I take maybe two weeks try to solve this problem but it's should be better to ask in this community. My work is to do the agar dilution for my organism Neisseria gonorrhoeae using GC agar base + 1% isovitalex according to CLSI guideline. I bought GC agar base from Oxoid company and isovitalex from BD. 

I prepare my GC agar base as suggested in the label of GC agar bottle by adding 18 g of GC agar base to 235 ml of distilled water (my water is RO) and boiling with stirring to dissolve the agar. After that I sterilize it by autoclaving and put it out to 50C water bath for cooling and adding isovitalex to be a 1% final concentration. This is how I make this medium.

But what I encounter for now is that my ATCC49226 that use for quality control in antimicrobial susceptibility testing cannot grow well after incubation 24 hrs in 5% CO2 at 37C and also my clinical isolates too. I ask this with the routine staff doing her job for preparing this media and she also told me that her media sometimes cannot grow too. 

So I wonder what I do a mistake or it is just typically happened in this media. does anyone has some suggestion? I try to do every step elaborately as suggested in CLSI guideline and manufacturer' instruction but still not good. 

I've prepare 100 ml of GC agar base and isovitalex 1 ml to make a 1% final concentration. BD will give you like 2 bottle, one for diluent and another one for powder. You have to reconstitute using 10 ml of diluent to dissolve the powder.

From my experience, ATCC 49226 and clinical isolates of Neisseria gonorrhoeae will grow well after 48 hrs incubation not 24 hrs. When incubation with 24 hrs, I can see colonies only first lane of streaking and sometimes I cannot see any grown colonies. What I should do because CLSI recommended 24 hrs of incubation.

Thanks for every suggestions.