Purpose: The focus of this experiment is to examine conjugation. The experiment has two goals. The first is to examine whether conjugation will occur between bacteria of different genera. For example, from Eschericia to Serratia. This will demonstrate whether conjugation is dependent upon the host in anyway. The second goal is to examine whether conjugation is able to occur from a Gram negative bacteria to a Gram positive. For more information on conjugation click here or for more information on bacterial cell walls click here.

Hypothesis: For the first part of this experiment, it is believed that conjugation will occur between bacteria of different genera. The reasoning for this is because the plasmid, the key player in conjugation, replicates independently of the host, and therefore should not need a specific host to conjugation with.  The hypothesis of the second part of this experiment is that conjugation will not occur through the Gram positive cell wall. The reasoning for this is the Gram positive cell wall possesses a relatively thicker peptidoglycan layer in the cell wall which will prevent a sex pilus from entering into the cytoplasm of the gram positive cell wall and thus prevent conjugation.

Equipment: Needed for this experiment was 6 strains of bacteria including 1 susceptible and 1 resistant to ampicillin strain of both Escherichia coli and Serratia marcesens, and 1 susceptible strain of both Staphylococcus aureus and Staphylococcus epidermidis. 4 different media will be required including Trypticase soy agar (TSA), TSA with .1mg/ml of ampicillin dissolved within (amp agar), EMB agar, & manitol salt agar. Other neccessary lab equipment include wire loops, bunsen burners, petri dishes, autoclave, and incubator set to 26 degrees celcius.

Procedure: The experiment entails 6 seperate experiments each crossing the different bacteria to attempt conjugation. The first part of this experiment involves a transfer between a Gram negative and a Gram negative bacteria. So the first two experiments will transfer between a resistant E. coli strain to a susceptible Serratia strain, and vice versa. General procedure includes streaking strains onto  amp agar to set controls (demonstrate resistance and susceptiblity), mixing the two bacteria onto a TSA to allow bacteria to conjugate. Then streaking from the mix plate, onto an amp agar test plate. If susceptible bacteria conjugated with resistant one, it should have picked up resistance and therefore should be viable on the test plate. When using susceptible Serratia, detection on the test plate is determined easily as the Serratia produces a pink pigment. When detecting for the E. coli, it requires an additional step of steaking from the test agar onto an EMB agar, which causes E. coli to produce a green metallic sheen. 
          The second portion of this experiment examines conjugation between Gram negative to Gram positive. This experiment is done by transferring from resistant E. coli or Serratia to susceptible strains of Staphylococcus aureus or epidermidis. Experiment is performed in the same manner, however, an additional step of streaking from the test plate onto a manitol salt agar, which selects for Staph. by killing off other bacteria with high salt concentrations. "streaking" refers to a process called the Asceptic technique which involves flaming a wire loop with a bunsen burner, placing bacteria onto the loop, and wiping back on forth on a different agar plate. 

Results: In the following table, "r" denotes resistance, "s" susceptibility

Experiment Performed	Observations; Did conjugation occur
E. coli (R) to Serratia marcesens (S)	Conjugation occured
Serratia marcesens (R) to E. coli (S)	Conjugation occured
E. coli (R) to Staphylococcus aureus (S)	Inconclusive: No growth on test plate
E. coli (R) to Staphylococcus epidermidis (S)	Conjugation did not occur
Serratia marcesens (R) to Staphylococcus aureus (S)	Conjugation did not occur
Serratia marcesens (R) to  Staphylococcus epidermidis (S)	Conjugation did not occur