Experiment Errors

[yousuf89]

I have done 2 experiments, one is Enumeration of Bacteria Using The Standard Plate Count and the other is Determination of Bacterial Growth By Optical Density. However my results didn't end well

the results of the Standard Plate Count is

 

DILUTION PLATED	ML PLATED	NUMBER OF COLONIES
1:10000	         1.0	      0
1:100000	        0.1	      1
1:1000000	       1.0	      4
1:10000000	      0.1	      0

 

and the theoratical result is

 

DILUTION PLATED	ML PLATED	NUMBER OF COLONIES
1:10000	         1.0	        >1000
1:100000   	     0.1	        536
1:1000000	       1.0	        47
1:10000000	      0.1	         6

 

For my results for the Determination of Bacterial Growth By Optical Density. my graph doesn't have a lag or stationary phase OR its not the graph that I wanted (the top is 3 ml and the bottom is 6 ml)

 

and it's suppose to be like this

 

according to my graph, it shows only the growth and death phase (no lag and stationary phase)

 

What errors that I've done in both of the experiments?

[CharonY]

The first experiment can go wrong due to a number of reasons. Without knowing the precise setup troubleshooting will be difficult. But some things to consider:

- was the initial count accurately determined and how

- were the cultures fresh

- was the dilution series made correctly, was the right buffer used

- was the medium OK

- did you let the scraper/spatula/whatever cool off (if heat sterilization is used) before plating the cells?

 

Regarding the second experiment, in the time frame that you measured (90 minutes) you cannot expect a growth curve. Even fast growers like E. coli or Shewanella have replication times of around 25 minutes under optimum conditions. So at most three cell divisions could have taken place in that time. However, depending on how and when you inoculated the batch you will start with the lag-phase (also the OD is already pretty high in your graph). And the results clearly indicate no growth as the changes are only minimal and can be easily the result of pipetting or mixing errors.

[yousuf89]

The first experiment can go wrong due to a number of reasons. Without knowing the precise setup troubleshooting will be difficult. But some things to consider:

- was the initial count accurately determined and how

- were the cultures fresh

- was the dilution series made correctly, was the right buffer used

- was the medium OK

- did you let the scraper/spatula/whatever cool off (if heat sterilization is used) before plating the cells?

 

Regarding the second experiment, in the time frame that you measured (90 minutes) you cannot expect a growth curve. Even fast growers like E. coli or Shewanella have replication times of around 25 minutes under optimum conditions. So at most three cell divisions could have taken place in that time. However, depending on how and when you inoculated the batch you will start with the lag-phase (also the OD is already pretty high in your graph). And the results clearly indicate no growth as the changes are only minimal and can be easily the result of pipetting or mixing errors.

 

I didn't understand the explanation of the second experiment

[CharonY]

In order to see a complete growth curve including lag, growth and stationary phase the experiment would have to run for several hours.