Isolation/Enrichment for virulent bacterial viruses from nature, in this case viruses from the chicken gut whose host is a known enteric gut bacterium. 

ISOLATION
We recently scooped a 3-4 gram sample of a messy fecal deposit left by a neighbor’s chicken.  The sample was put in a 100 ml bottle.  To the bottle we added about 70 ml 10-2M MgSO4 (PBS or water would also do just fine) and a magnetic stir bar added.  This was stirred for several hours (Picture 1). 
Picture 1. 

What a slurry of chicken poop looks like after several hours stirring. Not pretty.  Dirt and leaf litter have sunk to the bottom.   
Safety Precaution:

You can get an infection from gut microorganisms. For example there are such things as E. coli and Campylobacteria in chickens, and some may be pathogenic.  
We asked about the presence of enteric bacteria in the slurry, by streaking a loopful of the slurry on a MacConkey agar plate and incubating overnight at 37C.  MacConkey agar contains crystal violet dye and bile salts, which inhibit the growth of Gram positive bacteria thus enriching for Gram negative enteric bacteria. The agar also contains the sugar lactose and neutral red dye.  Lactose fermenting bacteria like E. coli form dark red colonies, and lactose negative bacteria form translucent, non-colored colonies.  A streak plate of the fecal slurry is shown in Picture 2.
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Picture 2. 

Fecal slurry streaked out on a MacConkey agar plate.  Most of the colonies are Lac+ (lactose fermenting), but a few white Lac- colonies are apparent at the right edge (see black arrow).
ENRICHMENT
Enriching for a virulent bacteriophage that uses an enteric bacterium as host. 

If you have a suitable lab strain of a bacterium known to be a gut microorganism, it is very likely that there will also be phages for that bacterium, in the typical gut.  We sampled the chicken fecal slurry described above, searching for phages that use E. coli as the host.  This was done by taking a 250 ml flask containing ~25-50 ml of sterile broth.  The bacterial host is then added.   We added 1 ml of an overnight growth of a lab strain of E. coli.  We used E. coli C1a, an E. coli C strain, though most any E. coli strain would work.  Finally we added 1 ml of the poop slurry from the 250 ml bottle shown in Picture 1.  The assumption is that the slurry will contain lots of different kinds of bacteria and lots of phages that can grow on those bacteria. 

The inoculated flask was shaken overnight at 37C.  Seeding the flask with a lot of E. coli ensures that E coli will likely be the dominant species after the overnight growth period. Further, because the added E. coli cells outnumber the other bacteria from the poop slurry, any E. coli phages in the slurry should have an easy time proliferating.  After shaking the flask overnight at 37C, a 1 ml sample was mixed with a few drops of chloroform in a small microfuge tube, and incubated at 37C for 10 minutes to sterilize it.  After a clarifying spin of 3 min at top speed in a microfuge, the supernatant was titered for phages, as follows.  A 1 uL aliquot of the supernatant was diluted into 1ml of
10-2 M MgSO4, and 1 uL of the dilution was added to 0.15 ml of an overnight broth culture of the E. coli C host cells.  Soft agar was added and the mix poured over a tryptone broth plate. When the soft agar had hardened, the plate was incubated overnight at 37C with the results shown in Figures 3 and 4 below.
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Picture 4.

Enlargement of Figure 3.  The resolution is not good, but the plaques are variable in size, have clear centers, and fuzzy edges.  It is unclear whether the plaque size variation is due to a single phage with variable plaque sizes, or because there is a diverse group of different phages.  [How could one test this?]
Picture 3. 

Plaques produced by phages from the enrichment culture on E. coli C. The ~250 plaques indicates that the titer of the overnight culture is 2.5 x 102 x 106  ≈ 2.5 x 108 phages/ml. 

Comments:

1.  This enrichment is an overnight phage-proliferation race.  If several phages can grow on the added host strain, the most fit, i.e., most prolific, phage will likely dominate the culture. There are strategies for finding other, less fit phages, such as isolating a host mutant resistant to the dominant phage, and repeating the enrichment with that mutant bacterium as host. 


2.  The host strain will likely have defenses, i.e., restriction systems, against some of the gut phages, and so those will be at a disadvantage.  One can use several independently isolated host strains, that may differ in defenses.  We’ve used an E. coli C strain, which lacks Type I and II restriction systems, and K-12 strains that have a Type I system, and both enriched phages from insect gut. 


3.  Other sources of enteric viruses are any creature with an alimentary canal.  Such creatures harbor enteric bacteria and their viruses.  The insect gut is an example, such as flies, crickets, and mosquitos. 


4.  In a lab exercise here, students dissected out the colon from crickets purchased at a pet shop.  The colon was mashed with a mortar and pestle, and used in an overnight selection as described above.  Phages were found, though not in every case. It is possible that domesticated crickets like those from a pet shop have less microbial diversity than wild critters, though this is a speculation.  An interesting extension of the insect gut experiment would be to weigh the dissected colon, and resuspend the homogenate in a defined volume, and then titer dilutions on broth agar and enteric-selective agar plates, i.e., Eosin-Methylene Blue or MacConkey agar plates. One could get an estimate of the number of enterics in an insect's gut.  Wild and petshop crickets are readily available and a comparison might be interesting. 

Selecting ‘Edit Text’ from this menu will also allow you to edit the text within this text boxWe.You can edit text on your website by double clicking on a text box on your website. Alternatively, when you select a text box a setWe asked about the presence of enteric bacteria in the slurry, by streaking a loopful of the slurry on a MacConkey agar plate and incubating overnight at 37C.  MacConkey agar contains crystal violet dye and bile salts, which inhibit the growth of Gram positive bacteria thus enriching for Gram negative enteric bacteria. The agar also contains the sugar lactose and neutral red dye.  Lactose fermenting bacteria like E. coli form d

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