Lab 25: Bacteriophage Flashcards
what is a bacteriophage or phage?
bacteria eaters or viruses that infect bacteria
in a plate lawned with E. coli, what does it mean when there are clear dots with no growth?
that means that those dots are where phages eat the e. coli
each phage is specific to ___ bacterial species
each phage is specific to 1 bacterial species
what can phages be used for?
to identify unknown bacteria
what is the importance of phages?
phages are important in maintaining ecological balances of bacteria (like in the ocean) - phages are a natural control of ecological balance
what is phage therapy?
phages are currently being explored for phage therapy - which is to treat antibiotic-resistant bacteria
what does the genome of phages contain?
DNA
what is the capsid? what is within the capsid?
The capsid is a protein and contains:
-Head
-Collar
-Tail
what does the tail contain within the capsid?
Fibers
Base Plate
Tube
what are the four steps of T4 (phage) replication?
- Attachment: phages attach to E. coli outer membrane lipids and proteins via tail fibers.
- Penetration: the phage tail tube drills a hole into the OM and CW; the tail contracts and injects its DNA into the cytoplasm.
- Synthesis: phage DNA and proteins are made.
- Assembly and release
what is phage typing?
using phages to identify unknown bacteria
what are two types of phages?
- Lytic phages
- Temperate phages
what do lytic phages do?
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
what do temperate phages do?
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.
what is vibrio cholerae?
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
what is the phage titer?
the number of phages in your sample
what is the purpose of our lab?
to determine the phage titer in our sample
what type of phage are we working with?
we worked with the T4 lytic phage that infects E. coli
what did we mix the T4 with?
we mixed the T4 phage with E. coli and then spread this mixture on an agar plate
what will the T4 cells do to the E. coli in the lab?
Each T4 phage will infect and kill E. coli cells and create a clear zone called a “plaque”
how do we determine the T4 phage titer in lab?
by counting plaques
what are the clear dots called in the E. coli lawn?
plaques
what happens if the number of phages in a sample is too high?
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.
what is a serial dilution?
when you dilute the sample of phages
since the sample has too many phages, what do we do?
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
what is a countable number of plaques?
between 25-250
what are the series of dilutions of our original phage sample?
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
what is the double agar layer technique?
-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.
what do we do after the soft agar plates are cooled?
-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.
what is the definition of plaques?
clear zones formed due to bacterial lysis by phages
usually, how many phages create 1 plaque?
Usually, 1 phage starts and creates 1 plaque
the total # of plaques can be used to find what?
phage titer
what is PFU and why do we use it?
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.
what do we do with the PFU?
we will calculate PFU/ml to find phage titer
what is the control plate?
no phages, bacteria only
when there are more than 250 plaques, what do we call that?
TNTC - too numerous to count
what is the formula to find phage titer with PFU?
PFU/ml = (# of plaques) / (volume * DF)
what is volume in the PFU formula?
the volume plated, which was 0.5 ml in our lab
what is DF in the PFU formula?
Dilution factor, which can be 10-2 for example
in our 10 -2 plate, we had 52 plaques. What is the PFU?
10,400 PMU - meaning that is the original number of plaques
