# Lab 8 Bacteriophages

## BACKGROUND

Bacteriophages are types of viruses that infect bacteria. Most bacteria have phages that can infect them. Phages play a significant role in bacterial typing, such as during the Salmonella outbreak associated with almonds in 2001, where Salmonella Enteritidis Phage Type 30 (PT30) was isolated. The lytic infection by phages destroys bacteria, leading to the formation of clear spots called "plaques" on a bacterial lawn, which can be counted to calculate the number of virus particles as plaque-forming units/ml.

Viruses infecting bacterial cells can follow different cycles: the lytic cycle, where the phage DNA replicates using the bacterial machinery leading to cell lysis, and the lysogenic cycle, where phage DNA integrates into the host genome and replicates along with it without killing the host. Under stressful conditions, the phage DNA might be excised from the bacterial chromosome and enter the lytic cycle.

Despite their role in fermentation failures, especially in the dairy industry, recent research has shown that endolysins from bacteriophages can target and control unwanted bacteria in food, offering an environmentally friendly approach to controlling infections and food contaminants. Bacteriophages are host-specific and do not infect eukaryotic cells or unrelated bacteria.
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## OBJECTIVES

- Become familiar with bacteriophage plating and enumeration techniques.
- Understand the calculation of virus concentrations.

## MATERIALS

### Cultures:
- 5 ml of fresh *Escherichia coli* culture (in LB broth)
- Bacteriophage T4

### Media:
- Luria-Bertani (LB) plates (5/group)
- Liquid LB agar (3ml/tube kept at ~50 ˚C)
- Saline solution (0.85% NaCl, 9 ml/tube for phage dilutions)
- Pipettes and tips

## PROCEDURES
### Procedures [VP for lab 8](https://drive.google.com/file/d/1vsfRPj7rh9rv80y5D-BZ0582k97NzOzz/view?usp=drive_link)
1. **Dilute your phage specimen** using the 9 ml saline solution tubes by transferring 1 ml of the stock or dilution into the next tube.
2. **Infection**: Mix 0.1 ml of each phage dilution with 0.3 ml of E. coli culture and keep the mixture at ambient temperature for 10 minutes for the phage to infect the bacteria.
3. **Plating**: After 10 min, mix the E. coli/phage mixture with 3 ml of liquid LB agar and pour the mixture onto the surface of the LB agar plate quickly to prevent solidification.
4. **Incubation**: Incubate the plates at 37 ˚C for 48 hours (right side up).
5. **Counting**: Count the plaques and calculate the concentration of bacteriophage as PFU/ml = (PFUs / Dilution of tube) × amount plated.

## RESULT

Record the dilution factors and the corresponding plaque forming units for each dilution. Calculate the final phage concentration in your sample.