E - In this lab, we used the versatile E. coli to understand growth curves and enumeration PDF

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In this lab, we used the versatile E. coli to understand growth curves and enumeration under controlled conditions. Using the spectrophotometer and the well plate reader to generate turbidity values at different dilutions and utilizing special plating techniques for inoculation, we were able to acq...

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Determining Generation Time for Escherichia coli using Turbidity and Viable Counts

Abstract Escherichia coli is a central strain of bacteria found almost everywhere. E. coli can be found on ample surfaces of our everyday environment, in meat, on produce and in the gastrointestinal tract of animals, including humans. In fact, there are hundreds of strains of E.coli with unique characteristics, from harmful to pathogenic. E. coli is the most studied bacteria on earth and plays a key role in our scientific understanding of microbial structure, function and growth (Gillespie, 2018). Since it can survive under variable conditions as well as reproduce and grow rapidly, E. coli has become the model bacteria for studying the growth and life cycle of microorganisms. In this lab, we used the versatile E. coli to understand growth curves and enumeration under controlled conditions. Using the spectrophotometer and the well plate reader to generate turbidity values at different dilutions and utilizing special plating techniques for inoculation, we were able to acquire an average count of Colony Forming Units (CFU’s). Using the rationale that each colony is the descendant of a single cell, we can use the CFU count to obtain an approximate concentration of cells per milliliter of suspension. By plotting this concentration versus time, we can determine the generation times for E. coli. The generation time was determined to be 39.14 minutes in exercise 5D, the sidearm flask, and 22.78 minutes in exercise 5E, the 96 well plate.

Introduction

The purpose of this exercise is to determine the growth curve for Escherichia coli using several conventional techniques for enumeration. The bacterial growth curve illustrates the stages of a bacterial population when they are grown in a closed system of single-batch microbial culture in a liquid medium of fixed volume, called a batch culture. The growth curve is a significant tool for analyzing and understanding bacterial population growth. There are several different techniques used in this exercise to for the enumeration (counting) bacterial cells: 

Direct Count Method using the Petroff-Hausser Counter With this method the researcher counts the number of bacterial cells observed under a phase contrast microscope in a small, predetermined segment of the specimen termed the Y square. After counting the individual cells in multiple Y squares the number is averaged and calculated with the dilution factor to estimate total cell count. However, it can be difficult during the observational count to determine which cells are alive and which are dead. This downside may lead to a direct count total that is much higher than the actual number of viable cells in the culture.

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Dry Weight Determination After conducting a direct count to adjust a suspension of bacteria to a desired number of cells/mL, the suspension is then centrifuged and washed, removing solutes and residues. Next, it is dried overnight at 100oC, then weighed. Once the dry weight is determined, one can calculate the individual cell weight in grams. This weight can then be used to determine the total number of cells in a suspension.

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Spectrophotometric Determination the using a Suspension’s Optical Density 2

Since bacteria scatters light as it is passed through a suspension, researchers can use this characteristic to estimate bacterial cell numbers. By using the turbidity, defined as a measurement of the degree to which a liquid loses its transparency due to the presence of suspended particulates, researchers can estimate bacterial cell numbers in a suspension. A spectrophotometer, the device used to measure turbidity, contains a light source, a photocell and a sample accepter. The spectrophotometer emits a light of a specific wavelength; as it passes through the suspension, the bacterial cells will scatter the light, allowing less light to reach the photocell for absorbance. The spectrophotometer provides a reading of optical density (OD), the unit of turbidity, for the wave length employed. With this, one can generate a growth curve plotted as viable culture count versus optical densities. This method is most effective when culture densities are in the range of millions to hundreds of millions per milliliter, outside of that range it is less effective. Also, since any particulate will scatter light, not just viable cells, it is possible that dead cells will affect the OD reading. 

Viable Cell Count This method uses serial dilutions, dispersion and plating to generate countable colonies of viable cells. Using a known amount of inoculum solution, sterile, serial dilutions and dispersion, via shaking, one can create a concentration of organisms that are suitable for counting. Once the desired dilution(s) are generated, the solution (s) can be inoculated onto sterile Tryptic Soy Agar (TSA) plates and incubated for growth. Each colony that grows on the TSA plate(s) is reasoned to have developed from a single cell in the initial inoculum solution. A countable plate will contain between thirty to three hundred colonies forming units (CFU). This method is the best for obtaining an accurate number

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of viable, living cells represented by CFU’s. However, it is time consuming in that a researcher must wait at least a day for colonies to grow and then one must properly dispose of possibly harmful agar plates. Using the data acquired via the above-noted methods, one can generate a growth curve for a bacterium, in the case of Exercise #5, E. coli. The typical growth curve for a population of cells can be divided into several distinct phases called the lag phase, log phase, stationary phase and death phase (see Figure 1.)

Citation: www.cs.montana.edu

Figure 1. Microbial Growth Curve in Batch Culture. Depicts the stages of growth as plotted as the log of the number of cells versus time. Lag phase, the initial period, is characterized by a lack of cell number increase. In the lag phase, the bacteria are adjusting to their new environment, having been recently added to a nutritious growth medium. This phase is followed by the exponential or log phase. In the exponential phase microbial growth is constant and balanced, doubling in cell number at unvarying intervals. Exponential phase provides that data to determine generation time, the time required for a doubling in bacterial cell number. Generation time is determined by dividing the total growth time in minutes by the growth constant times the quantity of the log cell concentration final

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minus log cell concentration initial (Plank, 1979). Following the exponential phase is the stationary phase. As its name suggests, this stage sees restricted growth, no increase in cell numbers is observed. Nutrients were depleted by the logarithmic cell growth of the exponential phase. Coupled with the build-up of toxic waste products from the exponential phase, the medium is no longer favorable for net cell growth. Finally, the death phase is characterized by exponential declination of viable cells. Using the previously-mentioned enumeration methods, we can generate a growth curve for E. coli. Although we did observe a previously prepared demonstration showcasing the PetroffHauser counting chamber and completed a mock dry weight calculation, it is the spectrophotometric determination and the viable cell count which will be the focus of this lab report and provide the necessary data to generate said growth curve.

Material and Methods Exercise 5C Spectrophotometric Determination Materials:         

E. coli cell suspension 16 x 150 and 13 x 100 mm tubes Pipettes Tryptic Soy Broth (TSB) Micro-pipettors P200 and tips 96 well plate Spectrophotometer Plate Reader

Procedure: 1. Pipette 4mL of Tryptic Soy Broth (TSB) into each 16 x 150 mm tubes (7). 2. Leaving one tube as a blank, prepare two-fold dilutions of the E. coli suspension provided for the remaining six tubes. 3. Transfer 3 mL of each dilution and 3mL of TSB (used as a blank) to 13 x 100 mm tubes. 4. Determine optical density (OD) with the spectrophotometer. 5

a. Blank the spectrophotometer using the blank tube. Determine the optical density at 595 nm for each of the six samples. b. Use this data to plot cell concentration versus the optical density. Each OD reading is correlated with a specific concentration of E. coli. 5. Determine optical density (OD) with the plate reader. a. Add 200 µL of each dilution (prepared in step 2) to 96 well plate. b. Add 200 µL of TSB to a well as a blank. c. Determine the optical density at 595 nm for each of the six samples using plate reader. d. Use this data to plot cell concentration versus the optical density. Each OD reading is correlated with a specific concentration of E. coli.

Exercise 5D Viable Plate Count Materials:          

Predetermined amount of E. coli suspension in exponential growth Sidearm flask containing 25 mL of sterile Tryptic Soy Broth (TSB) 50 mL of saline 36 TSA plates Microfuge tubes P200 micro-pipettor and tips P1000 micro-pipettor and tips Sterile glass beads Spectrophotometer (at 595 nm) Constant temperature water bath/shaker at 37oC

Procedure: Day One 1. Using aseptic technique, inoculate side arm flask by pouring the entire contents of the E. coli suspension into sidearm flask containing 25mL of TSB. 2. After blanking the spectrophotometer, take an OD reading at 595 nm of the culture in the sidearm flask. 3. Dilution Preparation: a. Prepare seven (7) microfuge tubes with 900 µL of saline. Label 10-1 through 10-7. Set aside. b. Mix culture by swirling or rolling between palms. Using a P200 and sterile tip, remove 100 µL of the culture sample and transfer to microfuge tube labelled 10-1. Discard tip. Immediately place the sidearm flask into shaker bath. c. Mix the 10-1 dilution by inverting several times. Using a P200 and sterile tip, remove 100 µL of the culture sample and transfer to microfuge tube labelled 10-2. Discard tip. 6

d. Repeat the dilutions for 10-3, 10-4, 10-5, 10-6, 10-7. e. Label six (6) TSA plates; two (2) for each 10-5, 10-6, 10-7, with dilution coefficients and time: time zero (t0) f. After mixing via inversion, transfer 100 µL in duplicate from the 10-5, 10-6, 10-7 dilutions. Pour 5-10 fresh, sterile beads onto the surface of each inoculated TSA plate. Swirl the beads with a figure eight motion on the surface of the plate to spread the microbes across the surface of the plate. 4. Repeat step 2 for OD reading and step 3 for dilution preparation at twenty (20) minute marks: t20, t40, t60, t80, t100 – ensuring to properly label the time on each set of TSA plates. 5. Incubate plates at 37oC.

Day Two (48 hours after Day One) 6. Generate a table with the number of colonies counted (CFU’s) for each dilution at each time interval. 7. Prepare two graphs by plotting the optical density over time and plotting cell concentration over time. Exercise 5E 96 Well Plate Procedure Materials:           

E. coli suspension optical density (OD) of 0.05 96 well plate 30 mL of saline 15 TSA plates, TSA Tryptic Soy Broth (TSB) Microfuge tubes P20 micro-pipettor and tips P200 micro-pipettor and tips P1000 micro-pipettor and tips Plate reader Incubator set at 37oC

Procedure: 1. Label petri-dishes for dilutions 10-4, 10-5, 10-6 for each time mark: t0, t30, t60, t90, t120 2. Dilution Preparation: a. Prepare five (5) microfuge tubes by labelling 10-2 through 10-6. b. Using a P1000 and sterile tip, dispense 990 µL of saline into microfuge tube 10-2 and 900 µL of saline into four additional microfuge tubes, 10-3, 10-4, 10-5, 10-6. c. Add 200 µL of E. coli culture with OD of 0.05 to wells B2, C2, D2 of 96 well plate. Wells B2 and C2 are blanks for OD readings and D2 will be used for sampling at each time mark. 7

d. Add 200 µL of TSB to wells C2 and D3 to act as negative controls. e. Using well plate reader, take initial OD reading. This is OD at time t0. f. Using a P20 and sterile tip, remove 10 µL from well D2 and transfer to microfuge tube labelled 10-2. Discard tip. Immediately place the 96 well plate incubator g. Mix the 10-2 dilution by inverting several times then transfer 100 µL to microfuge tube labelled 10-3 containing 900 µL of saline. Discard tip. h. Repeat the dilutions for 10-4, 10-5, 10-6 3. Plating a. Label five large TSA plates with t0, t30, t60, t90, t120 b. Spot 20 µL aliquots in duplicate onto the surface of large TSA plates for 10-4, 10-5, 10-6 dilutions only. Each plate should have six spots, two for each dilution 10-4, 105 , 10-6. c. Tilt the plate upright to allow spots to spread toward opposite edge of plate, but do not allow spot to spread far enough to touch side of dish. d. Incubate at 37oC. e. Repeat steps 2 and 3 for the time intervals t30, t60, t90, t120. Day Two 4. Generate a table with the number of colonies counted (CFU’s) for each dilution at each time interval. 5. Prepare two graphs by plotting the optical density over time and plotting cell concentration over time.

Results

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In Exercise 5C, it was determined that as the E. coli suspension was more diluted, more light was able to pass through the sample. This indicates that the solution is less concentrated with cells, as the dilutions progress. The spectrophotometer and the well plate reader are valuable tools in estimating the cellular concentration of a suspension. Using the standard curve graphs, generated from the data collected, can guide us in estimating bacterial cell concentrations. The standard curve graphs are linear and reveal a bacterial suspension that is growing exponentially (see Graph 1.). Graph 1. corresponds to the typical exponential (log) phase of E. coli growth.

Standard Curve Graphs Optical Density (OD)

0.350 0.300 0.250 0.200 0.150 0.100 0.050 0.000

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96 Well Plate ( Well Plate Reader)

Graph 1. Exercise 5C Standard Curve. The turbidity was measured for different dilutions of the E. coli suspension while the culture was in exponential (log) phase. The original suspension had a concentration of 2.0 x 108 cells/mL. By conducting serial dilutions of the suspension and taking optical density measurements with both the spectrophotometer and 96 well plate reader, a standard curve graph is generated.

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Time

10-5 CFU Count 1

10-5 CFU Count 2

10-6 CFU Count 1

10-6 CFU Count 2

10-7 CFU Count 1

10-7 CFU Count 2

0 20 40 60 80 100

51 50 35 82 198 187

59 82 75 112 199 151

7 13 3 14 20 8

3 8 4 6 14 35

2 3 0 2 3 2

1 1 0 1 0 1

Table 1. Exercise 5D: Sidearm Flask Viable Cell Count. The number of Colony Forming Units (CFU’s), which were inoculated in duplicate for each dilution on the TSA plates. The above dilutions were counted after incubation at 37oC. The values for each dilution are then averaged and use to determine colonies per mL.

Time

10-4 CFU Count 1

10-4 CFU Count 2

10-5 CFU Count 1

10-5 CFU Count 2

10-6 CFU Count 1

10-6 CFU Count 2

0 0 1 2 2 0 1 30 58 52 8 8 2 2 60 76 73 10 17 2 3 90 106 103 23 15 2 2 120 TNTC TNTC 111 89 22 23 Table 2. Exercise 5E: 96 Well Flask Viable Cell Count. The number of Colony Forming Units (CFU’s), which were inoculated in duplicate for each dilution on the TSA plates. The above dilutions were counted after incubation at 37oC. The values for each dilution are then averaged and use to determine colonies per mL. TNTC denotes “Too Many To Count”, where plates were too overgrown to collect accurate CFU counts.

In Exercise 5D, we used a dilution plate count and spectrophotometer measurements to generate a growth curve. By diluting and dispersing the suspension of E. coli, we can acquire a concentration of microbes that fitting to count and can be used to estimate total vital cell concentration. Table 1. displays the counts of the Colony Forming Units (CFU’s). By interpreting the results from the sidearm flask in Graph 2, one can deduce that in the first twenty minutes, the bacterial suspension was likely in the lag phase, barely reproducing (see Graph 2). After that, linear growth is observed, corresponding with the exponential (log) phase.

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Similarly, in exercise 5 E, we used a dilution plate count and the 96 well plate reader measurements to generate a growth curve. Again, after dilution and dispersion of the E. coli suspension, we were able to grow a manageable number of microbes that were convenient to count and could be used to estimate vital cell concentration. However, with the 96 well plate, the results did not match the expected outcome. The O.D. at T60 dropped below that of the O.D. at T30 (see Table 2). This is unexpected. One would expect to the O.D. reading increase over time as the turbidity changes from more cellular reproduction, like that of the sidearm flask (see Graph 2). The values from the Optical Density analysis were then plotted against time to generate the O.D. vs Time graph. The sidearm flask O.D. results were in line with the expected results ( Graph 2).

Optical Density (OD)

Optical Density (O.D.) vs. Time 0.800 0.700 0.600 0.500 0.400 0.300 0.200 0.100 0.000

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Time (minutes) Sidearm Flask (Spectrophotometer)

96 Well Plate (Well Plate Reader)

Graph 2. Exercise 5D and 5E Plot of Optical Density versus Time. Optical density measurements were taken of the E. coli suspension at regular time intervals, using both the spectrophotometer and the well plate reader. The graph depicts the turbidity over time.

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After obtaining the CFU counts from 5D and 5E, we were able to determine viable counts. This is accomplished by taking the average of the CFU counts of the duplicate dilutions plated, divide it by the volume plated per mL and multiply it by the dilution factor. From there, we took the log of the colonies per mL to prepare Graph 3. Example Calculation 1. Sidearm Flask Viable Cell Calculation. At time0: average for dilution 10-5 = 55 colonies (plated 100µL) 55/0.1 mL x 105 = 550 x 105 = 5.5 x 107 colonies per mL

Log (5.5 x 107) = 7.740363

Example Calculation 2. 96 Well Viable Cell Calculation. At time0: average for dilution 10-5 = 2 colonies (plated 20µL) 2/0.02 mL x 105 = 100 x 105 = 1.0 x 107 colonies per mL

Log (1.0 x 107) = 7.000000

Cell Concentration of Viable Cells/mL (Log)

Viable Cell Concentration of E.coli per mL 9.00

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Time (minutes) Linear (96 Well Plate)

Graph 3. Viable Cell Concentration of E. coli per mL. Since each CFU is assumed to have come from a single cell, we can use the CFU counts as a measure of cell concentration. The log of the viable concentration for the 10-5 dilutions, both the sidearm flask and the 96 well plate, at the regular intervals, is plotted versus time. 12

Determining Generation Time The growth rate constant (k) is the amount of generations per unit time and is regularly noted as generations per hour (Willey, 2017, page 163). It can then be utilized to comp...