Microbiology Lab Exam 1

When should hands be washed?

Wash your hands thoroughly with soap and water after handling living microbes and before leaving the laboratory each day. Also, wash your hands after removing gloves.

How do you clean your lab bench?

Wipe the desktop with a disinfectant (e.g., Coverage or 10% chlorine bleach) before and after each lab period. Allow disinfectant to evaporate; do not wipe dry

Why must Bunsen burners and incinerators be treated with caution even after you have finished using them?

It is a fire & safety hazard as well as an additional source of unnecessary heat in the room

When should you sterilize your loop?

Before and after use through Bunsen burner if BSL-1 or in enclosed electric incinerator if BSL-2 (also, sterile disposable loops/wooden sticks)

Describe 2 specialized waste containers that can be found in lab:

1) sharps/broken glass container: disposal of broken glass or any other item that could puncture an autoclave bag

2)autoclave container: disposal of plate cultures and other contaminated non-sharp disposable items

Describe appropriate attire that should be worn in lab:

1) Wear eye protection whenever heating chemicals

2)Wear disposable gloves while staining microbes and handling blood products

3) Lab coat

4) close-toed toes

Why must electronic devices and other personal items be put away before entering the lab room and not brought out before fully exiting the room?

Contamination of these items can be a health hazard as well as expensive.

What is the protocol if infectious material is spilled?

Cover any culture spills with paper towels. Soak the towels immediately with disinfectant and allow them to stand for 20 minutes. Report spill to the instructor. When you are finished, place towels in container for autoclaving

Where should gloves be disposed of? Why?

in container designated for autoclaving/biohazardous waste bin to reduce risk of contamination

The lysis of red blood cells in blood agar is indicative of the presence of which enzymes?

streptolysin O and S

When looking at a blood agar plate with bacterial growth, beta is complete lysis of the red blood cells, alpha is partial lysis, and gamma is no lysis.

True

An organism that obtains its nutrients from dead organic matter is a(n) __________.

saprophyte

A reservoir is

a source of microbial contamination.

Commensalism is best described as a(n)

relationship between two organisms where only one member benefits and the other is unharmed.

A pathogen is best described as

any microorganism that causes disease.

Staphylococcus bacteria are commonly present in the human nasal cavity but rarely cause disease of the upper respiratory system. This situation is an example of (commensalism/mutualism/parasitism).

commensalism

Normal microbiota may cause disease if conditions change in the body.

True

It has been determined that the temperature in an autoclave should reach __________ for sterilization.

121 degrees Celsius (and 15 psi for 15 minutes to sterilize small volumes) (

What role does pressure play in an autoclave?

Pressure is applied to boiling water to prevent heat from escaping as steam (Adding pressure to boiling water leads to higher water temperatures.)

How do scientists ensure that autoclaved materials were actually sterilized?

They may place a sterility indicator with a special dye to detect the growth of endospores. ( Some sterility indicators have dyes that change color in the presence of living bacteria, indicating that the correct sterilizing temperature and time have not been reached.)

What is the purpose of an autoclave?

to sterilize equipment and media

Autoclaves are used to ensure that equipment and media are sterile, or free of contamination

Where are you likely to find an autoclave?

in a dentist office

Why is autoclaving rather than boiling water used for sterilization?

Boiling water does not kill everything, including bacterial endospores and some protozoan cysts.

Boiling water reaches a maximum temperature of 100 degrees Celsius, compared with autoclaving where the temperature reaches at least 121 degrees Celsius. Boiling water is not hot enough to kill endospores or cysts in a reasonable amount of time.

Why do containers of liquid placed in an autoclave need to remain at least slightly open?

If they remain closed, they may implode.

The extreme temperature and pressure changes within the autoclave may cause closed containers to implode.

Why are endospores sometimes used in sterility indicators?

They are the hardest life form to kill.

If an autoclave kills endospores within a sterility indicator, it is safe to assume that everything else is sterilized.

Which of the following are the parameters for autoclaving?

15 minutes at 121°C and15 psi

In general, moist heat kills microorganisms in less time than dry heat.

True

Which of the following lists microbes or microbial structures in order from the hardest to destroy by heat to the easiest to destroy? (4 total)

bacterial endospores, fungal spores, vegetative bacteria, fungi

Which of the following techniques can be used to sterilize microbiological media?

autoclaving

Why are endospores used to measure the effectiveness of autoclave sterilization?

Endospores are very hard to kill

To solidify media used to grow microbes, usually __________ is added to the nutrient broth.

agar

The growth resulting in the elevation of a bacterial colony is due to the colony growth spreading vertically as well as horizontally.

True

A colony on an agar plate that is slightly and equally elevated and unbroken would be classified as ___________________

a raised, circular colony.

The growth characteristics of a bacterial culture streak on an agar slant will not be the same as if the streak were on an agar plate.

False

he cultural characteristics of a bacterial colony are almost entirely due to __________

genetic factors affecting growth patterns

What is an inoculum?

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.

Which of the following best describes aseptic technique?

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.

What is the correct order of loop sterilization? 6 steps

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

Why must both the wire and the loop portions of the inoculating loop must be heated until red-hot?

To be properly sterilized

The nonglowing wire could still contain live bacteria that might contaminate the student's cultures.

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?

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.

If a student forgets to flame the mouth of a broth culture tube before transferring culture to it, what might result?

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.

What's wrong with placing 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.

Why should you flame the mouth of a tube?

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.

Why should the lid should partially cover the plate while the student is removing inoculum?

This technique prevents airborne microbes from contaminating the surface of the agar.

Which hypothesis best explains why there is so little growth on the agar?

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.

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) Two loopfuls of inoculum must be transferred.

B) An inoculating needle is used to stab the inoculum into the agar.

C) The agar deep medium must be heated (melted) so that it is liquid when inoculated.

D) The inoculum must be taken from a broth culture.

B) 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.

The process of transferring microorganisms from one medium to another is known as:

subculturing

Microorganisms are typically present in which of the following locations?

-On laboratory surfaces

-In the air

-On your skin

-In the air, on laboratory surfaces, and on your skin

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

When should inoculating instruments be flame sterilized?

Before and after you transfer the culture

Which of the following statements is true regarding proper aseptic culturing techniques?

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

B) Blow gently on the sterilized inoculating loop to make sure it is cool enough to pick up the culture.

C) After removing the cap of the culture tube, place the cap onto the bench top to prevent contamination.

D) All of the above

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

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?

Avoid killing the bacterial cells with excess heat

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.

loop; needle

To prevent or minimize contamination, __________ is used in microbiology procedure.

aseptic technique

Before you use an inoculating loop or needle, it must be __________.

heated until red-hot

Aseptic techniques used in microbiology involve all the following EXCEPT __________.

-heating the mouth of the culture tube

-blowing on your loop to cool it

-heating the inoculating loop

-disinfecting the work area

-holding culture tubes at an angle

blowing on your loop to cool it

A nutrient agar slant is used in microbiology to __________.

transport and store microbes

Bacteria are routinely __________ into new culture media to study their growth and other characteristics.

inoculated

Which of the following media would NOT contain agar?

-semisolid deep

-broths

-solid deep

-Petri plates

-slants

broths

Which of the following is the proper technique for inoculating an agar slant?

-Streak the surface of the media with a back-and-forth motion using an inoculating loop.

-Streak the surface of the media with an inoculating needle.

-Stab the butt of the tube using an inoculating needle.

-Stab the butt of the tube using an inoculating loop.

Stab the butt of the media with glass rod.

Streak the surface of the media with a back-and-forth motion using an inoculating loop.

Unwanted microbes that find their way into our cultures are called

contaminants

Which of the following is the correct sequence for the aseptic transfer of a microbial inoculum?1) Remove the cap from the culture tube. 2) Reheat the mouth of the culture tube and replace the cap. 3) Get a loopful of the culture. 4) Sterilize the loop by heating it red-hot. 5) Flame the mouth of the culture tube.

4-Sterilize the loop by heating it red-hot

1- Remove the cap from the culture tube

5- Flame the mouth of the culture tube.

3- Get a loopful of the culture

2- Reheat the mouth of the culture tube and replace the cap

Visual inspection of a broth culture after 24 hours of growth will indicate whether it was incorrectly transferred using non-aseptic techniques and is now contaminated.

False

A microscope is needed to differentiate between bacterial species.

Environmental bacteria or fungi will not affect your results when culturing microorganisms.

False

How should you heat an inoculating loop in the Bunsen burner flame to ensure sterility?

loop and wire until they become red hot

After flaming the loop or the needle to sterilize it, you will need to allow it to cool for a few seconds so that the heat will not kill or damage the bacteria you are about to pick up with it out of the broth culture.

True

Why is the technique of subculturing an important one that a microbiologist should master?

Subculturing allows microbiologists to inoculate multiple tubes, broths, and agar plates, which helps them identify and characterize microorganisms in a laboratory setting.

If you are transferring bacteria from a broth culture to multiple tubes of different broths and agar types you should always flame the loop after transferring into each new tube.

True

Which of the following observations would suggest that a plate was inoculated with a pure (axenic) culture?

-Isolated colonies are all white in color and about the same size.

-Isolated colonies are all white in color, but some colonies are noticeably larger than others.

-Bacterial growth is apparent in all four quadrants.

-Bacterial growth is apparent along the streaks connecting each quadrant.

Isolated colonies are all white in color and about the same size.

Bacterial species tend to produce distinctive-looking colonies. If all colonies on the plate appear identical, then it's likely the inoculum was pure.

Imagine that you forgot to flame the loop before streaking the inoculum from the first quadrant into the second quadrant. What is the most likely consequence of this error?

Too much bacterial growth outside the first quadrant.

Proper streak plating technique deposits progressively smaller amounts of bacteria in each quadrant. This is done by having the loop pick up only a small amount of bacteria from the previous quadrant's streaks. Flaming the loop between streaks ensures that the loop starts clean and that only this small amount of bacteria is used to inoculate the next quadrant.

For a plate to be streaked correctly...

streak d must draw inoculum from streak c. In this example, streak d draws inoculum from streak a instead. This will draw too much bacteria into quadrant 4 and produce few, if any, isolated colonies.

Why should you lift the lid of the Petri plate only enough to get the loop inside?

keeps the agar protected and prevents airborne microbes from falling into your culture.

Where should you label your Petri plate with information about the culture?

All labeling should be done on the bottom portion of the agar plate. This ensures that the written information stays with your culture, even if the lid gets accidentally exchanged with another plate.

Imagine you're streaking a plate. You've just finished the first streak (streak a), closed the lid, and rotated the plate. Arrange the next steps of the procedure in their correct order.

1) Flame loop and let cool

2) Lift lid of petri plate

3) Streak across streak a and into Quadrant 2

4) Streak within Quadrant 2

5) Close petri plate lid

6) Rotate plate and flame loop

Why would there be colonies on this plate that have different color, size, and shapes?

This means that another bacterial species is present—a contaminant. Notice, too, that both colonies are growing along the streak lines. This suggests that both species came from the broth tube. Airborne contaminants introduced during streaking would, in contrast, be more randomly scattered about the plate.E. coli colonies should all have the same color, size, shape, and margins.

If a correctly streaked plate were INCORRECTLY incubated right side up (instead of upside down), which of the following plate results would you most likely see?

During incubation, water tends to condense in the lid of Petri plates. If the plate is incubated in a right-side-up position, this condensation will "rain" onto the surface of the agar. The water on the agar will create a soupy mess of bacteria instead of isolated colonies. Incubating the plate upside down keeps water condensation in the lid and away from your culture.

What happens when a plate is exposed to airborne contaminants?

the contaminating microbes settle all over the plate. After incubation, the colonies from these contaminants will be all over the plate also, not just following the streak lines.You want to spread only a tiny fraction of the bacteria in one quadrant to the next, so it's important to flame and cool the loop between each streaking step. This ensures thinning of the bacteria with each streak, which ultimately produces isolated colonies after incubation

Why is it important to flame the loop between quadrant streaks?

You want to spread only a tiny fraction of the bacteria in one quadrant to the next, so it's important to flame and cool the loop between each streaking step. This ensures thinning of the bacteria with each streak, which ultimately produces isolated colonies after incubation.

How would you know that the unknown culture contains two species of bacteria?

There are multiple distinct colonies along the streak lines consistently in each quadrant. This suggests that more than one microbe was present in the initial inoculum.

Streak plates are useful in microbiology to __________.

isolate bacteria and develop pure cultures

Which of the following techniques is NOT used to isolate bacteria and develop a pure culture?

-pour-plate technique

-spread-plate technique

-a mixed broth culture tube

-streak-plate technique

-None of the listed responses is correct.

a mixed broth culture tube

In the streak-plate technique, the intent is to isolate bacteria by dilution in theory by __________.

dilution on a solid surface

An unwanted organism in a streak plate is called a(n) __________.

contaminant

Which of the following isolation techniques does NOT require the preparation of sterile dilution buffer?

-pour-plate

-streak-plate

-spread-plate

-All of the listed techniques require sterile dilution buffer.

-None of the listed techniques require sterile dilution buffer.

streak-plate

The main advantage of a spread plate over a streak plate is that the spread plate __________.

allows for the calculation of cell numbers

In the streak-plate method, streaking the bacteria from region to region on the Petri dish serves the purpose of __________.

decreasing the numbers of bacteria in each area, producing individual colonies in the final area

What would a streak plate look like if you had not sterilized the loop between streaking into the new areas of the plate?

While the number of bacteria colonies seen in each area may decrease, the numbers would not decrease enough for individual colonies to form.

What could occur if, during a streak-plate inoculation, your loop went into the area of the first introduction of the bacterial culture and then into the final area?

The number of bacteria in the final area would be increased so much as to negate any dilution effects.

A streak plate would work best to isolate bacteria if ____________________

you think there may be a chance of a contaminate in your broth culture.

If you aseptically transferred a single colony from a streak plate to a test tube with a broth-growing medium, all of the bacteria in that broth would be genetically identical.

True

A compound microscope uses two lenses at once to magnify the image of a specimen. The _____ lens is found in the eyepiece and the _____ lens is found in the revolving nosepiece.

ocular; objective

Which component of the microscope is found directly under the stage, and contains two sets of lenses that collect and concentrate light as it passes upward from the light source into the lens systems?

Abbe condenser

Which property of the lens describes its ability to show two adjacent objects as discrete entities?

Resolving power

Which of the following is an INCORRECT association?

-Resolving power; expressed mathematically as a function of the wavelength of light and the numerical aperture

-Magnification; degree of enlargement of the specimen when using a specific lens or combination of lenses

-Iris diaphragm; used to raise and lower the objective lenses to bring the specimen into focus

-Stage; platform with an opening in the center that allows for the passage of light through the specimen

-Iris diaphragm; used to raise and lower the objective lenses to bring the specimen into focus

Loss of bent, or refracted, light results in a reduced numerical aperture, which diminishes the resolving power of the objective lens. Adding which of the following substances between the slide and the lens acts to decrease the refraction of light?

Oil

Which of the following objective lenses is the ONLY lens that should be used with oil immersion?

High-power lens (100X)

The use of clean lenses increases the efficiency of the microscope. While dry lenses should be cleaned with _____, the oil-immersion lens can be cleaned with _____, followed by 95% ethanol.

lens paper; xylol

Which of the following microscope parts should routinely be adjusted to control the light source and provide optimal illumination of the specimen?

Iris diaphragm

Why is a specimen smaller than 200 nm not visible with a light microscope?

Anything smaller than 200 nm cannot interact with visible light

What happens to the light rays when they hit the specimen?

They are reflected, refracted, or absorbed by the specimen.

What is the role of the ocular lens?

To recreate the image in the viewer's eye