Appendix 1: Sample activity for students. Appendix 1 The Investigation of the Op
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Appendix 1: Sample activity for students. Appendix 1 The Investigation of the Optimal Growth Conditions Your objective in this laboratory activity is to investigate the optimal growth conditions of three different microorganisms and apply your knowledge of physical factors that impact their growth to solve a problem. After completing this activity you will be able to: Explain how competition affects the growth of a microorganism when it is grown in a mi culture. * xed urately apply the following terms to organisms after evaluating their growth in media with different salt concentrations: halophile, halotolerant, and non-halophile. Differentiate a set of organisms growing under various physical conditions based on their morphology and growth response to those conditions Design a simple culture based on its known optimal growth conditions. method to isolate a specific microorganism from a mixed community You will be working with three microorganisms: Halobacteriam salinarium, Serratia marcescens, and Staphylococcus aureus. A description of the salinity and temperature range of each of these organisms is provided below Halobacterium salinarium is classified in the domain Archaea. When it is cultured in the laboratory it requires at least 15% (wtvol) salt in the growth medium. It grows optimally when the salt concentration is above 15%. This bacterium produces pink colonies when grown optimally, due to a pigment called rhodopsin. Its optimal temperature for growth is 37-42 degrees C. While it can grow at 25 degrees C, it requires a longer incubation time for growth and the growth is sparse. At 37 degrees C, a plate will take approximately 7.9 days to produce adequate growth with visible colonies. Serratia marcescens is classified in the domain Bacteria. It grows optimally at a 1% salt concentration in its media. It can tolerate salt concentrations up to 5%, but it doesnt grow very well when the salt concentration is above 3% (sparse growth and the colonies lack color). S. marcescens produces bright pink colonies when it is grown optimally, due to a pigment called prodigiosin. Its optimal temperature for growth is 25 degrees C. It also grows well at 37 degrees C, but produces slightly less pigment. At 25-37 degrees C a plate will take 18-24 hours to produce adequate growth with visible colonies Staphylococcus aureus is classified in the domain Bacteria. It grows optimally at a salt concentration that is between 1% and 5%. It can tolerate salt concentrations up to 10%, but the growth is sparse when the salt is above 7.5%. This bacterium produces creamy and sometimes yellowish colonies. Its optimal temperature for growth is 37 degrees C. It can grow at 25 degrees C, but does not grow as well at that temperature. At 37 degrees C a plate will take 18-24 hours to produce adequate growth with visible colonies.Explanation / Answer
Aim : to obtain pure culture of Halobacterium salinarium, Serratia marcescens and Staphylococcus aureus. from their mixed sample
Concept: In nature, microorganisms exists as mixed population, however to study microbial characteristics, we need to have them in pure form. This requires, dilution of the sample till individual microorganisms becomes separated on the agar surface. Such plate from which well separated microbial colony can be picked and pure cultured is called isolation plate. In the current scenerio the three different microorganisms can be morphologically identified on the basis of their pigment production at 370 C, and two different salt concentrations ( 1% and 15 %).
Protocol:
Serially dilute the sample using sterile saline solution.
Plate three consecutive dilution on growth medium prepared at two different concentration of salt (1% and 15%).
Streak plate or spin plate method can be used for isolation technique.
The plates are incubated at 370C till growth is seen.
Results: Considering the salt tolerance, Serratia marcescens and Staphylococcus aureus will not grow on the growth mediium with high salt concentration (15%). Halobacterium salinarium can be easily identified by their pink pigmentation under optimum growth conditions. (15% salt at 370C). Similarly on growth medium (with 1% salt) both Serratia marcescens and Staphylococcus aureus will grow as bright pink and creamy yellowish colony.
well isolated colony of each can be picked up from the isolation plate and transferred to new sterile medium and incubated again. The resultant bacteria will be the pure culture.
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