Serial Dilutions and Enterococcus faecalis

Div. 1 Plate Count A Plate Count B Mean Plate Count Mean Plat Count

Time CFU/ml CFU/ml CFU/ml Log CFU/ml

0 min 1.9 X 107 * 2.4 X 107*



1 min 1.3 X 107 -



3 min 4.7 X 106 -



5 min 6.4 X 105 -



10 min 3.4 X 104 2.7 X 104



15 min 1.9 X 103 -



This is the data table was used for the experiment. (the table below
is just an example)

QUESTIONS:

Take average of plate count A and B if both are available. If not, use
the one that is available. Write the mean plate counts in the table.

Convert the mean plate counts into Log counts and record the log counts
in the table . Please show work

Write a title of your table above, following the instruction provided to
you (Title should look like the one in the table below).

Please do the graph in Microsoft EXCEL. Plot your mean log CFU/ ml vs.
time (min). Use sample graph below as a guide.

If you know how to calculate linear regression, you may draw the death
curve accordingly.

If you don’t know what liner regression is, draw a best fit line that
is close to all the data points on the graph.

On the graph, determine the D value (the time needed for the population
to reduce by 1 log).

Briefly describe the result and write a conclusion.

If the data is not perfect, discuss what possible errors were and how
you would correct the problem if you ran the experiment again.

Use one paragraph to explain what the decimal reduction time (D) value
is, why it is important to determine D value of a microorganism. In
another paragraph, please write about the bacterium Enterococcus
faecalis if you can find relevant references.

Please write a title for this experiment. The tile must include all
the necessary information such as the name of the organism, the temp,
the method used, and what you determined—see the sample title in
reference #2: Determination of decimal reduction time (D value) of
chemical agents used in hospitals for disinfection purposes)

Finally, please write the objective of this study.

Sample Table (this is how the table above should look; this table is
just an EXAMPLE):

Table 1. Viable counts of S. epidermis in NB with 5% Lysol determined by
spread plate method on NA plates

Time (min) CFU/ml Mean CFU/ml Log CFU/ml

Trial 1 Trial 2



0 5.6 x 107 6.4 x 107 6.0 x 107 7.8

2 3.5 x 105 2.9 x 105 3.2 x 105 5.5

5 1.3 x 102 1.5 x 102 1.4 x 102 2.1

10 <10 <10 <10 <1



Sample Figure:



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subtilis D = 25 min (○). (2) ( Reference

My data was at heating time 15 minutes.

Assignment:

The class will work together to determine the D65°C of Enterococcus
faecalis. Students will work as pairs. Each pair of students will make
a serial dilution and determine the viable count of a sample after
heating using pour plate method.

The instructor will heat the bacterial suspension at 65°C. At time 15
min a sample will be taken out by the instructor and placed in a
microfuge tube. A pair of student will take the sample and determine
the viable count of the bacterium in the sample using standard plate
count –pour plate procedure.

Supplies (1/2)

4~6 sterile microfuge tubes, 4~6 sterile 1 ml pipettes, sterile
phosphate buffer saline (10 ml)

5 petri-dishes , 100 ml melted TSA (don’t remove it until you are
ready to pour plate)

Procedure:

Prepare 3 dilution blanks by adding 0.9 ml of PBS into each of the
sterile microfuge tubes. Label the tubes as –1, -2,

Label 5 sterile petri-dishes with the dilutions that you are going to
plate. (refer to the table 1)

After receiving the sample, mix the sample by vortexing for a few sec,
then make a serial dilution.

Aseptically transferring 100 μl (0.1 ml) of the sample into 0.9 ml
dilution blank labeled as “-1”. Mix the tube by vortexing for a few
seconds.

Repeat the procedure to prepare the remaining dilutions.

While preparing the serial dilution, transfer 0.5 ml of appropriate
diluted samples into petri-dishes.

Pour ~18 to 20 ml of melted TSA, which has been cooled to 50°C, into
each petri-dish and swirl the plates.

Allow the plates to solidify on your bench without disturbance.

Turn the plates up-side-down for incubation at 37°C after the TSA
solidified completely.

Table 1. dilutions to be plated.

Heating Time Dilutions to be plated

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