Angelina Raimonde

Microbiology 210
Lab Report #1
October 8, 2014
Background Information
Serratia marcescens is and aerobic, motile, Gram negative, bacillus shaped microorganism with
a red pigment. It is apart of the Enterobacteriaceae family, meaning that it is chiefly found
within the gastrointestinal tract. Unlike other organisms in that family, S. marcescens is able to
produce extracellular deoxyribonuclease, gelatinase, and lipase, which are all resistant to the
antibiotics: colistin and cephalothin. Its red pigmentation is what made it become a great
infection marker within the biological world. S. marcescens has recently become a popular
hospitalized pathogen that causes nosocomial respiratory infections, blood stream infections,
skin and soft tissue infections, and urinary tract infections (http://www.antimicrobe.org/b26.asp).
S. marcescens is seen red at a temperature of 25C and seen white at a temperature of 37C
(http://scienceline.ucsb.edu/getkey.php?key=434).

Micrococcus luteus is an aerobic, Gram-positive, cocci-shaped microorganism with a yellow

pigment. This bacterium is classified as an obligate halophile and performs optimum growth at
37C. M. luteus is most likely to be found on human skin, in the soil, in water, and in dust. This
organism tends to be harmful to humans, howerver, it can be harmful to humans with sensitive
immune systems, like patients with HIV. If HIV positive patients do contract this bacteria, it
will cause sever skin infections that result in pruritic eruptions of the skin
(https://microbewiki.kenyon.edu/index.php/Micrococcus).

Bacillus stearothermphilus is an aerobic, motile, Gram-positive, bacillus-shaped, spore-forming

microorganism. The temperature at which it grows optimally is 55C, but it can grow within the
30-75C ranges. It can be found in sand, ocean, vents, and hot spring environments. Fortunately,
this microbe has not yet been found to be pathogenic. But it is very popular in verifying
sterilization in the food industry. It also is very important in the dairy industry, with its main
function as being able to break down lactose
(https://microbewiki.kenyon.edu/index.php/Bacillus_stearothermophilus).

Pseudomonas fluorescens is an aerobic, Gram-negative, bacillus-shaped, bacteria that lives on

soil, water, and plant surfaces. It optimally grows at 25-30C. It produces a green fluorescent
pigment when iron concentration levels are low. P. fluorescens are nonpathogenic microbes. In
fact, they produce metabolites that may be used as antibiotics for soil-borne plant pathogens. It
also produces another type of antibiotics; know as Mupirocin, which aids in the healing of many
skin, ear, and eye disorders. They are also unique to the fact that they are able to break down
pollutants (https://microbewiki.kenyon.edu/index.php/Pseudomonas_fluorescens).

Catalase tests are used to test if a microbe is an aerobe or anaerobe. Aerobes are microbes that
must grow in the presence of oxygen and produce the catalase enzyme. This catalase enzyme is
what is able to break down poisonous forms of oxygen, hydrogen peroxide. Anaerobes lack this
enzyme and thus cannot grow in the presence of oxygen at all. During the procedure of a
catalase test, a small drop of hydrogen peroxide is added to an aerobe and anaerobe. The
presence of catalase is then shown by the production of bubbles, which signifies the release of
oxygen (BIO 210 Lab Handout, 2014).

There are two types of media that are used for growing microorganisms, selective and
differential. Selective medium is a medium that selects for bacterial growth with certain
qualities. For example, MacConkey Agar is a selective medium for the growth of Gram-negative
enteric bacteria. MacConkey Agar is able to do this by its make up of crystal violet, sodium
chloride, and bile salts. The crystal violet is the key component for determining if the bacteria is
Gram-negative or Gram positive. Differential medium is a medium that is able to distinguish
whether or not bacteria can perform fermentation or hemolytic activities. An example is Blood
Agar. BA is contains sheeps blood, which is used as an indicator of hemolytic activity. A clear
zone around the growth of the culture signifies that a complete break down of blood cells
occurred, which is known as beta-hemolysis. Alpha-hemolysis is the partial break down of
blood cells, and this cause a green zone to appear around the culture (BIO 210 Lab Handout,
2014).

Methods
Investigating the effect of temperature on the growth of different species of bacteria
Each lab bench should inoculate 5 Nutrient Agar slants of S.marcescens, M. luteus, and B.
stearothermophilus. The inoculation should be performed as followed. The Bunsen burner was
set up by plugging the tube into the gas nozzle, turning the gas nozzle on, and then using a striker
to strike a spark and light the fire. Next, flame inoculation loop until the loop and the neck of the
loop are fiery red to insure sterilization. Then, uncap the test tube of S. marcescens and flame
the lip of the test tube. Cool the inoculation loop on the inside of the test tube. Place the
inoculation loop into the S. marcescens and swirl the loop around, in order to obtain as much
bacteria as possible. Remove the inoculation loop, flame the lip of the test tube, and place the

cap back onto the test tube. Next, obtain a test tube containing Nutrient Agar. Uncap the test
tube, flame the lip of the test tube, and cool the inoculation loop on the inside of the test tube.
Then, gently rub the tip of the inoculation loop onto the nutrient agar, in a back and forth
movement. Once the inoculation loop has been removed, flame the lip of the test tube; place the
cap back onto the test tube. And place test tube into the correct test tube rack that is labeled at
the temperature for which the test tubes should be incubated at. Sterilize the loop by flaming the
inoculation loop until it is fiery red in color.

For the 4 remaining slants of S. marcescens and the remaining 5 slants of M. luteus and B.
stearothermophilus, the previous steps were repeated. Once all the slants have been inoculated,
test tube racks should be placed at the following temperatures: 4C, 25C, 30C, 37C, and 60C,
for 24-48 hours.

For the next part of this section, each lab bench should have inoculated 5 plates of Pseudomonas
agar with Pseudomonas fluorescens. The steps shown previously about obtaining the culture of
S. marcescens should have, again, been carried out to obtain a culture of P. fluorescens. Then, a
plate containing Pseudomonas agar was obtained. The plate was uncapped and streaked with the
inoculation loop filled with P. fluorescens. The cap was returned onto the plate and the streaked
plate was set aside to dry for 5 minutes before it was inverted. The inoculation loop was then
sterilized by placing the loop into the flame of the Bunsen burner until it was fiery red in color.
These steps were repeated for the remaining 4 Pseudomonas agar plates.

Once the streaks were all dry, the plates were flipped upside down and placed at each of the
following temperatures: 4C, 25C, 30C, 37C, and 60C for 24-48 hours. The following week,
results were recorded based on degree of culture growth and degree of pigment shown under a
UV light. The symbols that were used are +, ++, +++, and ++++ to indicate growth and
- for no growth.

Investigating the use of temperature to inhibit growth of microorganisms

Each lab bench should have divided the work evenly among the students. Two petri dishes were
labeled: 0 min, two of the plates 5min, and two of the plates 10min. The plates were also
labeled with the name of the bacterial culture, the date, and the initials of the people at the lab
bench.

Three Nutrient agar test tubes were removed from a 70C water bath and brought to the lab
bench. The 1 ml pipette added 0.1ml of 48 hour nutrient broth culture of E. coli to each of the 3
Nutrient agar test tubes. Two of the Nutrient agar test tubes were returned to the 70C water
bath. These test tubes were then timed: one at 5 minutes and the other at 10 minutes. The third
test tube was rotated to mix the contents and then immediately poured into the empty petri plates.
The dishes were set aside to cool and solidify for 15 minutes. Once the test tubes in the water
bath reached their desired incubation times, the Nutrient agar test tubes were removed and
rotated to mix the contents. And then the mixture was immediately poured into the remaining,
designated petri plates. The plates were also set aside to cool and solidify for 15 minutes.

The previous steps were again followed but with the culture of B. stearothermophilus. Once all
the plates have solidified, they were inverted, and placed in the incubator for 24-48 hours at
37C. The following week, the results were recorded based on the degree of cultural growth
present on the plates. The symbols that were used are +, ++, +++, and ++++ to indicate
growth and - for no growth.

Investigating the effect of oxygen on bacterial growth

For the Catalase Test the following method was carried out. The aseptic inoculation that was
described in the previous section occurred in this section as well. Once the culture of S.
marcescens was obtained, it was then spread onto a clean microscope slide. A small drop of
hydrogen peroxide was added onto the slide. From this reaction, the presence or absence of
catalase was determined. A positive reaction was illustrated by a presence of bubbles. And a
negative reaction was illustrated by the absence of bubbles.

The previous steps were repeated for the cultures of M. luteus, B. stearothermophilus, P.
fluorescens, and Enterococcus fecalis. Results were then recorded by using + for a positive
result and - for a negative result. After examination of the slides, they were discarded into jars
filled with disinfectant.

For the Oxidase Test, the following method was carried out. The aseptic inoculation technique
was used again. Once the culture of S. marcescens was obtained, it was spread onto a piece of
filter paper, via inoculation loop. A small drop of oxidase reagent was added to the culture on
the filter paper to determine the presence or absence of oxidase. The filter paper was allowed to

sit for 1 minute before it was examined. A positive reaction appeared purple/dark in color, while
a negative reaction appeared as no color change.

The previous explained steps were repeated for the cultures of M. luteus, B. stearothermophilus,
P. fluorescens, and Enterococcus fecalis. Results were recorded as a + for a positive results
and a - for a negative result. After examination, the used slides were then discarded into the
jars filled with disinfectant.

Microbiological media and their uses

Each lab bench was assigned one of the following pairs of cultures:
a. Escherichia coli and Staphylococcus edpidermidis
b. Enterococcus fecalis and Enterobacter aerogenes
c. Proteus mirabilis and Staphylococcus aureus
d. Proteus mirabilis and Streptococccus lactis
e. Enterobacter aerogenes and Staphylococcus aureus
f. Escherichia coli and Streptococcus lactis
A marker was used to divide each of the 6 agar plates (Nutrient agar, MacConkey agar, Mannitol
Salt agar, Blood agar, Eosin-Methylene blue agar, and Hektoen agar) into half sections. The
aseptic inoculation technique was again carried out to streak the assigned cultures onto all of the
halved agar plates and then repeated to streak the cultures onto the remaining halves of agar
plates. The streaks were allowed to dry for 5 minutes, and then they were inverted and stacked
on top of each other. Once the stack of agar plates are complete, the stack was taped together, so
that all the plates stayed together, and then labeled with the half lab benches name on it. The

taped stack was then placed into the incubator at 37C for 24 hours. The following week results
were obtained in a table based on presence + or absence - of growth, type of hemolysis
present, color of the colonies, and color of the medium around the edge of the colonies.

Results
Investigating the effect of temperature on the growth of different species of bacteria graph
B.
Temperature

S. marcescens

M. luteus

P. fluorescens
stearothermophilus

4C growth?

4C pigment?

Clear, no glow

25C growth?

+
Glows under UV

25C pigment?
Slightly red

Yellow

No color

light, creamy
yellow

30C growth?

+
Glows under UV

30C pigment?
Slightly red

Yellow

No color

light, creamy
yellow

37C growth?

++
Glow, creamy

37C pigment?
Bright red

Yellow

No color
yellow

60C growth?

60C pigment?

No color

No color

None

Optimum
Optimum Growth

temperature range

temperature range

for pigment

Bacterium

Classification?

production
S. marcescens

4-37C

30-37C

Mesophile

M. luteus

25-37C

25-37C

Mesophile

60C

60C

Thermophile

25-37C

25-37C

Mesophile

B.
stearothermophilus
P. fluorescens

Investigating the use of temperature to inhibit growth of microorganisms graph

Bacterium

0 min

5 min

10 min

E. coli

Slight growth

No growth

B.
stearothermophilus

The Catalase Test graph

Bacterium

Detection of Catalase enzyme

S. marcescens

M. luteus

B. stearothermophilus

P. fluorescens

E. fecalis

S. marcescens, M. luteus, B. stearothermophilus, and P. fluorescens all exhibited positive

results for the catalase test. This means that when the hydrogen peroxide was added to
the bacterium, bubbles appeared, signifying the presence of the catalase enzyme within
those bacterium.

The Oxidase Test graph

Bacterium

Detection of Oxidase enzyme

S. marcescens

M. luteus

B. stearothermophilus

P. fluorescens

E. fecalis

M. luteus, B. stearothermophilus, P. fluorescens, and E. fecalis all exhibited positive

results for the oxidase test. This means that when the oxidase reagent was added, it
turned the sample a dark/purple color and that signified the presence of oxidase enzyme.

Microbiological media and their uses graph

Bacterium

NA

MAC

MSA

BA

EMB

Gamma-

Metallic

hemolytic

green

HA
+

+
-

E. coli

Bright Purple

Orange

No growth
colonie

colonies on
orange media

S. aureus

White

colonies

Beta-

surrounded by

hemolytic

yellow media
+
+

S.

White
+

Gammacolonies on

epidermidis

hemoyltic
red media
+
Slight growth,

E. fecalis

Gammared media
hemolytic

P. mirabilis

Pink/Purple

No

Gamma-

colonies

fermentation

hemolytic

+
White/clear
colonies

Green
colonies with
black centers

E.

+
Red growth

Gamma-

White/clear

hemolytic

colonies

Pink/red

aerogenes

+
+

Orange

media, no
fermentation

colonies on
orange media

Discussion
Questions 1-3 on page 5 of lab handout
1. E. coli was the bacterium that was not able to survive exposure to a temperature of 70C
because 70C is higher than its optimum growth temperature which is 37C (BIO 210
Lecture Notes, 2014).
2. The structures that were produced by other bacterium that allowed it to survive exposure
to a temperature of 70C are endospores. Endospores are very resistant to changes in the
environment, such as, heat, radiation, and dehydration (BIO 210 Lab Handout, 2014).
The stain used to detect endospores is called the Endospore stain, which involves
malachite green stain, extreme heat, and safranin stain.
3. Based on the findings from the graph above, it was predicted that Clostridium spp. would
be more resistant to heat than E. coli because of the presence of endospores. E.coli has
an optimum growth temperature of 37C and can be classified as a mesophile (BIO 210
Lecture Notes, 2014). Whereas Clostridium has an optimum growth range of 43-47C,
which lies on the border between the mesophile and thermophile range. However,
though high temperatures can kill the Clostridium cells, the spores can thrive and produce
toxins at temperatures ranging between 21-48C
(http://www.foodsafety.unl.edu/pathogens/perfringens.html).
Questions 1-2 on page 8 of lab handout
1. The controls did give expected results because S. marcescens tested negative for oxidase
and P. fluorescens tested positive for oxidase. This allowed a definite showing of a
positive and negative result for which other results could be compared to
(http://www.yourdictionary.com/control-group).

2. The bacterium that was tested that can be classified as an enteric organism is S.
marcescens because it was characterized as Gram-negative, bacillus-shaped, oxidasenegative, and catalase positive (BIO 210 Lab Handout, 2014).

Questions 3-7 on page 2 of lab report 1

3. The bacteria that gave positive results for both the oxidase and catalase tests were M.
luteus, B. stearothermophilus, and P. fluorescens. It was concluded that these bacteria
can both metabolize poisonous oxygen forms, like hydrogen peroxide, and metabolize
normal oxygen forms. In fact, the presence of cytochrome c oxidase means that the
bacterium is an aerobe and uses oxygen as the final electron acceptor during respiration
(http://learn.chm.msu.edu/vibl/content/oxidase.html).
4. Selective and differential media that were used in the lab and their uses are as followed:
Blood agar is a differential medium that contains sheeps red blood cells. These red
blood cells allow this medium to show different types of hemolysis: alpha-hemolysis,
beta-hemolysis, and gamma-hemolysis. Alpha-hemolysis is characterized by a greenzone surrounding bacterial colonies on the blood agar. The green-zone signifies the
partial-hemolysis of red blood cells by that bacterium. Beta-hemolysis is characterized
by a clear-zone surround bacterial colonies. The clear-zone signifies the complete
hemolysis of red blood cells by that bacterium. And gamma-hemolysis is characterized
by no hemolysis present. Blood agar is known as differential media because of its ability
to differentiate between different hemolytic actions among different bacterial cultures
(BIO 210 Lab Handout, 2014).

MacConkey agar is an example of a selective medium because of its selection for Gramnegative, enteric bacteria. It is able to select for Gram-negative bacteria through its
containment of crystal violet stain, sodium chloride, and bile salts. These reagents select
for growth of Gram-negative bacteria and inhibit growth of Gram-positive bacteria.
However, MacConkey agar can also be classified as a differential medium because of its
containment of lactose, which allows the process of fermentation to be seen on the agar.
If fermentation does occur, the colonies will turn purple in color due to the presence of a
pH indicator within the medium. Contrastingly, if fermentation does not occur, then the
colonies will appear cream-white in color (BIO 210 Lab Handout, 2014).
Mannitol Salt agar is an example of a selective medium. This is because this medium
selects for the growth of staphylococci species only. Staphylococci bacteria are Grampositive and are able to grow in halophillic environments, while Gram-negative
organisms cannot. Because of the presence of mannitol, which is a carbohydrate, this
medium can also be considered differential because of the mannitol having the ability to
ferment. When fermentation occurs on this medium, growth of colonies are yellow in
color and the mannitol salt medium, itself, appears yellow. The medium turns yellow
because of the pH indictor that is involved in the medium, and when fermentation occurs,
the pH indicator turns the medium yellow because of the production of acid (BIO 210
Lab Handout, 2014).
Eosin-methylene blue agar is another example of a selective medium that is also
differential. This type of agar contains eosin and methylene blue, which are two reagents
that inhibit the growth of Gram-positive bacteria, and selects for the growth of Gramnegative, lactose fermenting bacteria. When lactose is fermented, the growths of the

colonies turn a dark blue/purple color or a metallic green color. Contrastingly, when
lactose is not fermented, the colonies appear cream-white or colorless (BIO 210 Lab
Handout, 2014).
Hektoen agar is also a selective and differential medium. This type of medium selects for
the growth of Gram-negative bacteria and differentiates between bacteria that can
ferment lactose and salicin and produce hydrogen sulfide. The fermentation of lactose
results in colonies with an orange/pink color, while nonfermenters of lactose appear
green/colorless. Salicin fermenters will have pink-zones around the colonies, while
nonfermenters of salicin will show no change in color. Hydrogen-sulfide producers will
produce colonies with black centers (BIO 210 Lab Handout, 2014).

5. In order to differentiate between S. aureus and P. mirabilis that were growing in nutrient
broth, the streak plate method on Nutrient agar, MacConkey agar, Mannitol Salt agar,
Blood agar, Eosin-methyelen agar, and Hektoen agar, would helpful. This would be
because P. mirabilis showed up as Gram-negative on MacConkey agar, while S. aureus
showed up Gram-positive. On Mannitol Salt agar, S. aureus showed white colonies on
yellow medium, which signified the presence of lactose fermentation, while P. mirabilis
showed growth, but no sign of lactose fermentation. On Blood agar, S. aureus had betahemolytic results, while P. mirabilis had gamma-hemolytic results. The Eosin-methylene
blue agar promoted for the growth of P. mirabilis and inhibition of growth for S. aureus.
Lastly, Hektoen agar showed no growth for S. aureus and showed green growth with
black centers for P. mirabilis, signifying the fermentation of lactose and the production of
hydrogen sulfide. The streak plate method would be a great technique, which was

introduced in lab one, to use to distinguish between the colonies of these bacteria. To
obtain pure cultures of these bacteria, Nutrient agar would be the most promising form of
medium because of its quality of being nonselective and allowing many different types of
bacteria to grow. Making 3-4 streaks of each bacterium on the nutrient agar would purify
the cultures. Continuously sterilizing the inoculation loop and obtaining bacteria from
the previous streak, allows the colonies to get purer and restricts the introduction of other
bacteria to occur. After 24 hours of incubation, the streak plates would illustrate isolated
colonies of each bacterium and this would allow the experimenter to visualize the
morphology differences (BIO 210 Lab Handout, 2014).
6. The results for the growth and appearance of E. aerogenes on selective and differential
media could predict the Gram reaction for this organism to be Gram-negative because the
MacConkey agar, Eosin-methylene Blue agar, and Hektoen agar select for growth of
Gram-negative organisms and E. aerogenes was able to grow on them. Contrastingly, E.
aerogenes was unable to grow on Mannitol Salt agar, which promotes for the growth of
Gram-positive organisms and inhibits the growth of Gram-negative organisms (BIO 210
Lab Handout, 2014).
7.

If cultures of Streptococcus pyogenes and Streptococcus mutans were grown in a tube of

tryptic soy broth and then streaked aseptically on Blood agar, it would be quite easy to
differentiate between the two organisms. S. pyogenes is characterized as being betahemolytic, which is the complete break down of red blood cells. It also requires bloodaugmented media in order to grow (http://textbookofbacteriology.net/streptococcus.html).
In contrast, S. mutans is normally characterized as being alpha-hemolytic, partial
breakdown of red blood cells, or gamma-hemolytic, no break down of red blood cells

(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC414143/). Therefore, the blood agar

plate containing S. pyogenes would have a clear-zone surrounding the colonies, while S.
mutans would have growth on the Blood agar, but would have a normal red colored
medium surrounding the colonies.
Questions 1-3 on page 12 of lab handout
1. The bacteria that were found to be Gram-positive were: Enterococcus fecalis,
Staphylococcus epidermidis, and Staphylococcus aureus because of their ability to grow
on Mannitol Salt agar (BIO 210 Lab Handout, 2014).
2. The only bacterium that was a producer of hydrogen sulfide was P. mirabilis because of
its ability to grow on Hektoen agar. The growth on the Hektoen agar showed up green in
color with black centers (BIO 210 Lab Handout, 2014).
3. The three bacteria that are identified as enteric bacteria are E. coli, P. mirabilis, and E.
aerogenes because of their ability to grow on MacConkey agar. This medium
specifically selects for the growth of enteric, Gram-negative bacteria (BIO 210 Lab
Handout, 2014).

Works Citied
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BIO 210 Lecture Notes, 2014.
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http://www.foodsafety.unl.edu/pathogens/perfringens.html. Date accessed: October13, 2014.
http://learn.chm.msu.edu/vibl/content/oxidase.html. Date accessed: October 13, 2014.
https://microbewiki.kenyon.edu/index.php/Bacillus_stearothermophilus. Date accessed: October
8,2014. Date last updated: November 1, 2011.
https://microbewiki.kenyon.edu/index.php/Micrococcus. Date accessed: October 8, 2014. Date
last updated: August 6, 2010.
https://microbewiki.kenyon.edu/index.php/Pseudomonas_fluorescens. Date accessed: October 8,
2014. Date last updated: April 22, 2011.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC414143/. Date accessed: October 13, 2014.
http://scienceline.ucsb.edu/getkey.php?key=434. Date accessed: October 8, 2014.
http://textbookofbacteriology.net/streptococcus.html. Date accessed: October 13, 2014.
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