Direct Isolation of Phages from Soil | Protocol 5.2 Demo

The plaque assay is a laboratory technique used to quantify bacteriophages by counting clear spots (plaques) formed on agar plates after incubation, where each plaque represents one infectious viral particle; the virus concentration is calculated by multiplying the plaque count by the dilution factor and dividing by the inoculum volume, expressed as plaque forming units per milliliter (PFU/mL).

To calculate phage titer, count plaques on a countable plate (30-300 plaques), then use the formula: titer = (number of plaques) / (infection volume in mL × dilution factor), expressing the result as plaque-forming units (PFU) per milliliter; for example, 39 plaques counted from a 10-microliter infection at a 1×10⁻⁹ dilution yields 3.9×10¹² PFU/mL.

Virus plaque assay is a double-layer technique used to enumerate bacteriophage particles by counting clear zones (plaques) formed on solid agar media when bacteriophages infect bacterial cells and cause lysis; each plaque represents one plaque-forming unit (PFU), and the original phage concentration is calculated by multiplying the plaque count by the dilution factor.

The bacteriophage plaque assay is a laboratory technique used to determine the concentration (titer) of bacteriophages in a sample by serially diluting the phage stock, mixing each dilution with a bacterial culture (E. coli) in soft agar, pouring onto petri plates, and incubating; the number of clear plaques formed indicates the original phage concentration, with more dilute samples yielding fewer plaques.

The plaque assay is a fundamental microbiological technique used to determine the concentration of bacteriophages (such as T4) in a sample by counting clear zones (plaques) formed when phages infect and lyse bacterial cells (E. coli K12 or strain B) on agar plates; each plaque represents one or more phage particles that infected a single host cell, and the titre is calculated by dividing the number of plaques by the dilution factor and volume of sample used, expressed as plaque forming units per milliliter (PFU/mL).

T4 bacteriophage host range is determined by two key properties: (1) Black morphology - the thickness of the bacterial lawn formed on agar plates, which reflects the efficiency of phage replication and host cell lysis; (2) Host range property - the ability of the phage to infect and complete its life cycle in different bacterial strains. Phages with broad host range can infect multiple bacterial strains, while those with narrow host range can only infect specific strains. The host range is determined by the interaction between phage tail fibers and specific receptors on the bacterial cell surface.

Phage host range determines which bacterial strains a phage can infect. Studies show that most isolated phages can infect a broad range of host strains, with only about 13.5% of samples failing to infect any tested host. This specificity is determined by the compatibility between phage receptors and bacterial surface structures. Understanding host range is crucial for selecting appropriate phages for therapeutic applications.

Phage host range (the range of bacterial species a phage can infect) is often narrower than previously thought. While phages like P1 are described as having narrow host ranges, they can actually infect multiple species within a bacterial family. Importantly, the host range for infection (adsorption) and the host range for completing the viral life cycle can differ - a phage may be able to inject DNA into many species but only complete its life cycle in a subset.

Comprehensive phage characterization includes: Efficiency of Plating (EOP)—serial dilution to find highest dilution producing confluent lysis; Routine Test Dilution—the highest dilution failing to produce confluent lysis (standard for future testing); Single Step Growth Curve—analyzing sigmoid curves to determine latent period (time to maximum growth rate) and burst size (phages released per cell); Host Range Determination—testing coverage across bacterial species within genus or family; PFGE for genome sizing; Electron microscopy for morphological identification. Different phages show varying lytic activities: clear confluent lysis, opalescent lysis, no reaction, individual plaques, or resistant mutants. For difficult-to-culture bacteria like Bacteroides, metagenomic approaches are used: extract all genetic material from the sample, sequence it, and use bioinformatics to distinguish phage sequences from host and human DNA. The CRASS phage was discovered this way in human fecal metagenomes—it is 97 kb and six times more abundant than all other known phages combined. Temperate phages can integrate their DNA into bacterial genomes (lysogeny), avoiding lysis. To detect lysogenic phages: Mitomycin C induction—add 4-8 μg/ml to bacterial cultures, incubate 2 hours, then incubate overnight with shaking; UV irradiation—expose diluted bacterial cultures (OD 0.2-0.3) to 6-30 mJ/cm² UV-C, then incubate overnight. Both methods induce the lytic cycle, allowing detection of previously latent phages through plaque formation on host lawns.

Characterization of the isolated phages revealed variable host ranges among the four identified phages. Each phage demonstrated different abilities to lyse Staphylococcus intermedius isolates, with host range ranging from 10 to 53 of the 60 Staphylococcus intermedius isolates in the collection. This variability was initially assessed through simple spot tests and validated using high-throughput turbidity reduction assays with a microplate reader, confirming the differential lytic capabilities of each phage isolate.

Comprehensive phage characterization includes: Efficiency of Plating (EOP)—serial dilution to find highest dilution producing confluent lysis; Routine Test Dilution—the highest dilution failing to produce confluent lysis (standard for future testing); Single Step Growth Curve—analyzing sigmoid curves to determine latent period (time to maximum growth rate) and burst size (phages released per cell); Host Range Determination—testing coverage across bacterial species within genus or family; PFGE for genome sizing; Electron microscopy for morphological identification. Different phages show varying lytic activities: clear confluent lysis, opalescent lysis, no reaction, individual plaques, or resistant mutants. Pulse Field Gel Electrophoresis (PFGE) extends DNA sizing beyond standard electrophoresis (0.2-50 kb to 200 bp-12 Mb). Procedure: mix phage suspension with low melting agarose, create plugs, release DNA by overnight shaking at 55°C, wash plugs, perform restriction digestion if needed, load plugs in agarose gel, run at appropriate voltage, stain with ethidium bromide. Transmission Electron Microscopy uses negative staining with uranyl acetate on carbon-coated grids. ~96% of examined phages are tailed phages with characteristic components: head, tail tube, sheath, base plate, spikes, and tail fibers. Different families have distinct morphologies: T4 phages have contractile tails, lambda phages have different tail structures, filamentous phages have long flexible tails. Modern sequencing technologies include Sanger (first generation, ~1000 bp reads), Illumina (next generation, ~300-400 bp fragments requiring assembly), and Nanopore (single-molecule real-time sequencing). Workflow: template preparation, fragmentation, barcoding, PCR amplification, sequencing, and bioinformatics assembly. Coverage of at least 9x ensures reliable reconstruction. Bioinformatics identifies open reading frames (ORFs) coding for structural proteins (capsid/head/tail assembly), enzymes (DNA synthesis), and other functions. Comparative genomics enables species identification and evolutionary analysis.

After isolating pure phage samples, researchers characterize them through multiple methods. They bulk up phage populations by growing large amounts of the phage and then precipitate and extract the DNA for sequencing. DNA sequencing reveals what type of phage it is and its properties. Additionally, samples are sent for transmission electron microscopy, which provides visual images of the phage structure. These images help identify the specific type of phage and determine which viral family it belongs to, providing both genetic and morphological characterization.

Phage genomic analysis involves extracting phage DNA and sequencing the genome. The process includes: (1) DNA extraction using methods that remove bacterial DNA contamination; (2) Sequencing using platforms like Illumina; (3) Genome assembly to reconstruct the complete phage genome. The JKT1 phage genome is 43,000 base pairs with 53.7% G+C content, containing 57 ORFs (open reading frames), of which 27 are known proteins and 30 are hypothetical proteins.

Isolating therapeutic phages involves a multi-stage process: collecting environmental samples (wastewater, soil, etc.), filtering to remove larger particles, amplifying phages by exposing them to target bacteria, and isolating individual phage variants through serial dilution and plating. Each isolate undergoes characterization including genomic sequencing to confirm it is lytic (non-integrating), testing against panels of bacterial strains to determine host range, and electron microscopy for morphological identification.

Phage therapy uses viruses (bacteriophages) to kill bacteria, offering an alternative to antibiotics in agriculture. This approach is particularly relevant for treating bacterial plant diseases. Thailand has companies developing phage-based solutions for agricultural applications, representing a promising direction for sustainable pest management.

Phage therapy, which uses bacteriophages (viruses that naturally attack bacteria) to treat bacterial infections, offers a promising alternative to antibiotics for combating multi-drug resistant infections. Bacteriophages are the oldest and most populous organisms on Earth, found wherever bacteria exist, and can be isolated from sources like sewage. Unlike antibiotics, phages can be continuously sourced from libraries to overcome bacterial resistance, and phage cocktails targeting different bacterial receptors make it harder for bacteria to develop resistance. The therapy has been used in the former Soviet Union and Eastern Europe since the 1920s-30s but was largely abandoned when penicillin became available in the West. Modern applications require genetic modification to ensure phages follow the lytic cycle (killing bacteria) rather than the temperate cycle (which can carry harmful genes).

Bacteriophages (viruses that infect bacteria) were discovered around World War I and used therapeutically for approximately 25 years before being largely abandoned in the 1940s when antibiotics became commercially viable; however, phage therapy and biocontrol have experienced a resurgence in recent decades, particularly for controlling foodborne pathogens and spoilage bacteria in the agri-food supply chain, with the FDA approving commercial phage preparations in the mid-2000s, offering promising solutions to antibiotic resistance challenges.

Bacteriophages are viruses that specifically target and destroy particular bacteria, offering a promising alternative to antibiotics for treating infections that have become resistant to conventional antibiotic treatments; unlike broad-spectrum antibiotics that kill both harmful and beneficial bacteria, phages are highly specific, targeting only the pathogenic bacteria causing the infection, which reduces the likelihood of resistance developing and preserves the body's natural microbiome.

Bacteriophage therapy emerged as an alternative to antibiotics due to antibiotic resistance. Phages are viruses that replicate within bacteria through the lytic cycle, ultimately killing them. Phages are ubiquitous and found wherever bacteria exist, with water environments (wastewater, lakes, rivers near agricultural areas) being the most accessible sources. Isolation involves standard microbiological techniques: sample processing, co-culture with target bacteria, and detection of bacterial lysis. Phage titration uses double-layer agar plates to visualize plaques. Further analysis includes electron microscopy and genetic sequencing. This foundational knowledge enables the development of phage-based treatments for bacterial infections.