Micro Lab -- answers to all previous quizzes

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What kind of an organism have you identified? Choose a category. for enterobacter cloacae

Coliform bacteria, Gram negative rod

What is the principle behind the use of a spectrophotometer to determine cell density?

The bacterial cells scatter the light as it passes through so the more cells, the less light gets through

Which one of these components is NOT a component of our classroom microscopes described on the webpage?

resolving lens

You have another organism you are trying to identify. You plate it on MSA, and here is what you observe the next day. Can this organism ferment mannitol? (Yellow petri dish with lines of growth)

yes

You are preparing a serial dilution and plan to plate 0.1 mL of your final dilution. Your original culture has a known concentration of 3.4 x 106. You take 4 containers and make four 1:10 dilutions in serial fashion. You add 0.1 mL of your final dilution to your plate with a sterile pipette and cover it up. The next day, you see several large blobs of bacterial growth on the plate, but no individual colonies. What went wrong?

you forgot to spread the drop across the plate with your loop

When performing Kirby-Bauer antimicrobial susceptibility testing, the area of clearing around the antibiotic disk that you see after incubation is called the:

zone of inhibition

Match the type of bacteria with the zone of the Winogradsky column where it occurs. 1. Sulfate-reducing bacteria 2. non-sulfur bacteria 3. heterotrophic bacteria 4. iron-oxidizing bacteria

1. anoxic 2. suboxic 3. oxic 4. suboxic

Put these 4 reagents that are used in the Gram stain in the correct order of use to perform the stain: safranin grams iodine crystal violet decolorizer

1. crystal violet 2. Gram's iodine 3. decolorizer 4. safranin

What is the dilution achieved in the following scenario, using 3 microfuge tubes: You add 99 drops of water to tube #1, and 9 drops of water to tube #2, and 9 drops of water to tube #3. Then you take one drop of a bacterial culture and add it to tube #1. You mix it, and take one drop from tube#1 and add it to tube #2. You mix it, and take one drop from tube#2 and add it to tube #3. What is the Final Dilution in tube#3?

10^-4

If you have a starting culture with a cell concentration of 4.1 x 108. You plan to plate 1 mL of the dilution that will provide countable number of colonies. Calculate the final dilution factor to achieve the countable results. Assume that you are plating on a petri dish with a diameter of 9 cm.

10^7

You also set up a test to see what pathway the organism uses for glucose fermentation. You set up the Voges-Proskauer test (the V of IMViC). After incubating at 37 degrees for 2 days, you add the Barritt's reagents 1 and 2 and look for a color change. You see the following reaction, as a reddish layer forms. Based on this reaction, which glucose pathway do you think this organism uses? (yellow throughout except for a red/orange layer on top)

2,3 butanediol pathway

We counted 22 colonies of lactobacilli on this plate. We plated 0.100 mL of a 10-6 dilution. What is the cell concentration of our original culture?

2.2 x 10^8 cells/ mL

We are setting up 2 plates from a dilution. We would like to have a 1/5 dilution. We only have 10 mL of liquid culture to work with, so we use half of it (5 mL). How much broth do we need to add to the 5 mL of culture to achieve a 1/5 dilution?

20 mL

You are trying to determine the cell concentration in a jar of lactobacilli. You make a serial dilution that has as its Final Dilution Factor 108. Then you take 1 mL and plate it on tomato juice-milk agar. After 2 days, you count 39 colonies. What is the original cell concentration of your culture?

3.9 x 10^9 cells per mL

We are setting up 2 plates from a dilution. We would like to have a 1/10 dilution. We only have 10 mL of liquid culture to work with, so we use half of it (5 mL). How much broth do we need to add to the 5 mL of culture to achieve a 1/10 dilution?

45 mL

You grow a Lactobacilli culture and see growth in a 1 liter jar. You perform several dilutions to obtain a countable plate: 1:100, 1:10, 1:10, 1:10, 1:10. After diluting, you plate a 1 mL aliquot. After 24 hours incubation, you count 57 colonies on your plate. How many cells per mL were in your original stock culture?

5.7 x 10^7

Which laboratory method of disinfection is most similar to using rubbing alcohol in the "residential lab"?

70% ethanol

You are performing a microbroth dilution test with an antibiotic, doxycycline, in order to determine the MIC when tested against Proteus mirabilis. You make a serial doubling dilution of the antibiotic, and then inoculate each well with a standard inoculum of your bacteria. Your concentrations from this dilution are (L to R) 16 ug/mL, 8 ug/mL, 4 ug/mL, 2 ug/mL, and 1 ug/mL. After 18 hours incubation, you check each well for growth. (Growth is indicated by the tan fill color; if the well is clear, there is no growth). What is the Minimum Inhibitory Concentration for this drug vs. Proteus mirabilis? photo: 16ug/mL clear 8 ug/mL clear 4 brown 2 brown 1 brown

8 ug/mL

These three slides show bacteria that stain dark purple (A) pink (B), and dark purple (C). One of these shows a Lactobacillus spp. Which is the Lactobacillus? Photo A: purple rods Photo B: Pink rods on green background Photo C: purple coccus grouped together

Most Lactobacillus species are aerotolerant anaerobes. Which tube would they likely be growing in?

A throughout the tube except for the little bit at the top

For the first step in your identification process you will use various media to see what carbohydrates your organism can ferment. Triple Sugar Iron Agar (TSIA) slant is a differential media that contains 3 sugars; Eosin Methylene Blue (EMB) is a selective AND differential media that selects for Gram negative organisms, and differntiates between lactose fermenters and lactose non-fermenters. MacConkey (MAC) is also a selective AND differential media that selects for Gram negative organisms, and differentiates between lactose fermenters and lactose non-fermenters. You will plate your unknown of the EMB and MAC, and inoculate the TSIA slant with a needle. After 18-24 hours incubation at 37 degrees, your TSIA looks like this. (You can ignore the little bit of red on the tip of the slant -- it might have incubated a little too long.) (Yellow butt that is broken halfway with the slant that is red/orange)

A/A with gas

A/A, H2S, no gas

You have isolated what you think is a member of the genus Staphylococcus. Which of these Gram stains would you expect to see? A- pink, rod shaped bacteria B. Purple, sphere-shaped bacteria

You want to test your organism -- that you think is a member of the Staphylococcus genus -- with the catalase test, so you add a drop of reagent to a glass slide and then add some of your organism. Which reaction do you expect to see? A- no bubbles/negative B- yes bubbles/ positive

Match the component of Mannitol Salt Agar (MSA) with the function of that component. carbon source=? selects for staph=? pH indicator=?

C source= mannitol selectivity = 7.5% salt pH = phenol red

Identify the special bacterial structure seen in this slide. The bacteria have been stained with Gram stain and stain Gram variable (both pink and purple), and the special structure is clear. (gram stain photo)

Endospores

Cyanobacteria obtain the energy, carbon, and electrons they need from ________

Energy from sunlight, carbon from inorganic compounds, and electrons from water

When testing water supplies for potability (drinkability), investigators will test for indicator organisms. These are organisms that are not pathogenic (disease-causing) in themselves, but that indicate when water might be contaminated. Which of the following is a typical indicator organism?

Escherichia coli

The dichotomous key on the webpage separates the organisms into two main groups. Which of the following organisms are lactose fermenters? Choose all that apply.

Escherichia coli AND enterobacter cloacae

Here is a picture of yeast that have been stained using the Gram staining technique. They are dark purple. Do yeast stain Gram positive or Gram negative? (purple background, dark purple circles)

Gram positive

Thee are only a few dark purple bacteria in this slide. Identify the Gram reaction and morphology. (green background with dark blue/purple circles)

Gram positive cocci

Identify the Gram stain reaction and the bacterial shape (morphology) of the dark purple bacteria you see in the following slide: (purple sticks)

Gram positive rods

What is an accurate description of Lactobacilli?

Gram positive rods that are aerotolerant anaerobes and can ferment lactose and glucose

A table of standard breakpoints is found here: (graph) This Kirby-Bauer result was obtained for Proteus vulgaris vs. Tetracycline. The zone of inhibition was measured at 15mm. What is the interpretation of this result, according to the breakpoints provided? Tetracycline row of graph [30ug] in disk <15= resistant 15-18= intermediate >18= susceptible

Intermediate

A table of standard breakpoints is found here: (graph) This Kirby-Bauer result was obtained for Staphylococcus aureus vs. Penicillin. The zone of inhibition was measured at 26 mm. What is the interpretation of this result, according to the breakpoints provided? Penicillin *for staphylococci* ros in chart [10 units] in disk <21 = resistant 21-28 = intermediate >28= susceptible

Intermediate

What kind of an organism have you identified? Choose a category. for shigella flexneri

Intestinal pathogen, Gram negative rod

The agar in the media we made serves what function?

Is a solidifying agent that provides a solid surface for bacteria to grow on.

What is the purpose of the small tube inside the larger tube?

It captures any gas formed by the fermentation process

For Kirby-Bauer testing with disk diffusion, we use Mueller-Hinton agar. Which one of the following is NOT a reason why this agar has been chosen as the standard for this type of testing?

It contains ions of mineral salts that are needed for the antibiotics to be effective

How is catalase production helpful for the bacteria?

It enable the organism to break down by products of oxygen metabolism

What is the purpose of the milk in the homemade media we are making?

It provides a carbon source (carbohydrate) for the bacteria

K/A, no gas

After incubating the indole broth and adding the reagent, you get a result. What is the reagent you added?

Kovac's reagent

You also plated your unknown on MacConkey media. This is how your plate looked after incubation. How would you record this result? (bright red/pink)

Lactose fermenter

You have also plated your organism on an EMB plate. This is how that plate looked after 18 hours incubation, with dark purple colonies. How would you record this result?

Lactose fermenter

Which of these staining reagents is used in staining endospores?

Malachite green

Many of the antibiotics currently in use are substances that are produced by microorganisms as a means of defense against other bacterial competitors. Which of the following is a true statement that follows from this observation?

Most antibiotics are substances that will target prokaryotic cells, but not eukaryotic cells (such as human cells)

You want to set up one more of the IMViC tests, so you inoculate a citrate slant with your organism. Here is your result. Is this positive or negative for citrate utilization? green slant and butt

Negative

What kind of an organism have you identified? Choose a category. for proteus vulgaris

Non-Enterobacteriaceae, Gram negative rod

You also plated your unknown on MacConkey media. This is how your plate looked after incubation, with clear or tan colonies. How would you record this result? (light yellow pink result)

Non-lactose fermenter

You also plated your unknown on MacConkey media. This is how your plate looked after incubation. How would you record this result? (yellow)

Non-lactose fermenter

You are working in a hospital lab and are working up an isolate from a urine sample. You plated your unknown isolate on MacConkey media. This is how your plate looked after incubation. How would you record this result? (yellow plate)

Non-lactose fermenter

You have also plated your organism on an EMB plate. This is how that plate looked after 18 hours incubation, with clear or tan colonies. How would you record this result? (brownish media still with white colonies)

Non-lactose fermenter

You have also plated your organism on an EMB plate. This is how that plate looked after 18 hours incubation. How would you record this result? (red plate)

Non-lactose fermenter

You are working with a mixed culture of bacteria. You have made several dilutions and have plate 1 mL of three different dilutions: 10-5, 10-6, and 10-7. Unfortunately, you forgot to label them and forgot which was which. You let the plates grow overnight, and then look at the plates. Now you can tell which dilution goes with which plate. Match the dilution with the Plate identified: Plate A: medium number of big red colonies and white colonies Plate B: a lot of both types of colonies Plate C: basically nothing growing

Plate A: 10^-6 dilution Plate B: 10^-5 dilution Plate C: 10^-7 dilution

At this point, you think you know what your unknown organism is, but you want to set up one more test. You set up the urease test. Here is your result. Is it positive or negative? (pink)

Positive

The catalase test is especially useful for distinguishing between which two groups of organisms?

Staphylococci and streptococci

Look at the following chart of characteristics of various antibiotics. Penicillin-cidal/Narrow-spectrum (mostly Gram positives) Streptomycin-cidal/Broad spectrum Tetracycline-static/Broad spectrum Rifampin-cidal (used to treat tuberculosis)/Broad spectrum Nitrofurantoin-static in most tissues/Broad spectrum Polymyxin B-cidal/Narrow spectrum (Gram negatives) You are trying to treat a leg wound where the specimen grew both Staphylococcus aureus and Escherichia coli. You would like to be able to treat the patient with one drug, so as to minimize the chances of side effects. What are all the drugs on this list you could consider for this treatment?

Streptomycin, Tetracycline, Rifampin, and Nitrofurantoin all the broad spectrum

We added an egg yolk to our Winogradsky column as a source of:

Sulfur as a source of energy and reducing power

You are trying to determine the cell concentration of a jar of lactobacillus culture. You remove one drop from your culture and use it to make a series of dilutions, and then plate 0.1 mL the final dilution. Your final dilution looks like this: (TMTC/Lawn growth photo)

Take another 1 drop sample from your jar of culture, and perform another dilution series, increasing the number of dilutions that you make. Then plate 0.1 mL of your final dilution of this new series.

Tetracycline and Nitrofurantoin the -static ones

Here is a picture of different bacterial species growing in thioglycollate media. This media is used to test bacteria's oxygen requirements. Match the description of bacterial growth to its oxygen requirement. obligate aerobes

They need oxygen for aerobic respiration and grow at the top of the tube

Below are the TSIA results for Proteus vulgaris (black looking butt and orange trail) and Salmonella Enteriditis (black looking butt and pink/red trail). Based on these results, what physiological characteristic do these two organisms share?

They both produce H2S

They can grow best in the presence of oxygen but can grow in anaerobic environments. Grow throughout the tube

They can grow in small amounts of oxygen a distance from the top since they are damaged by the concentration of oxygen found in the atmosphere

They cannot use oxygen and are harmed by its presence. Grow near the bottom of the tube

They don't use oxygen but are not harmed by it. Tend to grow near the bottom of the rube

You are preparing a Gram stained slide of lactobacilli. You perform the staining procedure, and then view the slide under the microscope. You see pink rod-shaped organisms. What could account for this result?

This is not the expected result. You may have left the decolorizer on too long.

You are preparing some media for your experiment. You wipe down your work surface with isopropyl alcohol, you boil the water you are using, you wipe the inside of your media container that you will pour the media into with an alcohol wipe. Oops! You dropped your container on the floor and the lid came off and rolled around. What is your best next step?

Use another alcohol wipe to clean the inside of the container and lid OR wipe the inside and outside of the container and lid, even though you need to use up an extra wipe to do it

Which tests require that a reagent or reagents be added before the test result can be read? Choose all that apply.

Voges-Proskauer and Indole

You are the laboratory coordinator for a large microbiology class. The experiment you want your students to do requires a diluted bacterial culture. When you tested the experiment, you made a dilution of a bacterial culture using 1 drop of culture and 9 drops of water. This worked fine, but now you need a much larger volume to dispense to your 250 students. You need a full liter of diluted culture to accommodate all the students, but you want the dilution to be the same. If you need a total of 1 liter of liquid, how much culture should you use and how much water should you use in order to achieve the same level of dilution as your original test?

You would have to do the same ratio that you did, but on a larger scale. You would need 100mL of the culture and 900mL of the diluent (in this case water) to make 1 total L of dilution.

You are collecting and testing samples of a pond to test for the presence of coliforms. You are looking for lactose-fermenting organisms, such as Escherichia coli. You are able to isolate some species of bacteria from your samples. One of these isolates you re-streak to an EMB plates and obtain the following result after 18 hours incubation: (red plate with green streaked colonies) Which of the following statements should appear on your report to the owner of the pond?

Your pond shows the presence of indicator organisms, indicating possible contamination from sewage or other waste sources.

A species of bacteria use fermentation to obtain useable energy (ATP) and carbon. It does not use oxygen as the final electron acceptor, but is not harmed by the presence of oxygen. It is classified as

an aerotolerant anaerobe

Bacteria that make their organic compounds from carbon dioxide are called:

autotrophs

Which ingredient makes MacConkey agar selective?

bile salts, since it selects for Gram negatives

You would like to sample your Winogradsky column to see what microbes are in the various layers of your column. You will use a straw inserted through your column in order to sample sediment containing bacteria from the layers, and then plate your samples. Three different colored layers have been identified on the image below. In your lab, you only have access to an aerobic incubator. Which color layer will probably not yield any growth if the culture is incubated in that incubator? black, purple or green

black

Urease and phenylalanine deaminase both fall into what category of biomolecules?

enzymes

Which is the phase of bacterial growth where the bacteria are dividing rapidly?

exponential phase/ log phase

T/F: Gram negative bacteria have a thick peptidoglycan layer in their cell wall that causes them to retain crystal violet and resist decolorization.

false

You are trying to determine which sugars an organism is able to ferment. You plate the organism on MacConkey and get this result: (yellow petri dish) Then you streak it on a TSIA slant and get this result: (black looking butt and orange streak) What sugar(s) can you definitely say that the organism ferments?

glucose and sucrose

What is the reagent used for the catalase test?

hydrogen peroxide

Which statement is true about the use of immersion oil with the mciroscope?

immersion oil should be used only with the 100x objective

What metabolic by-product causes the de-naturing of casein proteins in milk, when Lactobacillus culture is added to milk?

lactic acid

What metabolic by-products cause the de-naturing of casein proteins in milk, when Lactobacillus culture is added to milk?

lactic acid

As a Winogradsky column develops, microorganisms in some layers may produce gases that appear as bubbles in the column. Gas bubbles that appear in the bottom of the column are most likely to be:`

methane

You also set up a test to see what pathway the organism uses for glucose fermentation. You set up the methyl red test (the M of IMViC). After incubating at 30 degrees for 5 days, you add the methyl red reagent and look for a color change. You see the following reaction. This indicates which glucose fermentation pathway? (cloudy grey on bottom with pink/red/orange top layer)

mixed acid pathway

At this point, you think you know what your unknown organism is, but you want to set up one more test. You set up the urease test. Here is your result. Is it positive or negative? (orangie yellow)

neg

At this point, you think you know what your unknown organism is, but you want to set up one more test. You set up the urease test. Here is your result. Is it positive or negative? (yellow)

neg

You also set up a test that looks for the production of an enzyme that can deaminate phenylalanine (and amino acid). You streak the media, and then incubate. The next day you add ferric chloride and get no color change. Is this reaction positive or negative? (clearish slant with light yellow/green streaks in it)

neg

Your indole test result looks like this. Based on this result, is your organism positive or negative for indole production? (grey cloudy through the whole tube)

negative

You have isolated an organism that you believe is a streptococcus species. You plate it onto an MSA plate. What do you expect to see the next day?

no growth

A bacterium is classified as a chemolithoheterotroph. This means it:

obtains energy from preformed molecules, uses inorganic compounds as an electron donor, and uses organic compounds as a carbon source

You want to set up one more of the IMViC tests, so you inoculate a citrate slant with your organism. Here is your result. Is this positive or negative for citrate utilization? (blue slant and butt)

pos

You also set up a test that looks for the production of an enzyme that can deaminate phenylalanine (and amino acid). You streak the media, and then incubate. The next day you add ferric chloride and get a green color. Is this reaction positive or negative? (very distinctively green)

positive

What are the three sugars in Triple Sugar Iron Agar. Choose all that apply.

sucrose, lactose, and glucose

Which is the correct definition of "resolution" in the context of microscopy?

the power to see two objects as separate

When we made media for our experiments, we added some milk as a carbohydrate. The milk in our media has a function most similar to which component of the Winogradsky column?

the shredded paper

T/F: Lactobacilli and enteric lactose-fermenting organisms have this in common: both groups of organisms are able to produce energy without the use of oxygen as an electron acceptor.

true

After reading these initial results, you set up several of the IMViC tests. The first one is the Indole test, so you inoculate a broth with our organism. This test looks for the production of an enzyme that can split an amino acid into indole and pyruvic acid. What is the name of this amino acid?

tryptophan

What is one test that can help distinguish Proteus vulgaris and Salmonella Enteriditis?

Micro Lab -- answers to all previous quizzes

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