Cultivation of Escherichia Coli

Created: Sep 2021

Objective

In this experiment, Escherichia coli will be cultivated in a liquid LB culture medium for 120 minutes. The aim is to measure the multiplication (growth) of the E. coli cells in the cultivation process using a direct and an indirect method. In the direct method, the diluted culture liquid will be applied to LB medium plates every 60 minutes. By counting the number of colonies formed on the plate, the number of colony forming units per 1 ml of the bacterial liquid can be calculated. In the indirect method, the bacterial turbidity (OD600) will be measured every 30 minutes, absorbance value corresponding to the number of colony forming units of the culture liquid. Another objective is to identify the growth phases of E. coli by plotting the experiment results on a graph.

Materials and Method

Equipment and Reagents

• Escherichia coli C600 strain preculture

• Liquid LB culture medium, 2 L-tubes (one for measurements, one as blank)

• Agar LB culture medium, 15 plates

• 0.9% (w/v) saline solution

• Plastic cell

• Ice box

• Laboratory incubator shaker (37ºC)

Experimental Procedure

a) Preparation

Cultivate E. coli in LB culture medium at 37ºC for 12-16 hours. Set up the spectrophotometer and vortex mixer, prepare an ice box. Assign the roles of the turbidity measurements (Student 1) and clean bench tasks (Student 2).

b) Cultivation, sample collection, bacterial turbidity measurement

i. E. coli distribution

Working inside of the clean bench, place 0.3 ml of E. coli preculture into a 10 ml L-tube and mix.

ii. Commencing the cultivation

Immediately after the start of cultivation (0 min), place 1 ml of the bacterial liquid into a plastic cell for the bacterial turbidity measurement. Also, place 100 μl of the liquid in a microtube and place it in the ice box to prevent multiplication of the bacteria. Put the L-tube in the laboratory incubator shaker at 37ºC and start the stopwatch (cultivation time).

iii. Bacterial turbidity measurement (Student 1)

Cover the top of the plastic cell filled with the bacterial liquid with parafilm. Overturn the cell and measure the absorbance at 600 nm (use the liquid LB culture medium in the unused L-tube as the blank solution). Overturn the cell and repeat the measurements 2 more times (3 times in total). Calculate the average and use it as the sample’s absorbance value OD600 (optical density at 600nm).

iv. Clean bench procedure (Student 2)

Retrieve the microtube from the ice box and suspend the solution using the vortex mixer. Place 10 μl of the sample into a microtube containing 990 μl of saline solution (dilute 100 times) and mix by pipetting. Place 10 μl of the diluted sample into another microtube containing 990 μl of saline solution (dilute 10,000 times in total) and mix by pipetting. Put 50 μl of the sample diluted 10,000 times on the LB medium plate and spread it evenly. Apply the sample in the same way to 5 LB plates in total. Seal the plates and record the date, name, cultivation time (in this case 0 min) and dilution on the lid.

v. Further measurements

In addition to the turbidity measurement for cultivation time equal to 0 min, measure the bacterial turbidity at 30, 60, 90 and 120 min using the method from iii.. Also, apply the diluted sample to the LB plates at 60 and 120 min in the same way as for 0 min sample, following the method from iv. (5 plates per every time point, 15 plates in total). Place the LB plates with bacteria samples in the incubator at 37ºC and cultivate for several days.

c) Colony number

Count the number of colonies formed on the LB medium plates. Calculate the average colony number on the plate for every cultivation time. Taking into consideration the dilution of the sample used, calculate the number of colony forming units (cfu) per 1 ml of the original bacterial solution from b)i.. Plot a graph of time versus OD600 and try to determine the growth phases (lag phase etc.). Investigate if the bacterial turbidity and cfu values are proportional to each other.

Results and Discussion

Escherichia coli has been cultivated in a liquid LB culture medium for 120 minutes. The bacterial turbidity OD600 has been measured 6 times (every 30 minutes). The diluted culture liquid has been applied to LB plates 3 times (every 60 minutes) and the number of colonies has been counted.

In the experiment, 100 μl of culture liquid has been diluted 10,000 times and made into a 1 ml (1000 μl) solution. 50 μl of this solution has been placed on each of the LB plates. The objective is to obtain the number of the colony forming units per 1 ml of the culture liquid. Therefore,

Cfu per 1 ml = [number of colonies on the LB plate] × 10 × 10000 × 20 =

= [number of colonies on the LB plate] × 2 × 10⁶

The OD600, average colony number and cfu per 1ml (calculated using the method above) values are shown in Table 1. The relationship between cultivation time and OD600 is illustrated in Figure 1.

In Figure 1, two phases of E. coli growth can be differentiated. The lag phase lasts from 0 min till about 30 min of cultivation. This agrees with the theory, since E. coli cell division time is 20- 30 min, so the lag phase is also expected to last about this long. From 30 min on, E. coli starts to divide vigorously and its growth enters the logarithmic phase. Based on the experimental data, E. coli continued to divide at larger rate than dying until at least 120 min of cultivation time, since the stationary phase (horizontal line) has not been reached. Death phase (negative gradient) is also not shown on the graph.

In order to see the last two phases, the cultivation time could be lengthened. Alternatively, the nutrient concentration of the liquid LB culture medium could be lowered, so that the bacteria reach the stationary phase faster.

In addition to using the OD600 measurement (indirect method), the colony number has been counted (direct method) and cfu/ml values have been calculated. At the beginning of the cultivation, the cfu value was almost 100 million cfu/ml and OD600 was 0.121 (Table 1). After 60 min cfu hs increased 1.4 times and OD600 increased over 3 times. After 120 min cfu increased 7.4 times and OD600 increased 9 times (compared to the start of the cultivation). Therefore, while both cfu and OD600 have increased with cultivation time, signifying the growth of E. coli, it probably cannot be said that they are proportional to each other. However, it is a conclusion based on cfu and OD600 values for 3 cultivation times only. To obtain a better, more accurate picture of the relationship of cfu and OD600, cfu for more cultivation times (30 and 90 min) should be found. Moreover, since the colony number varied significantly from one LB plate to another and thus the measurement precision was low, more than 5 LB plates should be used for each cultivation time. This way, the accuracy and precision of cfu values could be increased and a stronger conclusion about the cfu and OD600 relationship could be drawn.

Extra Calculations

Calculating the number of E. coli cells in 100 µl of bacterial fluid at OD600= 1

From Table 1, the average OD600 at 60 min is 0.391, and cfu is 138,800,000= 1.388 × 10⁸

Number of E. coli cells in 100 µl of bacterial fluid when OD600 is 1 = (1.388 × 10⁸) / 0.391 = 3.55 × 10⁸

Calculating the turbidity increase rate µ and time needed to double turbidity

From Figure 1, the most vigorous growth (greatest gradient) occurs between 90-120 min of cultivation. Using the data from Table 1, turbidity increase rate can be calculated.

µ = [loge (OD600t2 / OD600t1)] / (t2- t1) = [loge (1.089/ 0.712)] / (120- 90) = 0.0142 min^-1 = 1.42 × 10^-2 min^-1

If the time needed to double turbidity is expressed as T = (t2- t1)

T = (t2- t1) = [loge (OD600t2 / OD600t1)] / µ = [loge(2)] / 1.42 × 10^-2 min^-1 = 48.8 min