Cause of the Glitch Edit
The extra Little Sisters bug is the result of scripted events that trigger a Little Sister and Big Daddy pair for a specific scene – typically a fight with Splicers. The triggers are usually as simple as entering a particular area (i.e. in Neptune's Bounty, it's the Upper Wharf; in Frolic it's the Atrium after visiting the Plaza the first time; in Arcadia, it's a Gatherer's Garden.) If the player avoids these trigger points until all Little Sisters have been dealt with, then entering the trigger area will spawn an extra Little Sister.
In other levels with these scripted events, it is impossible to avoid the trigger, because the player is required to pass through that particular area in order to advance the game. For example, in Hephaestus there is a Big Daddy defending a Little Sister at the entrance to the Core area the first time the player passes through, but since that is the only way into the facility, it is impossible to avoid triggering the event.
Extra Little Sisters In Each Level Edit
When the first Rosie is encountered in the dock area he will retrieve a Little Sister from the hallway before the dock and they start gathering ADAM from a corpse in a muddy area in the middle of the Lower Wharf. DO NOT attack Rosie until the scripted fight between a Rosie and the Nitro Splicer on the balcony starts (it happens as Big Daddy walks across muddy area). After this scripted fight you can kill Rosie and gather the ADAM from the Little Sister. Eventually, another Rosie will spawn and get another Little Sister from the vent in the Lower Wharf. After dealing with her, wait for THIRD Rosie in the same area and repeat extraction.
Don't talk to Peach Wilkins until all three Little Sisters have been dealt with, and wait for new Big Daddies in spawn location to appear. After all three have appeared, talk to Peach, he will open the gate to Upper Wharf, at which point the extra Little Sister will appear in the Upper Wharf. Head there and witness another scripted fight between Leadhead Splicer and Rosie, kill Rosie and gather FOURTH Little Sister. After she is harvested or rescued, a fourth Little Sister symbol should appear on the pause menu.
Save the game before fighting the last Big Daddy, since if the player is killed, the extra Little Sister may have time to "go home" permanently.
X-Box 360 & PS3 "Greatest Hits"Edit
In the PS3 "Greatest Hits" version of the game, the Little Sister (and her Rosie) in the Upper Wharf of Neptune's Bounty will respawn in the same area. First, kill the Rosie and deal with the Little Sister, then head back to the corridor between Jet Postal and The Fighting McDonagh's Tavern. As the player heads back toward the passage up to the Wharfmaster's quarters, a respawned Little Sister and her Rosie will appear. The player may need to go up to the Wharfmaster's Office area for this to occur, but do not get the camera until the extra Little Sister has been found.
There are normally two Little Sisters in Arcadia, but if players avoid the scripted fight between a Splicer and Big Daddy at the Gatherer's Garden outside the Grotto area, then a third can be found. If the player hears a Big Daddy fighting Splicers when passing around this area before dealing with the two Little Sisters, it means that the event has already been triggered and it will be necessary to restart from a previous save to try again.
To trigger an extra Little Sister, go directly from the Tea Gardens to the Lower Concourse, avoiding the Gatherer's Garden in the upper area. Staying in the canal, travel all the way through to the Storage Room, then up the stairs to the Upper Rolling Hills where there should be a Big Daddy and a Little Sister. The key to getting this to work correctly is to crouch down as you maneuver through the canal. Otherwise, you may trigger the scripted event on the way to the Storage Room. While traversing through the canal take note of a Houdini Splicer nearing the end of the canal. The player will notice him with triggered splashes that can be visible in the water. It is recommended to stick to the right side of the canal while moving slowly while remaining crouched to remain undetected. Fighting this Splicer while in the canal can tempt the player to stand which may trigger the event in Garden. Go to the Upper Rolling Hills. On the way you will encounter an RPG turret. Deal with it then continue past the turret downstairs and the player should run into the first Big Daddy on the way. Wait until he grabs a Little Sister then Kill him to deal with the first Little Sister.
At this point, Big Daddys have been activated across the entire map and the closest Big Daddy spawn point is by the health station you just passed. So turn around and you will see a lone Big Daddy there, give him a few minutes to lumber off and when he returns he will have a Little Sister in tow. Deal with them as you see fit and then head to the Gatherer's Garden to start the scripted fight with the Extra Little Sister. Alternatively:
After the first Big Daddy has been defeated and the Little Sister has been dealt with, trigger Andrew Ryan's radio message and release of the poison and go to Langford's lab, which will prompt the opening of the Tree Farm.
Go find the rose Langford wants. However do not enter through the area containing the rose using the door across the Garden as this may trigger the event. You will see - through the other doorway - several Splicers beating a burnt Houdini Splicer. Go through that doorway, get the rose, and leave the way you came in (NOTE: It is highly recommended that the player make back-up saves. Then test to see if the event triggers. If nothing happens in the Garden then the event has already happen, which will require the player to load a second previous save. Otherwise if the event has been triggered reload the last save. This is basically to test that the event hasn't been triggered.) Return the Rose to Langford and keep playing until the player receives the next goal to create the Vector. On the way out of the office the player should find a Big Daddy in the area leading up to the office. Kill him and deal with the second little sister.
After the first two Little Sisters have been dealt with, head towards the Gatherer's Garden machine. The Gatherer's Garden can be approached from the Tree Farm entrance or from the Tea Garden.
To find an extra Little Sister in Fort Frolic, do not return to the Atrium before three Little Sisters have been dealt with in Poseidon Plaza.
After gaining access to Poseidon Plaza, head to the plaza. Deal with all three Little Sisters within Poseidon Plaza, waiting for each Big Daddy to spawn after the last has been killed. When ready for the extra (fourth and last) Little Sister, head back to Sander Cohen's Masterpiece to find her. Her spawning is triggered by a fight between her Big Daddy and several Spider Splicers.
Note: If the player finishes exploring all of Poseidon Plaza once having dealt with three Little Sisters there before returning to the Atrium (i.e. fully investigating all rooms including the basement and [more importantly] completed all objectives in the area for Cohen), the triggered Big Daddy fight near the Masterpiece will be skipped and the extra Little Sister can not be found without reloading a save that meets these criteria.
The player receives gifts from Brigid Tenenbaum for every three Little Sisters that are rescued – seven gifts total and maximum – and extra Little Sisters count towards this number. Therefore, it is possible to get Tenenbaum's gifts early by saving the extra three Little Sisters. However, there is no extra eighth gift or message from Tenenbaum if 24 sisters have been rescued.
The ending cinematic the player receives after defeating Fontaine depends on the number of Little Sisters the player has saved or harvested. There are normally 21 Little Sisters in the entire game, but by successfully exploiting the glitch in each level there can be 24. Therefore, if the player has found extra Little Sisters, it is possible to harvest one without the game counting it toward the bad ending.