This achievement requires that you do not harvest ANY little sisters on your play through. As long as you do not Harvest any little sisters, you will fulfil the requirements for this achievements. Thus, you technically do not have to do save every little sister in the game, just save every one you encounter. However, although "dealing" with all Little Sister's is not required, it is highly recommended that you do as there are only 21 and by saving all of them you will unlock this achievement and the "Dealt With All Little Sisters" achievement
Main bit: PLEASE make sure you have a good save right before the final boss fight (at the very, very end of Proving Grounds before you ascend the lift to start the final area)
This is because this achievement has an annoying bug with a very easy fix; for some reason, I can confirm this will not pop unless the game is finished on the new hardest difficulty "Survivor", or at least this seems to be continuous with everyone I know who has gotten this achievement (why this happens I do not know.) Thus, you might unlock this when you should as it should unlock when you try for another difficulty related achievement anyway, so this isn't the end of the world.
To get "Brass Balls", you will be doing a trick where you play the entire game on easy and when you........
and switch to hard by pausing........
Do exactly this for Brass Balls, then reload your save and do the same except switch to survivor instead. This popped it for me and everyone I know first time, and is required anyway to get "Seriously Good At This", so you very well might not need this at all!
Thought I'd do the solution as there seems to be some confusion. To summarise, do the difficulty glitch where you switch difficulty.
For anyone interested, use the electric gel in the chemical thrower. Downs the boss 3 times in about 45 seconds (literally)