Microbiology Lab Exam

Name two ways in which you can enhance the resolving power.

...

Assume the diameter of the field of vision in your microscope is 2mm under low power. If one Bacillus cell is 2um, how many Bacillus cells could fit end to end across the field? How many 10um yeast cells could fit across the field?

1000 bacillus cells; 200 yeast cells

If a DNA molecule had the restriction sites A and B for a specific palindrome, how many fragments would be produced?

3

If the GAATTC palindrome is repeated four times on the same piece of DNA, and the restriction enzyme that recognizes that base sequence is present, how many DNA fragments will be produced?

5

How many fragments were produced by the restriction enzyme HindIII?

6

What is meant by control plate? What purpose does a control serve?

A control plate is a guide that is used to help you interpret the experimental results. In this experiment, both (-) pGLO plates are control plates. The LB/amp control plate can be compared to the LB/amp (+)pGLO plate. This comparison shows that genetic transformation produces bacterial colonies that can grow on ampicillin (due to the uptake of the pGLO plasmid and the expression of the ampicillin resistance gene). The (-) pGLO/LB control plate can be compared to any of the LB/amp plates to show that plasmid uptake is required for the growth in the presence of ampicillin. The (-) pGLO LB/amp plate shows that the starter culture does not grow on the LB/amp plate. Without this control one would not know if the colonies on the LB/amp (+) pGLO plate were really transformants

Describe the evidence that indicates whether your attempt at performing a genetic transformation was successful or not successful.

A successful experiment will be represented by the presence of colonies on the (+) pGLO LB/amp and (+) pGLO LB/amp/ara plates and the absence of colonies on the (-) pGLO LB/amp plate. Moreover, the colonies on the LB/amp/ara plate should fluoresce green. An unsuccessful experiment will show an absence of colonies on the (+) pGLO LB/amp and (+) pGLO LB/amp/ara plates. This could be a result of not adding a loopful of plasmid to the (+) pGLO tube or not adding a colony of bacteria to the (+) pGLO tube.

Results: What would you expect your experimental results to indicate about the effect of ampicillin on the E. coli cells?

Antibiotics usually kill bacteria (are bacteriocidic) or inhibit their growth (bacteriostatic). Thus, there should be few, if any, bacterial colonies present on theampicillin plate. The presence of any colonies on the ampicillin plate would suggest that those bacteria are resistant to the antibiotic ampicillin

On which of the plates would you expect to find bacteria most like the original untransformed E. coli colonies you initially observed? Explain your prediction.

Bacteria which resemble the non-transformed E. coli will be found on the LB/(-) pGLO plate. These bacteria were removed from the starter plate, did not have any plasmid added to them, and were replated on an LB plate. Thus, they are virtually identical to the non-transformed starter E. coli.

For a bacterium, what is the evolutionary advantage to forming a pellicle in a liquid medium?

Bacterium can gain easier access to oxygen. It can take in nutrients from the bottom while being near an oxygen supply

In bacterial research assignment, what is the unknown bacterium?

Bartonella henselae

Why are basic dyes more effective for bacterial staining than acidic dyes?

Basic dyes have a positively charged chromogen. In many instances, cell walls and bacteria nucleic acids have negative charges. Because they are negative, they attract and combine to the chromogen.

Recall that the goal of genetic transformation is to change an organism's traits (phenotype). Before any change in the phenotype of an organism can be detected, a thorough examination of its usual (pre-transformation) phenotype must be made. Look at the colonies of E. coli on your starter plates. List all observable traits or characteristics that can be described.

Color of colonies, number of colonies, distribution of colonies on the plate.

Describe how you could use two LB nutrient agar plates, some E. coli, and some ampicillin to determine how E. coli cells are affected by ampicillin

Equal amounts of E. coli cells could be plated on two different LB nutrient agar plates, one which contains just LB nutrient agar and one which contains LB nutrient agar ampicillin. The growth of the E. coli could be compared on the two plates. If ampicillin negatively affects the growth of E. coli, then there should be fewer colonies of bacteria on that plate. If ampicillin has no effect, there should be approximately equal numbers of colonies on both plates

True or false: The high-dry lens will be optimal for observing multicellular fungi or algae.

False

If it were possible to weigh each of the fragments, which one would be the heaviest? Why?

Fragment D would be heaviest because it is the largest piece of DNA and would thus have the greatest mass.

What was the largest organism you observed? The smallest?

Fungi/mold; bacteria

What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions?

Gene regulation allows for adaptation to differing conditions and prevents wasteful overproduction of unneeded proteins. Good examples of highly regulatable genes are the enzymes which break down carbohydrate food sources. If the sugar arabinose is present in the growth medium it is beneficial for bacteria to produce the enzymes necessary to catabolize the sugar source. Conversely, if arabinose is not present in the nutrient media, it would be very energetically wasteful to produce the enzymes to break down arabinose

Observation of a streak plate culture shows more growth in Quadrant 4 than in Quadrant 3. Account for these observations.

Human error: loop touched to a different area which can spread bacteria from one quadrant to another

During the performance of the simple staining procedure, you failed to heat fix your E. coli smear preparation. Upon microscopic examination, how would you expect this slide to differ from the correctly prepared slides?

I would expect there to be no bacteria present.

Why is it desirable that microscope objectives be par-focal?

If the organism is visible in one objective, it will be visible in all objectives

Why was the arrangement of Lactococcus in the broth culture different from the arrangement of the slant culture in the second period?

In the broth culture, many bacteria will grow in a small area and are close together.

How will the arrangement of the Lactococcus lactis in broth differ from the arrangement in the slant culture?

In the broth, the bacteria will grow in a small area and are closer together. In the slant culture, the bacteria will be more spread out.

Did all of the organisms living in or on the environments sampled grow on your nutrient agar?

No, because not all organisms are culturable at this point in time

Should you set the loop down to let it cool?

No, it could become contaminated.

During a coffee break, your friend spills coffee on your lab coat and the fabric is discolored. Is this a true biological stain or simply a compound capable of imparting color.

No, this is not a biological stain. Biological stains are used to view the structures of bacteria.

From your results, can you tell if these bacteria are ampicillin resistant by looking at them on the LB plate? Explain your answer.

No. You cannot tell if the bacteria are ampicillin resistant just by looking at them. Both types of bacteria (those that are ampicillin resistant and those that are ampicillin sensitive) look similar when cultured—think about the colonies on the LB starter plate and the colonies on the +pGLO LB/amp plate

Why do you think that this method of identification would be very exact and precise?

PCR primers are the exact duplicates of the

Which plates should be compared to determine if any genetic transformation has occurred? Why?

The LB/amp (-) pGLO and the LB/amp (+) pGLO plates should be directly compared. Cells which were not treated with DNA (-pGLO) should not be expressing the ampicillin resistance gene and will not grow on the LB/amp plates. Cells which were treated with DNA (+pGLO) should contain the pGLO plasmid and should express the ampicillin resistance gene—the corresponding LB/amp plate will contain transformed bacterial colonies.

What color are the bacteria?

The bacteria on the (+) pGLO LB/amp plate and the (-) pGLO LB plates should be whitish. The bacteria on the (+) pGLO LB/amp/ara plate should appear whitish when exposed to normal, room lighting, but fluoresce bright green upon exposure to the long-wave UV light.

How would you change the bacteria's environment to best tell if they are ampicillin resistant?

The best test would be to take some of the bacteria growing on the LB plate and streak them on an LB/amp plate. If the bacteria are viable on the LB/amp plate, then they are resistant to ampicillin. If no bacterial colonies survive, then they were not ampicillin resistant (they were ampicillin sensitive).

From the results that you obtained, how could you prove that these changes that occurred were due to the procedure that you performed?

The best way is to compare the control to the experimental plates. Cells that were not treated with the plasmid (LB/amp (-) pGLO and LB/amp/ara (-) pGLO plates) could not grow on ampicillin, whereas cells that were treated with the plasmid (LB/amp (+) pGLO and LB/amp/ara (+) pGLO plate) can grow on the LB/amp plate. Thus, the plasmid must confer resistance to ampicillin.

In this diagram. A and B are different palindrome sequences on a DNA strand. Only the restriction enzyme that recognizes site B is present. Explain why only two fragments would be produced.

The enzyme would cut at site B, producing 2 DNA fragments.

Where would the larger fragments — those with the greater number of base pairs — be located; towards the top of the gel or the bottom? Why?

The large fragments would be toward the top of the gel because it is more difficult for the larger pieces to strain through the gel.

Complete this rule for the movement of DNA fragments through an agarose gel:

The larger the DNA fragment, the slower it migrates through an agarose gel.

Which of the two possible sources of the fluorescence can now be eliminated?

The pGLO plasmid DNA and the original bacteria can be eliminated from providing the fluorescent source.

If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, then what can be inferred about the other genes on the plasmid that were involved in your transformation procedure?

The plasmid must express a gene for ampicillin resistance (the protein product of the bla gene codes for beta-lactamase, the protein that breaks down ampicillin).

What does this observation indicate about the source of the fluorescence?

The source of fluorescence is probably from some protein that the plasmid encodes.

What two factors must be present in the bacteria's environment for you to see the green color? (Hint: one factor is in the plate and the other factor is in how you look at the bacteria).

The sugar arabinose in the agarose plate is needed to turn on the expression of the GFP gene. The UV light is necessary to cause the GFP protein within the bacteria to fluoresce.

What do you think each of the two environmental factors you listed above are doing to cause the genetically transformed bacteria turn green?

The sugar arabinose turns on expression of the GFP gene by binding to a regulatory protein, araC, which sits on the PBAD promoter. When arabinose is present, it binds to araC, consequently changing the conformation of araC which facilitates transcription of the gene by RNA polymerase (see detailed description in Appendix D). Exposure to UV light causes GFP to resonate, thereby giving off energy in the form of green light.

If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your prediction

The transformed cells are found on the LB/amp and LB/amp/ara plates. Genetically transformed cells have taken up the pGLO plasmid which expresses the ampicillin resistance gene—these cells can survive on the plates which contain ampicillin

How much bacterial growth do you see on each, relatively speaking?

There should be multiple colonies on both the LB/amp and LB/amp/ara plates that received the pGLO plasmid (optionally ~ 75 colonies). There should be no growth on the LB/amp (-) pGLO plate. There should be a lawn of bacteria on the LB (-) pGLO plate.

Count how many bacterial colonies there are on each plate (the spots you see).

There should be optionally ~ 75 bacterial colonies on the two (+) pGLO plates. The lawn of bacteria on the LB plate contains an even spread of bacteria and individual colonies can't be counted.

Describe the growth pattern of a motile organism in a semi-solid deep.

There will be lateral growth of a motile organism. Growth will move away from the point of inoculation and will look like an inverted pine tree.

How can you tell that a media provided for this exercise were sterile?

There would be no growth on the media provided

True or false: The oil immersion objective is necessary to determine the morphology of prokaryotes.

True

How does increased magnification increase the field of vision?

When magnification increases, it decreases the field of vision

Can simple staining techniques be used to identify more than the morphology of logical characteristics of microorganisms?

Yes, it can also show the arrangement of bacterial cells.

Look again at your four plates. Do you observe some E. coli growing on the LB plates which do not contain ampicillin/arabinose?

Yes. The bacteria that did not receive the plasmid are growing on a plain LB plate.

Can a pure culture be prepared from a mixed-broth or a mixed-broth agar slant?

Yes. Transfer to an agar plate for growth. Then you can isolate colonies

How could you determine whether the turbidity in your nutrient broth tube was from a mixture of different microbes or from the growth of only one kind of microbe?

You could transfer the sample to a Petri dish with agar and see if different types of colonies grow

The portal of entry for Neiseria meningitides is by respiratory tract. Neiseria can be a causative agent of meningitis. If a throat swab is taken and came up positive for Neiseria, why wouldn't this indicate that a person has meningitis?

a positive test indicates that they are carriers of the bacteria; just because you're a carrier does not mean that you are infected with the disease

What are the advantages of the low-power objective for viewing fungi or algae?

able to see the entire organism instead of just a magnified portion of it

Morphology and cell arrangement of lactococcus lactis in broth culture

bacilli; dipolobacillus

Assume you are looking for microorganisms in a tissue sample from a lung biopsy. The microbes become apparent when you switch to 100x. What type of microbe is most likely?

bacteria

A white pellicle has formed on leftover spaghetti you put in the refrigerator a week ago. What is the pellicle?

bacteria such as mold

Why is it essential that smears be air-dried?

bacterial stain will wash it away if it is not dried

What color is gram positive?

blue

Based upon your previous answer, how might targeting these cellular sites in a microbe be responsible for any toxic effects that a disinfectant can have on humans?

can denature proteins, resistance for certain diseases, similar to targeted sites

Explain the medical significance of a capsule.

capable of producing disease

What are possible cellular sites in a microbe that a disinfectant can target?

cell wall, cell membrane, cytoplasm

What three shapes of bacteria did you observe?

cocci, spiral, bacilli

Morphology and cell arrangement of lactococcus lactis in broth culture

cocci; staphylococcus

Equation to determine microorganisms per mL of urine

colonies counted X Inoculating Loop Volume (0.01mL) = microorganisms per mL urine

How do you know if bacteria is present on your agar plate?

colonies of growth will appear

Which is the most crucial step in the performance of the Gram staining procedure?

decolorizing - gram negative will appear gram positive and vice versa

What is the primary use of deeps?

determine motility

What are the advantages of differential staining procedures over the simple staining technique?

easier to differentiate between different microorganisms or cellular components; can see shape, gram reaction, arrangement instead of just morphology

Why is agar preferable to gelatin as a solidifying agent in culture media?

few bacteria can degrade it

How many microorganism per mL could indicate a bladder infection?

greater than 100,000

What other methods can be used to determine motility?

hang drop, bacterial smear, wet mount

What factors can influence the effectiveness of a disinfectant when it is applied?

how old the disinfectant is, how long disinfectant is applied for, concentration, resistance of bacteria, kind and number of microorganism

Purpose of Primary stain in gram staining.

impart its color to the cells

Purpose of Mordant in gram staining.

increase a cell's attraction to the stain

Which controls on the microscope affect the amount of light reaching the ocular lens?

iris diaphram

Why must the loop cool before you touch it to a culture?

kill the bacteria or burn it

Why are thick or dense smears less likely to provide a good smear preparation for microscopic evaluation?

large, thick concentration of cells makes it hard for light to pass through the slide

Why do you think the presence of grease or dirt on a glass slide will result in a poor smear preparation?

may disrupt the amount of light coming through,; can interfere with organism and change its appearance

Why is aseptic technique important?

minimizes contamination and cross-contamination by different pathogens

Can you determine whether a broth culture is pure by visually inspecting it without a microscope? Deep culture? Agar plate?

no; no; yes

Will nutrient broth or agar provide more information on the variety of bacteria in an environment?

nutrient agar

Which objective focuses the closet to the slide when it is in focus?

oil immersion lens

Why should you be careful not to overheat the smear during the heat-fixing process?

overheating can destroy to organism and the morphology of the cell can be changed

Why is a needle used to isolate individual colonies from a spread plate or streak plate?

picks up less microbes, so less of a chance of picking up more than one microorganism/bacterium

What color is gram negative?

pink

Recall what you observed when you shined the UV light source onto a sample of original pGLO plasmid DNA and describe your observations

plasma did not fluroesce

What are bacteria using for nutrients in the agar?

protein within the agar

What is the primary use of broths?

provide large numbers of bacteria in a small space

What is the primary use of slants?

provide solid growth surface that is easy to transport

If the GAATTC palindrome repeats are randomly spaced along the DNA strand, then what can you say about the size of the fragments that will be produced?

random sized fragments

Explain the function of water in spore staining?

remove the excess primary stain

What happens when you use safranin as the primary stain and malachite green as the counterstain?

spores will stain red and cells will stain green

What happens if you use acid-alcohol as decolorizing agent?

spores, stain, and bacteria would be washed away from the slide

What happens if you did not apply heat during the application of the primary stain?

stain will not be able to penetrate the spore

Why is heat necessary in spore staining?

stains are not easily accepted in spores because of its impermeable coat. Heat is needed to penetrate the coats

In collected urine, what species could be present?

staphylococci, streptococci, enterococci, various bacilli, yeasts

What is the purpose of flaming the loop before use? After use?

sterilization

Suppose you had 500 pieces of each of the four fragments, how would the gel appear?

still would only have 4 bands present

How can you determine if the colony that you chose to isolate is a pure culture?

streak a sample to another agar plate and see if more than one type of colony grows

What is the purpose of agar?

support growth on the surface of a Petri plate and it is also used as a solidifying agent

Explain why some microorganisms could be missed by this sampling technique. (source of microorganisms 2 lab)

the general purpose agar supports the growth of many but not all microorganism

What would occur if water were accidentally used in place of mineral oil?

the image would not be as clear because light ray and the water do not have the same refractive index.

What is the value of Petri plates in microbiology?

they provide a solid medium to grow bacteria on

What was the purpose of the DNA Polymerase Reaction?

to amplify DNA samples so there are enough copies for the experiment

Purpose of Counterstain in gram stain.

to show if an organism is gram-negative or gram-positive

Why is it essential that the primary stain and the counterstain be contrasting colors?

to tell the difference between the cells (distinguishable)

How do you determine if the loop is cool?

touch to agar where there is no growth

When is a loop preferable for transferring bacteria?

transferring bacteria slants and Petri dishes

When is a needle preferable for transferring bacteria?

transferring bacteria to a deep

How can a throat infection lead to an infection of the middle ear (otitis media) or Pinkeye (conjunctivitis)?

traveling through ducts; eustachian tube connects throat to ear

Purpose of decolorizing agent in gram stain.

used to establish color contrast; might remove primary stain from from the cell or cell structures

Why can't smears be gently heated over a flame to speed up the drying process?

water will cause bacteria to sizzle and denature

Explain the function of copper sulfate in this procedure (gram staining)?

works as a decolorizing agent

Do you expect turbidity in the unsterilized nutrient broth that was incubated?

yes

Is your lens corrected for chromatic aberrations?

yes