Lab 5. Part 3. Re-Propagation, RNase Sensitivity testing and Genotyping F+ Coliphage
Isolates by Nucleic Acid Hybridization
Introduction.
F+ coliphages as fecal indicator viruses ican be distinguished on the basis of taxonomic
group as F+ RNA viruses of the family Leviviridae and F+ DNA viruses of the family
Inoviridae. The F+ RNA viruses can be further distinguished into 4 subgroups, I, II and
III and IV, which appear to have some specificity with respect to their animal host of
origin as being either human (Groups II and III) or other animals (Groups I and IV).
The F+ RNA and F+ DNA coliphages can be distinguished from one another by their
response to RNase, an enzyme that degrades RNA, when they are cultured in bacteria
host cells. When performing infectivity assays, such as plaque assays or spot plate
assays in agar media, F+ RNA viruses are inhibited by RNase and do not grow (no
plaques or lysis zones). In contrast, F+ DNA viruses are not inhibited by RNase and will
grow when RNase is present in the medium (plaques and lysis zones appear).
Once F+ coliphages are resolved as RNA or DNA viruses by their response to RNase,
the F+ RNA viruses can be further analyzed by immunological (serological) or nucleic
acid methods to determine their sub-group (I, II, II, or IV).
Besides immunological or serological grouping methods, such as the particle immunoagglutination assay you previously previously used the F+ DNA and various groups of
the F+ RNA coliphages can be grouped by methods based on detecting their specific
nucleic acid. A conventional method for this is a so-called nucleic acid hybridization
assay. In this assay a sample of nucleic acid extracted from the specific virus is mixed
with a nucleic acid “probe” that has a complimentary nucleotide sequence to that of the
virus group or sub-group (such as F+ RNA group I, II, III, or IV). When strands of target
(virus) nucleic acid and the probe are complimentary they form a hybrid (duplex)
molecule that can be physically separated from the unreacted singe strands of probe
nucleic acid. The hybrid molecules can then be detected by a signal-generating
molecule that has been incorporated into the nucleic acid probe. This signal generating
molecule can be radioactive, enzymatic or fluorescent, thereby allowing the hybrids to
be detected using analytical methods or instruments that detect the radioactivity,
enzymatic activity or fluorescence, respectively.
In order to carry out F+ coliphage identification by immunological or nucleic acid
methods, it is often useful to first isolate, purify and re-grow (in their host bacteria cells)
the separate coliphages that have been detected initially as plaques or in broth
enrichments. Each plaque on a plate from coliphage analysis of an environmental
sample (such as sewage) may be a different coliphage. In this lab exercise you will
take coliphages previously isolated and re-grown (enriched) from individual plaques of a
plaque assay of sewage, identify the plaques as F+ RNA or F+ DNA coliphages by
RNase susceptibility testing and then subject the F+ RNA and F+ DNA coliphages to
analysis by nucleic acid hybridization.
PURPOSE
F+ coliphages previously isolated and re-grown (enriched) from individual plaques of a plaque
assay of sewage will be:
(1) identified as RNA or DNA coliphages by RNase susceptibility testing and then
(2) further confirmed as F+ DNA coliphages and identified as F+ RNA coliphage sub-group (GI
= MS2-like), GII = GA-like, GIII = Q-beta-like), and GIV = SP- and FI-like) by nucleic acid
hybridization.
Materials for RNase Testing Nucleic Acid Hybridization (Genotyping) Lab:
A. For RNase Sensitivity Testing and Re-Spotting of F+ RNA and F+ DNA Coliphage Isolates
1. F+ coliphage isolates from previous lab; preferably as re-enrichments or as centrifuged reenrichments
2. E. coli Famp host, log phase or overnight culture; keep chilled on ice
3. Tryptiic soy agar with antibiotics
4. RNase stock, 100, 0.1 mg/ml (100 mg/l or 0.1%.
5. Petri plates, 100 mm diameter, sterile
6. Pipets, 1, 5 and 10 ml
7. Pipetter, 100 and 1000 ul capacity, with sterile pipet tips
8. Pasteur pipets, plugged, sterile
9. Pasteur pipet bulbs (small squeeze bulbs)
10. Coliphage diluent: either tryptic soy broth, peptone water or PBS
11. 50-ml capacity conical centrifuge tube, sterile
13. Test tubes, 13x100 mm or 16125 mm, sterile, glass, with sterile slip or screw cap
OR polypropylene tubes, 5-10 ml capacity, sterile, with screw cap
14. Microcentrifuge tubes, sterile, flip or screw cap
B.
1.
2.
3.
4.
5.
6.
7.
For Nucleic Acid Hybridization Assay
Coliphage isolates, as re-enriched cultures ; identified as F+ RNA and F+ DNA
Positive control F+ RNA coliphages (MS2 (GI), GA (GII), Qβ (GIII), SP (GIVa), and Fi (GIVb)
Positive control F+ DNA coliphages (M13 and Fd)
Pre-made single agar layer-E. coli Famp host cell plates in tryptic soy agar (made prior day)
Nylon Membranes - need 4 per 30 isolates
Blotting Buffer (4.0M Formaldehyde and 7.5x sSc)
Pre-hybridization Solution
 8x SSE
 5X Denhardt’s solution
 16mM Trinl Buffer pH 8.3
 0.1% SDS
 75 µg/ml salmon sperm DNA
8. Genius Buffer (Type I, II, III, and IV)
 Types I, III, and IV can be autoclaved and stored at room temperature.
 Type II must be heated on block to 60oC (NO AUTOCLAVE) and stored in refrigerator.
9. 6X SSC/0.1% SDS
10. NBT
11. X-phosphate
12. Membrane handling materials, trays and other containers for buffers and reagents
METHODS
Day 1 – RNase Testing
Prepare F+ coliphage isolate for spot plating.
1a.Obtain the previous enrichment cultures of picked F+ coliphage plaques that confirmed with
lysis when spot-plated. You may have up to 6 of them. Transfer about 0.3 to ml of each F+
coliphage-positive enrichment to a sterile microcentrifuge tube.
1b. If you do not have the enrichment cultures, obtain the spot plate from the previous lab and
locate those spots that are positive for lysis. Use a sterile 10 ml pipet to extract coliphages from
the lysis zone and resuspend this material in 0.2 ml of TSB or peptone water or PBS in a sterile
microcentrifuge tube. Repeat this step in a separate microcentrifuge tube for each lysis zone.
2. Centrifuge the microcentrifuge tubes with coliphages at top speed for 10-15 minutes to pellet
cells and debris.
3. Recover the supernatant from each centrifuged enrichment with a pipet and transfer to
another sterile microcentrifuge tube.
Prepare spot plates
4. “No RNase Plate”, 100 mm diameter: In a sterile 50 cc conical tube combine 20 ml of tryptic
soy agar and 1 ml of E. coli Famp, swirl or roll in your palms and pour into a 100 mm diameter
Petri plate. Allow to harden.
5. “RNase Plate”, 100 mm diameter: In a sterile 50 cc conical tube combine 20 ml of tryptic soy
agar,0.2 ml of 100X RNase and 1 ml of E. coli. Swirl or roll in your palms and pour into a 100
mm diameter Petri plate. Allow to harden.
6. “Hybridization spot plate”, 150 mm diameter. In a sterile 50 cc conical tube combine 40 ml of
tryptic soy agar and 1 ml of E. coli, seal cap, roll in your palms or gently invert several times to
mix, remove cap and pour into a 150 mm diameter petri plate. Allow to harden. Store at 4
degrees C.
Spot plating
6. One each plate, “No RNase” and “RNase”, mark a grid pattern on your plate for each F+
coliphage isolate that you centrifuged and from which you recovered the supernatant.
7. On each plate gently spot one drop (a few microliters) onto the surface of the agar medium
of each plate.
8. Allow spots to dry or seep into the agar medium and then incubate the plates overnight at
35-37 degrees C
9. Save microcentrifuge tubes with F+ coliphages for later use; store at 4 degrees C.
Next Day:
Morning:
10. Identify those spots on your “No Rnase” and “RNase plates” that are positive for lysis zones
and determine if each coliphage isolate is an F+ RNA or F+ DNA coliphage.
11. Retrieve two pre-poured 150 mm diameter spot plates, label one DNA coliphages and
another RNA coliphages and prepare a square grid pattern on the bottom of each plate.
12. Retrieve the microcentrifuge tubes of F+ coliphage supernatants and identify which ones ar
F+ RNA and which ones are F+ DNA. Label individual squares on the DNA and RNA plates
with identities of the specific coliphage isolates you will spot onto that square of the plate. Be
sure to reserve the top row of the plate for the positive control coliphages, either F+ DNA
or F+ RNA. See and follow the example grid pattern below.
13. On the F+ RNA plate, gently spot one drop (a few
microliters) of the appropriate F+ DNA coliphage onto
the surface of the medium of each plate. Be sure to
spot the positive control coliphages onto the squares
reserved for them. The first row of spots are
designated for the positive controls phages: MS2 (GI),
GA (GII), Qβ (GIII), and SP/Fi (GIV). The remaining
space is for 15-30 environmental isolates.
Spot the controls and isolates onto the agar from right
to left. Starting with MS2 in the upper right corner (refer
to figure 1).
14. For F+ RNA coliphages, repeat this procedure for
a total of 5 plates.
15. On the F+ DNA plate, gently spot one drop (a few
microliters) of the appropriate F+ DNA coliphage onto
the surface of the medium of each plate. Be sure to
spot the positive control coliphages onto the squares
reserved for them.
16. For F+ DNA coliphages, repeat this procedure for a total of 2 replicate plates.
17. Allow spots to dry or seep into the agar medium and then incubate the plates either until the
afternoon or overnight at 35-37 degrees C.
Methods for Nucleic Acid Hybridization:
In the afternoon or the following day, the phages in lysis zones of spot plates will transferred
from the lysis zones on the plate onto a nylon membrane for hybridization. For F+ RNA
coliphages four oligonucleotide probes will be used, with one nylon membrane required for each
probe. For F+ DNA coliphages, two probes will be used, with one nylon membrane required for
for each probe.
18. Remove spot plates from the incubator and place in the refrigerator at 4oC for at least 30
minutes to make the agar more solid and damp.
19) The nylon membranes used to capture the phages comes in sheets provided with protective
layers above and below. Plastic gloves should be worn at ALL times when handling the
membrane. This is to protect the membrane from any stray nucleic acids on the fingers.
20) Each group doing F+ RNA coliphages will need 4 membranes. Each group doing F+ DNA
coliphages will need 2 membranes. Mark the membranes lightly with a pencil on the protective
layer, to identify the sheets. Then, carefully cut the membranes with sharp scissors as
instructed.
21) After separating the membranes, stack them together and snip a corner off to serve as a
reference point.
22) Use a sharp pencil to lightly label each membrane in one corner with the number of the
probe (I, II, III, or IV for RNA and ____ and ____ for DNA) and the initial of one of the group
members.
23) Orient the nylon membrane over the plate so that the pencil labels are face down and the
notched corner is at the upper right (Be mindful of the snipped corner when labeling, so
confusion won’t result in the final genotype determination).
24) To best place the membrane on the agar, start at the bottom end and apply the membrane
by “rolling” it onto the agar surface.
25) For the first transfer from each of the duplicate RNA and DNA spot plates, the membrane
should be allowed to stay on the agar for two minutes.
For F+ RNA coliphages, the membranes used for these transfers should be the ones
marked I for MS2 and II for GA.
For F+ DNA coliphages, the membranes used for these transfers should be the ones
marked ___ for ___ and ___ for ___.
26. Remove the membranes carefully from the agar; do not “yank” the membrane off of the
agar. Pull the membrane off using the opposite motion you used to place it on the agar.
 DO NOT set membrane face down on lab bench.
27) For the second transfer for F+ RNA coliphages, the membranes marked III for Qβ and IV for
SP/Fi should remain on the agar for four minutes.
28) Wash each filter for 2 minutes in each of the following in succession:
1: 0.2MNaOH + 1.5M NaCl
2: 1M Tis-Cl + 1.5M NaCl
3: 2X SSC
To release and denature the nucleic acids and bond them to the membrane, first saturate the
membranes in a denaturing solution called blotting buffer (4.0M of formaldehyde and 7.5x SSC).
This is done as follows:
 Label a set of 50 cc conical centrifuge tubes so that you have one for each membrane (I,
II, III, and IV for RNA and ___ and ___ for DNA).
 Pipette the buffer into each 50 ml Corning centrifuge tube (make sure to saturate all
membranes).
 Lay each membrane face up on a nylon mesh and roll the membrane and nylon mesh
into a cylinder, taking care not to let any part of the surface of the membrane touch any
other part of the membrane surface.
 While rolling the membrane make sure not to place your fingers in the areas where the
coliphages are bonded. (NOTE: Handle the membranes from their outer edges).
 Insert the rolled membrane cylinder into its marked centrifuge tube containing blotting
buffer.
 Turn and invert each tube to soak the membrane in the buffer.
 Then immerse the tubes in a water bath at 65oC for 15 minutes (or boiling water for at
least 5 minutes).
This heating step in the water breaks down the proteins of the phage coats and releases the
nucleic acids. Once the nucleic acids have been released it is necessary to bond them to the
membrane.
29) Remove membrane cylinders from their tubes of blotting buffer, unroll, and exposed to long
wave ultraviolet light for 5 minutes. (The UV light bonds the nucleic acids to the membrane).
30) Dry membranes in a vacuum oven at 80oC for 15 minutes, as directed by the instructor.
(This step also removes formaldehyde from the previous saturation step).
31) Store the dried membranes at -20oC for use.