Spectrophotometer and Bacteria Growth

LAB 4 - Spectrophotometer and Bacteria Growth

Introduction

When cells are exposed to certain environmental conditions, they divide and reproduce at an accelerated rate. Understanding how quickly these bacteria divide in a laboratory allows for microbiologists and engineers to determine how quickly these same bacteria can reproduce in the field. This growth pattern can be appreciated via a growth curve. Growth curves are constructed by taking cultured bacteria, inoculating it, and then measuring its growth at regular time intervals. This time interval is normally an hour, and it done for the length of 24 hours to get the most accurate depiction of bacterial growth.

The purpose of this laboratory procedure is to use a spectrophotometer to determine the growth of bacteria, as well as understand how to use a hemocytometer  to count the number of bacterial colonies in an plate. This allows for a basic estimation for how many cells are present in an inoculating broth with bacteria present. Using the information from both the spectrophotometer and the hemocytometer, the bacterial growth curve can be made.

Materials and Methods

This lab was divided into two section, with Part 1 focusing on the proper measuring of absorbance of samples taken during different time periods. Part 2 was geared more towards using and being comfortable using the hemocytometer and counting bacterial colonies using the hemocytometer.

• Part 1 was conducted with 10 LB broth samples, ranging from having bacteria for 0 hours to 18 hours. A 1000 µL micropipettor was used to extract 1mL of broth with bacteria  and placing them in spectrophotometer cubettes. The cubettes were then placed into the spectrophotometer, and the absorbance of each sample was recorded. For samples whose absorbance was observed to be beyond a value of 1, PBS solution was used to dilute the LB broth sample and a dilution factor was used to scale up the new value measured in the spectrophotometer. This would allow the readings to be much more accurate.
• Part 2 was performed with a hemocytometer. A plate was loaded into the hemocytometer using each of the samples used in the spectrophotometer test. The number of cells or colonies were counted using the hemocytometer, and a specific counting procedure. Rules as to what to count and what not to count were decided to beginning the bacterial count. The number of cells was finally recorded.

Results and Discussion

Measuring absorbance for all of the samples proved to be time-consuming, so in order to save time, the groups of experimenters were asked to record absorbance values for three or four samples, which were assigned to them. The procedure to obtain the values was simple enough, as everyone involved in the procedure has a general knowledge on how to operate a spectrophotometer. Figure 1 below shows the proper volume of bacterial broth in a cubette, ready to be tested for absorbance.

Figure 1: Spectrophotometer Cubette with Bacterial Broth

The  issue that caused the most problems during Part 1 of the lab procedure was the values being obtained when diluting the samples with absorbance values greater than one. A dilution factor of 1:1 was being used in this case, which meant that half of the solution in the cubette was the bacteria and LB broth, while the other half was PBS buffer solution. DI water was not used to prevent the hypertonic bacteria from swelling and exploding. Table 1 below shows the measured absorbance values for all of the times samples, as well as the average and standard deviation of each sample.

Table 1: Absorbance of Bacterial Samples

Using the given data, a growth curve can be made. Experimenters were asked to point out the beginning and ends of the lag phase, log phase, and stationary phase. Figure 2 below illustrates the data as a growth curve. As can be appreciated, the lag phase lasted approximately an hour, while the log phase and stationary phase lasted 8 and 9 hours, respectively.

Figure 2: Growth curve of Bacteria 

Part 2 of the lab was originally to be done by the experimenters, but due to the lack of time, ended up being performed by the lab instructor. However, experimenters were asked to come up to the hemocytometer and understand how it works. Figuure # below depicts one such experimenter using the hemocytometer to count cells.

Figure #: Experimenter Jabari Lee Using a Hemocytometer

Overall, the laboratory procedure was performed satisfactorily and was a great experience. This lab helped students properly create a growth curve for any kind of bacteria, following the proper steps of inoculation and measuring absorbance. This lab also assisted students in understanding the importance of cell count and how to establish comprehensive guidelines and rules for counting cells in a hemocytometer.