Mastering: Aseptic Technique

Name the steps of loop sterilization in their correct order.

1. Attach burner to gas source. Adjust air intake. 2. Turn on gas. 3. Light burner with striker. 4. Adjust air supply. 5. Heat loop until entire wire glows red. 6. Cool loop.

Once you have sterilized your inoculating loop, it is important that you wait 10-20 seconds before using the loop to pick up a sample from your culture. What is the purpose of letting the inoculating instrument cool off? a) Ensure that you are taking your time and not rushing the transfer to avoid accidents b) Avoid burning the surface of the bench with the inoculating loop when you place it down on the bench top c) Avoid killing the bacterial cells with excess heat d) Avoid over-heating and damaging the instruments

c) Avoid killing the bacterial cells with excess heat

When should inoculating instruments be flame sterilized? a) You should never sterilize the inoculating instruments using a flame. d) After transferring the inoculum c) Before and after you transfer the culture d) Before picking up the inoculum

c) Before and after you transfer the culture

Which of the following statements is true regarding proper aseptic culturing techniques? a) Blow gently on the sterilized inoculating loop to make sure it is cool enough to pick up the culture. b) After removing the cap of the culture tube, place the cap onto the bench top to prevent contamination. c) It is necessary to hold the inoculating instrument in the Bunsen burner flame until it becomes red hot. d) All of the above

c) It is necessary to hold the inoculating instrument in the Bunsen burner flame until it becomes red hot.

What is an inoculum? a) The sterile medium to which bacteria will be transferred. b) The bacteria that are undesired within a culture. c) The bacteria transferred to a new medium. d) The inoculating instrument used to transfer bacteria.

c) The bacteria transferred to a new medium. The bacteria that are transferred during inoculation are called the inoculum. The new sterile medium receives, or is inoculated with, the inoculum. The inoculum, it is hoped, consists only of the desired species of bacteria and does not include any contaminants.

If a student forgets to flame the mouth of a broth culture tube before transferring culture to it, what might result? a) Condensation might occur at the top of the tube. b) The medium will not be warm enough for the culture to grow properly. c) The medium in the tube might become contaminated. d) Aerosols might form.

c) The medium in the tube might become contaminated. Heating the mouth of the test tube creates an air current that helps prevents airborne bacteria from falling into the tube while the tube's cap is off. If airborne bacteria do fall into the tube, they will contaminate the medium.

An inoculating _____ is generally used to obtain an inoculum from a broth culture, while an inoculating _____ is typically used to transfer microorganisms to an agar deep tube. a) needle; pipette b) pipette; loop c) loop; needle d) needle; loop

c) loop; needle

After incubating your culture for two days, you suspect that your culture is contaminated. Which method(s) would help you determine if this is the case? a) Differential staining techniques, such as the Gram stain b) Subculture onto an agar plate c) Microscopic examination of cellular morphology d) All of the above

d) All of the above

Microorganisms are typically present in which of the following locations? a) In the air b) On laboratory surfaces c) On your skin d) In the air, on laboratory surfaces, and on your skin

d) In the air, on laboratory surfaces, and on your skin

Which of the following describes the growth of Serratia marcescens in nutrient broth? a) The growth was concentrated along a distinct line through the middle where the bacteria was inoculated. b) The growth was concentrated as a pellet at the bottom of the tube while the media was clear. c) The growth was concentrated as a pellicle on the surface of the broth while the broth itself was clear. d) The growth was distributed both on the surface and throughout the broth, creating a turbid appearance.

d) The growth was distributed both on the surface and throughout the broth, creating a turbid appearance.

After removing the cap from the culture tube it is recommended that the neck of the tube is briefly passed through the flame of the Bunsen burner. What is the purpose of this step? a) This sterilizes the outer portion of the tube to prevent contamination from the surface b) Heating up the outer surface of the tube helps maintain optimal growth temperatures for the bacterial culture c) Heating up the outer surface of the tube helps loosen the bacteria from the media so they are easier to transfer d) This creates an upward movement of airflow out of the tube that prevents contaminating organisms from traveling into the tube from the outside air

d) This creates an upward movement of airflow out of the tube that prevents contaminating organisms from traveling into the tube from the outside air

Which type of media should be inoculated using a zig-zag motion across the surface of the media? a) Agar slant b) Broth c) Agar deep d) All of the above

a) Agar slant

Transfer to an agar deep involves a slightly different procedure from transfer to an agar slant. Which of the following steps is unique to inoculating an agar deep? a) An inoculating needle is used to stab the inoculum into the agar. b) The inoculum must be taken from a broth culture. c) The agar deep medium must be heated (melted) so that it is liquid when inoculated. d) Two loopfuls of inoculum must be transferred.

a) An inoculating needle is used to stab the inoculum into the agar. An inoculating needle is used for inoculating an agar deep. The agar deep medium is stabbed, not streaked. This technique is used in some biochemical tests to place the bacteria within the medium, rather than on the surface.

This video shows a student improperly sterilizing an inoculating loop. What is the student's mistake? a) The wire portion of the instrument was not adequately heated. b) The loop portion of the instrument was heated for too long. c) The student positioned the hand too close to the flame. d) The loop portion of the instrument was not placed in the correct part of the flame

a) The wire portion of the instrument was not adequately heated. To be properly sterilized, both the wire and the loop portions of the inoculating loop must be heated until red-hot. The nonglowing wire could still contain live bacteria that might contaminate the student's cultures.

What error in aseptic technique most likely caused this contaminant colony to grow on this agar slant? a) Failure to properly sterilize the loop. b) Failure to flame the mouth of tube. c) Failure to cool the loop. d) Digging into the agar.

b) Failure to flame the mouth of tube. This yellow colony most likely originated from an airborne bacterium that landed at the top of the agar. Flaming the mouth of the tube prevents airborne bacteria from entering the tube while it is uncapped. Airborne bacteria may fall anywhere on this slant and grow to form a colony.

What type of inoculating instrument is used to transfer a bacterial culture to an agar deep? a) Inoculating loop b) Inoculating needle c) Pipette tip d) None of the above

b) Inoculating needle

After incubating an agar slant containing S.marcescens, you observe growth lacking orange-red pigmentation. Is this growth necessarily that of a contaminating organism? a) Yes, incorrectly colored colonies always indicate contamination b) No, young S.marcescens sometimes have not accumulated sufficient pigment to appear orange-red c) Yes, all S.marcescens cells contain orange-red pigment d) No, colonies of S.marcescens can grow in all different colors

b) No, young S.marcescens sometimes have not accumulated sufficient pigment to appear orange-red

This video shows a student incorrectly obtaining an inoculum from a broth culture. What is his error in aseptic technique? a) The tube was not held correctly. b) The cap of the tube was not handled correctly. c) The mouth of the tube was not flamed correctly. d) The loop was not handled correctly.

b) The cap of the tube was not handled correctly. The student placed the cap from the tube on the bench top. This may have contaminated the cap. Once the cap is placed back on the tube, the bacteria on the cap may contaminate the culture within the tube. Always hold caps in the hand that holds the loop. This minimizes the chance that environmental microbes might contaminate the cap.

A student properly flames her loop but then forgets to let it cool. Instead, she immediately inserts the hot loop into her broth culture. What is the most important issue associated with this mistake? a) The hot loop may burn the student's hand. b) The hot loop may create aerosols when it touches the culture. c) The hot loop may distort the bacterial cells. d) The hot loop may contaminate the broth culture.

b) The hot loop may create aerosols when it touches the culture. When the hot loop touches the liquid broth culture, it will cause some of the broth (and bacteria) to boil briefly, creating a bacteria-containing aerosol. These airborne bacteria could enter the student's respiratory tract or land on her skin. If you hear a hissing sound when you place the loop into the broth culture, you'll know you haven't cooled the loop sufficiently.

This video shows a student incorrectly obtaining bacteria from a colony on a Petri plate. What has he done incorrectly? a) He removed only a small part of the colony. b) The lid is too far away from the plate. c) He is using an inoculating loop. d) The plate is sitting on the lab bench.

b) The lid is too far away from the plate. The lid should partially cover the plate while the student is removing inoculum, as shown in this photograph. This technique prevents airborne microbes from contaminating the surface of the agar.

A student inoculated an agar slant with inoculum from a colony on a Petri plate. The slant was incubated at an appropriate growth temperature for this species for 2 days. The results are shown in this photograph. Which hypothesis best explains why there is so little growth on the agar? a) The mouth of the slant tube was not flamed after inoculation. b) The loop dug into the agar during inoculation. c) The loop was removed from the flame before it was red-hot. d) The loop wasn't cooled before the student made the transfer.

b) The loop dug into the agar during inoculation. Notice the small gouge in the agar at the bottom of the slant, as indicated by the arrow in this photograph. The student most likely gouged the agar with the loop, depositing a good portion of the inoculating cells under the surface of the agar. After the gouge, very few cells were left on the loop to inoculate the rest of the slant surface.

Which of the following best describes aseptic technique? a) To flame the inoculating loop. b) To manipulate bacteria without introducing contaminants. c) To keep the environment sterile. d) To use chemicals to inhibit or destroy bacteria.

b) To manipulate bacteria without introducing contaminants. Aseptic technique is used to prevent environmental bacteria (e.g., from the air) from contaminating cultures. This is why we flame the mouths of the culture tubes before and after transferring bacteria.

The process of transferring microorganisms from one medium to another is known as: a) planting. b) subculturing. c) transmission. d) relocation.

b) subculturing.