BIOL 2730 — Lab 3 Exercise 8: Aseptic Technique

Flashcards covering Exercise 8 (Aseptic Technique) for BIOL 2730.

What is the main purpose of aseptic technique?

To prevent contaminating organisms from being introduced into culture materials during handling or inoculation, prevent handled organisms from contaminating the handler or others, and prevent contamination from remaining after work is completed.

What are the three learning outcomes for Exercise 8?

1) Aseptically transfer a bacterial culture from a broth tube to a sterile broth tube. 2) Aseptically transfer a bacterial culture from an agar slant to a sterile agar slant. 3) Aseptically transfer a bacterial colony from an agar plate to a sterile agar slant.

Does aseptic technique mean the work environment is sterile?

No. A sterile environment would require the absence of all microbes, including endospores and other resistant forms. Aseptic technique reduces the likelihood of contamination by removing or killing many microbes.

What kinds of contamination does aseptic technique help prevent?

It helps prevent contamination of culture materials, contamination of the handler/others, and contamination remaining after work with cultures.

What is the purpose of disinfecting the work area before working with cultures?

To kill microorganisms that may be present on the work surface.

What does the manual say disinfectants destroy, and what may they not destroy?

They destroy vegetative cells and viruses but may not destroy endospores.

When should the work area be disinfected?

Before beginning the procedure and again after all work is complete; disinfectant should also be used after any spill of live microorganisms.

How are inoculating loops and needles sterilized?

By inserting/flaming them in a Bunsen burner or incinerator until the entire wire becomes red-hot.

Why must the entire wire of the inoculating loop be heated to red-hot?

To incinerate contaminating organisms that may be present on the implement.

Why must a loop be allowed to cool completely before picking up inoculum?

So viable cells can be transferred rather than being killed by a hot loop.

What should you do with the loop after inoculation is complete?

Flame the loop or needle to destroy organisms remaining on it, then return it to its receptacle. Never place it on the desk.

How are flaming procedures different when working with agar plates?

Loops and needles are flamed as usual, but unlike the mouths of culture tubes, agar plates are never flamed.

How should a culture-tube cap be handled during aseptic transfer?

Remove it with the fingers of the hand holding the loop and keep it in that hand; never place the cap on the laboratory bench.

What should happen to the mouth of a culture tube after the cap is removed?

The mouth may be flamed before the loop is inserted, and it may be reflamed before the tube is recapped.

When using an incinerator instead of a flame, is the tube mouth flamed?

No. If an incinerator is used, the tube is not flamed.

How is a broth culture dispersed before taking a sample?

Gently shake the tube.

How is organisms' transfer into broth completed?

Insert the cooled inoculating loop into the culture and twist the loop several times so organisms on the loop are delivered to the liquid.

How is an agar slant inoculated?

Draw the loop up the surface of the slant from the bottom to the top in a zigzag pattern.

What should you avoid when inoculating an agar slant?

Do not gouge or disturb the agar surface.

How is a stab culture inoculated?

A needle is inserted into the agar medium by stabbing it into the agar.

What does 'clamshelling' mean when working with an agar plate?

Raising the plate lid and holding it diagonally over the plate to minimize contamination from the air.

Why is the agar-plate lid held diagonally over the plate?

To protect the agar surface from contamination in the air while the plate is open.

How should the inoculating loop be used on an agar plate?

Streak it gently over the agar surface without gouging or disturbing the agar.

Should an agar plate be completely uncovered while inoculating?

No. The cover should be only partially opened/clamshelled to reduce exposure to air contamination.

Where should an agar plate be labeled?

On the bottom of the plate—the part containing the agar.

Why should an agar plate be labeled on the bottom?

So the culture remains identifiable even if the lid becomes separated from the growing culture.

What nomenclature should be used when labeling an agar plate?

Binomial nomenclature, such as Escherichia coli or E. coli.

How are inoculated agar plates almost always incubated?

Upside down.

Why are inoculated agar plates incubated upside down?

To prevent moisture from condensing on the agar surface and spreading the inoculated organisms.

What is one major difference between working with a culture tube and an agar plate?

The mouth of a culture tube may be flamed; an agar plate itself is never flamed.

What culture is transferred in the broth-to-broth exercise?

Escherichia coli (E. coli) broth culture is transferred to sterile nutrient broth.

What materials are listed for the broth-to-broth transfer?

E. coli broth culture; sterile nutrient broth; inoculating loop; Bunsen burner or incinerator; disinfectant and paper towels; Sharpie marking pen.

What is the first step of the broth-to-broth transfer?

Disinfect the desktop according to the instructor's directions.

How should the sterile nutrient broth tube be labeled?

With your initials and E. coli, preferably toward the top of the tube.

What is done to the inoculating loop before taking E. coli from the culture?

Flame it until it becomes bright red; the entire wire must be heated, then allow it to cool completely.

What is done to the E. coli culture before removing the cap?

Gently shake the tube to disperse the culture.

How is the cap handled when removing E. coli from the broth culture?

Grasp it with the little finger of the hand holding the inoculating loop and remove it from the tube.

What happens after removing the cap from the broth culture?

Flame the mouth of the tube, unless an incinerator is being used.

After the loop enters the E. coli culture, what happens next?

Remove the loop containing the culture, flame the tube mouth again, recap the tube, and return it to the rack.

What is done to the sterile broth tube before inoculation?

Remove the cap while holding it with the little finger, flame the mouth of the tube, and then insert the culture-containing loop without flaming the loop.

How are organisms dispersed into the sterile broth?

Move the loop back and forth in the tube to disperse the organisms into the medium.

What is done after the sterile broth is inoculated?

Remove the loop, flame the tube mouth, recap the tube, flame the loop, return it to its holder, and incubate the culture at $37\,^\circ\text{C}$ for $24\text{--}48\text{ hours}$.

What culture is transferred in the slant-to-slant exercise?

An E. coli agar slant culture is transferred to a sterile nutrient agar slant.

How should the sterile nutrient agar slant be labeled?

With your initials and E. coli, preferably near the top of the tube.

What is the first step with the inoculating loop in the slant-to-slant transfer?

Flame it until it is red-hot and allow it to cool.

How do you obtain E. coli from the source slant?

Pick up the slant culture with the free hand, remove the cap with the little finger of the hand holding the loop, flame the tube mouth, and pick up culture with the cooled loop.

How is the sterile slant inoculated?

Flame the mouth of the sterile slant, insert the loop, and inoculate the surface using a zigzag motion from the bottom toward the top.

What must you avoid when streaking an agar slant?

Do not gouge the agar; allow the loop to glide gently over the gelatin-like agar surface.

What happens after streaking the slant?

Flame the tube mouth, recap the tube, flame the loop red-hot, return it to its receptacle, and incubate the slant at $37\,^\circ\text{C}$ for $24\text{--}48\text{ hours}$.

What materials are used for the agar-plate-to-slant transfer?

A nutrient agar plate with bacterial colonies, a sterile nutrient agar slant, an inoculating loop, and a Sharpie marking pen.

How is the sterile slant labeled for a plate-to-slant transfer?

With your name and the name of the organism being transferred.

How is the agar plate opened when picking up a colony?

Raise the lid just enough to access the colony; keep the lid partially open/clamshelled.

How is a colony picked up from an agar plate?

Use a sterile, cooled inoculating loop to pick up a loopful of organisms while the plate lid is raised only enough to access the colony.

What should you NOT do when picking organisms from an agar plate?

Do not gouge the agar with the loop and do not completely remove the lid.

How is the sterile agar slant inoculated from a plate?

After flaming the mouth of the sterile slant, insert the loop and streak the surface in a zigzag motion without disturbing the agar.

What is done to the loop after transferring organisms from a plate to a slant?

Remove it, flame it red-hot, and return it to the receptacle.

At what temperature and for how long are the inoculated agar slants incubated?

$37\,^\circ\text{C}$ for $24\text{--}48\text{ hours}$.

How can you tell whether the broth transfer was successful?

After incubation, growth/turbidity in the inoculated broth indicates that organisms were transferred and grew.

How can you tell whether a transfer to an agar slant was successful?

After incubation, visible bacterial growth along the inoculated streak on the slant indicates successful transfer.

What are possible errors if a transfer is unsuccessful?

Possible errors include failure to sterilize/cool the loop properly, contaminating the culture, failing to transfer organisms, improper tube/plate handling, or incorrect inoculation technique.

Why is it important that only the desired organism be transferred?

Ensuring only the desired organism is transferred is critical for identifying and characterizing an unknown bacterium.

Give three reasons aseptic technique is essential when handling microbial cultures.

It prevents contamination of culture materials; prevents organisms being handled from contaminating the handler or others; and prevents contamination from remaining after working with cultures.

Give two examples of how heat is used during inoculation of a tube culture.

Heat is used to sterilize the inoculating loop/needle by flaming it until red-hot, and to flame the mouth of a culture tube after removing the cap and again before recapping.

How is air contamination prevented when using an inoculating loop with an agar plate?

Keep the lid only partially open and hold it diagonally over the plate (clamshelling) while working.

Where should a label be written on an agar plate?

On the bottom of the plate, the part containing the agar.

Why are agar plates incubated upside down?

To prevent condensation from forming on the agar surface and spreading the inoculated organisms.

Which types of organisms may remain on a lab bench after disinfection?

Endospores may remain because disinfectants may not destroy them.

Compare bacterial growth in broth, slants, and agar plates.

Broth provides growth throughout a liquid medium; slants provide growth on the surface of a solid agar slope; plates provide growth as colonies on a broad solid agar surface.

What is an advantage of broth?

It provides a liquid environment in which growth can occur throughout the medium.

What is an advantage of an agar slant?

It provides a solid surface for growth in a tube while requiring less surface area than a plate.

What is an advantage of an agar plate?

It provides a broad solid surface that allows colonies to be observed and separated by streaking.

Can you tell from an incubated broth alone whether it is a pure culture?

Not necessarily. The manual's question asks you to consider how purity could be determined; growth in broth alone does not show whether only one organism is present.

How could you determine whether an incubated broth or slant is a pure culture?

The manual's question asks how purity could be determined; a culture can be examined for evidence of more than one type of growth, such as by examining/streaking it to obtain separated colonies.

Multiple choice: When is a disinfectant used on the work surface?

All of the above: before the beginning of laboratory procedures, after all work is complete, and after a spill of live microorganisms. (Answer: e)

Multiple choice: How should a culture-tube cap be handled when retrieving a sample with an inoculating loop?

It should be removed with the fingers of the hand holding the loop and held in that hand. (Answer: a)

Multiple choice: How is an inoculating loop or needle sterilized using heat?

Until the entire wire is bright red. (Answer: c)

Multiple choice: Which is a correctly labeled agar plate?

S. aureus on the bottom. (Answer: b)

Multiple choice: What did Noah do wrong when transferring S. aureus from broth to an agar plate?

He did not handle the agar plate correctly: he opened the lid rather than maintaining it partially open/diagonally (clamshelling). (Answer: c)

Figure 8.1: What does the illustration show?

Sterilizing an inoculating loop in the flame of a Bunsen burner; the loop must be heated until the wire is red-hot before transferring a bacterial sample.

Figure 8.2: What does 'clamshelling' look like?

The agar-plate lid is held diagonally over the plate while the plate is accessed, minimizing contamination from air.

Figure 8.3: What is the overall procedure illustrated?

Removing organisms from a broth culture with an inoculating loop: heat loop red-hot; disperse culture; remove cap and flame tube mouth; remove a loopful; flame mouth again; recap.

Figure 8.4: What is the overall procedure illustrated?

Inoculating a sterile nutrient broth: remove cap and flame mouth; insert inoculating loop with culture; remove loop and flame mouth; recap; flame loop and return it to its receptacle.

Figure 8.5: What is the overall procedure illustrated?

Inoculating a sterile agar slant from a slant culture: sterilize/cool loop; remove cap and flame mouth; pick up culture; flame sterile slant mouth; inoculate in zigzag; recap; flame loop and incubate.

Figure 8.6: What is the overall procedure illustrated?

Inoculating a nutrient agar slant from an agar plate: sterilize/cool loop; clamshell plate and pick up colony; flame sterile slant mouth; streak slant; recap; flame loop.

What do Figures 8.3–8.6 have in common?

They demonstrate aseptic transfer procedures designed to move microorganisms while minimizing contamination.

What is the general order for handling a culture tube with an inoculating loop?

Sterilize and cool the loop → remove cap without setting it down → flame tube mouth (when applicable) → obtain/transfer inoculum → flame tube mouth again → recap → re-sterilize the loop.

What is the general order for finishing an inoculation?

Reflame the tube mouth when applicable → recap the tube → flame the loop/needle red-hot → return it to its receptacle → incubate the inoculated culture as directed.

What should never be done with an inoculating loop after use?

It should never be placed on the desk surface.

What should never be done to the surface of agar while transferring organisms?

Do not gouge, tear, or otherwise disturb the agar surface.