Purpose 

The purpose of the experiment is to quantitate the virus – host interactions using a plaque assay with the aid of crystal violet. Lambda phage and E.coli LE392 have been used to study these interactions. The primary goal was to determine the viral titer and gain insights into the dynamics of virus – host interactions. 

Introduction 

The viral samples can be enumerated by plaque assay. Viruses are dependent on host cells for their growth. Some viruses lyse the cells in which they replicate. A plaque assay enumerates viruses that lyse their host cells (Libre texts, 2021). In this assay, the hosts and virus are incubated together to allow the virus to attach and enter the host. This mixture is plated in a semi-solid agar. This semi-solid agar is poured onto a bottom agar that serves nutrients to the host cell. The plates are incubated for 24 hours and checked for plaques. “The plates containing an immediate number of viruses will have plaques, clear or partially clear circular areas in a turbid background of cellular growth.” (Libre texts, section 6.6, 2021) Each plaque is a representative of one virus known as plaque forming unit or PFU. In the experiment, bacterial viruses have been used referred to as phage, Lambda phage. The host cell is E. coli LE392. In this experiment, crystal violet, a dye that stains dead cells, is used to aid in plaque visualization. Crystal violet staining can help enhance the contrast between the clear plaques and the surrounding cellular growth, making it easier to count and quantify the plaques (Libretexts, 2021). 

Methodology 

The performers have utilized E. coli LE392 culture and 
λ 
𝜆
 
 
phage sample. The bacterial culture was prepared overnight by technicians in 0.5% (w/v) yeast-extract enriched LB with 20% maltose and 10mM MgSO4. The phage stock was the plate lysate phage sample that was prepared in the first half of the semester. 10 petri plates containing bottom agar, 10 tubes containing 5mL molten top agar, 10mL 
λ 
𝜆
 
 
broth supplemented with 1:1 mixture of 20% maltose and 1M MgSO4 were used for the project. 2mL aliquots of 20% maltose and 1M MgSO4 were provided in the lab. Eppendorf tubes, P10, P100 and P1000 micropipette and tips were used throughout the experimental steps.   

Some steps and values in the protocol were changed for the efficiency of the experiment. The changes are explained as following: 

Media Preparation: 

Media was prepared following the protocol (Rabeneck, 2023, https://learn.senecacollege.ca/ultra/courses/_684187_1/cl/outline ). 

The above protocol was used to prepare 300mL 
λ 
𝜆
 
 
broth agar and 70mL 
λ 
𝜆
 
 
top agar. 

Plate lysate 

The lysate was prepared previously. The protocol (Rabeneck, 2023, https://learn.senecacollege.ca/ultra/courses/_684187_1/cl/outline ) was followed. 

E.coli LE392 overnight stock provided. 4mL worth pellet collected, each 1mL centrifuged at 8000rpm × 2 mins. 

The Pellet resuspended in 0.5mL yeast extract – enriched LB broth supplemented with 20% maltose and 10mM MgSO4. 

0.1mL of resuspended culture transferred to 4 new Eppendorf tubes and 25µL phage stock added into each tube. 

Incubation and plating as per page 2 of the protocol. 

For Step 9, 2mL suspension media was added to each plate for 3X to scrape off the top agar, and 0.15mL chloroform was added at 2% (v/v) concentration to the final volume of 7.5mL. 

Plate lysate tube balanced by a water sample weighing 24.1170g and 24.1168g for centrifugation in Step 12 of the protocol. 

Plaque Assay 

Follow the plaque assay for plate lysate (Rabeneck, 2023, https://learn.senecacollege.ca/ultra/courses/_684187_1/cl/outline ). 

The concentration of the plate lysate phage stock was at 
1.5×1010pfumL
1
.
5
×
10
10
�
�
�
�
�
 
. 

The goal in the assay was to get 200 and 100 pfu. A diluting scheme was proposed to achieve the same from the stock. The scheme is explained in the table below. 

1:1 mixture of maltose and MgSO4 was prepared. This mixture was prepared by adding 700µL of 20% maltose to 700µL 1M MgSO4. 

HK performed the dilutions and JP plated the dilutions. 

The last two dilutions were plated. To achieve accuracy, the dilutions were plated in triplicates and then averaged at the end. 

The dilutions were performed in yeast extract – enriched LB broth supplemented by 20% maltose and 1M MgSO4. 

0.1mL of viral stock dilutions 4 and 5 were each taken and transferred to new Eppendorf tubes. This step was performed in replicates to achieve titer accuracy. 0.3mL of bacterial culture was added to each of the tubes followed by their incubation as mentioned in protocol step 8 and 9, page 3.  

The controls for the experiment were made in duplicates and incubated with the sample tubes. Positive control tube contained 0.3mL culture with 10µL plate lysate stock. Negative control contained 0.3mL bacterial culture with 0.1mL supplemented LB broth. 

Post-incubation, the contents from each tube were added to each of the tubes containing 5mL molten top agar. To all the tubes, 0.1mL of 1:1 mixture of 20% maltose and 1M MgSO4 was added successively. 

The mixture was vortexed and poured onto the 
λ 
𝜆
 
 
agar plates. The plates were incubated for 24 hours in a 37oC incubator. 

Apply the above-mentioned numerical values in the referenced protocol. Follow along the steps 1 – 12, page 1 to 3 in the lab manual with the mentioned modifications and determine the titers of the viral samples. 

Table 1: Dilution scheme used for plaque assay. 

Dilution Number 

1 

2 

3 

4 

5 

Dilution 

1100
1
100
 
 

175
1
75
 
 

1100
1
100
 
 

110
1
10
 
 

12
1
2
 
 

Total Dilution 

1102
1
10
2
 
 

175×102
1
75
×
10
2
 
 

175×104
1
75
×
10
4
 
 

175×105
1
75
×
10
5
 
 

1150×105
1
150
×
10
5
 
 

Dilution factor 

102
10
2
 
 

75×102
75
×
10
2
 
 

75×104
75
×
10
4
 
 

7.5×106
7
.
5
×
10
6
 
 

1.50×107
1
.
50
×
10
7
 
 

Diluent Volume (µL) 

990 

740 

990 

900 

500 

Viral plate lysate (µL) 

10 

10 

10 

100 

500 

Viral Titer 

pfu1mL
�
�
�
1
�
�
 
 

1.5×108
1
.
5
×
10
8
 
 

 2 × 106 

2 × 104 

2 × 103 

103 

Viral titer 

pfu0.1mL
�
�
�
0
.
1
�
�
 
 

1.5×107
1
.
5
×
10
7
 
 

2 × 105   

2 × 103 

200 

100 

The above table provides rationale for the dilution scheme used in plaque assay for the enumeration of viral titers from plate lysate. Dilution 4 and 5 were plated in triplicates. Controls were prepared. Both positive and negative control were plated in duplicates. 

Crystal Violet Staining 

Follow the crystal violet staining protocol (Mendoza, 2020, https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cpmc.105 ). 

0.1% crystal violet solution was prepared in 20% ethanol. 

From each set of dilutions poured, two plates from the triplicates were stained, and each positive and negative control was stained with crystal violet dye. 

Prior to staining, plates were washed with 10X TBS to get rid of residual substances like culture media, bacterial debris and unwanted phages that can interfere with the staining process. 

Plates were flooded with TBS for 5 minutes followed by disposal into the biohazard liquid waste. 

Prepared 8mL crystal violet dye was added onto the washed plates and kept untouched for a minute. 

The stain was rinsed with lots of double distilled water into the biohazard waste beaker. 

Results 

Table 2: Titer enumeration for plate lysate before staining 

Total Dilution of viral stock plated 

17.5×106
1
7
.
5
×
10
6
 
 

11.5×107
1
1
.
5
×
10
7
 
 

Volume diluted viral stock plated (mL) 

0.1mL 

0.1mL 

PFU replicate 1 

88 

39 

Calculated titer (pfu/mL) replicate 1 

6.6×109
6
.
6
×
10
9
 
 

5.85×109
5
.
85
×
10
9
 
 

PFU replicate 2 

72 

36 

Calculate titer (pfu/mL) replicate 2 

5.4×109
5
.
4
×
10
9
 
 

5.4×109
5
.
4
×
10
9
 
 

PFU replicate 3 

86 

35 

Calculate titer (pfu/mL) replicate 3 

6.45×109
6
.
45
×
10
9
 
 

5.25×109
5
.
25
×
10
9
 
 

Average titer 

5.8×109
5
.
8
×
10
9
 
 

 

Table 3: Titer enumeration for plate lysate after staining 

Total Dilution of viral stock plated 

17.5×106
1
7
.
5
×
10
6
 
 

11.5×107
1
1
.
5
×
10
7
 
 

Volume diluted viral stock plated (mL) 

0.1mL 

0.1mL 

PFU replicate 1 

107 

48 

Calculated titer (pfu/mL) replicate 1 

8.0×109
8
.
0
×
10
9
 
 

7.2×109
7
.
2
×
10
9
 
 

PFU replicate 2 

91 

53 

Calculate titer (pfu/mL) replicate 2 

6.8×109
6
.
8
×
10
9
 
 

7.95×109
7
.
95
×
10
9
 
 

PFU replicate 3 

N/A 

N/A 

Calculate titer (pfu/mL) replicate 3 

N/A 

N/A 

Average titer 

7.5×109
7
.
5
×
10
9
 
 

The above table enumerates plate lysate results before and after staining. The results are obtained after performing plaque assay on the proposed dilution scheme in triplicates to obtain average concentration in pfu/mL. From the triplicates of each dilution, two plates were taken to perform crystal violet staining. Table 3 enumerates plate lysate results after staining. A notable difference in average titer can be seen before and after staining the plaques. 

Sample Calculation 

PFU (replicate 1, before staining): 88 

Titer for replicate 1:  
(#pfu)(dilution factor)vol. plated
#
�
�
�
�
�
�
�
�
�
�
�
 
�
�
�
�
�
�
�
�
�
.
 
�
�
�
�
�
�
 
= 
(88)(7.5×106)0.1mL
88
7
.
5
×
10
6
0
.
1
�
�
 
= 
6.6×109 pfumL
6
.
6
×
10
9
 
�
�
�
�
�
 
 

 

 

Figure 1: Plaque assay results before and after staining. 

From each set of triplicates of a dilution, two plates have been stained by crystal violet dye; third one acts as the control of that specific dilution in the staining procedure. The plaques were counted prior to staining for quantitative analysis. 8mL of 0.1% crystal violet was used to stain each plate. It was left untouched for a minute for the dye to act with the host cell monolayer followed by rinsing with double distilled water. 

 

Figure 2: Variation in calculated lambda phage titer (pfu/mL) in Jetul’s and Harleen’s plaque assay from Experiment 1. 

The above table summarizes and compares the viral titers obtained in the plaque assay from experiment 1. The comparison is made based on the viral titers per 
109
10
9
 
 pfu/mL. The average titer for JP was 
3.70×109
3
.
70
×
10
9
 
pfu/mL with a standard deviation of 0.883 whereas for HK, the titer came out to be 
5.75×108
5
.
75
×
10
8
 
with deviation of 0.0212.  

Figure 3: Variation in lambda phage titer (pfu/mL) in samples before and after crystal violet staining. 

The above table summarizes and compares the viral titers obtained before and after crystal violet staining. Dilution 4 and 5 were plated in triplicates and then averaged to enumerate the viral titer. From the triplicates, two plates for each dilution were used for staining purposes and compared with the previous results. The obtained results were averaged and enumerated to get the viral titer in pfu/mL.



Read the above information and write me a detailed conclusion based on the above information