Berlinda Juste

MCB 3020L
Section 4
Lab Experiment 5
Isolation of Bacteria: Viable Titer and Pure Culture
Purpose
The purpose of this experiment is to isolate pure cultures and obtain viable titers in three lab
sessions. First, we serially diluted a soil sample. Then, the sub-samples were spread plated on the
agar surface by a sterile glass rod through aseptic transfer. Two different types of agar were used
in spread plating: M-9 agar, chemically defined ingredients and amounts in agar are known and
Nutrient agar, ingredients and amounts in agar are unknown. We incubate agars at 37 degree
Celsius. In lab session two, the Colony Forming Units were counted beginning with the highest
dilution to determine the viable titers on each plate. The colonies that were over 200 colonies
were discounted. The colonies in our M-9 agar and Nutrient agar with the sample of 10^-4
dilution as well as 10^-6 dilution were used as viable titers. We took the average of both viable
titers from each agar. Three different colonies from each viable M-9 agar and Nutrient agar used
in Gram staining. Next, we selected a yellow colored colony from a Nutrient agar with 10^-4
dilution that seem to be pure. We use the streak plate technique on a Nutrient agar and a M-9
agar plate. We isolated a pure colony to streak in our agar slant.
Part 1
Countable Colonies Per Dilution in Two Different Media
Dilution M-9 Agar Nutrient Agar
10^-2 TNTC TNTC
10^-3 TNTC TNTC
10^-4 27 48
10^-5 4 12
10^-6 55 26

Viable titer for M-9 Agar: 55.27 10^4 CFU per microliter
Viable titer for Nutrient Agar: 26.48 10^4 CFU per microliter

Part 2: Two Different Media

Nutrient Agar
 Figure 1: At 100X magnification in oil immersion, this
sub-sample of a white streaked colony in 10^-4 dilution
from a Nutrient Agar plate shows a Gram-negative
bacterium and a Gram-positive bacterium of two
Unknown species overlapping each other after Gram
staining. Both bacteria are shaped in long rods and
chains. These two species of bacteria may be competing
or coexisting with each other for the same resources in
the original biofilm.

Figure 2: At 100X magnification in oil

immersion, this sub-sample of a yellow streaked
colony in 10^-4 dilution from Nutrient Agar
shows a Gram-negative bacterium of Unknown
species in long rods and chains after Gram-staining. This bacterium can be aerobic or facultative
anaerobic depending on whether the soil sample was taken on the topsoil or deep in the soil.
Figure 3: At 100X magnification in oil immersion, this sub-sample of a colony in 10^-6 dilution
from Nutrient Agar shows Gram-positive bacteria of Unknown species in long rods and chains.
They appear purple in color. There are some clear areas, almost light below the magnification in
which the bacteria resist Gram-staining. PHB (poly-beta hydroxybutyrate: bacterial fat),
glycogen and other bacterial cell inclusions can be depicted as those clear areas. It can indicate
the presence of endospores in The Spore Stain procedure and the presence of a waxy wall in the
Acid-Fast Stain procedure (indicator for Gram-positive bacterium). Figure 3 illustrates the ability
of the bacteria to survive in unfavorable conditions, ex. lack of the right carbon source (inorganic
or organic), energy source (light or chemicals), temperature or pH optimum growth, habitat, etc.
M-9 Agar

Figure 4: At 100X magnification in oil immersion,

this sub-sample of a colony in 10^-6 dilution from
M-9 Agar shows Gram-positive bacteria in small
round clusters and Gram-negative bacteria in long
rods and chains of two Unknown species. The Gram-positive bacteria is shown in limited
amounts, perhaps, the defined ingredients for growth of this bacteria in the M-9 Agar is limited.
 Figure 5: At 100X magnification in oil immersion,
this sub-sample of a colony in 10^-4 dilution from
M-9 Agar consist mainly of Gram-negative bacteria
of Unknown species crowding the magnification.
They are composed of long rods and arranged in
chains. The M-9 Agar has the defined ingredients
and amounts for this bacterium to survive.

Figure 6: At 100X magnification in oil

immersion, this sub-sample of a colony in 10^-6
dilution from M-9 Agar shows Gram-negative bacteria of Unknown species. They are in long
rods and arranged in chains. They appear dark red in color.
Part 2
We selected a yellow colored colony from the Nutrient agar in 10^-4 dilution from Figure 2 and
streak it on two agar plates, the Nutrient agar and the M-9 Agar.
Part 3
Our results showed that the Nutrient agar on the re-streak contain yellow colored colonies and
after Gram-staining, were identical to the colony in Figure 2, the same Gram-negative bacteria
with the same long rods and chains. In contrast, the M-9 Agar had colonies that were of white
color, far different than the colonies of the Nutrient Agar. We can conclude that the M-9 Agar
cannot support the growth of the yellow colored colonies and can also infer that the carbon
sources found in the Nutrient agar allowed for the yellow colored colonies to grow. The colony
from the M-9 agar is depicted as Gram-negative cocci and in grape-like clusters. The pictures
below illustrate this:
M-9 Agar and Nutrient Agar of the yellow colored colonies
The M-9 agar did not produce any yellow colored colonies because it does not have the
ingredients to grow this specific Gram-negative bacterium.

Under the Microscope

Gram-stain from Nutrient Agar
Identical to Figure 2:

Gram-stain from M-9 Agar

Conclusion
I expected more colony forming units in the Nutrient agar with a dilution of 10^-6 and a viable
titer greater than the M-9 agar. The spread plating technique was not done properly: the loop was
too hot from sterilization and the agar was not solidified, which in turn, made for a low viable
titer. We were unsuccessful in obtaining a reasonable colony count for the dilution of 10^-6 in
Nutrient agar. Nutrient agar supports growth for a wide range of bacteria and M-9 agar only
supports bacteria that use certain known chemical ingredients. Therefore, the number of colony
forming units in the Nutrient agar in each dilution is predicted to be higher than that of the M-9
agar. To conclude, we isolated a pure culture and the colonies on the re-streak are identical to
Figure 2 as well as its identical morphology: yellow in color, Gram-negative bacteria, red in
color, arranged in chains and in long rods.
Questions
1. State a good reason for cooling the melted agar to 45C before pouring into petri plates.
Agar acts as a solidification agent for most microorganisms to grow on. Therefore, if it is not
cooled down, the bacteria will become denatured due to heat.
2. Are all the bacteria in soil counted by this procedure? Is this an overestimate or underestimate
of the actual number of viable bacteria in soil?
Not all bacteria in soil are counted by this procedure. It is an underestimate. The ingredients of
the M-9 agar and the Nutrient agar plate, whether known or unknown, limit the growth of all
bacteria found in soil.
3. What are possible reasons that the CFU underestimates the number of bacteria in soil?
A colony could have arisen from two or more cells stuck together
A mixture of two different bacteria, Gram-negative and Gram-positive bacteria in one
colony (impure colony)
A mixture of different strains of one bacteria in one colony
Manual miscalculations when counting the number of colonies
4. What is pure culture? How do you prove a culture is pure?
A pure culture is defined as the progeny from one cell, in other words, that one cell of a single
species or strain gave rise to a colony. All the colonies on the re-streak should be identical, and
Gram-staining these to demonstrate all the cells in the resulting colonies are identical and the
same as those on the original plate.
5. Why is it important to obtain pure culture?
It is important to obtain pure culture to identify the genus of one bacterial species, its phenotype,
morphology (shape, size, color, etc.), physiological functions, energy sources and carbon sources
used in growing this bacterium. Basically, we want to identify one bacterium and its properties.