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Unformatted text preview: Experiment 6-4: (Read Only pgs 226-229)/ Handout: Isolation of Bacteriophage from Sewage
Purpose: To learn about bacteriophage and to isolate them from sewage. Viruses vs. Bacteria
- viruses are smaller, non-cellular, and intracellular parasites - viruses can not be grown on normal media because they require a host since they have their own DNA or RNA but lack the cellular machinery needed to replicate - viruses can infect all types of cells: prokaryotic and eukaryotic Phages - viruses that infect bacteria; also called bacteriophages Phages that parasitize E. coli are referred to as coliphages.
Tail: provides for attachment of virus to host and for exit of nucleic acid Head/capsid: composed of protein and contains either DNA or RNA Lysis vs. Lysogeny
-some viruses have both properties Lytic viruses - considered virulent; viruses enter the cell, take over the cell, replicate, then lyse the cell for release Lysogenic viruses - considered temperate; viruses enter the cell, integrate into the DNA of the host; cell replicates normally then eventually viruses replicate and lyse the cell to be released Quantification of Viruses: -done to gain understanding of viruses and replication -Viruses can be observed with an electron microscope -Normally, viruses are quantified by their effect on the host cells, so you would count the VIU's (virus infection units) -VIU is the smallest unit causing an effect with a host -We utilize the technique of a Plaque assay: -Plaque- zone of lysis/ growth inhibition (clear areas denoted by arrows in figure) in a lawn of bacteria -1 plaque = 1 virus -PFU = Plaque Forming Units -Efficiency of count from the assay is always less then with an electron microscope because efficiency of infection by viruses is not 100% 10-7 dilution of an enriched sewage sample plated on TSA using E. coli as the host Isolation of bacteriophage from Sewage: Three Steps:
1. Enrichment:(performed in rows) - enrichment increases the number of phage in the raw sewage a. Performed by combining sewage (source of phage), E. coli (host of phage), and Deca Strength Phage Broth (DSPB) b. E. coli not only acts as a host, but it also selects only for phage that infects E. coli so the number of coliphage will be increased c. The DSPB is not broth for phage since they can not grow in media without a host d. The DSPB is used to increase the number of E. coli which indirectly increases the number of phage Materials: 125ml Erlenmeyer flask containing 45 ml sewage water (premeasured) DSPB tube (5ml) Latex gloves TSB culture of E. coli (5ml) Methods: 1. Pour 5 ml of DSPB medium and 5 ml of E. coli into the flask of sewage. 2. Incubate the flask in your TA incubator at 37oC for 24 hours. 1 2. Filtration: (performed in rows) - separates the E. coli from the phage a. Uses a 0.2m pore size filter; allows viruses to pass but not the bacteria; can also use 0.45m b. The sample must be centrifuged prior to filtration to remove all the cellular debris so that the membrane doesn't get clogged. c. The centrifuged, enriched sewage is filtered using a Nalgene filtration apparatus (don't filter all of the sewage because it will clog the filter - only 4ml is needed for the entire class) d. EMB plates: (three plates per filtration group) i. Plate the filter ii. Plate 0.1ml of the unfiltered, enriched sewage iii. Plate 0.1ml of the filtrate iv. EMB is selective and differential 1. Methylene blue - inhibits Gram + bacteria 2. Eosin - acidic dye which gives coliforms like E. coli a green metallic sheen 3. Contains lactose and sucrose: if lactose and sucrose are both fermented=> whole colony is purple if only lactose is fermented=> center of the colony is purple Seeding: (performed in pairs) -plate the dilution of the filtrate with E. coli to see evidence of phage and to find the concentration of phage in the original filtrate -Soft Nutrient Agar is used to prepare an overlay of the E. coli and phage on top of a TSA plate -The tubes of Soft Nutrient Agar are liquefied and in the waterbath just inside the microscope room. -After adding the 0.1ml of the diluted filtrate and 0.3 ml of E. coli, pour the entire contents of the tube over the surface of a pre-warmed TSA plate. -The TSA plates (15ml) are pre-warmed in the TA incubator to prevent the Soft Nutrient Agar from solidifying as soon as it hits the plates. - Allows the Soft Nutrient Agar to flow over the surface of the TSA plate -Once the Soft Nutrient Agar has solidified, the plates should be placed in the basket on the TA bench at the front of the lab so that all of the plates can be incubated at the same time. -Plaques will appear on the plates within 2 to 2.5 hours -Longer periods of incubation will cause the plaques to become confluent making it difficult to count the individual plaques Diluting of the Bacteriophage (filtrate) and Seeding: 0.1ml 0.1ml 0.1ml 0.1ml 9.9 ml 0.85% NaCl 9.9 ml 0.85% NaCl 9.9 ml 0.85% NaCl 9.9 ml 0.85% NaCl Bacteriophage 0.1 ml + 0.3 ml E.coli B 0.1 ml + 0.3 ml E.coli B 0.1 ml + 0.3 ml E.coli B 0.1 ml + 0.3 ml E.coli B 5 Softml Soft Nutrient Nutrient Agar Agar 5 ml 5 Softml Soft Nutrient Nutrient Agar Agar 5 ml 5 Softml Soft Nutrient Nutrient Agar Agar 5 ml 5 Softml Soft Nutrient Nutrient Agar Agar 5 ml 15 ml TSA 15 ml TSA 15 ml TSA 15 ml TSA Place plates in the basket on the TA bench for incubation. DO NOT put them in the incubator. 2 ...
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