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    Lab 10 Bacterial Growth Curve

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    Sanjiv Gautam
    1. The document describes a laboratory experiment to determine the generation time of bacterial growth through serial dilutions and spectrophotometric measurements taken at regular intervals. Students will monitor the growth of E. coli cultures over several hours, recording optical density and colony forming units. 2. By plotting optical density versus time, students can indirectly calculate generation time from the exponential growth phase. They will also perform serial dilutions and plate cultures to directly count colony forming units at each time point. 3. The goal is to collect multiple growth curves from different starting cultures to construct a complete curve characterizing the lag, exponential and stationary phases of E. coli growth under standardized conditions.

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    Lab 10 Bacterial Growth Curve

    Uploaded by

    Sanjiv Gautam
    585 views4 pages
    1. The document describes a laboratory experiment to determine the generation time of bacterial growth through serial dilutions and spectrophotometric measurements taken at regular intervals. Students will monitor the growth of E. coli cultures over several hours, recording optical density and colony forming units. 2. By plotting optical density versus time, students can indirectly calculate generation time from the exponential growth phase. They will also perform serial dilutions and plate cultures to directly count colony forming units at each time point. 3. The goal is to collect multiple growth curves from different starting cultures to construct a complete curve characterizing the lag, exponential and stationary phases of E. coli growth under standardized conditions.

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    msc microbiological

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    1. The document describes a laboratory experiment to determine the generation time of bacterial growth through serial dilutions and spectrophotometric measurements taken at regular intervals. Students will monitor the growth of E. coli cultures over several hours, recording optical density and colony forming units. 2. By plotting optical density versus time, students can indirectly calculate generation time from the exponential growth phase. They will also perform serial dilutions and plate cultures to directly count colony forming units at each time point. 3. The goal is to collect multiple growth curves from different starting cultures to construct a complete curve characterizing the lag, exponential and stationary phases of E. coli growth under standardized conditions.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    585 views4 pages

    Lab 10 Bacterial Growth Curve

    Uploaded by

    Sanjiv Gautam
    1. The document describes a laboratory experiment to determine the generation time of bacterial growth through serial dilutions and spectrophotometric measurements taken at regular intervals. Students will monitor the growth of E. coli cultures over several hours, recording optical density and colony forming units. 2. By plotting optical density versus time, students can indirectly calculate generation time from the exponential growth phase. They will also perform serial dilutions and plate cultures to directly count colony forming units at each time point. 3. The goal is to collect multiple growth curves from different starting cultures to construct a complete curve characterizing the lag, exponential and stationary phases of E. coli growth under standardized conditions.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 4
    At a glance
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    The passage describes the stages of bacterial growth and how to measure generation time using direct and indirect methods.

    The four phases of bacterial growth are lag phase, exponential phase, stationary phase, and death phase.

    Generation time can vary among species and within a species based on environmental factors like nutrient availability and waste accumulation.

    Miramar College

    Biology 205 Microbiology


    Lab Exercise 10: Bacterial Growth Curve & Serial Dilutions
    Background
    Bacterial population growth studies require inoculation of viable cells into a sterile broth medium and incubation of the
    culture under optimum temperature, pH, and gaseous conditions. Under these conditions, the cells will reproduce
    rapidly and the dynamics of the microbial growth can be charted by means of a population growth curve, which is
    constructed by plotting the increase in cell numbers versus time of incubation and can be used to delineate stages of
    the growth cycle. It also facilitates measurement of cell numbers and the rate of growth of a particular organism under
    standardized conditions as expressed by its generation time, the time required for a microbial population to double.
    The stages of a typical growth curve (Figure 1) are:
    1. Lag phase: When the cells are adjusting to their new environment. During this phase, cellular metabolism
    is accelerated, resulting in rapid biosynthesis of cellular macromolecules, primarily enzymes, in preparation
    for the next phase of the cycle. Although the cells are increasing in size, there is no cell division and
    therefore no increase in numbers. 2. Exponential phase: Under optimum nutritional and physical
    conditions, the physiologically robust cells reproduce at a uniform and rapid rate by binary fission. Thus
    there is a rapid exponential increase in population, which doubles regularly until a maximum number of
    cells is reached. The length of the log phase varies, depending on the organisms and the composition of
    the medium, although the average may be estimated to last 6 to 12 hours. 3. Stationary phase: During this
    stage, the number of cells undergoing division is equal to the number of cells that are dying. There is no
    further increase in cell number and the population is maintained at its maximum level for a period of time.
    The primary factors responsible for this phase are the depletion of some essential metabolites and the
    accumulation of toxic acidic or alkaline end-products in the medium. 4. Death phase: Because of the
    continuing depletion of nutrients and buildup of metabolic wastes, the microorganisms die at a rapid and
    uniform rate. This decrease in population closely parallels its increase during the log phase. Theoretically,
    the entire population should die during a time interval equal to that of the log phase. This does not occur,
    however, since a small number of highly resistant organisms persist for an indeterminate length of time.

    Figure 1: Growth curve for an hypothetical population. Note the four phases of growth: lag; exponential; stationary and
    death.

    Construction of a complete bacterial growth curve requires that aliquots of a 24-hour shake-flask culture be measured
    for population size at intervals during the incubation period, however, such a procedure does not lend itself to a regular
    laboratory session. This experiment is designed to include only the lag, log and possibly stationary phases of population
    growth. Upon completion of this experiment, you will plot the data collected during this experiment by using two
    values for the measurement of growth. The direct method requires that you use serial dilution to plate out cells at 30
    minute intervals in order to calculate the number of colony forming units (CFUs) at a given time. The indirect method
    uses spectrophotometric measurements of the developing turbidity at the same 30-minute intervals, as an index of
    increasing cellular mass (assumed to correlate with an increase in the number of cells).
    You will determine generation time with indirect methods by using data you collect, once it has been plotted onto a
    graph like the one shown below. Indirect determination is made by simple extrapolation from the log phase as
    illustrated in the figure below. Select two points on the optical density (OD) scale, such as 0.4 and 0.8, that represent a
    doubling of turbidity. Using a ruler, extrapolate by drawing a line between each of the selected optical densities on the
    ordinate and the plotted line of the growth curve. Then draw perpendicular lines from these end points on the plotted
    Lab Exercise 10: Bacterial Growth Curve & Serial Dilutions

    Page 1 of 4

    line of the growth curve to their respective time intervals on the abscissa (Figure 2). With this information, determine
    the generation time using the formula GT =t(O.D. 1) - t(O.D. 2).

    Figure 2: Graphical representation of the indirect method of measuring bacterial growth, optical density plotted vs. time.
    Generation time is determined using two points within the exponential portion of the growth curve where the population
    doubles in size, in this example: GT =t(O.D. 1) - t(O.D. 2) = 263 minutes - 222 minutes =43 minutes, so the generation time of this
    hypothetical population is 43 minutes.

    Introduction
    In this experiment you will be determining the generation time of a bacterial culture. Because of the amount of
    time needed for these cultures to reach the stationary phase of growth, the cultures will be started before class
    begins. You will receive instruction as to when the cultures were started, what organism was used for the
    inoculation, and what media these organisms were inoculated into. The class as a whole will be testing Escherichia
    coli with each table having a culture started at different times. In this way, we hope to collect data that can be
    plotted together and yield a growth curve sufficient to determine the generation time of this organism.
    These cultures will be incubated throughout the experiment in a shaking incubator. The incubator will be set to
    the optimal temperature for your specific bacterial species and will shake in order to keep your culture properly
    aerated. These conditions will allow for optimal growth of your culture via aerobic respiration. You will take
    control of this culture when lab begins.
    At thirty minute intervals, you will take measurements of the cultures growth. You will do this directly using
    serial dilutions and spread plating, and indirectly using OD readings. This means that you will be performing
    the protocols outlined below at thirty minute intervals.
    Objectives
    1. Take metered spectrophotometric readings of a growing culture.
    2. Perform serial dilutions and spread plating at metered intervals.
    3. Plot growth data and determine generation time of a bacterial species.
    Protocol
    Team Supplies

    Class Supplies

    bacterial culture

    shaking incubator

    Spec20 spectrophotometer
    sterile broth to zero the Spec20 spectrophotometer
    ice bath
    16 nutrient agar plates
    4 test tubes containing 9.9 ml H2O
    16 test tubes containing 9.0 ml H2O
    200l mechanical pipetter and tips
    1000l mechanical pipetter and tips
    waste container for used tips
    glass Petri dish and alcohol
    spreader, plate spinner

    Lab Exercise 10: Bacterial Growth Curve & Serial Dilutions

    Page 2 of 4

    Day One: Setting Up


    1. Properly zero your Spec 20, refer to Lab Exercise 10a: Using a Spectrophotometer and Pipetman if necessary.
    2. LABEL YOUR MEDIA: For each time interval you will be taking measurements (e.g., time 0, time 120, time 150, etc.)
    label five test tubes with the time period and 10-2, 10-3, 10-4, 10-5 and 10-6. Label four Petri dishes: 10-4, 10-5, 10-6 and 10-7.
    You should have five test tubes and four Petri dishes for each time period.
    Day One: Collection of Time Interval Specimens
    Note: This section explains how to collect the specimens that will be diluted and plated in the following section.
    3. AT 30 MINUTE INTERVALS (specified in step 3 above) perform the following steps. This procedure is also pictured
    below:
    1. Collect your flask from the shaking incubator.
    2. Remove 100l of your bacterial culture from the flask and put it in the test tube labeled 10-2. Put this
    test tube into your ice bath. Note: placing this tube on ice effectively halts binary fission and means
    that you can take your time in setting up the remaining steps of the serial dilution and plating
    experiment.
    3. Give your flask a swish and then take an OD reading using the Spec 20. It is not necessary to remove
    any of the culture for this step, simply tilt the culture to pour enough of the liquid to fill the attached
    test tube with culture. Place this test tube into the Spec 20 and record the OD600nm.
    4. Quickly put the culture back into the 37C shaking incubator.

    Day One: Serial Dilution and Spread Plate Protocol


    Note: These steps will be performed for EACH INCUBATION TIME, using the tubes collected during the Collection of
    Time Interval Specimens section above.
    1. Remove your 10-2 test tube from the ice bath, and mix the culture by rolling it carefully in your hands. Perform
    the serial dilutions and plate a portion of each of these dilutions onto your four Petri dishes as directed in the
    figure below.

    Lab Exercise 10: Bacterial Growth Curve & Serial Dilutions

    Page 3 of 4

    2.

    Spread the bacterial inoculates onto each of the four plates using the protocol outlined in the figure below. Note:
    heat is not the sterilizing step in this procedure- alcohol is. The only reason you are using heat is to remove the
    alcohol once it has sterilized your spreader.
    Note: There may be some residual liquid on the spreader even after you have flamed it. This is water, not alcohol.
    Do not worry about removing it- its sterile. If you leave the spreader in the heat for longer than it takes to ignite the
    alcohol, the spreader will be too hot to spread the culture without killing the cells.
    Be sure also to keep a safe distance between the dish of alcohol and the flame so you do not cause the alcohol to
    ignite in the dish. Never put a hot spreader into alcohol as the flash point of alcohol is relatively low (~56o F) and it
    will easily ignite.

    3.

    After you have spread the inoculates onto the Petri dishes, leave them agar side down in the 37C incubator.

    Day Two: CFU counts


    1. Collect your plates from the 37C incubator. Count the number of distinct colonies on each plate.
    2. For each time set, keep only those plates which contain between 30-300 colonies, discard all the rest. If more than
    one plate contains 30-300 CFUs, average the numbers and record only one for each time set.
    3. Determine the number of CFUs/ml of the original culture for each of the time sets.
    Results and Data Analysis
    1. Record the optical densities and corresponding cell counts in the chart below.
    Clock Time

    Incubation Time
    (min. past)a

    Optical Density
    (600nm)

    CFU Counts
    (CFU/ml)

    This column records the actual number of minutes passed from the time zero inoculation.

    The following questions can be completed after Lab Exercise 14: Data Analysis and Presentation I.
    2. Plot the CFUs/ml on the Y axis and incubation times on the X axis of the provided semi-log paper (you may also do
    this using computer software). Plot the OD600nm on the Y axis and incubation times on the X axis of the provided
    graph paper.
    3. Identify the log phase for each of the graphs and determine the generation time of your bacterial culture using
    both the indirect and direct methods described in the introduction to this lab exercise. Remember that the log
    phase is represented by the straight-line portion of the curve.
    Discussion
    1. Does the term growth convey the same meaning when applied to bacteria and to multicellular organisms? Explain.
    2. Why do variations in generation time exist:
    a. among different species of microorganisms?
    b. within a single microbial species?
    3. Is generation time a useful parameter to indicate the types of media best suited to support the growth of a specific
    organism? Explain.

    Lab Exercise 10: Bacterial Growth Curve & Serial Dilutions

    Page 4 of 4

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