When I spot the phage lysate on MG1655, I see the clearance. But, to calculate the phage titer I mix the lysate (100ul) with MG1655 as background strain (100ul) in 3ml 0.5% agar. In this case, I am not getting any plaques. What could be the possible reasons?
I diluted my overnight culture of the background strain to OD 0.1 and then used this in the top agar method.
Are you making serial dilutions of your phage lysate? The titer of your lysate will be orders of magnitude too high to give you individual plaques to count.
I did not make dilutions. But if titre was high, there shouldn't be any growth of background host. I observe conplete lawn of host growth in plate.
True but you are always going to get some growth in the lawn regardless. Are you sure that the spots you saw were really lysis from phage and not just some growth inhibition or water spots? You might want to see if a 10-fold and 100-fold dilution also give you spots (they should if you really have phage)
Because absence of microorgnism or lack or defect in stain crystal violet or inubation temperatue.
Thanks. I shall try to use exponential phase culture and also overnight culture with dilutions. I am also worried if the phages are sensitive to chloroform, which I used to clean the lysate and also few drops before storing them at 4 degrees.
Many phages are resistant to chloroform but certainly there are some phages that can be inactivated by choloroform. If you want to be certain, then you can sterilize your lysate using a syringe filter (0.45 or 0.22 micron). Do you know what phage you are working with?
I do not know what phages. I am trying to induce active prophages using ciprofloxacin and then characterize the plaques and likely phages.
Do you know your putative lysogenic strain is an E. coli K12 you are trying to induce? If not then I would recommend you use a restriction minus host to screen for plaques instead of MG1655.
I induce prophages from environmental E. coli strains and then plate these lysates on MG1655.
I would suggest that you use a strain that is Restriction minus (hsdR- or hsdS-). There are some variants of MG1655 carrying this mutation but the standard MG1655 is wild type for the restriction system. You could also use any other strain that is hsdR- instead (but be sure it is RecA+ or some phages won't grow).
Otherwise the phage from many environmental strains might have plating efficiencies reduced by 10e4-10e6 fold due to restriction.
Thanks. But, I do not have restriction minus strain. Nor I can buy it.
If there are any good E. coli labs at your university they should have something. It might still work without but you will need to have good titers of phage to overcome restriction.
1) Why I am getting a lawn of bacterial growth in the center of the spot?
2) Also, when plating the same lysate using the top-agar method along with MG1655 as background strain, I see heterogeneous colonies and some clearance around them. However, these colonies are yellow in color.
3) Prophage induction using Ciprofloxacin sometimes yields a high phage titer and sometimes no plaque. What could be the problem?
4) When spotted with dilutions (10^0 to 10^12 dilutions made in SM buffer), I observed no plaques in any of the dilutions except in 10^0 dilution.
Somehow and somewhere my technique is wrong.
How do I get this right?
THank you.
for Your first question, which I presume is about the bottom picture, the most likely explanations are either that your phage is lysogenizing your playing cells at high efficiency and the growth in the center are the lysogens OR your phage lysate is not sterile and those are cells from the lysate spot. I think the second one is the most likely. You can easily test by spotting a few microliters of the phage lysate on a fresh plate with no lawn and see if you see growth.
the top picture is hard to interpret. It could be either your plating culture was contaminated or top agar was too hot. You should always prepare a nonphage lawn to be sure the lawn itself looks look every time. (The lawn in the bottom picture looks good). It may be the same problem that your lysate is not sterile. And the colonies with clear spots around them are the original lysogens still releasing some phage.
Again you should always do control plates until you are confident of your technique, phage only, cells only, and the dilution series.
I did control plates.
1) Plate with only MG1655
2) Plate with only lysate
The Lysate-only plate does not show any bacterial colonies. But, spotting shows a lawn at the center.
Also, I am getting this small yellow colony contamination every time I do plaque assay. Yellow colonies show clearance around them but this is not likely E. coli (since E. coli colonies look colourless).
The yellow colonies are probably some contaminant with a zone of inhibition around them.
Steriliity test for the lysate is to just spot 5ul or 10ul direct into an LB plate
Is there a possibility to find the phage-type or predict what kind of phage is it from the plaque morphology?
I have direct and short answer that you phage is temperate.That's it.
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