Lab 25: Bacteriophage

Question 1

what is a bacteriophage or phage?

Answer 1

bacteria eaters or viruses that infect bacteria

Question 2

in a plate lawned with E. coli, what does it mean when there are clear dots with no growth?

Answer 2

that means that those dots are where phages eat the e. coli

Question 3

each phage is specific to ___ bacterial species

Answer 3

each phage is specific to 1 bacterial species

Question 4

what can phages be used for?

Answer 4

to identify unknown bacteria

Question 5

what is the importance of phages?

Answer 5

phages are important in maintaining ecological balances of bacteria (like in the ocean) - phages are a natural control of ecological balance

Question 6

what is phage therapy?

Answer 6

phages are currently being explored for phage therapy - which is to treat antibiotic-resistant bacteria

Question 7

what does the genome of phages contain?

Answer 7

DNA

Question 8

what is the capsid? what is within the capsid?

Answer 8

The capsid is a protein and contains:
-Head
-Collar
-Tail

Question 9

what does the tail contain within the capsid?

Answer 9

Fibers
Base Plate
Tube

Question 10

what are the four steps of T4 (phage) replication?

Answer 10

1. Attachment: phages attach to E. coli outer membrane lipids and proteins via tail fibers.
2. Penetration: the phage tail tube drills a hole into the OM and CW; the tail contracts and injects its DNA into the cytoplasm.
3. Synthesis: phage DNA and proteins are made.
4. Assembly and release

Question 11

what is phage typing?

Answer 11

using phages to identify unknown bacteria

Question 12

what are two types of phages?

Answer 12

1. Lytic phages
2. Temperate phages

Question 13

what do lytic phages do?

Answer 13

They infect bacteria, replicate, and lyse the cells to release hundreds of new phages. The phages kill the cells and leave.
1. Attachment of phage and inject viral genome into the cell.
2. Degradation of the host genome.
3. Replication of viral genome and synthesis of viral proteins.
4. Assembly and release.
5. New viruses

Question 14

what do temperate phages do?

Answer 14

They have a lytic and lysogenic cycle (the ability to integrate into the host genome as prophages)
1. The phage attached to the cell surface of a bacterium.
2. Phage DNA enters the bacterial cell.
3. Phage DNA integrates into the bacterial DNA as a prophage.
4. The integrated prophage replicates when bacterial DNA replicates.
5. These cells may exhibit new properties.

Question 15

what is vibrio cholerae?

Answer 15

a bacterium that causes cholera through a toxin that it gained from a prophage! it remains as a prophage until an agent triggers the lytic cycle

Question 16

what is the phage titer?

Answer 16

the number of phages in your sample

Question 17

what is the purpose of our lab?

Answer 17

to determine the phage titer in our sample

Question 18

what type of phage are we working with?

Answer 18

we worked with the T4 lytic phage that infects E. coli

Question 19

what did we mix the T4 with?

Answer 19

we mixed the T4 phage with E. coli and then spread this mixture on an agar plate

Question 20

what will the T4 cells do to the E. coli in the lab?

Answer 20

Each T4 phage will infect and kill E. coli cells and create a clear zone called a “plaque”

Question 21

how do we determine the T4 phage titer in lab?

Answer 21

by counting plaques

Question 22

what are the clear dots called in the E. coli lawn?

Answer 22

plaques

Question 23

what happens if the number of phages in a sample is too high?

Answer 23

if we put the sample with many phages - onto an E. coli lawn, we may not see any isolated plaques since there will be complete destruction and clearing of the E. coli from phages.

Question 24

what is a serial dilution?

Answer 24

when you dilute the sample of phages

Question 25

since the sample has too many phages, what do we do?

Answer 25

before plating the phage sample, we have to dilute this sample using the serial dilution method. This ensures that we get a countable number of plaques on at least one of our plates

Question 26

what is a countable number of plaques?

Answer 26

between 25-250

Question 27

what are the series of dilutions of our original phage sample?

Answer 27

1:10 (10-1) = 1 ml of sample + 9 ml of sterile water

1:100 (10-2) = 1 ml of 10-1 sample + 9 ml sterile water

1:1000 (10-3) = 1 ml of 10-2 sample + 9 ml of sterile water

1:10,000 (10-4) = 1 ml 10-3 sample + 9 ml of sterile water

Question 28

what is the double agar layer technique?

Answer 28

-When we mix each dilution of phage with E. coli cells in tryptone (which is a soft agar of 0.7%)
-We pour the mixture onto a regular agar plate (1.5%)
-When cooled, the soft agar is solid enough to immobilize bacteria but pliable enough to let small phages diffuse, infect bacteria, and undergo lytic cylce.

Question 29

what do we do after the soft agar plates are cooled?

Answer 29

-We incubate the plates
-Each phage will infect a bacterial cell releasing hundreds of phages, which will in turn infect neighboring cells - resulting in plaque formation.

Question 30

what is the definition of plaques?

Answer 30

clear zones formed due to bacterial lysis by phages

Question 31

usually, how many phages create 1 plaque?

Answer 31

Usually, 1 phage starts and creates 1 plaque

Question 32

the total # of plaques can be used to find what?

Answer 32

phage titer

Question 33

what is PFU and why do we use it?

Answer 33

Plaque Forming Unit.
There is a possibility that more than 1 phage created 1 plaque. To take this into account, we call each plaque a PFU.

Question 34

what do we do with the PFU?

Answer 34

we will calculate PFU/ml to find phage titer

Question 35

what is the control plate?

Answer 35

no phages, bacteria only

Question 36

when there are more than 250 plaques, what do we call that?

Answer 36

TNTC - too numerous to count

Question 37

what is the formula to find phage titer with PFU?

Answer 37

PFU/ml = (# of plaques) / (volume * DF)

Question 38

what is volume in the PFU formula?

Answer 38

the volume plated, which was 0.5 ml in our lab

Question 39

what is DF in the PFU formula?

Answer 39

Dilution factor, which can be 10-2 for example

Question 40

in our 10 -2 plate, we had 52 plaques. What is the PFU?

Answer 40

10,400 PMU - meaning that is the original number of plaques