What happens to bacteria that were used in a procedure that have been incubated for a longer period of time?

this bacteria was used for a Gram staining prodcedure. Before it was used, the bacteria was incubated for a long period of time. Would this alter the result in anyway? Does the bacteria cell walls get stronger or weaker with time?

What happens to bacteria that were used in a procedure that have been incubated for a longer period of time?
Unless you were constantly adding nutrients to your cell culture, (and unless you have some really expensive equipment, you were not), the bacteria would stop growing.





When that happens, the cell wall would become weaker.





The Gram stain uses Crystal violet dye which stains the thick peptidoglycan layer in the cell walls of Gram positive bacteria. If bacteria do not have this layer, they will not keep the crystal violet stain when washed with your decolorizer (usually acetone or alcohol). So the cells would only take up your counterstain (safranin, a red/pink dye). This would normally indicate that the bacteria are Gram negative.





Therefore if you have an old culture, the peptidoglycan of the cell wall would not hold the crystal violet dye. So no matter whether your bacteria are Gram positive or negative, you would only see pink bacteria.





So this would cause gram positive bacteria to appear gram negative.
Reply:This really depends on the type of culture and nutrient base used to colonize the bacteria. If the nutrient base runs out the microbes typically turn to one another for survival. In which case most will die as a result. If you there is enough nutrient base and environmental temperatures/pH is able to sustain microbial life you will have constant colonization.