Identifying Unknown Microorganism by Differential Staining and Metabolic Testing

Mariya Dineva
Dr. Williams
Microbiology Lab 311 G6

Introduction
The preliminary steps of identifying bacteria always start with Gram staining. Gram
staining is a differential technique, meaning that it can distinguish between various types of
bacteria. The process of gram staining involves applying four chemical reagents. Each chemical
is washed off with water, after the application. The first step is to apply the primary stain, which
is crystal violet, and all organisms turn purple in color. Followed by the application of Grams
iodine which helps the crystal violet molecules to form a complex with the substrate to which
they are attached. The sample is then decolorized, which forces the complex to detach from
certain cells than others. Finally, the fourth reagent which is safranin is applied, and it acts as a
counter stain, staining the organisms that were previously decolorized. The microorganisms that
retain the purple color are Gram positive, and those who appear pink are Gram negative. Gram
positive bacteria have a thicker peptidoglycan cell wall, and Gram negative bacteria have a
thinner peptidoglycan cell wall with lipids embedded in it.
Gram staining alone is not enough to identify an unknown organism, although it cuts
down the list. Gram staining is only able to differentiate between different cell walls, thus
separating bacteria into two groups those who have a thick peptidoglycan cell wall are Gram
positive and those who have a thin peptidoglycan cell wall with lipids embedded are Gram
negative.
Metabolic tests have to be performed in order to identify the unknown bacteria. Bacteria
are able to perform numerous biochemical processes, thus they are metabolically active.
Microorganisms utilize a wide variety of substrates, by obtaining them from their environment,
then metabolizing them, and producing an end product. Such metabolic processes in bacteria,
occurring both inside and outside the cell, are carried out by enzymes. The product is used to
identify the bacteria, knowing that the enzymes are very specific for certain species.

Methods
For the Gram staining procedure, only four major steps are involved. After every major
step of staining, the slide is washed with water. A slide of an unknown was prepared for both

unknown A and unknown B, using aseptic technique, air drying, and heat fixing. The first step of
Gram staining is to flood the slide with crystal violet and let it stand for one minute. The second
step is to flood the slide with Grams iodine, and let it stand for one minute. The third step is to
decolorize using 95% ethyl alcohol for 5-15 seconds, not longer. The final step is to apply
safranin for one minute. The slide is then dried with bibulous paper.
Figure 1 shows the dichotomous key that was prepared for all the possible species that
the unknown can be. It is very useful to use a dichotomous key because all the information about
the different stains and test in regards to the 10 possible bacterial special can be organized and
the unknowns can be easily identified. The dichotomous key used was constructed by first
determining the Gram stain reaction, and morphology. After knowing the gram stain and
morphology, the results of the three metabolic tests were used and the bacteria that test positive
and negative for those tests.
Gram results
Positive
Bacilli

Coccus

Gelatinase
(+)

S.aureus

Hydrolysis of Gelatin

Lactose

(-)

B.subtilis

Negative

(+)

(-)

C.xerosis

Urease

M.luteus

(+)

(-)

Positive

Negative

S.marcens

Lactose

(+)
Gas present

S.faecalis
E.coli

(-)
Urease

Yes

No

(+)

(-)

E.aerogenes

P.vulgaris

P.aeruginosa

Fig 1. Dichotomous key for unknown bacteria identifying.

Three metabolic tests were used for both unknown A and unknown B- hydrolysis of urea,
hydrolysis of gelatinase, and lactose fermentation. The hydrolysis of urea metabolic test involves
inoculating a urea agar slant which contains phenol red pH indicator, with each unknown.

Following incubation period, if the organism is able to produce urease and break down urea,
ammonia is released, changing the pH level, thus changing the color to a red or cerise color. If
the microorganism is not able to catalyze the reaction, there is no color change. The hydrolysis of
gelatinase metabolic test involves inoculating the gelatin deep tube by stabbing down towards
the bottom of the tube with the unknowns, using aseptique technique and a inoculating tube. The
gelatin deeps are then incubated in a refrigerator providing a temperature below 25 degrees
Celsius. This tests whether or not the bacteria produces the enzyme gelatinase and hydrolyzes
gelatin. If it does, after incubation the gelatin deep should liquefy and remain liquid. The lactose
fermentation metabolic test involves inoculating the unknowns in a lactose broth tube, which
contains a pH indicator and a Durham tube. The pH indicator is useful in identifying whether or
not the organism has produced an acid by turning yellow. A Durham tube is used to trap any gas
that the organism might produce during fermentation, which gets trapped at the top of this small
inverted tube. If the organism does not ferment lactose, there is no change in color. If the
organism does ferment lactose, the color changes to yellow, with or without the presence of gas
bubbles. Both unknowns are inoculated using aseptique techniques and a inoculating needle,
followed by incubating the lactose broth tubes.

Results
Unknown A
Gram stain results
The color of the microorganisms after Gram staining was pink. The Gram stain
reaction is therefore negative. The morphology of the bacteria was bacilli, and they were
arranged in single rods. (Figure 2)

Fig 2. Gram negative, E.coli.

Metabolic Test results
For the hydrolysis of urea, there was no color change, the results are negative, so
the microorganism did not produce urease. The hydrolysis of gelatin test, there was no
liquidation, the results are negative, so the microorganism does not produce gelatinase. The
lactose fermentation test, there was a yellow color change, and gas bubbles were present, the
results are positive, thus this microorganism can ferment lactose.
Unknown B
Gram stain results
The color of the bacteria after Gram staining remained purple. The Gram reaction
is positive. The morphology was cocci, and the bacteria were arranged in staph. (Figure 3)

Fig.3 Gram positive, S.aureus.

Metabolic Test results
For the hydrolysis of urea, there was a slightly orange color change, the results are
negative, so the microorganism did not produce urease. The hydrolysis of gelatin test, there was
no liquidation, the results are negative, so the microorganism does not produce gelatinase. The
lactose fermentation test, there was a yellow color change, and no gas bubbles were present, the
results are positive, thus this microorganism can ferment lactose.

Conclusion
Based on the results of Gram staining and the metabolic tests performed, both unknown A
and unknown B were identified. Unknown A is Escherichia coli. Using the dichotomous key,
metabolic tests, and the results from the Gram staining, it was very easy to identify this bacteria.
Being that it is a gram negative, pink in color, bacilli and single rod arrangement, and only
positive for lactose fermentation, and producing gas bubbles, it is E. coli. An additional test that
could have been performed to confirm the results is glucose fermentation, which would also
result in a positive result, because E. coli is able to produce the needed enzymes and ferment
glucose.

Unknown B is Staphylococcus aureus. Some complications occurred during the

hydrolysis of urea metabolic test, resulting in a negative result. The correct result should have
been a positive of urease production. The bacteria might have not been incubated long enough or
might have not been provided the optimum temperature at which it functions, thus resulting in
negative results. However, based on the gram stain being positive, the bacteria colored purple,
cocci and arranged in staph, and being positive for lactose fermentation, without producing gas,
it is S.aureus. An additional metabolic test that could have been performed to confirm the results
is the activity of catalase test, which would result in a positive result, because S.aureus can
produce catalase and break down hydrogen peroxide.
Escherichia coli are a large group of bacteria, with many different strains. Most of the
E.coli strains are harmless, however some can make you ill causing diarhia, urinary tract
infections, respiratory illness, pneumonia and other illnesses. This bacteria is usually found in the
intestines of humans and animals, making it an important part of the healthy stomach flora.
Those that are pathogenic and cause illness can be transmitted through a contaminated water or
food, or by being in contact with an already ill animal or person. E. coli is a gram negative, pink
in color, bacilli arranged in single rods.
Staphylococcus aureus is commonly found on peoples skin and noses without harming
them. However, if it does enter the body this bacteria causes an infection, either minor such as
boils and pimples, or a major one such as blood infections. S. aureus is usually spread through
direct contact with an infected person. If an infection does occur, it is usually treated with an
antibiotic. Some strains of staph have become resistant to antibiotics such as methicillin. This
can be an issue because staph infections could potentially not be able to be treated with the
current antimicrobials available. S. aureus is a gram positive, purple in color, cocci and arranged
in staph.

References:
Gorelick, Melvin. Microbiology Lab Manual. 2014. Print
Pommerville, C. Jeffrey. Alcamo's Fundamentals of Microbiology. 2011. Print.