Pràctiques de Virologia 1

Unit of Microbiology
Department of Biology

VIROLOGY
Laboratory Exercises

Biology/Biotechnology
2015-2016

Name:

Group:

Week:
2 Virology Lab Exercises

“ONE STEP GROWTH” AND POST-INFECTION HOST DYNAMICS

Multiplication of lytic viruses in sensitive hosts follows a model kinetics called “One Step
Growth”. This model is characterized by different phases:

a) Eclipse: This period immediately follows the penetration of viral particles

into the host cell. Eclipse is characterized by the incapacity to detect free
virions since viruses are actively transcribing and replicating inside the
host. The eclipse usually lasts from minutes (bacteriophages) to hours or
days (animal or plant viruses).

b) Intracellular accumulation: During the eclipse period all structural

proteins and viral genomes have been produced and massively
accumulated in the cytoplasm of the host cell. Both components self-
assembly to form new viral particles that accumulate
intracytoplasmatically.

c) Latent period: It is the sum of eclipse and intracellular phases. This

phase has nothing in common to the lag phase of the bacterial growth!

d) Release: After assembly and maturation of new viral particles they must
exit the host cell. Bacteriophages usually lyse the host cell using their
own lytic enzymes. If the viral infection has been synchronous, the
progeny release can be easily detected by a step increment in viral titre
(the “one step” rise in phage abundance).

The “One Step Growth” experiment allows the calculation of both the duration of the
different phases (see above) and the yield of the viral cycle (also known as burst size,
that is, the number of virions produced by each infected cell). This value can be directly
extrapolated from a semilogarithmic plot where the number of viral particles normalized
by the initial number (Nt/N0) is plotted at different time intervals (Fig. 1).

The main goals of this experiment are:

 To monitor the growth of a host cell after infection by a lytic

bacteriophage (post-infection host’s growth dynamics).

 To monitor the one step growth kinetics to calculate the OSG parameters
(burst size and duration of different phases) as well as the growth and
death rates for the host cell culture.
Pràctiques de Virologia 3

Figure 1: One step growth curve.

MATERIAL
– Pipettes – Water bath
– Luria-Bertani (LB) agar plates – Ice bath
– Semi-solid agar (BBL) tubes – Sterile Eppendorfs
– Centrifuge – Monod Erlenmeyers
– Spectrophotometer – MgSO4 0.1 M
– Phage lysate – Host culture (E. coli strain B)

PROTOCOL

1. Cultivation of host and infection

1.1. Growth monitoring: We will begin by inoculating a 1 mL aliquot of an exponential growing

culture of E. coli to triplicate cultures (Monod Erlenmeiers containing 10 mL of liquid LB
(Luria-Bertani broth, see Annex 1)). These three experimental cultures will be labelled as:
1) CONTROL (C1)
2) INFECTION CONTROL (C2)
3) ONE STEP GROWTH (OSG)
… and incubated under the same conditions (37 °C, no agitation).

1.2. Measure the absorbance at 550 nm (OD550) every 15 minutes approx. to monitor cell growth.
8 –1
1.3. When OD550 reaches 0.2 AU (~10 cfu mL ) we will infect C2 and OSG cultures by using a
phage T4 lysate.

1.4. Infection: Calculate the appropriate volume of T4 lysate to obtain a MOI = 1.

4 Virology Lab Exercises

– Use infected culture C2 to monitor post-infection host dynamics and its OD550

– Place OSG culture in an ice bath until processing (see next). This culture will be used to
analyse OSG kinetics.

1.5. Cultures C1 and C2 will be monitored until the end of the experiment and their OD550 will be
measured at regular time intervals (15 minutes approx.) until the end of the experiment.

1.6. Plot OD550 values of C1 and C2 versus time in the graphic of page 7.

2. OSG culture processing

2.1. Centrifuge culture OSG at 10,000 rpm for 15 minutes.

2.2. Discard the supernatant and resuspend the cell pellet in 10 mL of MgSO4 0.1 M by vortexing.

2.3. Repeat steps 2.1.

2.4. Discard the supernatant and resuspend the cell pellet in 10 mL of fresh LB medium by
vortexing.

2.5. Place the cell suspension into a 90 ml of LB medium and place in a water bath at 37 ºC.

3. POST-INFECTION DYNAMICS and OSG processing

3.1. TIME 0. Collect two aliquots of 1 mL using an automatic pipette and two sterile Eppendorf
tubes. Remember to properly label the tubes!
a) Make a serial dilution bank for each of the samples collected (see Table 1 for details about
the dilutions recommended at each time interval). Use the three last dilution tubes to quantify
the number of infectious phage particles at time 0 (see protocol for quantification in Annex 1).

CAUTION: This sample is precious. It is then recommended to carry out all

manipulations with extreme care.

3.2. OSG SAMPLING: From now on, we will collect samples at 5-minute intervals (see Table 1).
These samples will be processed as follows:
a) Collect two replicas of 1 mL (A and B) at the recommended time using an automatic
pipette and sterile Eppendorf tubes. Remember to properly label the tubes!
b) Place the Eppendorf tubes on an ice bath until centrifugation (see next).
c) Centrifuge the samples at 10,000 rpm for 10 min.
d) Use the proper volume of the supernatant to make a serial dilution bank of each sample.
e) For each sample replica, use the last three dilutions to quantify the number of infectious
phage particles at each time interval. Use LB agar plates and BBL melted agar tubes as
indicated in the quantification protocol (Annex 1).
Pràctiques de Virologia 5

4. PLAQUE COUNTING

4.1. After 24 hours, count the lytic phage plaques in each of the plates spread. Calculate the
number of infectious viral particles by using the formula in the protocol (Annex 1). Keep a
record of your results in the dedicated tables (see next section).

4.2. Plot the average number of infectious phage particles (normalized by the value at time 0, Nt/N0)
at each time interval using a semi logarithmic graph (see template in page 10).

Table 1: Sampling times and recommended dilutions for spreading

*
Group Time (min) Dilutions

1 0 (N0) –2, –3, –4

2 5 –2, –3, –4

3 10 –3, –4, –5

4 15 –3, –4, –5

5 20 –3, –4, –5

6 25 –4, –5, –6

1 30 –4, –5, –6

2 35 –4, –5, –6

3 40 –5, –6, –7

4 45 –5, –6, –7

5 50 –5, –6, –7

6 55 –5, –6, –7

7 60 –5, –6, –7

8 70 –5, –6, –7

*
Recommended dilutions for each sample replica at each time interval.
6 Virology Lab Exercises

RESULTS GROUP Nº______

Experimental conditions

Host:
Bacteriophage: [lysate] (pfu/ml):
mL required for infection at MOI 1:

Absorbance (OD550nm) versus time

Time C1 C2 OSG
Hour
(min) (OD550) (OD550) (OD550)
 Pràctiques de Virologia 7

1
Absorb‡ncia550

0.1

0.01

0 30 60 90 120 150 180 210 240 270 300 330 360

Temps (minuts)
8 Virology Lab Exercises

COUNT of INFECTIOUS PHAGE PARTICLES

Infectious particles (replica A)

Time Nt (A)
Dilution Lytic plaques –1
(min) (pfu mL )

Infectious particles (replica B)

Time Nt (B)
Dilution Lytic plaques –1
(min) (pfu mL )

Infectious particles (average)

Time Nt
–1
(min) (pfu mL )
Pràctiques de Virologia 9

GLOBAL RESULTS

Time Nt
Group –1 Nt/N0
(min) (pfu mL )

1 0 (N0)

2 5

3 10

4 15

5 20

6 25

1 30

2 35

3 40

4 45

5 50

6 55

7 60

8 70
 10 Virology Lab Exercises

100
Nt/N0

10

0 5 10 15 20 25 30 35 40 45 50 55 60

Temps (minuts)
Pràctiques de Virologia. Annexos 11

Annex 1: Growth media

1. Luria-Bertani (LB) agar: Composition in gL–1

Tryptone 10,0
NaCl 10,0
Yeast extract 5,0
Agar* 15,0
pH 7,2

• Dissolve the ingredients in 500 mL of distilled water.

• Bring the volume to 1,000 mL and adjust the pH.
• Mix thoroughly and sterilize by autoclaving.
• Plate.

2. BBL semisolid agar: Composition in gL–1

Tryptone 10,0
NaCl 10,0
Agar 6,0
pH 7,2

• Dissolve the ingredients in 500 mL of distilled water.

• Bring the volume to 1,000 mL and adjust the pH.
• Distribute in plastic tubes (3 mL/tube) and plug with cotton.
• Sterilize by autoclaving.
12 Pràctiques de Virologia: Anexos

QUANTIFICATION OF INFECTIOUS PHAGE PARTICLES

MATERIAL

– Luria-Bertani (LB) agar plates – Tubes of soft BBL medium

– MgSO4 0.1 M solution – Water bath at 55 °C
– Sterile Eppendorf tubes – Incubator at 37 °C
– Vortex – Automatic pipettes and sterile tips

Quantification

(1) Design a serial dilution procedure taking into account the estimated virus titre in the viral lysate.
The dilution steps should be done using a sterile solution of MgSO4 0.1 M.

OBSERVATION: After each dilution step, remember to mix the tubes by vortexing.

(2) Only the last three dilutions of the dilution series would be use for spreading. Prepare a Luria-
Bertani agar plate and one tube of soft BBL medium for each dilution to spread. Keep the
BBL tubes melted in a water bath at 55 ºC. Label each plate with the corresponding dilution,
experiment name a group number.

(3) Add 0.1 mL of the corresponding bacterial host and 0.1 mL of the corresponding dilution into a
tube of melted BBL medium.

(4) Mix by vortexing and quickly pour the content on a LB plate. Distribute homogeneously on the
plate until obtaining a uniform overlayer (Fig. 1).

CAUTION: The tube content must be poured quickly on the plate and slowly move the entire
plate to distribute uniformly the overlayer.

(5) Keep the agar plates on the desk for 10 minutes to allow agar solidification. Do not invert the
plates.

(6) Incubate the plates at 37 ºC for 24 h.

(7) After 24 h, count the number of lytic plaques. Only plates with a number of viral plaques
between 30 and 300 should be considered for future calculations. The original virus titre can
be obtained by using the following formula:

Virus titre (pfu/mL) = Nº of plaques / (Dt x Vd)

where: Dt: dilution titre; Vd: Volume of dilution used.

Pràctiques de Virologia. Annexos 13

Figura 1. Schematic representation of the spreading process.