One-step growth experiment

Dolah Dalee
Department of Biology
Faculty of Science & Technology
Yala Rajabhat University
VIRUSES: One-Step Growth E 2
Viruses Don't 'Grow'
Unlike all living organisms, virus particles
(virions) do not 'grow' or undergo division.
Instead, they are produced from the assembly
of pre-formed components inside infected
cells. How do we know this ?
The "One Step Growth Curve".
VIRUSES: One-Step Growth E 3
The "One Step Growth Curve"
In the 1930's, Emory Ellis and Max Delbrck
performed a classic experiment which revealed the
fundamental nature of virus replication. If you'd like
to read the paper they published, click on this link
(Ellis E.L, Delbruck M. 'The growth of bacteriophage'
J.Gen.Physiol. 22: 365-384, 1939). If not it's OK, just
carry on and do the experiment.
Although this experiment involves a bacteriophage,
the principles it demonstrates apply to ALL viruses,
(but not all viruses show such clear-cut or rapid
replication cycles, e.g. HIV).
VIRUSES: One-Step Growth E 4
So how does it work?
Yesterday evening, I prepared a 100ml culture of Escherichia coli (E.coli ) bacteria.
This was grown overnight at 37C.
Earlier today, I prepared a sterile 2 litre flask containing 900ml L-broth (bacterial
growth medium), 10ml 1M MgSO4, 10ml 1M CaCl2.
(Why is the addition of Mg and Ca necessary?)
1 hour ago, I added the overnight E.coli culture to the pre-warmed flask, which
has been incubated at 37C with rapid shaking. The bacteria are now in the log
phase (i.e. growing exponentially).
To start the experiment, I'm just about to take 1ml of the culture (which now
contains about 1x109 bacteria) and add 1x1010 plaque forming units (pfu =
'infectious units') of bacteriophage. This will give a multiplicity of infection
(m.o.i.) of 10: i.e. 10 virus particles/cell.
I will incubate this for 1minute at 37C. (Why?)
Then I will quickly spin down the cells (discarding the supernatant), resuspend
them and add them back to the 1000ml log (exponential) phase culture culture.
This is the most critical part of the whole experiment - and the smart bit that
Ellis and Delbrck came up with - since it synchronizes the infection of the cells.
(How?)
It's important that you understand how this works, so you might want to re-read
the above section and think about it for a while.
VIRUSES: One-Step Growth E 5
Shaking the mixture of culture
VIRUSES: One-Step Growth E 6
What next ?
Since the sampling intervals are critical, we will
perform this as a group experiment.
I will take samples from the bacteriophage-infected
culture at 3 minute intervals.
Half of each sample will be lysed with chloroform to
assay the total p.f.u. (i.e. free phage plus any
infectious intracellular phage particles). The other
half will be used to assay the free (extracellular) p.f.u.
(N.B. This bacteriophage is resistant to chloroform,
but the bacterial cells are not!)
You will analyse the free/total p.f.u. from a single
time point - 60 minutes. At the end of the
experiment we'll pool all the results so that you can
plot graphs.
VIRUSES: One-Step Growth E 7
(Replication)
You are provided
with the following:
E.coli cell suspension
('plating cells')
12 agar plates
12 tubes of molten soft
agar (in 45C
waterbath)
12 dilution tubes
Phage dilution buffer
('PDB')
VIRUSES: One-Step Growth E 8
Here are your samples
VIRUSES: One-Step Growth E 9
Making 10-fold dilution
To measure the titre (concentration) of bacteriophage particles in your two
samples, you need to make serial 10-fold dilutions.
How are you going to do this? (click on the correct option below):
Method 4:
a.Dispense 1ml phage dilution buffer into each of 6
dilution tubes.
b.Label the tubes: 10
-1
, 10
-2
, etc. to 10
-6
.
c.Add 0.1ml phage suspension to the 10
-1
tube, mix.
d.With a clean pipette, transfer 0.1ml to the 10
-2
tube
and mix.
e.Repeat this procedure through the whole dilution
series.
Method 3:
a.Dispense 0.9ml phage dilution buffer into each of 6 dilution
tubes.
b.Label the tubes: 10
-1
, 10
-2
, etc. to 10
-6
.
c.Add 0.9ml phage suspension to the 10
-1
tube, mix.
d.With a clean pipette, transfer 0.1ml to the 10
-2
tube and
mix.
e.Repeat this procedure through the whole dilution series.
Method 2:
a.Dispense 0.9ml phage dilution buffer into each of 6
dilution tubes.
b.Label the tubes: 10
-1
, 10
-2
, etc. to 10
-6
.
c.Add 0.1ml phage suspension to the 10
-1
tube, mix.
d.With a clean pipette, transfer 0.1ml to the 10
-2
tube
and mix.
e.Repeat this procedure through the whole dilution
series.
Method 1:
a.Dispense 9.9ml phage dilution buffer into each of 6 dilution
tubes.
b.Label the tubes: 10
-1
, 10
-2
, etc. to 10
-6
.
c.Add 0.1ml phage suspension to the 10
-1
tube, mix.
d.With a clean pipette, transfer 0.1ml to the 10
-2
tube and
mix.
e.Repeat this procedure through the whole dilution series.
VIRUSES: One-Step Growth E 10
Next
Add 0.1ml E.coli
suspension (plating
cells) to each of the
dilution tubes. (Why?)
Remove one tube of soft
agar from the 45C
water bath.
Add 0.1ml of the 10-6
phage dilution to the
agar.
VIRUSES: One-Step Growth E 11
Next
Mix thoroughly but gently (no
bubbles!) and pour onto one of
the agar plates.
Swirl gently to distribute the
agar evenly across the plate.
Repeat this operation for each
of the phage dilutions in this
series.
Repeat the entire procedure
above to test for total p.f.u.
from the chloroform-lysed
sample.
VIRUSES: One-Step Growth E 12
Put your plates into the incubator overnight
at 37C
VIRUSES: One-Step Growth E 13
Here are your plates
Free pfu total pfu ditlution free pfu total pfu dilution
10
-1
10
-4
10
-2
10
-5
10
-3
10
-6
VIRUSES: One-Step Growth E 14
Next
Count the number of plaques on each of your
plates.
Calculate the titre of your phage samples
VIRUSES: One-Step Growth E 15
Calculate the titre of your phage samples:
Express your answer in terms of 'plaque forming
units (pfu)/ml'
(i.e. number of plaques x dilution factor x 10).
No
plaques
No
plaques
8 plaques
85
plaques
Too many
plaques to
count
(~800)
Confluent
phage
plaques
Total
p.f.u:
No
plaques
No
plaques
8 plaques
83
plaques
Too many
plaques to
count
(~800)
Confluent
phage
plaques
Free
p.f.u:
10
-6
10
-5
10
-4
10
-3
10
-2
10
-1
Sample:
VIRUSES: One-Step Growth E 16
Practise this exercise
Now practice calculating titres by using the data for the 42
minute samples:
Express your answer in terms of 'plaque forming units
(pfu)/ml'
(i.e. number of plaques x dilution factor x 10).
No
plaques
3 plaques 8 plaques
34
plaques
330
plaques
Confluent
phage
plaques
Total
p.f.u:
No
plaques
No
plaques
No
plaques
1 plaque
15
plaques
140
plaques
Free
p.f.u:
10
-6
10
-5
10
-4
10
-3
10
-2
10
-1
Sample:
VIRUSES: One-Step Growth E 17
Here are the results for the whole experiment
8.7 x 10
5
8.9 x 10
5
66
8.8 x 10
5
8.6 x 10
5
63
8.5 x 10
5
8.3 x 10
5
60
8.8 x 10
5
7 x 10
5
57
8.5 x 10
5
6.1 x 10
5
54
7.7 x 10
5
4 x 10
5
51
5.2 x 10
5
3.6 x 10
5
48
4.4 x 10
5
2 x 10
5
45
3.3 x 10
5
1.4 x 10
4
42
1.6 x 10
5
3.4 x 10
4
39
7.5 x 10
4
3 x 10
4
36
4.4 x 10
4
1.8 x 10
4
33
1.6 x 10
4
1.3 x 10
4
30
1.6 x 10
4
1.2 x 10
4
27
1.6 x 10
4
1.2 x 10
4
24
1.6 x 10
4
1.2 x 10
4
21
1.5 x 10
4
1.3 x 10
4
18
1.5 x 10
4
1.2 x 10
4
15
1.4 x 10
4
1.3 x 10
4
12
1.4 x 10
4
1.1 x 10
4
9
1.2 x 10
4
1.1 x 10
4
6
1.3 x 10
4
1 x 10
4
3
Total p.f.u: Free p.f.u: Time:
VIRUSES: One-Step Growth E 18
Here are the results for the whole experiment plus the logs
5.94 8.7 x 10
5
5.95 8.9 x 10
5
66
5.94 8.8 x 10
5
5.93 8.6 x 10
5
63
5.93 8.5 x 10
5
5.92 8.3 x 10
5
60
5.94 8.8 x 10
5
5.85 7 x 10
5
57
5.93 8.5 x 10
5
5.79 6.1 x 10
5
54
5.89 7.7 x 10
5
5.60 4 x 10
5
51
5.72 5.2 x 10
5
5.56 3.6 x 10
5
48
5.64 4.4 x 10
5
5.30 2 x 10
5
45
5.52 3.3 x 10
5
5.15 1.4 x 10
4
42
5.20 1.6 x 10
5
4.53 3.4 x 10
4
39
4.88 7.5 x 10
4
4.48 3 x 10
4
36
4.64 4.4 x 10
4
4.26 1.8 x 10
4
33
4.20 1.6 x 10
4
4.11 1.3 x 10
4
30
4.20 1.6 x 10
4
4.08 1.2 x 10
4
27
4.20 1.6 x 10
4
4.08 1.2 x 10
4
24
4.20 1.6 x 10
4
4.08 1.2 x 10
4
21
4.18 1.5 x 10
4
4.11 1.3 x 10
4
18
4.18 1.5 x 10
4
4.08 1.2 x 10
4
15
4.15 1.4 x 10
4
4.11 1.3 x 10
4
12
4.15 1.4 x 10
4
4.04 1.1 x 10
4
9
4.08 1.2 x 10
4
4.04 1.1 x 10
4
6
4.11 1.3 x 10
4
4.00 1 x 10
4
3
Log Total p.f.u: Total p.f.u: Log Free p.f.u: Free p.f.u: Time:
VIRUSES: One-Step Growth E 19
Here the plotted graph
VIRUSES: One-Step Growth E 20
The one-step growth curve comes