Hi Nishant, 

#For a phage to enter a cell,  its absorption is the first requirement.  When it comes to absorption of phage to bacterial cells few salts play a major role in it .  Like Calcium chloride, Magnesium salts.  Just look in the journals regarding the usage of salts.  

# Incubate the phage and bacterial cells for 5min before adding to the soft agar.  

# Age of the bacterial culture also should be taken care of .  Try to use a bacterial culture which is in early log phase. Inoculate the phage suspension during early log phase.  

# Also the count of phage in the suspension also plays a major role when you perform the plaque assay.  To determine the Pfu do spot test initially. Make a soft agar with the host bacteria. Then do a serial dilution with the phage solution.  Add 10 micoliter of the diluted phage suspension to the bacterial lawn.  

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Many potential variables here. More information will help in answering this question. What type of phage (if you know)? What is the target bacteria? If you grew the phage in a broth solution with your target and then plated, it's very possible for complete lysis of the entire bacterial culture so there would be no visible plaques. It's always good to start with a pure bacterial culture and include at least one 'control' plate to ensure growth occurs. Then perform your serial dilution with the phage.

Some phage benefit from having oxygen bubbled through the broth. It may also be a factor of switching between lytic and lysogenic stages. It is unlikely that this is related to attachment factors or salt concentrations unless you changed the composition of your medium.

thanks for your reply 

I have already add the CaCl2 in broth but still that phage in not propagating in broth. The lysate added for propagation is showing plaque on agar plate but when the same lysate added in broth it was not propagating or increasing titre in broth. I have checked the titre/propagation of incubated broth through plating on agar plate and observed that agar plate was not showing any plaque.  I will be happy to receive any scientific reasons for the  same.

Dilute your phage before Double layer agar plating and can you show your plates 

Hi Nishant, do you know how much phage do you have on your lysate? bear in mind that from a single plaque picked and resuspended in 1mL of the approppriate broth, you can get up to 1 x 10E8 Pfu/mL, so if you've made the lysate from a scrap plate full of plaques, your lysate can be easily at 1 x 10E11 pfu/mL, (I'm not sure how you're titring your phage suspension where you say you don't find plaques), but if you do a plaque assay to get single plaques, you'll need to dilute several times (maybe about 7 times) your sample (10 fold dilutions), so you'll end up getting countable plaques, if you don't dilute the appropriate times you'll find just a bacterial lawn disrupted or almost cleared by phage lysis. All of it on agar plates, pouring a soft top agar layer containing the phage-host dilution :)

Hi, when you inoculate your phage to the broth medium inoculated with bacteria, do you agitate it during incubation or not? If you use high agitation rates, phages may not be able to attach to the bacteria..

Thanks for reply

I am incubating at 100 rmp shaking condition.

Actually, I am not getting plaque after incubating the LB broth and host with 100 ul phage lysete. But lysete is showing plaque on agar plate means phage in lysate.

I have diluted and plate is showing lawn of host . Any specific conditions is required to propagate the phage in broth. Pl guide me. 

I think 100 rpm is high speed. I incubate phage-bacteria without shaking or with shaking at 20-30 rpm. 

Was your plaque clear or turbid?

Hi Nishant, I'm wondering if you have solved your problem. I have the same problem lately with a naturally isolate E. coli phage. it can form big and clear plaques but can not propagate well in liquid culture

Hi Nishant, I have the similar problem with my phages. Have you managed to solve the issue? Can you, please, share your experience? Thank you in advance!