Lab exam 2 questions from lab manual

Which types of bacteria can grow on MacConkey agar, on EMB, on T-7? Why?

-MacConkey, EMB, and T-7 agars are all designed for use in the differentiation of Gram-negative bacteria. -Most types of Gram-positive organisms will not grow on these media because they are inhibited by one or more of the substances present.

What is a bacteriophage; a virion; a plaque?

A bacteriophage is a virus that infects bacterial cells. A virion is a virus particle as it exists outside the host cell, or free in the environment. It is the complete, infective form of a virus. A plaque is a cleared area or "window" visible in a lawn of bacteria on a plate. It is due to cell lysis caused by a lytic bacteriophage present in the culture.

What are dideoxynucleoside triphosphates (ddNTPs) and how do they affect DNA replication if they are incorporated into growing nucleotide strands?

A dideoxynucleoside triphosphate is an analog of a nucleoside triphosphate (e.g., dATP, dGTP, dCTP, etc.) that lacks a hydroxyl group (OH-) on the #3 carbon of its sugar. When these molecules are incorporated into growing nucleotide strands during replication, they terminate the strand (stop the replication process) because new, incoming bases cannot bind to them (they do not have the required 3' OH- group).

The plasmids pUC19, pGEM, and pGLO carry marker genes that code for resistance to what type of antibiotic?

All have the bla gene which codes for β-lactamase. This enzyme confers resistance to ampicillin.

Why is amplification of 16S ribosomal DNA (rDNA) likely to yield a product that can be used in the identification/ classification of unknown bacteria?

All known bacteria contain ribosomes and produce 16S ribosomal DNA. This DNA is highly conserved (has not changed much through evolutionary time) so serves as a good indicator of the evolutionary relationships between bacteria.

What is an electropherogram?

An electropherogram is a four-colored chromatogram generated by an automated sequencing machine. Each electropherogram displays sequencing results as a series of peaks accompanied by the machine's interpretation of this data presented as a text file.

What are antibiotics? How do broad and a narrow spectrum drugs differ?

Antibiotics are antimicrobial agents that, at least initially, were produced by living organisms. Broad-spectrum antibiotics will kill or inhibit the growth of a wider range of microorganisms (more different types) than will narrow spectrum drugs.

What are antimicrobial agents?

Antimicrobial agents are a group of chemotherapeutic agents that can be used systematically in the treatment of infectious disease.

What do the letters BLAST stand for and how was the BLAST algorithm used in association with this exercise?

BLAST = Basic Local Alignment Search Tool, and was used to compare student nucleotide sequences with those available in the public database of NCBI.

How can you determine if an organism is motile by observing its growth pattern in SIM media?

Bacteria that are non-motile cannot "swim" through the SIM medium, and so grow only along the line of inoculation. If a distinct line is observable, the organisms in question are not motile. Motile organisms will distribute themselves throughout the medium, and no line of inoculation will be visible.

Why might investigators want to remove plasmid DNA from bacterial cells, i.e., what purpose does this procedure serve?

Bacterial plasmids can be used as cloning or expression vectors. Typically, investigators remove plasmid DNA from host cells, cut the plasmids open using restriction enzymes, insert pieces of DNA (genes) that are being investigated, and then reintroduce the plasmids as closed loops containing new pieces of DNA. Since the plasmids replicate themselves (and because their host cells replicate rapidly), they can be used to produce large quantities of the particular genes being studied. Removing plasmid DNA from host cells is often the first step in genetic engineering or gene manipulation experiments.

What is bioinformatics?

Bioinformatics is a science that involves the application of computer technology to the study of various aspects of living organisms.

What are the differences between blunt and cohesive termini? Which of these is produced when DNA is cut with EcoRI and which when DNA is cut with AluI?

Blunt ends are formed when a restriction enzyme cuts both strands of a DNA molecule at the same location (straight across), leaving no unpaired bases. The enzyme AluI forms blunt ends. Cohesive termini are formed when a restriction enzyme cuts the two strands of a DNA molecule at different points within the recognition sequence leaving one strand longer than the other by one or more bases. The unpaired bases form the cohesive termini or sticky ends (because these can bind to complimentary strands through hydrogen bonding). The enzyme EcoRI forms cohesive termini four bases in length.

What are cloning vectors and expression vectors and what are they used for in genetic manipulation experiments?

Cloning vectors are pieces of DNA that can initiate their own replication when inserted into host cells (bacteria, yeast, or others). They are used to carry a gene into bacteria in Recombinant DNA Technology. Expression vectors are similar to cloning vectors, but have signals that allow the host cell to also express the gene spliced into the vector in addition to replicating it.

What are competent cells?

Competent cells are those able to take in DNA from the environment. Although in nature such cells can only take in DNA from closely related cells, under laboratory conditions cells can be made to take up DNA from any source.

Why do DNA molecules migrate when subjected to an electric current? In which direction do they travel (toward the anode or cathode)?

DNA molecules migrate because they have a net negative change. They tend to travel toward the positive pole.

Which of the bacteria strains tested was a better host for the coliphage T2? How did you know?

E. coli strain B was the better host for the coliphage T2 as indicated by the number of plaques formed when a specified concentration of virions were used to infect a population of cells. When the host strain E. coli C was exposed to the same type of virus, at the same concentration, fewer plaques were formed.

Which of the bacteria strains tested was a better host for the coliphage X174? How did you know?

E. coli strain C was the better host for the coliphage X174 as indicated by the plaques formed when a specified concentration of virions were used to infect a population of cells. When the host strain E. coli B was exposed to the same virus, no plaques were formed.

What is gel electrophoresis? What is this procedure used for?

Gel electrophoresis is a procedure that uses an electrical current to separate DNA fragments into discrete size categories. In gel electrophoresis, the DNA fragments are first loaded into wells in a slab of agarose gel, and then subjected to an electrical current. The smaller DNA fragments travel faster through the gel, and so tend to move away from the larger fragments. The procedure has many applications, but in this laboratory is used to indicate the success of the miniscreen and PCR procedures.

What morphological differences can you detect between the colonies growing on MacConkey's agar and EMB?

Gram-negative bacteria growing on MacConkey's agar appear opaque pink if they are lactose-positive (can ferment lactose), and "clearish" if they are not. Colonies on EMB appear dark, or have dark centers, often with a metallic green sheen, if they are lactose-positive, and are "clearish" if they are not.

A student streaks his unknown onto a Mueller-Hinton Agar plate. Following incubation, the entire plate around the colonies is green. What does this tell him about his organism?

His unknown produces a pigment.

Hydrogen sulfide (H2S) is produced by certain bacteria that are able to catabolize what type of organic compounds?

Hydrogen sulfide is produced by certain bacteria that are able to catabolize certain amino acid molecules.

What does a gamma-hemolysis result on blood agar tell you about an organism? What does a gamma-hemolysis result look like?

In a gamma-hemolysis result, there will be no change to the medium around the colonies and means that the organism cannot breakdown red blood cells. (no hemolysis)

Were the chemical agents you tested more effective in the control of Gram-negative organisms or Gram-positive organisms? What explanation can you give for the variation in results obtained?

In general, Gram-negative organisms are less sensitive to chemicals than are Gram-positive organisms, but different chemicals will have variable effects on the organisms being tested.

What function do restriction endonucleases have in vivo?

In vivo, these enzymes appear to be involved in the protection of bacteria against viral invasion and in the maintenance of species specificity.

What were the approximate sizes of the bacteriophage lambda DNA fragments generated during this exercise? Which of the various plasmids used is larger in size?

Lambda DNA cut with HindIII will yield eight fragments that are 23,130, 9,146, 6,557, 4361, 2322, 2027, 564 and 125 base pairs in length. The plasmids pGEM and pUC19 are roughly the same size (~3000bp), but pGLO is larger than both (~5000bp).

What is lysogeny? Did any of your bacteria become lysogenic? What is the condition of the viruses within cells that have undergone lysogeny?

Lysogeny is a condition or relationship between a bacteriophage and its host cell in which the virus has become incorporated into the host cells chromosome. You may or may not see evidence of lysogeny. If you see evidence of lysogeny (turbid plaques or colonies within plaques), the cells growing within these are carrying viruses that have become prophages, i.e., have become incorporated into the host cells' chromosome.

Were more bacteria able to grow on the ampicillin plates, or on the TSA plates? Why would you have expected these results?

More bacteria grew on the TSA plates. This would be expected since only those cells that had been transformed (had picked up the plasmid DNA) would be able to grow on the ampicillin plates. All cells should be able to grow on the TSA plates.

What do the letters NCBI stand for in association with this exercise, and what kinds of information are available at the NCBI website?

NCBI is the National Center for Biotechnology Information and is a public database containing a tremendous volume of information e.g., nucleotide and amino acid sequences obtained from a wide variety of organisms, the human genome project and scientific articles from multiple publications, many freely accessible.

A student streaks her unknown onto a bile esculin slant. Following incubation, the slant is brown and no growth is visible. What does this tell her about her organism?

No growth is visible, so her unknown is sensitive to bile salts. It is not possible with this medium to know if the organism can hydrolyze esculin (as it is not growing).

A student determines that his A and B organisms are both Gram-positive? What does he do next?

No student should have two Gram-positive unknowns. He should begin by performing a second KOH test on each organism to make sure that he interpreted those results correctly. If that doesn't help, then he should consult his instructor for help.

What percentage of cells in a bacterial population are usually made competent by treatment with ice cold calcium chloride?

Only about 10% of the viable cells in a population are made competent to receive DNA.

Why is it possible to separate plasmid DNA from chromosomal DNA using fairly simple centrifugation steps?

Particles of significantly different sizes will tend to separate when subjected to centrifugation, because the larger, heavier particles settle to the tube bottom more quickly than will the smaller, lighter particles.

How many different types of plaques were visible on your phage isolation plate? How many different types of virus appear able to infect E. coli strain C?

Phage isolation plates will show considerable variation, however at least 3-4 different types of plaques are likely. Each different looking plaque type represents a different type of virus.

What is phage typing, and why can viruses be used to identify specific types of bacteria?

Phage typing is a bacterial identification method that involves the use of known bacteriophages (viruses that infect bacteria) to identify unknown bacteria. Viruses can be used to identify specific types of bacteria because they are host specific, i.e., each type of virus will infect only a certain type of host cell.

Why is plasmid DNA frequently used in transformation procedures?

Plasmid DNA is fairly easily removed from cells via miniscreen procedures, and so is readily available. Plasmids can be used to carry genes into host cells and will reproduce themselves there, thus replicating the genes they carry.

How does plasmid DNA differ from chromosomal DNA?

Plasmid DNA occurs in loops that are much smaller than the loops formed by bacterial chromosomal DNA. (A plasmid model the size of a hula-hoop would compare fairly accurately to a chromosome the size of a standard college-size track).

What are primers, and why are they necessary in the PCR, i.e., what function do they serve?

Primers are short segments of DNA (or RNA) usually 18-20 bases in length, that are able to hybridize with (anneal to) specific regions of DNA. They provide the free 3' ends required by Taq polymerase to build new complimentary strands of DNA. Remember, DNA polymerases cannot attach to single-strand nucleic acid, so a primer allows the Taq to attach.

What is RFLP and what role does this technique play in bacterial identification?

RFLP (Restriction Fragment Length Polymorphism) is a technique or method used in the analysis of DNA, and involves cutting a sample of DNA with a restriction enzyme and subjecting the resulting fragments to gel electrophorsis. The pattern created is sometimes called a DNA fingerprint, but may also be referred to as a RFLP pattern.

What are restriction endonucleases? Where are they normally found?

Restriction endonucleases are enzymes that catalyze the "site-specific" cleavage of DNA molecules. They are normally produced, and therefore found, within bacteria cells.

What function do restriction enzymes have in recombinant DNA technologies?

Restriction endonucleases are used to "cut" chromosomal DNA into small fragments and to cut loops of plasmid DNA so that the small fragments of chromosomal DNA (carrying genes) can be inserted into them.

Why was it necessary to "flip" or "reverse" the 1530-Reverse sequence before copying it to the word file? What would occur if you failed to do this?

Reverse sequences are constructed on primers that anneal to DNA strands complimentary to those used for building Forward sequences. For this reason, unless Reverse sequences are "flipped" or "reversed" they cannot be combined with Forward sequences. It would be impossible to obtain a contiguous 300 sequence of maximum length if the Reverse sequence was not "flipped" or "reversed" before it was copied.

A student determines that her A and B organisms are both Gram-negative? What does she do next?

She should inoculate an O/F test for each organism, as one of them is her enteric (and will be O/F positive) and the other is not (O/F negative). Then she can consult the dichotomous key to determine what the next step is.

What name would be given to the second restriction endonuclease system found in association with Bacillus stearothermophilus strain ET?

Such an enzyme would be called BstEII.

What is Taq polymerase and where does it come from?

Taq polymerase is a type of enzyme (DNA polymerase) produced by hyperthermophilic bacteria (Thermus aquaticus) that live in the hotsprings of Yellowstone National Park. This enzyme is frequently used to replicate DNA in polymerase chain reactions.

Which of the following DNA fragments would you expect to travel a greater distance from the loading well within a given period of time; a 500 base pair fragment, or a 300 base pair fragment? Why?

The 300 base pair fragment will travel the greater distance because it is smaller, and can more readily pass between the molecules in the gel.

Is the sample of PCR product DNA larger or smaller than the plasmid samples used?

The PCR product DNA is smaller than the plasmids. It is only ~1500bp in length.

Following incubation, a TSI slant has a yellow slant and a black butt. What does this result tell you about the organism?

The black indicates that the organism can produce H2S from amino acids. Although the pH of the butt cannot be determined, the slant is yellow which indicates that the organism ferments more than one of the sugars present in the tube.

The presence of hydrogen sulfide is indicated in TSI and SIM media by the formation of what?

The black precipitate present is iron sulfide and forms when the sulfur from H2S binds with iron in the medium.

What is burst size as described in this exercise, and how is it determined?

The burst size is the number of free virions released from each infected cell in the population. It is determined by dividing the maximum number of PFU present at the end of the latent period by the initial number of PFU (infected bacterial cells) present at the start of the exercise. In order to determine these numbers, it is necessary to know the dilution factor for each plate being counted, and to recognize that a PFU (plaque forming unit) can be either a free virion or an infected cell

What is the burst time as described in this exercise and how is it determined?

The burst time is the time required for a virus to complete its life cycle within a host cell, and escape from that cell (the time from adsorption/attachment to release). To determine the burst time, look for a sharp increase in the number of PFU as plotted on semilog paper. The time elapsed between time zero and the point of this sharp increase is the latent period.

Why is it necessary to inoculate a control tube containing glucose without any amino acid whenever you are testing a culture's ability to decarboxylate lysine?

The control tube contains the same glucose that is present within the lysine tube, but lacks the lysine. For this reason, it gives a clear indication of whether or not the bacteria being tested can ferment the glucose. If the glucose cannot be fermented (control tube stays purple) the lysine test is invalid, because there is no way to determine if the purple color is due to amine formation, or if it is due to no reaction at all. Note that it is essential to inoculate both the control tube and the lysine tube with a visible amount of the bacteria being tested, and that both tubes must be sealed with vaspar.

What is the key ingredient of the catalase test? What does a positive catalase result look like?

The key ingredient is 3% H2O2. A positive result will show obvious bubbles as soon as the organism is mixed with the hydrogen peroxide.

How would you interpret the results of an O/F test if the sealed tube showed no change in color and the unsealed tube was yellow only near the surface of the medium and not within the Durham tube?

The organisms present are obligately aerobic and cannot utilize the carbohydrate present under anaerobic conditions (they cannot ferment). They are able to produce some acid when utilizing the carbohydrate aerobically.

What is the overall effect of high temperatures (heat) on bacteria suspended in fresh water? Does exposure time influence this effect?

The overall effect of high temperatures (heat) is to denature proteins and stop metabolic activity. If temperatures are high enough, the exposed organisms will die. After one minute of exposure to boiling water, most vegetative cells were killed, but endospore-forming bacteria and some other forms survived. After five minutes of exposure, few if any of the bacteria present remained viable. Exposure time does influence the effects of temperature.

What is being tested for in the oxidase test? What does a positive oxidase result look like?

The oxidase test will determine if an organism has cytochrome C (cytochrome C oxidase) in its electron transport chain. A smear of organism on the test paper will turn blue within one minute in a positive reaction.

What is the pH indicator present in Simmons Citrate agar? What happens to the pH of this medium when an organism uses citrate?

The pH indicator present in Simmons citrate agar is bromothymol blue. This indicator appears greenish in neutral media, but turns a deep blue if the pH 298 increases. This medium contains sodium citrate as the only carbon source. If bacteria are able to utilize citrate there is a decrease in acid content within the medium (thus a rise in pH) and the agar turns blue.

How do the plaques formed by the coliphage X174 differ from those formed by the coliphage T2?

The plaques formed by the coliphage X174 are larger than those formed by the coliphage T2, and therefore easier to see.

What is the PCR and what is it being used for in this exercise?

The polymerase chain reaction is a unique process that allows specific segments of DNA to be amplified (replicated millions of times) in vitro within a few hours. It is an extremely powerful tool with multiple applications in biotechnology. In this exercise, the PCR is being used to amplify segments of 16S ribosomal DNA (rDNA) from bacteria.

Why must the DNA polymerase used in PCRs be thermostable?

The polymerase chain reaction requires that DNA be heated to around 94oC, multiple times (around 30-35) during processing. Exposure to such high temperatures will often inactivate (denature) enzymes. If the Taq polymerase were not thermostable, the PCR would not work.

What is a genome and how is it related to genomics?

The term genome is usually defined as the total DNA content of the chromosome(s) within a cellular organism. It may be RNA in some viruses and is RNA in viroids. Genomics is the study of genomes from multiple different organisms as a single functional unit, i.e., all at once.

An unknown organism is growing on MSA and the medium around its colonies is yellow. What does this result tell you about the physiology of the organism?

The unknown organism can tolerate 7.5% salt since it is growing. The yellow medium indicates that it can ferment mannitol.

What is a zone of inhibition, and what is meant by minimum inhibitory concentration?

The zone of inhibition is the cleared region around the sensitivity disc where microbial growth is inhibited. The minimum inhibitory concentration is the lowest concentration of the drug being tested that will inhibit the growth of the microbe. The MIC is found at the outermost edge of the zone of inhibition.

What is transformation?

Transformation (as it occurs naturally) is the process by which DNA is transferred from dead donor cells to living recipient cells. Under experimental conditions the DNA being transferred is artificially extracted and often modified prior to being introduced into new host cells. The efficiency of transformation is also greatly increased.

Indole is produced when the amino acid ______________________ is degraded by certain bacteria.

Tryptophan

What is ultraviolet radiation? What wavelength of UV radiation is most effective in controlling microorganisms?

Ultraviolet (UV) radiation is electromagnetic radiation with a wavelength of less than 400 nm. (usually designated as 4-400 nm). Ultra violet radiation with a wavelength of 260-270 nm is considered to be the most effective against microorganisms because it is known to be strongly microbicidal and mutagenic.

What pH indicator is present in urea agar? What chemical change is responsible for the color change apparent in this medium when urea is catabolized?

Urea agar contains the pH indicator phenol red. This indicator appears as a peach color prior to inoculation (pH 6.8), and becomes "hot" pink if the urea present is catabolized. The hydrolysis (catabolism) of urea yields ammonium, which is basic (pH 8.1 or greater), and this is responsible for the color change.

What is vaspar? Why is it necessary to use this material with the lysine decarboxylation test?

Vaspar is a mixture of Vaseline and paraffin wax. It is used during the lysine decarboxylation test to seal the tubes and thus prevent the escape of volatile amines. Without some type of seal, most of the amines produced will leave the solution, and the test results will appear negative. In addition, it keeps oxygen out of the tube, so the bacteria will ferment.

What would a scientist/technician want to know how many viable bacterial cells are present in a sample? Give one example of a situation in which this information would be important to know

Viable cells counts are performed routinely on materials intended for human use or consumption; water and milk for example. Though low numbers of certain bacteria are considered acceptable in milk arriving from various dairies, Salmonella species are not tolerated. Viable cells counts are used in research and clinical laboratories.

When the polymerase chain reaction is used to amplify DNA in vitro, the components of the reaction mixture are subjected to three different temperature settings during each reaction cycle. These temperatures cause DNA to be denatured, to anneal and then to extend. Explain what is occurring during each of these steps.

When DNA is denatured, the hydrogen bonds between complimentary bases break and the two strands of the double helix separate (come apart). Primers anneal to template DNA, which means they bond with or hybridize with the specific regions of DNA they are complimentary to. Hydrogen bonds form between bases of primer and template DNA. The dNTPS also hydrogen bond to their complementary nucleotides in this step. Taq polymerase is responsible for extension. It will attach to the DNA at the primer, and then move 5' to 3' forming phosphodiester bonds between nucleotides (extension).

Why does treatment with ice-cold calcium chloride increase the efficiency of transformation?

When present at temperatures at or near 0o C, calcium chloride (or other cations) will bind to the negatively charged phosphate groups of the cell membrane, thus shielding the negative charge on the surface of the cell. Since DNA is a negatively charged particle, it will now be attracted to the cell surface rather than repelled by it.