Aseptic Technique

Question 1

Define ubiquitous
As in “bacteria are ubiquitous.”

Answer 1

Everywhere. Bacteria are found almost everywhere!

Question 2

What is the importance of aseptic techinque to creating a sterile environment?

Answer 2

aseptic technique is used to remove or destroy living organisms that are present to create a sterile environment.

Question 3

What are some environmental factors that influence microbial growth?

Answer 3

temperature,
osmotic pressure,
atmospheric gases,
pH,
moisture.

Question 4

Describe the moderate conditions that most microbes prefer.

Answer 4

Microbes can exist in harsh environments, but most prefer moderate conditions.
* temperatures between 20 to 40°C
* moist conditions rather than very dry conditions
* an oxygen-rich environment
* an environment close to neutral pH

Question 5

Briefly describe how agar plates are made.

Answer 5

Agar plates are made by pouring hot agar into a petri dish and allowing it to cool.

Question 6

How and why are petri dishes positioned?

Answer 6

Condensation often forms which could fall down onto the agar surface causing contamination or dispersion of any bacterial colonies. To prevent this, plates are always incubated upside down.

Question 7

At what temperature are bacteria usually incubated?

Answer 7

Most bacteria are mesophiles (grow between 20–40°C) and are incubated at 37°C (human body temperature).

Question 8

Where and why are petri dishes labeled?

Answer 8

Agar plates are labeled on the bottom in case the lid becomes separated from the actual culture.

Question 9

Define:
Agar media

Answer 9

Bacterial culture medium that contains agar as a solidifying agent and is used for growing microorganisms.

Question 10

Define:
Agar plate

Answer 10

Bacterial culture medium containing agar poured into a petri dish.

Question 11

Define:
Agar slant

Answer 11

Bacterial culture medium containing agar in a test tube that is allowed to solidify at an angle and forms a solid, slanted surface.

Question 12

Briefly describe aseptic technique as it relates to lab work.

Answer 12

A set of procedures employed in the handling of microbes without causing contamination from outside sources.

Question 13

Define:
Colony

Answer 13

A visible mass of microorganisms formed by replication of a single microorganism on an agar surface, often in a petri dish.

Question 14

Define:
Inoculating loop

Answer 14

A sterilizable tool made of wire, used to implant or move microorganisms.

Question 15

Define:
Inoculum

Answer 15

A small amount of microorganism used to start a new culture.

Question 16

Define:
Liquid broth culture

Answer 16

A liquid nutrient medium used for the propagation of microorganisms.

Question 17

Define:
Petri dish

Answer 17

A shallow, transparent glass or plastic dish that is covered by a larger lid.

Question 18

Define:
Pure culture

Answer 18

A bacterial culture containing only one strain of microorganism.

Question 19

Define:
Sterile

Answer 19

An environment devoid of any living cells.

Question 20

When working with aseptic techinque where may contaminating organisms come from?

Answer 20

the environment,
your hands,
tools used

Question 21

Give examples of when pure cultures are necessary.

Answer 21

when trying to identify or study a particular microorganism

Question 22

What tools are used to transfer microbial cultures from one medium to another?

Answer 22

inoculating loops or needles.

Question 23

What tools are used to transfer an inoculum from tubes of broth?

Answer 23

Inoculating loops

Question 24

For what kind of analysis is the broth medium limited?

Answer 24

A broth is limited in that one cannot see individual cells or colonies so pure cultures cannot be created in this medium.

Question 25

What is the importance of using the correct culture media when growing microbes?

Answer 25

The use of culture media with a specific composition and consistency helps **grow microbes in a controlled manner**.

Question 26

When are inoculating tools to be sterilized?

Answer 26

Inoculating tools must be sterilized both before and after transfer.

Question 27

Briefly describe how inoculating tools are sterilized.

Answer 27

by heating to red hot in the flame of a Bunsen burner or in a microincinerator.

Question 28

After heating an inoculating tool, what step comes next and why?

Answer 28

The inoculating tool must be allowed to cool before picking up cells to be transferred, or they will be destroyed in the process.

Question 29

Describe how to treat the **mouth** of a glass culture tube.

Answer 29

The mouth of a glass culture tube should be passed through the flame of a Bunsen burner in order to reduce contamination when the cap is first removed and again before it is replaced.

Question 30

Describe how to treat the **cap** of a glass culture tube.

Answer 30

The cap of a tube should never be placed on the bench; instead, it should be held by the little finger of one hand.

Question 31

Describe how to innoculate an agar slant.

Answer 31

bacteria are **spread** on the slanted surface using an inoculating tool. Alternatively, a needle is used to **stab** into the agar down to the bottom of the tube.

Question 32

Describe a primary use of agar plates.

Answer 32

Agar plates allow for isolation of individual bacteria to form colonies.

Question 33

Describe good practice for handling plates when transferring bacteria.

Answer 33

For agar plates it is good practice to raise only one side of the plate lid from the plate when removing or adding bacteria to the surface.

Question 34

Describe the advantage of using a broth medium.

Answer 34

The advantage of a broth medium is the ability to support rapid and large bacterial population growth.

Question 35

Name a common use for broth mediums.

Answer 35

Broths are also commonly used for **antimicrobial sensitivity assays**.

Question 36

Name common uses of agar slants.

Answer 36

Many **biochemical tests** involve the use of specialized media in slants. This type of medium is also an excellent choice for **storage** of stock bacterial cultures.

Question 37

Name a common use for agar plates.

Answer 37

Plates provide a large surface area for microbial growth and are often used to **isolate** individual bacterial colonies.

Question 38

Explain why the inside of a banana does not have any microbes.

Answer 38

Plants and animals have many ways of excluding microorganisms. It is common for the internal tissues of organisms, like plants and animals, to be an environment where microorganisms are not found.

Question 39

Explain why the inside of a sealed beverage does not have any microbes.

Answer 39

beverages are **pasteurized**, which would remove all microorganisms for as long as the can was sealed. additionally, some may have an **acidic pH** and/or **high concentrations of sugar** that serve to prevent microbial growth.

Question 40

What effect did hand washing and cleaning the bench with alcohol have on the number of bacteria growing on the plate?

Answer 40

* The plates sampling the hands and bench after the alcohol treatment had fewer colonies growing on them than the plates that sampled the unwashed hands or bench. * Hand washing or alcohol did not completely eliminate microorganisms, however, since some colonies still grew on the post-washing plates.

Question 41

Briefly describe why aseptic technique is important to use of pure cultures.

Answer 41

Aseptic technique prevents the introduction of unwanted microbes or contamination of a culture. Therefore, aseptic technique is an important part of making pure cultures.