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SEA-PHAGES lab > Lab Practical > Flashcards

Lab Practical Flashcards

1
Q

What is used to wipe down the bench at the beginning of every lab?

A

At least 70% ethanol

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2
Q

How does the bunsen burner work? Why is it used?

A

The flame creates an updraft. Because heat rises, microogranisms and dust particles will be forced upward and away from the immediate work area.

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3
Q

What is the difference between aseptic and sterile?

A 
Sterile= free of all living microorganisms
Aseptic= an area whose contamination has been minimized

Supplies used are often sterile, and aseptic technique is used to minimize contamination by undesired microorganisms.

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4
Q

Why should you never set the cap of sterile objects down?

A

Any area inside the cap of a sterile bottle or sterile tube is considered a “sterile field.” Setting the cap down will compromise the sterile field.

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5
Q

Why is “flaming” done to bottles and tubes?

A

Flaming gently heats the air at the bottle opening and creates a mini-microbiological force field because the air is more likely to come out of the bottle than to fall into the bottle when the air at the open end is heated.

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6
Q

What is the goal of keeping a lab notebook?

A

To create a record that anyone can use to perform the same procedures and obtain the same results.

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7
Q

What should a lab notebook include?

A
  • Date
  • Aims and purpose
  • Materials
  • Procedures and protocols
  • Results
  • Analysis and Interpretation
  • Future Plans
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8
Q

What are bacteria?

A

Prokaryote, the simplest free-living life-form

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9
Q

How do bacteria replicate?

A

Binary Fission

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10
Q

What are three properties of bacteria?

A
  1. They grow rapidly
  2. They are susceptible to antibiotics
  3. They are ubiquitous
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11
Q

What are the four phases of bacterial growth?

A
  1. Lag Phase: When put into fresh media, bacteria take some time to recover without growth
  2. Logarithmic (exponential) growth phase: Number of bacteria doubles one every time period
  3. Stationary Phase: Once the nutrients are used up, the bacterial number will remain constant
  4. Decline (death) Phase: Bacteria begin to die and the numbers decline
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12
Q

What are the inner cellular machinery in bacteria?

A
  • Nucleoid that contains DNA

- Ribosomes to translate DNA into proteins

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13
Q

What are bacteriophages?

A

-They are a class of viruses that are specific to bacteria

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14
Q

What are some properties of phages?

A
  • They cannot replicate outside their host bacteria
  • They are not susceptible to antibiotics
  • They are ubiquitous
  • Most abundant life form on earth
  • Can survive in almost any environment
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15
Q

What are the three basic components of phages?

A
  1. Capsid (head that contains genetic material)
  2. Genetic material
  3. Tail (Allows the phage to attach to bacteria; DNA passes through here to get into the bacterium)
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16
Q

Who discovered phages?

A

Felix d’Herelle in 1910

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17
Q

What happens when a phage infects a bacterium?

A
  • The phage binds to a receptor on the surface of the bacterium
  • The phage infects its DNA
  • The DNA makes and assembles copies of itself using the bacterium’s replication machinery
  • The host bacterium lyses and new phages exit
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18
Q

What instrument can you use to see bacteria? What instrument can you use to see phages?

A

Standard light microscope, electron microscope

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19
Q

What are bacterial colonies?

A

Visible collections of bacteria that arose from one bacterium

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20
Q

How can you grow a bacterial colony?

A

When a bacterium is inoculated onto an agar plate containing all the nutrients required for growth, it will replicate and produce many bacteria.

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21
Q

How are phage plaques formed on a plate?

A

Growing bacteria are inoculated into top agar and poured on top of an agar plate. This creates a visible suspension of bacteria. If a phage is present, it will infect one of the bacteria cells, replicate in the cell, and lyse the bacterium.

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22
Q

What is burst size?

A

The number of new phages released after lysis of a bacterium.

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23
Q

What is a plaque?

A

A clearing zone that results from lysis of bacteria. Because of lysis, there is no cloudy suspension of bacteria radiating outward from the area that contained the phage.

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24
Q

What are three reasons to study phages?

A
  1. Genetics (phages can be used as tools to move DNA aroun for cloning or mutation)
  2. Epidemiology (some phages make their host bacterium deadly)
  3. Therapeutics (scientists want to use phages to kill antibiotic-resistant bacteria that cause disease)
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25
Q

What are the two types of phages?

A

Lytic and Temperate

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26
Q

What kind of life cycle are most bacteriophages in?

A

Temperate

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27
Q

What is the plaque morphology of a lytic phage?

A

Clear plaques in which very few or no bacteria remain intact

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28
Q

What is the plaque morphology of a temperate phage?

A

Cloudy or bullseye plaques in which some bacteria remain intact

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29
Q

What is the phage genome incorporated into bacterial DNA called?

A

Prophage

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30
Q

The mechanism by which a bacterial cell becomes infected with prophage is called _________.

A

Lysogeny

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31
Q

What is the life cycle of a lytic phage?

A

Infection of the bacterium, viral assembly, and viral release through lysis

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32
Q

What is the life cycle of a temperate phage?

A

Infection of the bacterium, can lyse the host bacteria or enter a dormant state by incorporating their genetic material into the DNA of the host bacterium

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33
Q

What is lysogenic growth?

A

When a temperate phage incorporates its DNA into the host bacterium’s DNA

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34
Q

What three things does a temperate phage accomplish through lysogenic growth?

A
  1. Lets the bacterium live
  2. Ensured its own survival
  3. Ensures its DNA is passed along
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35
Q

Why should the TA not sit in the pipette more than a few moments?

A

The agar will begin to solidify

36
Q

What is the purpose of the plaque streak protocol?

A

To purify a single phage population from samples with a potentially mixed phage population by diluting the phage.

37
Q

Which streak is the most dilute zone in a plaque streak protocol?

A

The third streak (designated by an X)

38
Q

During the plaque streak protocol, where was the TA and bacteria added?

A

The mixture of bacterial cells and top agar is added to the plate at the most dilute point and allowed to spread to the more concentrated regions of the plate.

39
Q

How many plaque streaks were done?

A

3

40
Q

What is the purpose of the Phage-Titer Assay?

A

To determine the concentration of phage particles in a given solution (pfu) and conduct successive rounds of purification of a single phage.

41
Q

What is a titer?

A

Counting the number of plaque-forming units per milliliter of original phage sample. It is the concentration of solution as determined by titration.

42
Q

During the serial dilutions, what was added to each tube?

A

90 microliters of phage buffer in the 10^-1 through 10^-4 tubes, and 100 microliters in the 10^0 tube. 10 microliters of undiluted phage sample in the 10^0 tube was added to the 10^-1, 10 microliters was taken from 10^-1 and added to 10^-2, and so on.

43
Q

What is done during serial dilutions in between the addition of 10 microliters of phage/PB to each successive tube?

A

The tube is vortexed to mix the phage and PB thoroughly

44
Q

What does a web plate look like?

A

Wispy M. Smeg. growth, in which most of the M. smeg has been lysed

45
Q

Which plate is counted in the phage titer assay?

A

The plate below the web plate in the dilution series

46
Q

What is the formula to calculate the titer pfu/mL?

A

pfu/microliter X 1000 microliters/mL X Dilution factor

47
Q

If the plate that was counted for the titer is the 10^-3 dilution, what is used as the dilution factor?

A

10^3

48
Q

Why were multiple plaque assay procedures done?

A

Some plates my have multiple phages that are difficult to isolate and purify. It is very important that the final plaque purification contain a single phage.

49
Q

Why is a plate with 20-200 plaques counted?

A

Counting more than 200 takes a long time, and counting fewer than 20 plaques is less accurate

50
Q

What is MTL, how is it made, and was is it used for?

A

Medium titer lysate made by flooding a lawn of pure, isolated phage-infected bacteria with phage buffer. The resulting MTL is used to purify higher numbers of filter-sterilized phage.

51
Q

What is HTL and what is it used for?

A

It is a large batch of pure phage that is titered to determine the approximate range needed for emperical determination of the number of pfu required for lysis of a bacterial lawn on a standard agar plate.

52
Q

Where is the phage genomic DNA that is isolated and purified retrieved from?

A

The phage head, which is virtually all protein

53
Q

What must be removed from the phage head before protective coat proteins of the phage head are disrupted?

A

The nucleic acids, DNA and RNA

54
Q

What are nucleases?

A

Enzymes that disrupt the structures of nucleic acids

55
Q

What does resin do?

A

Denatures the coat proteins of the capsid.

56
Q

Why is resin used during extraction of the nucleic acids and disruption of the capsid?

A

While the capsid proteins are being denatured, the phage genomic DNA binds to the resin.

57
Q

What is used to remove the denatured proteins and salts from the resin?

A

Isopropanol

58
Q

What is used to remove the phage genomic DNA from the column to prepare it for analysis?

A

Hot phage buffer (80 degrees celsius)

59
Q

Why must you change gloves in between after adding nuclease mix?

A

The enzymes can contaminate all DNA-containing solutions and ruin the sample.

60
Q

What is used to determine the concentration of the DNA?

A

Spectrophotometer

61
Q

What is done during restriction analysis of the DNA?

A

The DNA is digested and the fragments are separated using agarose gel electrophoreses

62
Q

What does it mean to “digest” DNA, and what is used to do this?

A

The DNA is cut, or restricted, through use of restriction enzymes

63
Q

Why is tracking dye added to the samples of digested DNA?

A

To determine how long the gel should be run

64
Q

Restriction enzymes are a kind of __________.

A

Nuclease

65
Q

Why should the soil sample tube only be about half full?

A

If there is too much soil in a sample tube, it will be hard to aspirate (draw fluid) enough liquid to filter sterilize

66
Q

If there is a large yellow spot in the middle of the plate, what happened?

A

TA was not added

67
Q

What does it mean if you see completely clear TA?

A

Either bacteria was not added or it was completely lysed

68
Q

If the TA is uneven and some is stuck to the stop of the plate, what does this mean?

A

The TA was not completely solidified before the plate was inverted

69
Q

How do you know if a phage is pure?

A

When the same assortment of plaque morphologies are yielded each time

70
Q

Why is M. Smeg used as the bacteria?

A

It is fast growing and non-pathogenic

71
Q

What is some external cell structures of bacteria?

A

Flagella and Cell membrane

72
Q

What is the approximate size of a phage?

A

100-200 nm

73
Q

What is a “ghost” Phage?

A

Phages that have inserted their DNA into their host bacterium. They are no longer infectious, their capsids are empty, and they remain attached to the bacterium

74
Q

What shape is phage DNA? Why is it in this shape?

A

The DNA is circular. This is because the DNA is double stranded, linear DNA whose strand ends have complementary base pairs that allow it to produce a circular molecule

75
Q

What are Repressors?

A

Repressors are a phage coded protein that stops the expression of lytic genes. Temperate phages have these, which is why they do not lyse their host.

76
Q

What is Induction?

A

The switch of a temperate phage from lysogenic growth to lytic growth

77
Q

During plaque streaking, why is the TA/bacteria mixture added to the most dilute area first?

A

So that the phage in the lesser dilute areas do not spread to the most dilute area

78
Q

Why are .22 mm filters used?

A

Larger molecules are filtered out while the phage particles are smaller and can filter through

79
Q

If a different kind of bacteria was used other than M. Smeg, would you expect the same results?

A

No, phages are host specific

80
Q

During the DNA isolation procedures, what DNA are you trying to isolate?

A

Phage genomic DNA

81
Q

Why does the separation of fragments occur on the agarose gel?

A

Differences in fragment size

82
Q

Why is a web pattern better than complete lysis?

A

A web pattern is proof that replication has occured and that phages are present

83
Q

Why were the restriction enzymes spun? What might happen if they weren’t spun?

A

They were spun to mix the contents so that the restriction enzymes can digest the DNA. If this is not done, the DNA might not be digested

84
Q

Do viruses have both DNA and RNA?

A

No, either DNA or RNA

85
Q

What is a virus?

A

A small, non-living thing that cannot reproduce by itself (parasitic). All living things have viruses.

86
Q

What is a serial dilution?

A

A dilution of a substance several times by the same amount each time

SEA-PHAGES lab (1 decks)

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