one stepgrowth (1in1)

8/12/2019 One StepGrowth (1in1)

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One-step growth experiment

Dolah DaleeDepartment of Biology

Faculty of Science & Technology

Yala Rajabhat University


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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 assemblyof pre-formed components inside infectedcells. How do we know this ?

The "One Step Growth Curve".


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The "One Step Growth Curve" In the 1930's, Emory Ellis and Max Delbrck

performed a classic experiment which revealed thefundamental nature of virus replication. If you'd liketo 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, justcarry on and do the experiment.

Although this experiment involves a bacteriophage,

the principles it demonstrates apply to ALLviruses,(but not all viruses show such clear-cut or rapidreplication cycles, e.g. HIV).


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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, whichhas been incubated at 37C with rapid shaking. The bacteria are now in the logphase (i.e. growing exponentially).

To start the experiment, I'm just about to take 1ml of the culture (which nowcontains 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 thatEllis 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-readthe above section and think about it for a while.


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Shaking the mixture of culture


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What next ?

Since the sampling intervals are critical, we willperform 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 toassay the total p.f.u. (i.e. free phage plus anyinfectious 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 singletime point - 60 minutes. At the end of theexperiment we'll pool all the results so that you canplot graphs.


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(Replication)

You are providedwith the following:

E.coli cell suspension('plating cells')

12 agar plates

12 tubes of molten softagar (in 45Cwaterbath)

12 dilution tubes

Phage dilution buffer('PDB')


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Here are your samples


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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 6dilution 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 tubeand mix.e.Repeat this procedure through the whole dilutionseries.

Method 3:

a.Dispense 0.9ml phage dilution buffer into each of 6 dilutiontubes.

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 andmix.e.Repeat this procedure through the whole dilution series.

Method 2:

a.Dispense 0.9ml phage dilution buffer into each of 6dilution 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 tubeand mix.e.Repeat this procedure through the whole dilutionseries.

Method 1:

a.Dispense 9.9ml phage dilution buffer into each of 6 dilutiontubes.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 andmix.

e.Repeat this procedure through the whole dilution series.


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Next Add 0.1ml E.coli

suspension (platingcells) to each of thedilution tubes. (Why?)

Remove one tube of softagar from the 45Cwater bath.

Add 0.1ml of the 10-6

phage dilution to theagar.


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Next Mix thoroughly but gently (no

bubbles!) and pour onto one ofthe agar plates.

Swirl gently to distribute theagar evenly across the plate.

Repeat this operation for eachof the phage dilutions in this

series. Repeat the entire procedure

above to test for total p.f.u.from the chloroform-lysed

sample.


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Put your plates into the incubator overnightat 37C


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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


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Next

Count the number of plaques on each of your

plates.Calculate the titre of your phage samples


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Calculate the titre of your phage samples:

Express your answer in terms of 'plaque formingunits (pfu)/ml'(i.e. number of plaques x dilution factor x 10).

Noplaques

Noplaques

8 plaques85plaques

Too many

plaques tocount

(~800)

Confluent

phage

plaques

Totalp.f.u:

No

plaques

No

plaques8 plaques

83

plaques

Too many

plaques to

count(~800)

Confluent

phageplaques

Free

p.f.u:

10-610-510-410-310-210-1Sample:


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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).

Noplaques

3 plaques8 plaques34plaques

330plaques

Confluent

phageplaques

Totalp.f.u:

No

plaques

No

plaques

No

plaques1 plaque

15

plaques

140

plaques

Free

p.f.u:

10-610-510-410-310-210-1Sample:


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Here are the results for the whole experiment

8.7 x 1058.9 x 10566

8.8 x 1058.6 x 10563

8.5 x 1058.3 x 10560

8.8 x 1057 x 10557

8.5 x 1056.1 x 10554

7.7 x 1054 x 10551

5.2 x 1053.6 x 10548

4.4 x 1052 x 10545

3.3 x 1051.4 x 10442

1.6 x 1053.4 x 10439

7.5 x 1043 x 10436

4.4 x 1041.8 x 10433

1.6 x 1041.3 x 10430

1.6 x 1041.2 x 10427

1.6 x 1041.2 x 10424

1.6 x 1041.2 x 10421

1.5 x 1041.3 x 10418

1.5 x 1041.2 x 10415

1.4 x 1041.3 x 10412

1.4 x 1041.1 x 1049

1.2 x 1041.1 x 1046

1.3 x 1041 x 1043

Total p.f.u:Free p.f.u:Time:


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Here are the results for the whole experiment plus the logs

5.948.7 x 1055.958.9 x 105665.948.8 x 10

5

5.938.6 x 105

63

5.938.5 x 1055.928.3 x 10560

5.948.8 x 1055.857 x 10557

5.938.5 x 1055.796.1 x 10554

5.897.7 x 1055.604 x 105515.725.2 x 10

5

5.563.6 x 105

48

5.644.4 x 1055.302 x 10545

5.523.3 x 1055.151.4 x 10442

5.201.6 x 1054.533.4 x 10439

4.887.5 x 1044.483 x 104364.644.4 x 10

4

4.261.8 x 104

33

4.201.6 x 1044.111.3 x 10430

4.201.6 x 1044.081.2 x 10427

4.201.6 x 1044.081.2 x 10424

4.201.6 x 1044.081.2 x 104214.181.5 x 10

4

4.111.3 x 10

4

18

4.181.5 x 1044.081.2 x 10415

4.151.4 x 1044.111.3 x 10412

4.151.4 x 1044.041.1 x 1049

4.081.2 x 1044.041.1 x 1046

4.111.3 x 1044.001 x 1043

Log Total p.f.u:Total p.f.u:Log Free p.f.u:Free p.f.u:Time:


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Here the plotted graph


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The one-step growth curve comes

one stepgrowth (1in1)

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