Katelyn Algas

BIO 210-101 Lab

February 12, 2014
Wednesday, 8:30am-11:30am
Lab 3: Microbial Growth
Introduction
The goal of examining the growth of microorganisms at different temperatures was to see
the minimum, maximum, and optimum growth temperatures of microorganisms. Growth was
directly dependent on how temperature influenced cellular enzymes. Enzyme activity increased
until the denaturation of the protein structure of the enzymes occurred, which stopped the
growth. Enzyme inactivation occurred as the temperatures decreased toward the freezing point.
The microorganisms observed had various temperature requirements.
The growth of various microorganisms was observed on differential and selective media.
Selective media allowed certain organisms to grow while other organisms were not able to.
Differential media did not prevent the growth of organisms, but it did cause specific colonies to
grow differently from other organisms that were present. By having microorganisms grow on
differential, selective, or differential and selective media, it helped isolate the properties of the
different microorganisms and identify the microorganisms, such as what selective agents in the
media affected the microorganisms or whether the microorganisms were Gram-positive or Gramnegative.
An oxidase test was performed on different microorganisms to see whether they had the
enzyme cytochrome c, which was an enzyme that assisted in cellular respiration. A catalase test
was performed on microorganisms to see if they had the enzyme catalase, which was made by all
actively growing aerobic microbes. These tests showed the properties of different
microorganisms. For example, the oxidase test can differentiate between oxidase-negative gram-

negative enteric bacterial rods and bacterial rods that belonged to the genera Pseudomonas and
Aeromonas. The catalase test can distinguish between bacteria with similar morphological
characteristics but different metabolic activities.
Materials and Methods
Escherichia coli, Bacillus stearothermophilus, Pseudomonas fluorescens, and Serratia
marcescens were streaked onto four Trypticase soy agar plates that were separated into quadrants
to see the effects of temperature on growth. Each plate was incubated at either 4, 20, 37, or 60C.
Saccharomyces cerevisiae was also streaked onto four soy agar plates, and each plate was
incubated at either 4, 20, 37, or 60C. All of these plates were observed the next day for growth.
Various cultures were streaked in pairs onto five types of media to observe their growth on
different types of media. The pairs were Escherichia coli and Staphylococcus aureus,
Escherichia coli and Enterobacter aerogenes, and Staphylococcus aureus and Enterococcus
faecalis. Each culture was streaked in a pitchfork pattern onto the five types of media, which
were blood agar, MacConkey agar, mannitol-salt agar, Hektoen enteric agar, and Trypticase soy
agar. All of the plates were incubated at 37C and were observed the next day for growth.
The oxidase test was performed on Escherichia coli and Pseudomonas fluorescens. A small
amount of each culture was spread on its own paper square, and one drop of the oxidase reagent
was placed on top of each culture. If there were a color change, this meant that there was
oxidase, cytochrome c was present, and aerobic respiration occurred. The catalase test was
performed on Escherichia coli. A small amount of the culture was spread on a microscope slide,
and one drop of the catalase reagent was placed on top of the culture. If there were bubbles
present, this meant that there was catalase present.

Results
Table 1: Effects of Temperature on Growth of Microorganisms
Microorganisms

Temperature
4C
25C
37C
60C

E. coli

B. stearothermophilus

P. fluorescens

S. marcescens

No growth
Little
growth
A lot of
growth
No growth

No growth
Little growth

No growth
Growth

No growth
Growth

Little growth one

colony
Growth

Growth

Growth one
colony
No growth

No growth

S.
cerevisiae
No growth
Growth
Growth
No growth

Table 2: Growth on Selective and Differential Media

Medium Type
Bacterial Species

Differential
Blood Agar

S. aureus

Growth
Beta
hemolysis
Clear
zones
around
colonies
Growth
Gamma
hemolysis
No zones
around
colonies
Growth
Beta
hemolysis
Clear
zones
around
colonies
Growth
Gamma
hemolysis

E. faecalis

E. coli

E. aerogenes

Differential and Selective

MacConkey Mannitol-Salt
Hektoen
Agar
Agar
Enteric Agar
No growth
Growth
No growth
Mannitol
fermenter
Yellow
zones

Trypticase Soy
Agar
Growth

Partial growth
Lactose
fermenter
Pink

Growth
Mannitol
fermenter
Yellow
zones

No growth

Growth

Growth
Lactose
fermenter
Pink

No growth

Growth
Lactose
fermenter
Salmonpink
colonies

Growth

Growth
Lactose
fermenter

No growth

Growth
Lactose
fermenter

Growth

No zones
around
colonies

Pink

Orange
colonies

Table 3: Oxidase and Catalase Test Reactions

Bacterial Species
E. coli
P. fluorescens

Oxidase Result
Oxidase negative cytochrome c
absent
Oxidase positive cytochrome c
present cellular respiration

Catalase Result
Catalase positive catalase present

Discussion
In the temperature experiments, all the bacteria exhibited at least some growth at 25C, but
Pseudomonas fluorescens, Serratia marcescens, and Saccharomyces cerevisiae grew the best. At
37C, Escherichia coli exhibited a great amount of growth, and Pseudomonas fluorescens and
Saccharomyces cerevisiae grew at that temperature as well. If the temperature were above the
maximum temperature of cellular enzymes, there would be no microbial growth because most
cell enzymes would be destroyed. There would be microbial growth if the temperature were
below the maximum temperature of cellular enzymes, which would depend on the
microorganism. If the temperature were above the minimum temperature of cellular enzymes,
there would be microbial growth, and if the temperature were below the minimum temperature,
then there would be no microbial growth. Cells would be inactive metabolically. A psychrophilic
microbe, which grows between -5C to 20C, would most likely not be pathogenic in warmblooded animals because typical body temperature is 37C, and psychrophilic microbes grow
best below that temperature, specifically -5C to 20C.
Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Enterobacter
aerogenes all grew on the blood agar. Although each bacterium broke down the hemoglobin in
the blood agar differently, all of the bacteria had hemolysins, which were extracellular enzymes

responsible for breaking down the hemoglobin. On the MacConkey agar, Staphylococcus aureus
did not exhibit any growth because this bacterium was a Gram-positive coccus, which was
inhibited by the selective agents in the MacConkey agar, which were bile salts and crystal violet.
Enterococcus faecalis, Escherichia coli, and Enterobacter aerogenes grew on the MacConkey
agar because they were Gram-negative rods and caused lactose fermentation. A change in pH and
lactose fermentation caused the medium to change color. Staphylococcus aureus and
Enterococcus faecalis grew on the mannitol-salt agar because the use of the agar was to isolate
staphylococci, as well as a few kinds of enterococcus bacteria. They were both able to survive
the salty conditions. Mannitol was also fermented, which caused the pH to change, as well as the
color of the medium. Escherichia coli and Enterobacter aerogenes did not grow on the mannitolsalt agar because they were Gram-negative, and the high salt concentration prevented them from
growing. Staphylococcus aureus and Enterococcus faecalis did not grow on the Hektoen enteric
agar because they were Gram-positive. The bile salts and the dyes brothmthymol blue and acid
fuchsin inhibited the growth of these two bacteria. Escherichia coli and Enterobacter aerogenes
grew on the Hektoen enteric agar because they were Gram-negative and they fermented lactose,
which changed the color of the colonies to salmon-pink or orange. Staphylococcus aureus,
Enterococcus faecalis, Escherichia coli, and Enterobacter aerogenes all grew on the Trypticase
soy agar because the soy agar is neither a differential nor a selective medium. Anything can grow
on it.
Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Enterobacter
aerogenes exhibited hemolysis. Staphylococcus aureus and Escherichia coli showed beta
hemolysis, and Enterococcus faecalis and Enterobacter aerogenes showed gamma hemolysis.
These results showed that the bacteria that exhibited beta hemolysis completely destroyed red

blood cells in the agar, and the bacteria that exhibited gamma hemolysis did not destroy any red
blood cells in the agar. Out of all the bacteria that were streaked on blood agar, Staphylococcus
aureus and Escherichia coli would most likely be pathogens because they would completely
break down red blood cells and infect the host. Enterococcus faecalis and Enterobacter
aerogenes, which showed gamma hemolysis, would not be pathogens because hemolysis would
not even occur.
Escherichia coli showed a negative result in the oxidase test. This showed that the enzyme
cytochrome c was absent, which meant that respiration did not occur in this bacterium or a
different method was used for respiration. Pseudomonas fluorescens showed a positive result in
the oxidase test, which showed that cytochrome c was present and respiration via an electron
transport chain did occur in this bacterium. Escherichia coli showed a positive result in the
catalase test, which showed that the bacterium did go through respiration but by a different
method.
From the information given, this new pathogen was Gram-negative. Because the bacterium
grew in the human gastrointestinal tract, it would grow best at body temperature, or 37C. Out of
the blood agar, MacConkey agar, mannitol-salt agar, Hektoen enteric agar, and Trypticase soy
agar, the bacterium would grow on all the media but the mannitol-salt agar, which only
staphylococci and certain enterococcus bacteria grow on there. The other media would be fair
game because each different type of medium would be incubated at body temperature. The
Hektoen enteric agar would be specifically used because it is used for fecal specimens. From
there, quick lactose-fermenting organisms can be found, which sounded like this pathogen. If the
oxidase test were done, the results would be positive because this bacterium sounded like it
moved and infected quickly, which would show that cellular respiration occurred. The catalase

test would be positive as well, since it was predicted that cellular respiration occurred. Overall,
this pathogen would need to be stopped quickly.