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Micro Lab Report

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					        Introduction


        Unlike the common perception of growth as being an enlargement of size, microbial

growth counts the number of individual colonies to predict the increase in volume of viable cells.

In most cases, microbial growth happens through binary fission, where one organism divides into

two distinct organisms. The growth is studied in both batch cultures and continuous cultures, the

differences being the method of providing nutrients for growth. The growth is measured using

either the total cell count or the viable cell count.2


        The microbial growth curve is a great way to represent the trend that microorganisms

tend to follow throughout the life cycle. Both prokaryotes and eukaryotes follow the same

general trend throughout a time span in batch culture. The main phases are lag, log, stationary,

and death5.



                                 Microbial Growth Curve5

                                           stationary


      Log #                                                              death
      of
      viable                      log
      cells                       (exponential)

                   lag




                                            Time 
        In the lag phase, microorganisms have just been introduced into a fresh medium; no real

increase in cell number happens here. However, the cell is in preparation for the log phase by

synthesizing components for growth. Eventually cell number will begin to increase.5


        This leads to the log (exponential) phase. During this, cell number increases

exponentially as it reaches its maximum possible growth rate. This determines the generation

time, which is the time for the number of cells to double in quantity. The batch culture will

maintain a steady rate of growth throughout this phase, until it cannot maintain this rate due to

environmental conditions or the growth medium.5


        Next comes the stationary phase. In this, the batch culture has reached the peak capacity

on its ability to support the number of organisms in it. Growth (increase in the number of viable

cells) does not occur, but the batch culture maintains the number of cells currently living in it.

This could be the result of either a lack of cell division but maintenance of an active metabolism

or a balance between cell death and cell division overall in the batch culture.5


        The final stage is the death phase. Here, microbial growth is negative, which means that

the rate of generation of new cell is less than the rate of cell death. Sherman et al5 suggests that

this could be related to two different causes. The first is known as viable but nonculturable and

occurs in periods of starvation. It states that some of the viable cells become dormant until better

conditions are established for growth. The other supports programmed cell death, which states

that it is in cell’s genetic makeup to die after a certain length of time; this provides nutrients to

other cells in the batch culture that do not have the genetic programming for cell death.5


        The prokaryotic organism being studied in this experiment is Vibrio natriegens. This is a

marine bacterium that was chosen because it theoretically displays the standard microbial growth
curve.1 The eukaryotic organism examined in this experiment is Saccharomyces cervisiae. It will

be grown on two different types of medium, Yeast Nitrogen Base (YNB) and Yeast Extract

Peptone Dextrose (YEPD) in order to determine if there is a difference in generation time based

upon growth medium.3 V. natriegens has a much faster generation time of about 30 to 60 minutes

as compared to the eukaryotic organism, which has about a 2 hour generation time.3




Materials and Methods:


This procedure follows methods stated in The Microbes Around Us2 and Online Exercise 3.4


       The data collected here was obtained during two different laboratory periods. The first

experiment dealt with prokaryotic growth and used the microbe Vibrio natriegens. The

absorbance and CFU/mL data were obtained for time points 0 through 120 minutes. The second

experiment examined the eukaryotic growth of Saccharomyces cervisiae in two different

mediums, YNB and YEPD. The absorbance, cell number, and CFU/mL data was obtained for

time points 0 through 36 hours.


       To begin the prokaryotic growth procedure, the class divided into groups of 2 to examine

the growth at one time point in the span of 0 – 120 minutes in increments of 10. The medium of

100 mL Vibrio natriegens with one bacterial colony from an isolated, streaked plate grown for 6

hours at 37*C was pre-prepared before lab. Then 10 mL of the culture grown for 6 hours is

inoculated into 300mL of V. natriegens.


       The BioPhotometer was zeroed at 595nm with 100 microliters of V. natriegens from the

flask with 300 mL. To begin testing, 1 mL of culture was removed from the batch. Each time
point of the culture was serial diluted until a 10^-6 dilution was achieved. For time points 0, 10,

and 20 minutes, agar pour plates of 10^-3 through 10^-6 were prepared using the respective

time’s sample. 100 microliters of the sample removed from the incubator at the respective time

period was placed in a cuvette to measure the absorbance with the BioPhotometer.


       Between the times of 30 through 70 minutes, the same procedure as before was used,

except that dilutions of 10^-4 through 10^-7 plates are prepared from serial dilutions. With time

points 80 minutes and above, the same procedure as above was followed with an exception that

plates of 10^-5 through 10^-8 are prepared. The purpose of diluting the plates to a greater

dilution for the longer time periods takes into account the accumulation of cell volume that has

begun to occur in the culture. This way, a more accurate plate sample will theoretically be

obtained. The plates then were incubated at 30*C for 24 hours. The following lab period, the

plates were examined to determine the number of colonies per plate. This was calculated using

Formula 1 below. In order to compare the data more effectively, the CFU/mL are put into

Formula 2 before plotting.


       Formula 1: CFU/mL = CFU/ (volume*dilution)


       Formula 2: log (CFU/mL)


       The cell count for each of the two types of growth medium of the Eukaryote organism,

Saccharomyces cervisiae, was determined using the counting chamber method. Using aseptic

technique and ensuring the culture was vortexed before using, the culture was diluted according

to a protocol based upon incubation time. The purpose of vortexing was to ensure that cells have

not settled on the bottom of the test tube and an accurate representation of cell components was

obtained.4
       Then, a drop of the culture was added to the chamber on the hemocytometer slide and

covered with a cover glass. In the center of this is a grid, which assists with counting the number

of cells per unit area. The sample was viewed under the microscope at 100X magnification.

Using Formula 3 to determine the number of cells, the bacteria in one square was counted and

multiplied by 25, which is the number of squares in the grid. It is then multiplied by 50, since the

chamber is .02mm deep. This will give a representation of the number of cells in a cubic

millimeter. Using Formula 4 to find the number in a cubic centimeter, the previous amount

should be multiplied by 10^3. Using Formula 5, the log of Formula 4 should be taken to

adequately compare the results in an easier to read manner. 5


Formula 3: Cells/ mm^3 = (Cells/ square)(25 squares)(50)


Formula 4: Cells/ cm^3 = (Cells/ square)(25 squares)(50)(10^3)


Formula 5: = log (Cells/cm^3)


       .1mL of the diluted culture used in the hemocytometer cell count was then plated using

the spread plate technique on both YEPD and YNB to determine the number of viable cells in

the sample rather than the total number of cells. Hopefully by using two different types of

growth medium for S. cervisiae will show different results in the growth curve. Again, aseptic

technique was used.4 Then the plates were inverted and incubated at 30*C for 48 hours. The

optical density was measured by dispensing 1.5mL of the sample culture in a cuvette and placing

it into the BioPhotometer. In the next lab period, the number of cells that grew on the plates

were counted and recorded for observation. To determine plate count, Formula 6 was used.


Formula 6: Plate Count = # of CFU/ (volume X dilution)
For the cell count values to determine generation time, Formula 7 is used. In order to figure out

the number of generations, Formula 8 is used. 5


Formula 7: g = (t-t0)/n


Formula 8:    n= [log(N)-log(N0)]/log(2)


*t = time

*n = the number of generations

*N = population at time t

*No= initial population




Results


       The absorbance data for the S. cervisiae cultures is displayed below in Figure 1. The

curve shows the lag (0-8 hours) and exponential (8-16 hours) phases of the microbes growth very

plainly. However, neither the stationary phase, nor the death phase is present. There are no

significant differences between the cultures growing in the nitrogenous base versus the dextrose

base. Also notable is the low value for both mediums at 30 hours.
Figure 1: Eukaryotic Absorbance vs. Time



                        Absorbance vs. Time
    A 2.5
    b
    s   2
    o
      1.5
    r
    b   1                                                                 YNB
    a
                                                                          YEPD
    n 0.5
    c
        0
    e
            0           10           20           30            40
                                 Time (hours)




         The cell count data for the eukaryotic growth in rich and minimal media is shown below

in Figure 2. The cell count number was plotted in a log scale to provide a better visual

comparison of the data. This data does not adequately represent the pattern that should be

observed here. There is no evidence of a lag phase, it is questionable whether there is evidence

of a log phase, and there is no stationary or death phase. This figure should show a lag phase, log

phase, and stationary phase. No death phase should be observed because this method does not

test for viable cells. Again, there is no significant difference between the two types of growth

medium. This graph also shows two notable and unexpected low values at 12 hours and 20

hours.
Figure 2: Eukaryotic Cell Count vs. Time



                       Cell Count vs. Time
     l 1.20E+01
     o 1.00E+01
     g
       8.00E+00
     # 6.00E+00
                                                                       YNB
       4.00E+00
     c                                                                 YEPD
     e 2.00E+00
     l 0.00E+00
     l
                0          10          20         30         40
     s
                                  Time (hours)




       The plate count data for the S. cervisiae growth in rich and minimal media is shown

below in Figure 3. There is no significant observation gained from this figure, but it should still

be presented to show why a conclusion on this data is difficult. All plates contained more CFUs

than what is acceptable to use for data collection, (over 300 CFUs). The 3 bars that exceed 300

CFUs in the 30-36 hour range had much higher values than displayed on the chart. However, the

data is not useful in comparing CFUs/mL, so the actual values are not displayed on the chart.
Figure 3: Eukaryotic Plate Count vs. Time



                       Plate Count vs. Time
          450
          400
     #    350
          300
     C    250
     F    200                                                          YNB
     U    150
          100                                                          YEPD
     s
           50
            0
                 0     4     8    12     16     20   30    36

                                 Time (hours)




         The absorbencies of the data for the V. natriegens are displayed below in Figure 4. This

begins by clearly showing the lag phase from 0 to 10 minutes and the log phase from 10 to 20.

The data does not strongly suggest that the organism enters stationary phase after 20 minutes, but

it could be speculated that stationary phase has taken over after 20 minutes. The only

discrepancy is time point 80. This drops very significantly, but it is such an unusual point that it

must be examined for accuracy.
Figure 4: Prokaryotic Growth Absorbance vs. Time



            Prokaryotic Growth Optical Density
                         vs. Time
  1.6
  1.4
  1.2
    1
  0.8
  0.6
  0.4
  0.2
    0
        0        20         40         60          80       100        120




        The CFU/ml values for the V. natriegens culture are displayed below in Figure 5. There

is no stage of the growth curve represented by this data. The data set’s second time point shows

the highest CFUs/mL, which according to the previous figure is still a part of the lag phase.

These two data sets do not agree. Again, the time point 80 is very unusual and contributes greatly

to the insignificant findings of the experiment.
Figure 5: Prokaryotic CFUs/mL vs. Time



                Prokaryotic CFU/ mL vs. Time
       14
     L
     o 12
     g 10
        8
    C
        6
    F
    U   4
    /   2
    m   0
    L       0       20        40            60      80     100       120
                                      Time (min)




        The generation times determined from the data are shown below in Figure 6. The time

point chosen for each calculation was based on where the graphs above represent the log phase to

occur. As notable from the negative generation time in the eukaryotic organism, the data does

not support the microbial growth curve. The data for plate numbers to determine generation time

is not shown because no useable data was obtained from the plates.


Figure 6: Generation Times determined by data results


                                   S. cervisiae (t=8)             V. natriegens (t=10)

Cell Count                         3.76E6                         2.99E11

Generation time                    -1.996                         8.527
Discussion


        The eukaryotic growth experiment did not effectively show the trend of a microbial

growth curve as stated in the purpose. Looking at the data obtained for generation time of S.

cervisiae, it is clear that this data is insignificant because it is impossible to have a negative

generation time. However, the prokaryotic growth curve experiment does provide somewhat

useful data. Since there was an apparent log phase, it was possible to be more accurate

determining the generation time using Formulas 7 and 8. It is shown that V. natriegens has a

generation time of roughly 8.5 minutes, which is reasonable for a prokaryote.


        If the plate count values were usable, it should show that in the later time stages where

cell death is occurring that the plate counts decline as cell counts rise. By looking at the

hemocytometer, it is impossible to determine which of the cells are live and dead. However, the

plate count only represents viable cells.


        If the eukaryotic data obtained was more accurate, a longer generation time for S.

cervisiae compared with V. natriegens should be apparent. Also, the culture in the minimal

media should show a longer generation time than that in the rich media, which is also not

apparent by looking at Figure 2. This is due to the circumstance that the minimal media culture

must generate metabolites that are already provided in the rich media, therefore slowing the

generation time due to the increased workload on the cells.


        None of the figures followed the expected growth curves, which is likely due to operator

error. One error could have been that some groups counted the cells in a non-diluted solution, but

others counted those in a diluted solution. Also, some groups may have not diluted the culture

correctly. Probably, as evidenced by the plate counts, no group diluted the culture to the correct
level. Therefore, there were too many cells being added to the plates, so the cells surpassed the

acceptable level of growth too quickly.


          In the sets of data, there are several points that represents data very unusual compared to

the rest of it. One example of this is the time point of 30 hours in absorbance vs. time in both of

the eukaryotic samples. This is extremely low, so it was most likely due to an inaccurate method

of checking the absorbance. Another example of an odd point would be the time point of 80

hours in the prokaryotic growth culture. In this case, since the CFUs/mL and the absorbance is

extremely low, there was probably a problem preparing the sample once it was taken out of the

shaker.


          This experiment was successful in orienting students to the techniques used to view and

analyze microbial growth. Although results were not as expected, the understanding of what

should happen in the experiment creates a good opportunity for students to learn by hands-on

techniques. This data set was very useful in providing confusing results, which allowed for

exploration into the science to determine what may have gone wrong. Through completing this

experiment, I was successfully introduced to characteristics of prokaryotic and eukaryotic

growth, along with understanding that the medium used for growth impacts the generation time

of the organism.
References


1. Berdalet, E. Packard, T. Lagace, B. Roy, S. St-Amand, L. Gagne, J. 1995. CO2 production,

O2 consumption and isocitrate dehydrogenase in the marine bacterium Vibrio natriegens.

Aquatic Microbial Ecology. Spain. 9:211-217


2. McPherson, E. The Microbes Around Us: Second Edition. Pearson, 2010.


3. Meneau, I. Sanglard, D. Billie, J. Huaser, P. 2004. Pneumoncystis jiroveci dihydropeteroate

       synthase polymorphisms confer resistance to sulfadoxine and sulfanilamide in

       Saccharomyces cervisiae. American Society for Microbiology. 48(7): 2610-1616.


4. Online Exercise 3: The Growth Curve


5. Sherwood, L. Woolverton, C. Willey. 2009. Prescott’s Principles of Microbiology. New York

       McGraw-Hill. 8:130

				
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