Question

List several advantages and disadvantages between the techniques of replication plating or using an agar-gradient plate preparation for detection of spontaneous streptomycin resistance.

Answer 1

Spontaneous resistance simply refers to strains of microorganisms that undergo spontaneous mutation to develop resistance against a particular antibiotic(here streptomycin). These mutant strains can be easily detected by some techniques as the mutant strains can grow on antibiotic concentration which inhibits the growth of normal (non-mutant) strains of microorganisms.

A) Replica plating technique - this technique was developed by Ether and Joshua Lederberg in 1952. The microorganisms colonies are grown on the original or control plate. These plates contain growth medium like nutrient agar on which multiple colonies develop. These patterns are then replicated on a velveteen like sheet and these are imprinted on the other plates (secondary plates). The secondary plates may contain the antibiotic resistance to be tested. So the strains which grow on streptomycin containing secondary plate are the resistant strains as the non-mutant strains do not survive. Multiple secondary plates can be made to test for the development of spontaneous streptomycin mutation. If the mutant strain surviving in the second plate also shows growth on the third plate, it affirms that the strains are indeed mutant. The advantage of this method is that antibiotic-resistant strains can easily be isolated and the type of mutation can also be verified through multiple secondary plates and comparing to the control plate. However, this is a time consuming and cost-incurring procedure as if on the third plate, no growth is observed, the whole procedure becomes useless as the results would be inconclusive.

B) Gradient Plate technique - In this technique, a gradient plate consisting of two layers is used. The top layer has streptomycin added nutrient agar while the bottom layer consists of nutrient agar as the control layer. A gradient is created by diffusion of streptomycin from top to bottom layer. The strains are then introduced in the gradient plate and incubated for 24-72 hours. After 72 hours, the gradient is observed. The colonies of strains of microorganisms in the top layer(streptomycin containing agar) indicate these are streptomycin resistant strains as they grow in the presence of the antibiotic. This method is relatively quick and the gradient plate indicates a clear separation of the two different nutrient agar layers, giving clear results. This technique provides more sensitive results for streptomycin spontaneous mutant strains. However, we cannot make replicates of the grown colonies in the gradient plate easily as it might be difficult to isolate the mutant strains and further verify the spontaneity of the mutation of these strains by growing them in a separate nutrient-rich media. Also, it depends on various environmental conditions like pH, temperature etc.

Hence, both methods have their own advantages and disadvantages.