Unformatted text preview: LABORATORY 3 – THE BACTERIAL GROWTH CURVE Objectives for Week 3 ‐ After completing these exercises, you should:
1. Be able to follow the growth of a liquid bacterial turbidimetrically. 2. Be able to determine viable cell numbers by plate counts during growth. 3. Understand how to plot and analyze bacterial growth data. Reading: Madigan et al (2015): Ch 5: pp 144; 149‐152 and 155‐160. Growth may be defined as an increase in cellular constituents, in some organisms. It leads to a rise in cell number when microorganisms reproduce by processes like budding or binary fission. In the latter process, individual cells enlarge and divide to yield two progeny of approximately equal size. Growth also results when cells simply become longer or larger. However, it is not usually convenient to investigate the growth and reproduction of individual microorganisms because of their small size. Therefore, when studying microbial growth, microbiologists normally follow changes in the total population number. Population growth is studied by analyzing the growth curve of a population in a confined space – broth culture (for bacteria) or in culture flasks (for tissue culture) for instance. When microorganisms are cultivated in liquid medium, they usually are grown in a batch culture or closed system. Since fresh medium is not provided during incubation, nutrient concentrations decline and concentrations of waste products increase. The resulting curve has four distinct phases (Fig. 3.1). Fig. 3.1 Bacterial growth curve in batch culture Lag Phase: During this phase, cells in a new culture adjust to the medium. Initiation of gene expression and subsequent increases in enzyme production and cell size occur. Exponential or Logarithmic Phase: During this phase, metabolic activities proceed at a constant rate, and cell mass as well as the number of cells double at a constant rate. The time required for the population to double is called the generation time (g) or doubling time and is usually expressed in hours. Because the population is doubling every generation, the increase in population is always 2n where n is the number of 38 generations. The resulting population increase is exponential or logarithmic. The rate of growth during the exponential phase in a batch culture can be expressed in terms of the growth rate constant (k) or the number of generations/unit time. k = ln2/g and is often expressed as generations/h. Stationary Phase: During this phase, waste products that can be toxic or alter the environment (i.e. make it more acidic) accumulate and/or the availability of nutrients decreases causing the cells to increase their generation time. Eventually division stops and the population reaches a plateau. Death Phase: Cells of the population enter this phase when toxic substances accumulate and/or cell starvation occurs. The rate of decline becomes exponential with time. Measurement of bacterial population growth can be determined by a number of methods. These include microscopic counts, plate counts, turbidimetric measurements, nitrogen or dry weight determinations, and biochemical activity measurements. In this laboratory, you will monitor bacterial growth using turbidimetric and plate counting methods. Before coming to the lab, consult the literature to determine the optimum temperature for:
Escherichia coli (DH5) You will work in pairs. Since it is impossible to follow an entire growth curve in a 3 hour lab period, most of your measurements will involve only the exponential and (perhaps) lag phases. To use your laboratory time efficiently and effectively, there are several things you must do ahead of time. 1. Read the procedures very carefully and be sure that you understand what has to be done. 2. Practice calculating dilutions. 3. Prepare you data tables (i.e. those below) so that you can fill in the data as you track the bacterial growth. Table 1: Turbidimetric growth results Tube 1 2 3 4 5 6 7 Growth time (min) 0 20 40 60 80 100 120 Dilution Dilution Factor (DF) Observed OD600 Undiluted OD (calculation) 39 Table 2: Plate count results Tube 1 2 3 4 5 6 7 Growth time Final plated (min) Volume (ml) 0 0.1 20 0.1 40 0.1 60 0.1 80 0.1 100 0.1 120 0.1 Final plated Dilution Final plated DF Number of Colonies Calculated cfu/ml THE MATERIALS AND PROCEDURES FOR TURBIDITY AND PLATE COUNT MEASUREMENTS (EXERCISES 1 AND 2 RESPECTIVELY) WILL BE DESCRIBED SEPARATELY BUT WHILE YOU ARE PERFOMING THAT LAB, YOU WILL COLLECT SAMPLES FOR BOTH TYPES OF MEASUREMENTS AT THE SAME TIME. THE PLATE COUNT STEPS CAN BE CARRIED OUT IN INTERVALS AFTER THE TURBIDIMETRIC SAMPLING AND READINGS. NOTE: Dilution Factor = Final volume / Solute volume Example: You want to make a 1:5 dilution of a culture in a final volume of 5 ml Using this formula, you would ultimately add 1 ml of your culture to 4 ml of diluent (e.g., LB broth or 0.95% saline solution). EXERCISE 1: Turbidimetric Measurements Of Bacterial Growth In Laboratory 2, you learned how to measure the turbidity of a bacterial culture using a spectrophotometer (Spec20). Materials and Methods Materials: 1. 5ml of Escherichia coli broth culture (37°C) in LB medium (OD600 = 0.2‐0.4). 2. Flask of pre‐warmed (37°C) LB broth. 3. 150 ml sterile LB broth to standardize the Spec20 and to dilute the bacterial culture for OD readings. 4. Sterile test tubes (18x150mm) for dilutions. 5. 13mm tubes for Spec20 readings (1 for the blank, others for the samples). 6. Sterile 5 and 10ml pipettes and a P200 pipette with tips. 7. Incubator set at 37°C. 8. Ice in a bucket. 40 9. Semi‐log (1‐cycle or 2‐cycle) and standard graph paper (not provided). Procedure 1. When you enter that lab, your Spec20 will have already been turned on for you to give it time to warm up. 2. Immediately prepare your blank tube (LB medium) and take a sample from your culture and make a measurement (the sample measurement will be your time point zero measurement – record the reading in table 1). 3. Remove 100µl (0.1ml) of this t0 sample and place it in a microcentrifuge tube (labelled t=0). Place the tube on ice, it will be used to complete exercise 2. 4. Place your flask in the assigned incubator (record the time you did this since samples must be taken at 20 minute intervals). 5. In the 20 minutes you are waiting to take the next sample, label the remaining microcentrifuge tubes for spread plating of the timed samples (you will use these in exercise 2). You already have the time 0 sample; you will need 6 more microfuge tubes for the plating exercise (see exercise). Start labelling these tubes with a 20 minute time point and label the rest with 20 minutes intervals up to the final tube which should be labeled as 120 minutes (i.e., label the tubes as 20min, 40min, 60min etc.). 6. Place the labeled tubes on ice. 7. As the 20 minute intervals progress, remove your two samples each time (5ml For the OD600 reading and 100µl for the spread plate samples in exercise 2). 8. When measuring the samples for the OD600 readings: When the OD600 has not yet reached a value of 0.5, measure the turbidity of the culture by aseptically removing 5ml of culture from the flask and placing the sample directly into a 13mm test tube. Measure the turbidity and record the OD reading and time in table 1. 9. Return the flask to the incubator immediately. 10. When the OD600 does reach 0.5, you must dilute your sample before taking a turbidity measurement. To do this, dilute your next sample 1:4 (i.e., add 1ml of culture to 3ml sterile LB broth). Do not return your diluted sample to the main culture flask. You may have to use a higher dilution for later samples. Be sure to record your dilution factors. 11. Stop sampling at 120min. 12. For each time point: plot OD600 reading (y‐axis) vs. elapsed time (x‐axis) on semi‐log graph paper. EXERCISE 2: Plate Count Measurements of Bacterial Growth Standard Plate Counting Technique (Viable Cell Count) The standard plate count (SPC) is used to determine the number of living (viable) organisms in various samples such as water, milk, and foods, and during the various stages of bacterial growth curves. The 41 technique is simple to perform and produces excellent reproducible results. It is based on the assumption that each viable cell will form one colony. Thus, the number of resulting colonies is an indication of the number of viable cells in the original sample. The SPC technique consists of diluting a sample (because the number of organisms present may be too numerous to count accurately) and then plating the dilutions. Plating can be performed using either the pour or spread plate techniques; in this laboratory, you will use the spread plating method. For purposes of accuracy and reliability, after incubation only plates with 30 to 300 colonies are typically counted. (This is a guideline – where there are fewer than 30 colonies, there is an increased chance of error; where there are >300 colonies, it may be difficult to determine if you are counting single colonies). The number of organisms in the original sample/ml is determined by multiplying the number of colonies formed by the dilution factor(s) for the particular plate(s) being used. Plot the Standard Curve as Log (Viable Cell Count, cfu/mL) versus OD pf the sample. Materials and Methods Materials: 1.
6. 100µl samples from Exercise 1. LB broth. 28 Nutrient agar plates. Sterile 5ml and 10ml pipettes. Microcentrifuge or glass tubes Ethanol and a glass spreader. Procedure for the preparation of Spread plates (see Fig. 3.2) 1. Label the bottom of all plates with your initials, the appropriate dilutions and the time points. Pipette the required volume (usually 0.1ml) of the diluted culture onto the medium in the middle of the plate. Do this in duplicate. 2. Sterilize your glass spreader. To do this: a. Dip the end of the spreader into ethanol b. Ignite the alcohol (by passing the spreader through your Bunsen burner flame once) c. Allow the alcohol to burn away from the flame d. Let the spreader cool in the air (near the flame, in the sterile region). 3. Check that the spreader is cool by touching the inside of the plate lid. 4. Spread the bacterial sample over the surface of the agar evenly by moving the spreader back and forth while rotating the plate. Use a turntable or rotate the plate by hand, a quarter turn at a time on the bench top (your TA will demonstrate). 5. Continue to spread the plate until the surface of the medium is completely dry (this prevents colonies from “running”). 6. Place the spreader back into the ethanol to avoid contaminating your bench. 7. Invert your plates and incubate them at 37°C for 24 hours. 42 Fig. 3.2 Spread plating method Procedure for counting the viable cell (use the 100µL samples drawn in Experiment 1): 1. Since you can only accurately count plates that have between 30‐300 colonies, you will have to prepare serial dilutions of the 100µl of samples you collected in exercise 1. Do this as follows: 2. Spread 100µl of each sample onto the surface of the nutrient agar plates. 3. Incubate plates at 37°C for 16‐24 hours. Count colony forming units (cfu) the following day. 4. For each time point keep only the plates that have between 30‐300cfu. If for a certain time point, you find that more than one plate contains 30‐300cfu, average the counts of these plates and record this in the table. 43 ASSIGNMENT: In this assignment, each student must write an independent submission. Follow the guidelines below to prepare the assignment for Lab 3. The report should include the components listed below. The discussion section should be brief, and only address what has been requested. Mark breakdown: [14 marks total] – DUE ONE WEEK AFTER COMPLETION OF LAB 3. Materials and Methods Results: Tables 1 and 2 (show measurements + calculations) Growth curves Generation time (g) in hours and growth rate constant (k) calculations Standard curves Discussion References 0.5 mark 2 marks 3 marks 1 mark 3 marks 4 marks 0.5 mark Detailed Lab 3 Marking Rubric Materials and Methods [0.5 mark]: Cite lab manual, indicating any changes made. Tables 1 & 2 [2 marks]: Tables should include your measurements and calculations. They should be neat and labeled appropriately. Do not include information that is not necessary. Growth Curves [3 marks]: Drawn by hand on semi‐log graph paper. Should be neat, clear, labeled properly. Plot both OD and viable cell count on one graph. Identify lag phase and/or log phase if possible. Calculations [1 mark]: Calculate generation time and growth rate constant. Standard curves [3 marks]: Using linear graph paper, construct the standard curves and produce the graph by hand and using a computerized method. Discussion [4 marks; 2 page max]: Brief analysis/explanation of your results. Compare your results to what has been reported in literature. If relevant, identify possible sources of error. Comment on the accuracy of your dilutions. Compare your growth curve to that of another bacterium published in peer reviewed literature* (include a copy of this other growth curve and include a complete reference/citation). Discuss aspects of these bacteria (environments, physiology) that would lead to differences. *Growth Curve from the Literature: Include copy of bacterial growth curve figure from research article (published in peer‐reviewed journal), with complete reference citation. References [0.5 mark]:See p.8‐9 for referencing formats. 44 ...
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