[SunilNagpal]

Hi Ozana,
Apologies for the delayed response. Your query was assigned to a subject matter expert at IIT Delhi, but being out for a conference she has not been able to respond. So, I thought to try my knowledge of the long left wet lab.?

Well, following could be a logical flow for troubleshooting :
a) My dyes are working fine or not (use a different batch of dyes). If that's not a problem then:

b) Resuspending in 2 batches: One in PBS, and other in Medium broth itself (to ascertain whether there was a problem with PBS or not).
If that's not a problem then:

c) OD monitoring before and after incubation period (to ascertain that there was no problem with growth conditions).
If that's not a problem then:

d) I am using sufficient active inoculum or not.

Looking at the protocol, it's indeed intriguing that you are getting this problem. In all its probability, it seems a problem of step a or b.

Hope it helps.

Do let us know what was the issue once you figure it out.

Cheers