Suppose that you are staining an old culture (more than 24 hrs) of gram-positive organisms. how will the age of the culture affect the staining results? why?

the old bacteria start to loose proteins of their membrane

Explanation:

In a healthy cell, the environmental surrounds it, will no cause lost of proteins, but as the cell get old, by natural means, the proteins  responsible to be stain using the violet color for stain, will be less, so in way you can confuse the result of gram positive into gram negative, even is a positive bacteria but with lack of proteins.  

substitution is the answer in this case