_Lab 4

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Kibria 1 DEPARTMENT OF CHEMISTRY AND BIOLOGY FACULTY OF SCIENCE FA Assignment and Lab Cover Page Please complete all the fields. Course Name Microbiology______________________________________________ Course Code BLG151_ __________ Section Number 142__________ Lab day and time (for lab submissions only) Wednesday and 5:00pm-8:00pm_______________________ Professor or TA to whom you are submitting (generally TA for labs ) ___Samantha Poon___________________ _______________________________ First Name Last Name Assignment Name: Laboratory 4: Enumeration of Bacteria________________ Submission Date Wednesday, November 1, 2023____________ Total Pages (including this page) 17___________________________ Student Information Rafia______________________ First Name Kibria____________________ _ Last Name 501185422___________________________ Student ID Number

Kibria 2 Table of contents Abstract………………………………………………………………………….. 3 Introduction………………………………………………………………………4-5 Materials and Methods………………………………………………………….. 6-7 Results……………………………………………………………………………8-11 Discussion..……………………………………………………………………..12-14 Conclusion…..…………………………………………………………………15-16 References.………………………………………………………………………17

Kibria 3 Abstract Binary fission is a reproductive process used by many microorganisms that involves a single parent cell splitting into two identical daughter cells. Population growth refers to the increase in number of cells or the biomass of a population of cells (Victorio-Walz and Gilbride, 2023). In this lab, population growth was observed through different methods of bacterial culturing. The objective of this lab was to view bacterial growth and practice counting colonies. Several methods of enumeration for bacterial colonies were introduced. Turbidimetric estimation was used to count the number of colonies in a liquid medium by observing how much light will pass through a sample. The pour plate method was used to visibly count the number of colony forming units inside and outside of a nutrient agar plate. The membrane filtration method was used to view colonies that have been filtered out of heat sensitive liquid. The method of probable numbers was used to qualitatively analyze a liquid medium for enumeration. The pour plate method provided the most accurate results with the smallest margin of error.

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Kibria 4 Introduction The objective of this laboratory experiment was to employ various enumeration techniques to determine the number of bacterial cells in a culture. Enumeration of bacteria is the process where bacteria is counted and can be performed within different methods. The crucial role this technique plays in the subject of microbiology is that it helps lead to discoveries about microbial growth, quality of different products, and can also aid in safety measures like safety of water sources. Though there are many ways to perform this approach, in this experiment four methods are taken into account so the advantages and disadvantages they offer can be explored and compared. When counting, it is important to consider that there are two possible ways to implement this procedure, one being total count and the other being viable count. The main difference between these is that total count focuses on both dead and living cells whereas viable just centers around enumerating the living cells. Total count is obtained by counting the individual cells or by using a spectrophotometer and this is part of the first experiment known as the turbidimetric estimation. The rest of the three measures are executed in terms of viable counts as they are obtained through plating and culturing of samples. A serial dilution is needed for viable counts as they aid in the reduction of cell density which helps achieve countable plates. Only 30-300 colonies is considered a “countable plate” whereas less than 30 is too few to count and in contrast, 300 is too many to count. By successfully counting and enumerating microorganisms in a sample, maintaining product quality can be assured as contaminants can be assessed and stopped. Since there's multiple methods for this technique, by performing this lab it should become understandable what method is the best choice to use considering circumstances like the type of sample, time accuracy etc. Overall, this laboratory experiment is broken down into four major parts where each part corresponds to the method being introduced. The first mechanism the lab focuses on is Turbidimetric Estimation, which is where a spectrophotometer is used to measure the scattered light caused by bacteria so cell concentration can be calculated. By inserting a tube of Escherichia coli in the spectrophotometer, the amount of light transmitted as a percent of the total light entering the sample is recorded, and so, if more light is scattered, then less light is transmitted leading to a greater number of cells counted. This is because the amount of light that passes through a culture is inversely proportional to the number of the bacterial cells. A single cell in isolation can give rise to a single colony where this colony can be related to the bacterial number. This is the second approach that is performed to

Kibria 5 acquire the enumeration of bacteria and it is called the Pour Plate Method. This method relies on colony-forming units (CFU’s) on agar plates by preparing dilutions of the culture and pipetting them onto the petri plates for them to form. By multiplying the number of colonies and the dilution factor of the culture that is pipetted in and dividing it by the volume inoculated, the colony forming units of the original culture is calculated. This can also be done by multiplying the number of colonies with the final dilution factor. Membrane Filtration Method deals with trapping bacteria within a filter so they can grow and be counted on the nutrient agar by a redox indicator known as triphenyl tetrazolium chloride which changes to a red color after incubation to be easily counted. Since this process consists of a dilution, it only focuses on the living cells and is therefore a viable count. The method that uses successive dilutions of a sample to inoculate broth tubes is the Most Probable Number Method.

Kibria 6 Material and methods All procedures were adapted from BLG 151 Lab Manual (Gilbride, K and Victorio-Walz, L., 2023). In experiment 4.1 , the enumeration of bacteria was explored through the total count method known as turbidimetric estimation. The materials needed in order for this lab to be successfully conducted were kimwipes, a spectronic 20 spectrophotometer, and an Escherichia coli culture sample. In this method, both living cells and dead ones were enumerated by firstly calibrating the spectrophotometer by setting the wavelength to 660nm and placing the cuvette with a sterile nutrient broth and adjusting it to 100% transmittance. Once it was calibrated, the Escherichia coli ( E.coli) culture sample was ready to be placed into the machine where the percent transmittance was read and recorded and this was done a total of three times so an average percent transmittance can be calculated and added to the class enumeration data table. Experiment 4.2 focused on the pour plate method of enumerating bacteria and because this was a viable count, only the living cells were focused on. The materials used were four test tubes containing a 9.9mL sterile dilution buffer, 100mL of molten nutrient agar, five empty sterile petri dishes along with five pipettes and a culture of Escherichia coli. Using these items, preparation of 1/100 dilutions of the cultures were made where either 0.1mL or 1.0mL was aseptically transferred to the corresponding final dilution factor (FDF) petri plate. Once all the plates had the diluted cultures, molten agar was added to half-fill the bottom of the dish where this was then left to solidify by incubation at 37 degrees celsius. Experiment 4.3 introduced the membrane filtration method where trapping bacteria was the goal needed to accomplish in order to obtain the enumeration of bacteria. The materials included in this part were forceps, 10 6 dilution of E.coli, 99 mL sterile dilution buffer, sterile 0,45 m membrane filter, filter apparatus, and nutrient agar plates with TTC. The first step needed to µ be done was transferring 1.0 mL from the 10 6 dilution of E.coli to the 99 mL of sterile buffer and 50 mL of this was filtered through the membrane filter. Using the forceps, the filter grid was transferred side up on the nutrient agar plate and this was then left for incubation at 37 degrees celsius.

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Kibria 7 In experiment 4.4 , the most probable number method was followed and to execute this, materials such as, a 10 -8 dilution of the bacterial culture , 10 mL/1.0 mL pipettes, 3 tubes of 10 mL double-strength nutrient broth, 3 tubes of 9.0 mL/3 tubes of 9.9 regular-strength nutrient broths. Once the materials were collected, labeling of the test tubes was crucial to avoid mixups. These tubes were inoculated where the 10 ml tubes were inoculated with 10 mL of culture, the 9.0 mL tubes with 1.0 mL of culture and lastly the 9.9 mL tubes with 0.1 mL of culture. Once this was done, the tubes were left for incubation at 37 degrees celsius.

Kibria 8 Results: Figure 1: Growth curve of E.coli The growth curve depicted in the graph showcases the typical stages of E. coli bacterial growth. Initially, there is a 20-minute lag phase where the population prepares for growth. This is then followed by a steady logarithmic growth phase lasting around 50 minutes. During this phase it can be implied that the bacterial population of the E.coli multiplied rapidly. The population then lastly enters a stationary phase but the graph only shows the point where it starts as the death phase is not included since its points were not plotted. Ultimately a growth curve is a fundamental tool in microbiology for studying bacterial responses to changing environments and their growth kinetics.

Kibria 9 Figure 2: CFU/ mL vs % Transmittance for E. coli This graph illustrates the relationship between CFU/mL and Absorbance for E. coli. It appears that the E. coli population decreases as it progresses in the percent transmittance, giving an overall negative exponential trend. The CFU/mL number increases exponentially with absorbance and therefore is inversely proportional to the transmittance, hence, explaining the trend of the graph. Table 1: Enumeration using Turbidimetric Estimation and Standard Growth Curve of Escherichia coli. % Transmittance of E. coli sample Corresponding CFU/mL Class Average CFU/mL 42.66 → 43 1.3x10 9 8.5x10 8 Table 1: In the turbidimetric estimation method, the E. coli sample exhibited a transmittance percentage of 43%. Based on this transmittance percentage that was determined from the spectrophotometer, an absorbance value of 1.6335 was calculated. The colony-forming units per milliliter (CFU/mL) was determined to be 8.5E+08.

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Kibria 10 Calculating Average % Transmittance: Trial 1: 47 Trial 2: 41 Trial 3: 40 Average % Transmittance: 47 + 41 + 40 = 42.66% Calculating Absorption using % Transmittance: 𝐴????????? = − ???? 𝐴????????? = − ???(0. 43) 𝐴????????? = 1. 6335 Table 2: Enumeration of Escherichia coli using the Pour Plate Technique. Plate ID Dilution Factor Number of Colonies Volume of Inoculation (mL) Final Dilution Factor (FDF) CFU/mL Class Average (CFU/mL) 2 10 -4 TMTC 0.1mL 10 5 TMTC / 3 10 -6 TMTC 1.0mL 10 6 TMTC / 3 10 -6 37 0.1mL 10 7 3. 7𝑥10 7 1.6x10 8 4 10 -8 TFTC 1.0mL 10 8 TFTC / 4 10 -8 TFTC 0.1mL 10 9 TFTC / Calculating CFU/mL of Plate ID 3 (0.1) : 𝐶𝐹?/?? = (?????? ?? ?????𝑖?? 𝑥 ?𝑖???𝑖?? ??????) ?????? ?? 𝑖???????𝑖?? 𝐶𝐹?/?? = (37 ?????𝑖?? 𝑥 10 7 ) 0.1 ?? 𝐶𝐹?/?? = 3. 7𝑥10 8 Therefore, the CFU/mL using the pour plate technique was 3. 7𝑥10 8 Table 3: Enumeration of Escherichia coli using the Membrane Filtration Technique.

Kibria 11 Method of Enumeration Dilution Number of Colonies Volume of Inoculation (mL) CFU/mL Class Average (CFU/mL) Membrane Filtration 10 8 8 50 1. 6𝑥10 6 6.1x10 7 Table 3:As a result, the enumeration of E.coli was 1.6x10 6 whereas the class average was 6.1E+07. Calculation of CFU/mL: 𝐶𝐹?/?? = (?????? ?? ?????𝑖?? 𝑥 ?𝑖???𝑖?? ??????) ?????? ?? 𝑖???????𝑖?? 𝐶𝐹?/?? = (8 ?????𝑖?? 𝑥 10 8 ) 50 ?? 𝐶𝐹?/?? = 1. 6𝑥10 6 Therefore, the CFU/mL using the membrane filtration technique was 1. 6𝑥10 6 Table 4: Enumeration of Escherichia coli using the Most Probable Number Technique. Number of Positive tubes in Dilution MPN per 100 mL Dilution MPN/mL Class Average MPN/mL 10.0 mL 1.0 mL 0.1 mL 3 3 1 460 10 ⁸ 4.6x10 ⁸ 3.5x10 8 Table 4:As a result, the most probable number of cells per milliliter was 4.6x10 ⁸ whereas, the class average was 3.5E+08. Calculation of MPN/mL: ?𝑃?/?? = (?𝑃? ?????/100??) 𝑥 ?𝑖???𝑖?? ?????? = (460 ?????/100??) 𝑥 (10 8 ) = 4. 6 × 10 8 ?𝑃?/??

Kibria 12 Discussion In this four-part experiment different techniques were used to successfully determine microbial numbers or in other words, enumeration of bacteria. The involvement of both dead and living cells was included and processes of serial dilutions were performed before carrying out the techniques 4.2-4.4 used in this experiment. Serial dilution is the process of dilutions created of specific solutions that take place with a corresponding dilution factor (). For 4.1 both viable and dead cells were counted and this was through the method of turbidimetric measurements, where the presence of solid particles in a nonhomogeneous solution is measured. (). This measure is commonly conducted with a spectrophotometer, and this works as the suspended particles scatter light which ultimately reduces the amount of light causing detection based on the amount of scatteredness (). Figure 1, the growth curve graph of E. coli, represents the turbidimetric measurements that took place in experiment 4.1. It shows how from the span of 0-20 minutes the population of the bacterium were in the lag phase, implementing that the growth was constant and that during this phase the bacteria were adjusting to the environment. This lag phase was followed up by the logarithmic phase and according to the graph it lasted around time intervals of 20-90 minutes. During this phase, bacteria started to increase in a logarithmic fashion and this gave insight that the bacteria was consuming nutrients which made sense to why the rapid growth occurred (). The most eye-catching trend was the log phase as the relationship between the number of cells and time was exponential making it the most important phase as well. Consequently, the E. coli went into a stationary phase but figure 1 only showed the starting of the stationary phase and did not show the last phase of bacterial growth known as the death phase. During these two phases, the growth of the E.coli would stop increasing and develop at a constant rate which would last for a few minutes and finally start declining, which would cause the curve to sharply fall. In comparison to this, figure 2, the standard calibration curve graph shows the correlation between CFU/mL and Transmittance in terms of E. coli . The E. coli calibration curve showed a pattern of an overall negative exponential trend. Due to absorbance being inversely proportional to transmittance, it also helped conclude that the CFU/mL value would rise exponentially when corresponding to the absorbance due to its relationship with transmittance. In the turbidimetric estimation method, the E. coli sample was observed to have a transmittance percentage of 43% when the average of all three trials was taken. Using a

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Kibria 13 spectrophotometer to measure the initial transmittance %, an absorbance value of 1.6335 was computed. The class average for the colony-forming unit per milliliter (CFU/mL) was 8.5x10 8 whereas the one measured from the experiment was 1.3x10 9 CFU/mL. This was not expected as this insinuates an error occurred which caused the bacterial suspension of the light scattering and cell concentration to be different than the class.. The pour plate was done in 4.2 and though this is a very common method used, it was a lot more open to errors and inaccuracies forming. Five petri dishes were used where specific dilutions of the microbial culture were added and according to table 2, the enumeration of E.coli was only able to be counted in one dish. The petri dish presented 37 colonies which were in the range of 30-300 whereas the others were either too few or too many. This petri dish had 0.1 mL inoculated and once calculated, had a CFU/mL of . In contrast to the class average, the 3. 7𝑥10 7 value calculated differed a lot as the class average was 1.6x10 8 and so was comparatively a lot smaller. This could be due to the number of colonies counted as the class may have had a broader number counted and therefore a higher CFU/mL. The third part of the laboratory experiment focused on the membrane filtration method The CFU/mL using the membrane filtration technique was 1.6x10 6 whereas the class average was 6.1x10 7 . As seen in previous results, once again the class average consisted of a much higher CFU/mL and this could have been due to many errors made when performing this. For instance, the membrane filtering process consists of very fragile filter paper which could have easily caused contamination or failure of equipment, letting cells pass through instead of blocking. Lastly, the most probable number (MPN) approach was performed where successive dilutions of samples to inoculate tubes of broth was done (). Though this is the least accurate method, but based on table 4 the results begged to differ, as the MPM/mL calculated versus the class average were very similar. The class average was 3.5x10 8 MPM/mL where on the other hand the one calculated in this experiment was 4.6x10 8 . This was expected as when calculating the MPM/mL while knowing the class median, it's understandable to expect a similar result and this helps indicate accuracy levels while executing the procedure. The reason why this method has more risk factors is because it can take a lot longer to get the results and because many mistakes can occur during the dilution process, ultimately affecting the presence of colonies. Enumeration methods in microbiology offer different advantages and drawbacks, catering to various research needs. The turbidimetric estimation method, renowned for accuracy, provides

Kibria 14 total bacterial counts but falls short in distinguishing between living and dead cells or pinpointing specific growth stages. The pour plate method, while simple and reliable, carries a substantial margin of error due to the potential for bacterial cell destruction caused by excessively hot agar, making complete prevention challenging. Similarly, the membrane filtration method simplifies enumeration but is susceptible to error, primarily due to contamination risks in the buffer and filtration system. On the other hand, the most probable numbers (MPN) method, known for its simplicity and qualitative focus, yields less accurate results, limiting its ability to make precise conclusions about bacterial growth dynamics. When conducting and performing labs for the growth of bacteria, choosing the best method based on these strengths and limitations should align with specific objectives to ensure the best results obtained.

Kibria 15 Conclusion In this laboratory experiment, various enumeration techniques were employed to determine the number of bacterial cells in a culture, shedding light on the strengths and limitations of each method. Four distinct methods were explored, including turbidimetric estimation, the pour plate method, membrane filtration, and the most probable number (MPN) method. Each method offers unique advantages and disadvantages, which can be leveraged based on specific research needs. The turbidimetric estimation method, which provides total bacterial counts, is accurate but cannot distinguish between living and dead cells or identify specific growth stages. The pour plate method, despite its simplicity and reliability, is susceptible to error, primarily due to the potential for bacterial cell destruction when using excessively hot agar. The membrane filtration method simplifies enumeration but can be prone to contamination issues. The MPN method is straightforward but less accurate, limiting its ability to draw precise conclusions about bacterial growth dynamics. The outcomes of the experiment varied for each method, and it's important to note that the calculated values sometimes deviated from the class averages. For instance, the turbidimetric estimation method yielded a CFU/mL value of 1.3x109, while the class average was 8.5x108. In the pour plate method, one dish was counted with a CFU/mL of 3.7x107, whereas the class average was 1.6x108. The membrane filtration method produced a CFU/mL of 1.6x106, with a class average of 6.1x107. The MPN method resulted in a value of 4.6x108, quite close to the class average of 3.5x108. These discrepancies can be attributed to experimental error or variations in technique. In real-life applications, the enumeration of bacteria is vital for diverse fields, including microbiology, food safety, and environmental monitoring. Accurate bacterial counts are essential for assessing product quality, ensuring safety in water sources, and identifying trends in microbial growth. The choice of enumeration method depends on the specific goals of the study and the trade-offs between accuracy and ease of use. Researchers can benefit from a comprehensive understanding of these methods and their limitations to make informed decisions. Regarding valid errors or limitations, it's important to acknowledge that variations in technique, equipment, and experimental conditions can lead to discrepancies in results. For example, contamination, issues with sample handling, or minor errors in pipetting can affect the accuracy of CFU/mL values. Future improvements could involve rigorous quality control measures, enhanced training for students, and more robust experimental protocols to minimize potential sources of error.

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Kibria 16 In conclusion, this laboratory experiment has provided valuable insights into bacterial enumeration techniques. While each method has its strengths and limitations, the knowledge gained from this experiment is crucial for microbiological research and practical applications in fields such as food safety, environmental monitoring, and product quality assessment. Addressing potential errors and continuously refining experimental protocols will contribute to more accurate and reliable results in future studies.

Kibria 17 References Department of Chemistry and Biology, Gilbride, K. A., & Victorio, L. (2023). Microbiology I, BLG151 Lab Manual . Toronto Metropolitan University. ';;' . (2019, March 9). ';;' - YouTube. Retrieved November 2, 2023, from https://www.bing.com/search?PC=N186&q=most+probable+method&FORM=N186DF &showconv=0 Absorbance vs. Transmittance - What's the Difference? (n.d.). This vs. That. Retrieved November 2, 2023, from https://thisvsthat.io/absorbance-vs-transmittance Bacteria | Cell, Evolution, & Classification . (2023, October 28). Britannica. Retrieved November 2, 2023, from https://www.britannica.com/science/bacteria Bacterial Enumeration: Definition, Methods & Example - Video & Lesson Transcript . (2022, January 19). Study.com. Retrieved November 2, 2023, from https://study.com/academy/lesson/bacterial-enumeration-definition-methods-example.htm l Daugherty, E. (2017, November 28). Bacterial Growth Curves using a Spectrophotometer (Turbidimetric Determination) . G-Biosciences. Retrieved November 2, 2023, from https://info.gbiosciences.com/blog/bacterial-growth-curves-turbidimetric-determination Log Phase Of Bacterial Growth . (n.d.). BYJU'S. Retrieved November 2, 2023, from https://byjus.com/neet/log-phase/ N, S. (n.d.). What is Membrane Filtration Method? Definition, Summary & Method . Biology Reader. Retrieved November 2, 2023, from https://biologyreader.com/membrane-filtration-method.html

Kibria 18 physical chemistry - Measuring turbidity using a spectrophotometer . (2016, December 7). Chemistry Stack Exchange. Retrieved November 2, 2023, from https://chemistry.stackexchange.com/questions/64056/measuring-turbidity-using-a-spectr ophotometer Rafferty, J. P. (n.d.). Log phase | biology . Britannica. Retrieved November 2, 2023, from https://www.britannica.com/science/log-phase Sapkota, A. (2023, September 7). Serial Dilution: Formula, Calculator, Method, Uses, Examples . Microbe Notes. Retrieved November 2, 2023, from https://microbenotes.com/serial-dilution/#Serial%20Dilution%20Formula%20and%20Ca lculations Tankeshwar, A. (n.d.). Pour Plate Method: Procedure, Uses, (Dis) Advantages • Microbe Online . Microbe Online. Retrieved November 2, 2023, from https://microbeonline.com/pour-plate-method-principle-procedure-uses-dis-advantages/

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