Bio 113 Unknown Lab Report
Andrew Thornton
Introduction
The Purpose of this assignment is intended to educate by accomplishing bacterial isolation and
identification to the genus and species level by using multiple selective and differential media types. (1)
The list of potential bacteria for said assignment is listed in Table 1.
Bacteria need be studied for a myriad of utilities beneficial to humankind. (2) Bacteria were first studied
for reasons related to production of yeasts to cause bread to rise, to ferment sugars to produce alcohols
in beer and wine, and to prevent infection in cuts and wounds. Ancient Egyptians used loaves of bread
that were allowed to become moldy to treat injuries. Additionally, understanding of microbiology has
allowed major advances in technology, genetics, and immunology. Before it was even known, a
mutualistic relationship existed between humans and bacteria in the digestion of unusable foodstuffs in
the gut. Even the study of pathogenic microbes can help understand controlling infection; Processes
such as pasteurization exist because of biotechnological study, and are of major importance in modern
medicine.
Bacteria are classified into groups that are gradually more selective and specific. Bacteria are classified,
in the science world, the same way that all organisms are classified. Starting with Domain, organisms
then proceed to small groups of kingdom, phylum, class, order, family, genus, and species. The term
“bacteria” is specific only to the Domain Bacteria, and as specific an organism many believe “bacteria” is,
an abundance of diversity exists within the term. Based on a multitude of characteristics that are too
numerous to list, bacteria are classified into said groups that gradually come down to a single type of
organism. For example, the type of sugar a bacteria is able to metabolize, or the structure of colony that
is produced may play a role into classification into more and more specific categories. A species of
bacteria is then referred to using binomial nomenclature. In this assignment, this classification plays an
integral role, and the types of media used selectively allow one to narrow a bacterial culture to one
species type.
However, to rely on media so heavily in the exercise, one must consider both major advantages and
disadvantages of utilizing said biomechanical tests. The major advantage is obviously convenience, in
that artificial media allows one to keep a culture of bacteria on hand in the laboratory setting. Media
also allows selectivity based on factors in the media that allows certain types of media to grow, while
inhibiting the growth of others. If one came across a culture of multiple bacteria, media would allow one
to easily isolate a sample of bacteria down to one species. However convenient, biotechnological media
simply is not a realistic environment, and may cause bacteria to react unreasonably, or become
stressed. When bacteria are studied too often in an artificial environment, it may become unpredictable
when it is occurred naturally.
Clinically, one’s ability to differentiate between bacteria (similar to this exercise) is of dire importance in
treatment. For example, if one were to use an antibiotic to treat a fungal ailment in a clinical setting, the
treatment would fail. A very simple example, but in essence, knowing what type of pathogen one is
working with in the clinical setting is of dire importance to how to treat it.
Materials and Methods
Table 2 shows a dichotomous key showing the process of how the bacteria were isolated. Specifically
however, minor changes were untaken to create the correct subculture within the experiment. First and
foremost, the original culture was highly contaminated. After a gram stain that appeared
overwhelmingly gram positive, the bacteria intended for identification were identified as gram negative.
To establish a less tainted culture, MacConkey’s agar was inoculated to get a purely gram negative
subculture to work with. All other tests were then inoculated from samples from this culture, or a
another culture put on a simple TSA media, subcultured from the MacConkey’s agar culture, to avoid
bacterial stress, and to provide plentiful nutrients. Every test was inoculated using a procedure that
eliminated possibility of outside contamination; Loops and needles were sterilized before inoculation,
and cultures were isolated from the outside to, again, avoid contamination.
The first test used was a gram stain. The bacteria are heat-fixed, and stained to allow differentiation
with two chemicals. (1) These two chemicals are crystal violet, which has an affinity to the cell walls of
Gram positive bacteria, and safranin, which has an affinity to the cell walls of Gram negative bacteria. In
between staining with crystal violet and safranin, iodine is applied, and the bacteria are “washed” with
an agent to decolorize, in this case alcohol was used. Alcohol is used again after safranin is applied.
Secondly, a Sulfide Motility Indole test is used to determine three separate qualities. First, the bacteria
are inoculated into the media by stabbing with a needle, and allowed to incubate for 24 hours. After
incubation, H2S production is determined by the iron in the medium. If the gas is produced, it reacts with
the iron to produce ferrous sulfide, and a black color. Motility is determined by stabbing the medium,
and the media showing deviance from the stab. If the bacteria are motile, they possess the ability to
move away from the stab, which is visible to the eye when viewing the media. Indole, however is slightly
more complex. Kovac’s reagent is added, to allow one to determine if Indole is present, showing a red
color. Kovac’s reagent, which contains acid, DMABA, and n-amyl alcohol, react with indole to produce a
red color.
Methyl Red- Voges-Proskauer test, or MR-VP, determines both whether the bacteria uses mixed acid
fermentation and if acetoin is produced. The bacteria are simply inoculated in a liquid broth and allowed
to incubate for 24 hours. The Methyl Red portion detects pH, and is related mixed acid fermentation. If
the nutrients in the media are metabolized, an acidic environment occurs, and methyl red, when added
will remain red. The Voges-Proskauer test utilizes KOH to indicate the presence of acetoin production by
the bacteria, by oxidizing it to create diacetyl. This produces a red color to indicate a specific type of
metabolism.
The Urea test simply tests whether a bacterial sample can metabolize urea. The test is a liquid broth,
and the bacteria is inoculated with a needle, and allowed to incubate for 24 hours. Phenol red acts as
the indicator in the test, and when in a basic environment like when urea is metabolized and ammonia is
produced, phenol red turns the media a bright pink.
The Catalase test is used to determine if the bacteria in question employs the enzyme catalase. Simply,
the bacteria are allowed to grow on a media, and peroxide is applied. A positive reaction results in
bubbling, and indicates catalase, if no bubbling occurs, the bacteria does not employ catalase.
Results
Initial Gram Stains indicated a small amount of Gram negative bacteria due to pink coloration of safranin
affinity. Bacteria were certainly circular as opposed to rod-shaped or spiral.
Pseudomonas aeruginosa
Gram Stain
Morphology
Sulfide production
Indole
Motility
Methyl Red
Voges-Proskauer
Urea
Catalase
Negative
Bacilli
Negative
Negative
Positive
Positive
Negative
Positive
Positive
Enterobacter
aerogenes(unknown #55)
Negative
Bacilli
Negative
Negative
Positive
Positive
Positive
Negative
Positive
Results were indicative of Enterobacter aerogenes simply by the tests listed above in the chart, that
compares results with the closest bacteria, Pseudomonas aeruginosa. All tests went well, and were all a
relatively strong positive or negative.
References
1. Chess, Barry. (2009). Laboratory Applications in Microbiology: A Case Study Approach. TheMcGrawCompanies, Inc.
2. Cowan, M. K., & Talaro, K. P. (2008). Microbiology: A Systems Approach (2 ed.). New York: McGrawHill Science/Engineering/Math.
Table 1
Shigella
Proteus vulgaris
flexneri
Bacillus cereus Enterococcus
faecalis
Micrococcus
Streptococcus
luteus
pyogenes
Thermus
Pseudomonas
aquaticus
aeruginosa
Bacillus
Staphylococcus
megaterium
aureus
Clostridium
Moraxella
sporogenes
catarrhalis
Citrobacter
Pseudomonas
freundii
fluorescens
Lactococcus
Escherichia coli
lactis subsp.
lactis
Staphylococcus Bacillus subtilis
epidermidis
Campylobacter Proteus mirabilis
jejuni
Salmonella
Halobacterium
typhimurium
salinarium
TA 98
Enterobacter
Bifodobacterium
aerogenes
longum
Treponema
Klebsiella
pallidum
pneumoniae