Lab #5
Enumeration of Microorganisms
Introduction
The purpose
for this lab was to become comfortable with being able to understand the total
viable count for enumerating microorganisms. This experiment will allow you to
realize just how many microorganisms are harbored in the environment. Now what I
mean by viable count is being based on the ability to place any microorganism
in a nutrient beneficial environment to watch it grow in population. To do this
we use different types of media that closely resemble the environment in which
the microbes thrive best. In order to count the amount of microorganisms in the
media, we literally use a counting method that resembles just looking at the
plate and counting the large specs on the plates. This is one of the oldest
techniques in the books and still is currently being used.
After
collecting a sample of water we would like to test, we can dilute the sample to
help counting the plates later on. The goal is to produce one series of plates
of about 30 to 300 colonies that have grown on the plate. Thus, sometimes we
have to seriously dilute the water sample down 3 separate times at which each
is tenfold. However, the more you dilute
the water the lest accurate the counting will be. Now in order to get them to
grow we have to plate them on an agar which is nutrients. Then we use a pour
method in which we take 1 mL of our solution and pour it on the plate and use a
sterile hockey stick like device to spread the sample on the nutrient filled
agar.
Methods and
Materials
Me
being the designated water boy for this lab. I collected a sample of lake water
next to my apartment out in BFE for this lab. We had taken this sample and
tested it in two different types of media. The first is a Luria-Bertani (LB) Agar (nutrient
rich) which has several components such as 10
g tryptone , 5
g yeast extract, 10 g sodium chloride and 15 g agar per
liter. The second media is our R2A Agar (low-nutrient) that consist of 0.5 g each of yeast extract, protease
petone, casamino acids, glucose, and soluble starch; 0.3 g dipotassium hydrogen
phosphate; 0.05 g magnesium sulfate heptahydrate; 0.3 g sodium
pyruvate; and 15 g agar per liter.
We needed to dilute our sample by placing 0.9 mL of
buffer mixed with 0.1 mL of the surface water sample. From there, we placed 0.1
mL of that mixture into a sterile cuvet and mixed it with 0.9 more mL of
buffer, repeating this process several more times until we had a 10-7dilution.
We used the first 3 dilutions for our test (10-1 ,10-2 , and 10-3) This was because of
the said microbes in freshwater lakes to be 103 to 105 therefore we only diluted up to 3 times for an
accurate reading. We then did 3 samples for each of the diluted samples thus
ending with 9 total samples for each media. In order to spread the sample on
the plates however, we used an old method of spreading with a glass sterile
hockey stick like piece. We stroked the liquid on the agar carefully to not rip
the agar and made sure it covered the top layer in the plate. We needed to incubate
the samples to allow ample amount of growth. After we waited for the growth, we
then counted the CFU in each sample. Below is a sample of what they looked like
as they progressed down in the dilution process.
Results
The results of our CFU's counting are listed below. The
charts show how many CFU's were counted at each dilution stage.
|
LB CFU’s
|
||
|
Dilutions
|
||
|
10-1
|
10-2
|
10-3
|
|
83
|
37
|
4
|
|
TMC
|
85
|
1
|
|
138
|
98
|
21
|
|
R2A CFU’s
|
||
|
Dilutions
|
||
|
10-1
|
10-2
|
10-3
|
|
56
|
9
|
1
|
|
80
|
11
|
16
|
|
85
|
26
|
6
|
Analysis
Of
course when picking a media, you have to pay attention to environmental and physiological
factors such as how dirty the water is and where you get it from. You have to
also keep in mind on what you aim to find in the sample that you wish to find.
Each broth has a better result for certain types of bacteria. LB for the faster
growers and R2A for the more diverse results. However, in our data above we seemed
to get the best results using the LB agar due to the fungi that takes over the
R2A agar. However, each plate of the 3 samples are different per dilution, so
it was beneficial to make 3 of each because we may not have had any decent data
to show otherwise.
Now
we had only plated 1 mL of our diluted sample per plate because if we had
plated any more than that then we would have flooded the agar and it would have
just not absorbed into the agar thus rendering our data, if not enough then we wouldn’t
be able to spread over the entire plate. After we incubated the plates upside
down to prevent condensation from tampering with our results.
The
streak plate technique normally yields higher counts due to the type of
bacteria that thrive in freshwater lakes and other surface waters. The pour
plate technique could potentially wipe out some aerobic bacteria and kill some
heat sensitive bacteria due to the temperature of the agar when its poured as
well as the anaerobic conditions that some bacteria like.
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