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.

Streak Plate, Culture Transfer Instruments and Techniques, Isolation and Maintenance of Pure Culture

LAB 3 - Streak Plate, Culture Transfer Instruments and Techniques, Isolation and Maintenance of Pure Culture

Introduction

Culturing bacteria in a controlled setting is extremely important in understanding the processes taking places on a microscopic level. Observing and analyzing the behavior of bacteria during culturing allows doctors to fight against deadly illnesses, microbiologists to understand microorganism growth and reproduction, and engineers to properly evaluate and improve water quality. In order to test on a certain bacteria, a single bacteria colony must be cultured from a large population, and the only way to extract a single colony out of the millions and millions of bacteria available is through the use of a streak plate. Microorganism growth can then be measured by isolating and incubating that bacteria colony, while it is inoculated into a broth.

The purpose of this laboratory procedure is to become comfortable and proficient in applying a bacteria onto a streak plate, extracting a colony, and inoculating the bacteria in an LB broth. Due to the nature of this lab being more operational than observational, results will not be heavily discussed. However, execution of provided instructions and perceived comfort or proficiency of execution will be discussed.

Materials and Methods

This lab was divided into three section, with Part 1 discussing how to deposit microorganisms into a streak plate, autoclave operation and how culture medias work, while part 2 was focused on transferring cultures from the agar plate to the inoculating broth, and part 3 focusing on the maintenance of pure cultures once the bacterial colony has been extracted.

• Part 1 was conducted with an agar plate, a tube of E. coli, and a set of streak toothpicks. Prior to receiving the streak plate, the work surface and experimenter's gloved needed to be sterilized using ethanol. Upon sterilization, streak plate was sent on the clean work surface and the streak toothpicks were opened, making sure one end remained sterile at all times. The sterile end of the toothpick was then dipped into the E. coli sample and was then streaked onto the plate, making sure the agar didn't tear. The toothpicks were then disposed of in a bio-hazard waste basket, while the agar plate was then placed into an incubation chamber.
• Part 2 was performed with an agar plate with colonized E. coli bacteria, streak toothpicks, and an inoculating broth. Upon incubation of the streak plate, the E. coli microorganisms in the agar multiplied. Colonies were formed during the incubation process. A single colony was then extracted from the agar plate using the sterile end of the streak toothpick. That end of the toothpick was then dipped into the inoculating broth and was stirred to ensure the complete removal of E. coli bacteria from the toothpick. The toothpick was then disposed into a bio-hazard waste basket, while the broth was placed on an incubating stirrer for 24 hours.
• Part 3 focused on the maintenance of pure colonies after extraction. This part was conducted by the lab instructor. It was explained that frozen pure cultures are maintained to allow for repeated testing. Frozen cultures are known to last several years, which is convenient for a laboratory that regularly performed micro-bacterial experiments. Proper inoculation of pure cultures in a broth allow for growth curves to be formed, and can help determine the growth rate for many organisms.

Results and Discussion

Part 1 of the lab, the proper streaking of bacteria onto the agar plate, proved to be more difficult from a first-person perspective than was anticipated. Figure 1 below provides a visual representation of the proper way to streak a bacteria sample onto the plate.

Figure 1: Proper Streaking onto Agar Plate

One of the most important aspects is preventing the agar from tearing, because this allows the bacteria to a previously unexposed surface, the surface of the agar plate is not rich enough in nutrients to allow the bacteria to grow properly, and the experimenter is risking a non-sterile surface being exposed when the agar tears. Figure 2 below shows an experimenter streaking an E. coli strand onto the agar plate, and the result of that experimenter's performance. Tears did happen when streaking the E. coli bacteria onto the agar, due to it being a new experience. As it was done more repeatedly, however, the nerves of not wanting to tear the agar went away, allowing for the bacteria strand to be deposited onto the agar plate without tearing the agar.

Figure 2: Jose Castano streaking E. coli bacteria onto agar plate

Extracting the bacterial colony was much easier, since the idea of the idea behind the extraction is to stab the bacteria onto the toothpick and then transferring the bacteria onto the inoculating broth. This was done successfully, and the broth was set into a stirrer. An autoclave tape was then set onto the top of the broth cap, made of aluminum foil, to make sure the broth underwent a sterile inoculation process. Figure 3 below shows an experimenter inoculating an E. coli colony into the broth, as well as the broth once the E. coli was inserted.

Figure 3: Inoculating broth with presence of E. coli

Because maintenance of pure microorganism cultures is kept strictly under the supervision of lab managers and instructors, this part of the lab was mostly discussed verbally. Interesting things that were explained during this discussion were that one can order cultures directly from the ATCC, or the American Type Culture Collection, or one can solicit a culture from a nearby University of institution of higher learning for a sample. It was also learned that cultures can be preserved for several years when frozen. Growth curves can be made after only eight hours of inoculation, although it is normally done for 24 hours. This is done as a simulation of what normally happens on an everyday basis. Some bacteria grow in a few hours, while others take days to reproduce. Growth curves help microbiologists, engineers, and doctors see how quickly a bacteria or possible pathogen grows as a population or colony.

Overall, the laboratory procedure was performed satisfactorily and was a great experience. This lab did help very much in helping students hone their skills in streaking an agar plate with bacteria, as well as inoculating a colony of bacteria and maintaining pure cultures. Additional pictures were taken and can be appreciated below, for documentation and observational purposes. 

Additional figures: