MKBS221 PRACTICAL 4

TITLE: ISOLATION OF BACTERIOPHAGES FROM SEWAGE WATER THROUGH ENRICHMENT,

CENTRIFUGATION AND PLAQUE-PLATE TECHNIQUE.

Name and Surname: Student Number:

Group number:

Total marks:

TOTAL: 60

RUBRIC

ITEM Mark

1 Abstract [5]

2 Relevance of introduction and [5]

In-text referencing [5]

3 Aims and objectives [5]

4 Method used [5]

5 Results [15]

6 Interpretation of results and relevance of discussion [5]

In-text referencing [5]

7 Conclusion [5]

8 Reference [5]

Total [60]

Abstract
Introduction
Viruses are classified as obligatory intracellular parasites since they require a
specific host cell to replicate (Carlton, 1999; Mayer, 2005). Bacteriophage (phage)
viruses only infect and reproduce within bacterial cells. Phage typically connect to
the surface of their host cell via specialized structures known as tail fibers (Figure
One). When the bacteriophage attaches, it injects nucleic acid into the bacterium.
The viral nucleic acid is reproduced and integrated into its protein capsid using the
host cell's replication, translation, and transcription machinery. The escape of mature
viruses from the host cell stresses the plasma membrane, eventually killing the
bacterium.

The procedure necessitates the application of a Double-Layer Agar (DLA)

technology or an overlay method. As a base layer, hard agar is employed, and the
host cells and phage particles are combined with warm melted soft agar. This is
used to cover the firm agar base. The vulnerable cells form a lawn of cells in which
the bacteriophages can adsorb and multiply. Newly released phage particles will
infect nearby cells, forming a circular area of reduced turbidity known as a plaque. A
plaque might be clear or a region of slow-growing cells, depending on the
bacteriophage. When a phage stock is diluted and plated with susceptible cells, the
concentration of virus particles within the stock can be determined by seeing
individual plaques on a bacterial lawn.

If the phage stock is diluted to the point where individual and well-isolated plaques
form on the bacterial lawn, each plaque is thought to be the consequence of one
phage infecting one cell. Titering, also known as the plaque assay, is the procedure
of measuring phage concentration by diluting and plating with susceptible cells. The
number of viable phage particles in a stock suspension is determined using this
method. A plaque-forming unit (PFU) is a bacteriophage capable of infecting a cell
productively. Sewage has a high concentration of fecal coliforms, which are
potentially very harmful enteric bacteria. Coliforms are gram-negative, facultative
anaerobic bacteria that digest lactose in 48 hours at 35°C. Escherichia coli and
Enterobacter aerogenes are examples of fecal coliforms. Enteric bacteria are
normally harmless in their natural habitat, but they can cause severe illness
symptoms when consumed by susceptible persons, notably children and those with
compromised immune systems (Davis, 2005).
Aim and objectives
The purpose of this experiment is bacteriophage isolation and purification from
sewage. The objectives of this study are to enrich and culture bacteriophages from
sewage effluent, demonstrate bacteriophage replication within a vulnerable host cell,
show how to use the double-layer agar technique, and determine the phage
suspension's titer.

Materials and method

Escherichia coli log phase cells, coliphage sample, saline buffer, 7×50 ml centrifuge
tubes, and 7×dry nutrient agar plates were supplied. The centrifuge tubes were
labeled as follows: one for control, one as 1 00, and the remaining five as 1 0−1
through 1 0−5. This was also done to the nutrient agar. To centrifuge tubes numbered
−1
10 to 1 0−5, 0.9 ml (900 μl) of saline buffer was added. The tubes were diluted with
0.1 mL of coliphage sample. Removed 0,8 ml from 1 0−1 to 1 0−5 tubes to throw in the
garbage container. Each test tube received 0.02 ml of Log phase Escherichia coli.
The tubes were combined and incubated at room temperature for 10 minutes to
allow for adsorption. 0.01 ml coliphage and 0.02 ml Escherichia coli were introduced
to tube 1 00 and the control tube, mixed, and incubated at room temperature for 10
minutes. Each test tube received 5 mL of melted top gar ( 1 00 to 1 0−5 including the
control) and was gently stirred by rolling the tubes between palms. emptied the
contents of each of the seven tubes onto a dry nutritional agar plate. The plates were
incubated at 370°C overnight.

Results
Write the results in the table and show the calculations:
To obtain the concentration of phage in the stock suspension, divide the number of plaques counted
by the dilution factor.
number of plaques counted
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝ℎ𝑎𝑔𝑒𝑠/𝑚𝑙 =
dxV

Where: d = dilution

V=volume of diluted virus added to plate

Table 1: …………….

Dilution Plaque count PFU/100µl PFU/ml

10-0 >300 3 1×1 03
10-1 >300 30 1.5×1 0 4
 10-2 >300 300 1.5×1 05
10-3 290 2900 1.45×1 06
10-4 199 19900 9.95 ×1 0
6

10-5 98 98000 4.9 × 10

7

Average=247.8 Average=2.0×1 0 4 Average=1.0

7
×1 0
Control 0 0 0

Calculation of PFU/100 μl :
0
300× 1 0
=3
100
1
300× 1 0
=3
100
Discussion (10)

Conclusion (5)

References (5)