Mastering Micro: First lab exam

When you look through the ocular lenses, you see this image of the endospore-forming bacteria Bacillus anthracis under oil. Which of the following qualities is most lacking in this image?

contrast. Contrast, which is lacking in this image, refers to the difference in light intensity between the specimen's features and between the specimen and the background. You can enhance contrast by adjusting the lamp intensity dial and the aperture diaphragm on the condenser.

condenser aperture diaphragm

controls angles at which light rays strike the specimen

The use of clean lenses increases the efficiency of the microscope. What should be used to clean the lenses of the microscope?

lens paper

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

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

objective lens

primary lens that magnifies the specimen

condenser focus knob

raises and lowers the condenser relative to the stage

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

resolving power

revolving nosepiece

rotates objectives into position

arm

supports the lenses

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.

Which of the following best describes serial dilution?

Each tube in a set of tubes is a dilution of the previous tube. In a serial dilution, the sample is sequentially diluted by transfer from one dilution to another, as shown in this diagram. Each dilution contains fewer bacteria than the previous dilution, from which it was created.

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

High-power lens (100X)

If you view a specimen at 40X total magnification with a 10X ocular lens, then you are viewing the specimen with what objective?

4X. Total magnification is calculated by multiplying the magnifying power of the objective lens by the magnifying power of the ocular lens: 4X objective x 10X ocular lens = 40X total magnification.

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

Par focal

stage

where the specimen is placed for viewing

Imagine you and a partner are preparing a serial dilution of a sample of pond water. While you were watching your agar, your partner labeled several test tubes with the numbers below. Which of these numbers correctly expresses a dilution?

10-4. A dilution is created by measuring a volume of a sample and adding it to a volume of sterile water, thus making the resulting solution less concentrated than the original. The resulting solution contains a fraction of the original sample. Therefore, it must have a negative exponent. A dilution of 10-4 means that dilution has 1/10,000 or 1/104 (also write as a fraction as above) as many bacteria per ml as the original.

If using a 40X objective with a numerical aperature of 0.65, what is the resolving power of the objective lens?

423 nm

You should begin viewing a specimen with what objective lens?

4X

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

While looking through a microscope at 400X total magnification, you see this image. To improve the quality of this image, what should you do?

Adjust the condenser aperture diaphragm. The specimen is in focus, but contrast is poor. You can enhance contrast by moving the aperture diaphragm toward the closed position. This restricts the angles at which light strikes the specimen and thereby tends to eliminate the bright, washed-out appearance of the image. This is a better alternative than reducing lamp intensity. Reducing the lamp intensity would make the image less bright, but contrast would remain poor.

You have just rotated the 40X objective into position after viewing the specimen with the 10X objective. What would your next step most likely be?

Adjust the fine focus knob. After changing objectives, you should always check to make sure your image is as well focused as possible. Your objectives are most likely parfocal, so not much adjustment should be necessary. The working distance between the 40X objective and the specimen is very small, so adjusting the fine focus (rather than the coarse focus) will help avoid smashing the objective into the slide.

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?

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.

Immersion oil should be cleaned off an objective with which product?

Lens paper. Using lens paper helps ensure the lens won't be scratched during cleaning. In addition, lens paper is essentially lint-free; no fibers will be left on the lens that could hurt the objective's performance.

Which of the following images shows how to properly open a Petri plate to streak the agar?

Lifting the lid of the Petri plate only enough to get the loop inside keeps the agar protected. Most of the lid remains right above the agar, and this prevents airborne microbes from falling into your culture.

What error in aseptic technique most likely caused this contaminant colony to grow on this agar slant?

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.

In a compound light microscope, what is the function of the condenser?

To concentrate light on the specimen. Light diverges through the air as it exits the light source. The condenser gathers this divergent light and concentrates it on the specimen.

A food microbiologist carried out a serial dilution procedure using a 1-gram sample of ground meat. After incubation, the plates had the results shown in this photograph. What was the bacterial concentration of the original sample of ground meat?

The 10-6 plate is the correct plate to use for counting colonies. There were 58 colonies on this plate. Using the formula below, Concentration (cfu/ml OR cfu/g) = # of colonies X reciprocal of dilution counted concentration is 58 colonies X 106 cfu/g, or 5.8 X 107 cfu/g

A food microbiologist is testing ground meat to estimate the bacterial concentration. He carries out the following serial dilution scheme. What is the total dilution of Plate D's sample?

If 1 ml inoculum is used, the total dilution of the plate is equal to the total dilution of the tube from which it came. The total dilution of the last tube in this dilution scheme is the product of the transfer dilution (10-1) and the total dilution of the dilution bottle from which the transfer came (10-5). Total Dilution = Current Dilution X Previous Dilution = 10-1 X 10-5 = 10-6

Which of the following is an INCORRECT association?

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

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

Isolated colonies are all the same color, have the same colony characteristics and are 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.

A student is counting the number of bacteria in a sample. She uses two methods: Counting bacterial cells with a microscope and A plate count method using serial diluted sample. Why does the plate count have an advantage?

It takes into account only viable (live) bacterial cells, giving a closer approximation to the actual number of cells present, whereas counting cells with a microscope wouldn't distinguish living cells from dead cells. Only living cells that are plated will result in colonies. Therefore, the plate count can be a valuable tool in sanitary and medical microbiology, where viable (living) cell counts are required. The microscope cannot distinguish between living and dead cells.

With respect to the image of a microscope specimen, refraction of light by the objective lens enhances __________.

Magnification. Magnification refers to the apparent size of the specimen when viewed through the microscope. Light bends at the convex surfaces of the objective lens, causing light rays to diverge and radiate outward. This divergence of light creates an image of the specimen that is larger than what would normally be seen by the naked eye.

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.

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

On the bottom of the plate. 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.

Why do we use the term colony-forming units, instead of cells, when expressing concentration of bacteria in a sample?

Sometimes a group of cells forms a single colony. If a cluster of cells is close enough together, they may form just a single colony. Thus, the term colony-forming unit describes both this situation AND the situation where a single cell grows to form a colony. It is a more accurate term to represent the concentration.

A student INCORRECTLY streaks the plate according to the diagram below, creating streaks in the order: a, b, c, d. Which of the following is a streaking error shown in the illustration?

Streak d does NOT draw inoculum from streak c. 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.

A student is learning how to perform serial dilutions, using a stock solution of E. coli. He was instructed to use the dilution scheme shown in the diagram. The stock solution was turbid, as shown below. He delivered 1 ml from each tube into a separate sterile Petri dish and poured melted agar into each dish. When he examined the plates after 48 hour incubation at 37oC, he found no growth in any of the seven plates. What could explain these results?

The agar was not at the correct temperature when poured. If the agar was not cooled to at least 50oC, then the extreme heat would kill the bacteria. This could result in no growth on any of the plates, including the one from the stock sample itself, which is turbid.

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.

This video shows a student incorrectly obtaining an inoculum from a broth culture. What is his error in aseptic technique?

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.

What is the total dilution of tube 5 in this serial dilution scheme?

The dilution of 1 ml from tube 4 into 9 ml of tube 5 is 10-1. To calculate the total dilution of tube 5, multiply this dilution by the total dilution of tube 4, which is 10-4. Total Dilution = Current Dilution X Previous Dilution = 10-1 X 10-4 = 10-5

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.

This video shows a student incorrectly obtaining bacteria from a colony on a Petri plate. What has he done incorrectly?

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?

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.

A student performs a streak plate using inoculum from a broth culture tube. The culture tube was labeled "Escherichia coli," indicating that the culture was composed of a single bacterial species. Which of the following best explains this unexpected plate result seen after incubation?

The loop wasn't flamed 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.

A student is testing a milk sample, and carries out the dilution scheme in the diagram below. What is the total dilution of Tube C?

The total dilution of Tube C is the product of the dilution being made in Tube C (10-1) and the total dilution of Bottle B (10-3). Remember to add the exponents when multiplying these two values. Total Dilution = Current Dilution X Previous Dilution = 10-1 X 10-3 = 10-4

A streak plate was prepared from an unknown culture. Which of the following best explains this plate result seen after incubation?

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.

This video shows a student improperly sterilizing an inoculating loop. What is the student's mistake?

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.

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.

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.

light intensity dial

adjusts light coming from the source

condenser

focuses light through the specimen

coarse focusing knob

for coarse focusing

fine focusing knob

for fine focusing

ocular lens

further magnifies the image and makes it viewable

base

supports the microscope