating microbes on a streak plate?

Answer 31

to isolate pure cultures of bacteria, or colonies, from mixed populations

Question 32

What are the steps in order to perform a successful streak?

Answer 32

1. Heat an inoculate loop to red-hot and then allow to cool for at least 5 seconds.
2. Remove the cap of the bacterial culture tube and flame the neck of the tube, then remove a loopful of the culture using the sterilized inoculating loop.
3. Flame the mouth of the culture tube, return the cap to the tube, and return the tube to the test-tube rack.
4. Streak the plate with the inoculating loop being careful not to gouge the agar, flame the loop, allow it to cool and finish streaking the plate.

Question 33

Why do we enumerate bacteria?

Answer 33

To determine the growth and death rates of bacteria

Question 34

What is the 30-300 rule?

Answer 34

The countable plate has between 30 and 300 colonies