Identification of Staphylococcus species using API Staph commercial kit

Jian Diana B. Timbol, Jose Angelo S. Trinidad, Maria Cherisse P. Tuazon

College of Science, University of Santo Tomas, Espaa Boulevard, Manila
Abstract
Staphylococci is a genus of Gram-positive bacteria that comprises and represents a vast
population of the microflora. An unknown Staphylococcus spp. was isolated from skin folds and
was subjected to the different tests present in the API Staph commercial kit. The unknown
Staphylococcus spp. was determined to be Staphylococcus hominis with a 67.0 % match.
Introduction
Staphylococci is a genus of Gram-positive bacteria that comprises and represent a vast
population of the microflora--microorganisms naturally living on and in the body--of mammals
and birds. However, despite the fact that they normally live inside the body, some species of
Staphylococcus are frequently isolated from various human and animal infections.
Staphylococci are groups of bacteria that cause a crowd of illnesses. When it is observed
with a microscope, staphylococcus bacteria are round in shape and are clustered together. They
can cause diseases directly or indirectly by infection or through the products they produce, such
as toxins which are responsible for food poisoning and toxic shock syndrome.
The Analytical Profile Index is a tool that consists of microtubes containing dehydrated
substrates. These microtubes are hydrated by adding a small amount of inoculated broth in each
of them. API is used for the classification of different bacteria based on conducted experiments
allowing rapid identification of an organism. In this experiment, the API Staph--an identification
system for staphylococci, micrococci, and other related genera--was used. In the API Staph, there
are twenty different microtubes, each corresponding with a test. These tests are used to identify
the ability of an organism to ferment sugars, specifically hexose sugars such as glucose and
mannose; ketose sugars such as fructose; disaccharides such as maltose, lactose, and trehalose;
mannitol, xylitol, melibiose, raffinose, xylose, sucrose, methyl-alpha-D-glucopyranoside, and N-

acetylglucosamine; to reduce nitrate to nitrite using the Nitrite test; and production of acetyl
methyl carbinol, alkaline phosphatase, arginine dehydrolase, and urease.
The aim of this study is to determine the most probable isolated Staphylococcus species
using the API Staph test.
Methodology
A sterile normal saline solution was prepared. A sterile cotton swab was dipped in the
normal saline solution and was swabbed on different folds of the body. After swabbing on the
body, the cotton swab was then streaked on a 5% sheeps blood agar and was left for incubation
for

24

hours

in

37C.

After incubation period, culture was isolated and subcultured in a 5% sheeps blood agar
and was subjected to Gram-stain and catalase test. The microorganism isolated proved to be
pure, testing positive for both Gram-stain and catalase test. The pure microorganism was
inoculated into a broth and was left for incubation for 18 hours at 37C. After less than 24 hours
of incubation, a homogenous bacterial suspension was prepared using A water chamber was
created by adding sterile distilled water into the small wells using a micropipettor. After the
water chamber has been created, using a different pipette tip, the inoculated broth was carefully
pipetted into the small tubes. The inoculated broth was filled only until the tube portion of the
microtubes as any overflow or underflow can result to a false-positive or false-negative result.
Formation of bubbles was avoided by tilting the strip slightly forwards. Anaerobiosis was
ensured in the ADH and URE tests by filling the cupules with sterile mineral oil. The incubation
box was closed and incubated for 24 hours at 37C.
After 24 hours of incubation, a drop of the following reagents were added for the
following tests: VP 1 and VP 2 reagents for VP test, NIT 1 and NIT 2 reagents for NIT test. The
PAL test was left unreacted, leaving a result of negative. Wait 10 minutes after adding a drop of
each of the reagents to see a reaction.

Results and Discussion

Staphylococci is a bacterial genus found normally as microflora. However, when the
environment persists, some species, specifically Staphylococcus aureus, can cause various

infections. Staphylococcus aureus can cause superficial skin lesions such as boils and styes,
deep-seated infections such as endocarditis and osteomyelitis, nosocomial infections through
surgical wounds, food poisoning, and many more. It is important then to determine first the
species of Staphylococci one is dealing with to know the precautionary measures to be done.
Two tests are done prior to using the API Staph test to ensure that the organism being
used is a staphylococci. These are the Gram-stain and the catalase test. Staphylococcus is both
positive for Gram-stain and catalase test. Another test, the coagulase test, is used to differentiate
Staphylococcus aureus and other staphylococcus species. Staphylococcus aureus shows positive
result which is coagulation of plasma in the test tube and production of clumps, while another
group of staphylococcus, namely the Coagulase Negative Staphylococcus (CoNS), produces no
coagulation nor clumps, which is a negative result.
The unknown organism was subjected to Gram-stain. The Gram-stain slide showed
purple round clusters, which means that the organism is Gram-positive. The same culture was
used to test for the presence of the enzyme catalase, wherein a drop of hydrogen peroxide was
added on a smeared slide. Formation of bubbles was observed, meaning that the organism
possesses catalase. The unknown organism, however, tested negative for coagulase, which means
the possibility of it being a Staphylococcus aureus is eliminated.
After confirmation of Gram-stain, catalase, and coagulase test, the organism was
subjected to the API Staph test. Table 1 presents the results of the API Staph test.
Table 1. Results of API Staph test
Tests

Result

Glucose fermentation

negative

Fructose fermentation

negative

Mannose fermentation

positive

Maltose fermentation

negative

Lactose fermentation

negative

Trehalose fermentation

negative

Mannitol fermentation

positive

Xylitol fermentation

positive

Melibiose fermentation

positive

Nitrate test

positive

PAL test

negative

Vogues-Proskauer test

positive

Raffinose fermentation

positive

Xylose fermentation

positive

Sucrose fermentation

negative

MDG fermentation

negative

NAG fermentation

positive

ADH test

positive

Urease test

positive

I. Sugar fermentation tests

The unknown organism showed a change in color (from red to yellow) in glucose,
fructose, mannose, maltose, lactose, trehalose, and sucrose tests. However, no color change was
observed in mannitol, xylitol, melibiose, raffinose, xylose, and methyl-alpha-D-glucopyranoside.
A change of color from red to yellow means that the organism was able to ferment the sugar
component, having an acidic pH; the unknown organism was able to use these sugars as it
primary source of carbon. Mannose, although showed color change, was considered negative as
it was noted in the API Staph instructions that when mannose is preceded or followed by a
positive result, an orange color is to be considered negative.
II. Nitrate reduction test
The unknown organism showed a color change from light pink to red after a drop of
Nitrogen reagent 1 (sulfanilic acid) and a drop of Nitrogen reagent 2 (dimethyl-alpha-

naphthylamine). A color change from light pink to red 10 minutes after dropping the reagents is a
positive result. Nitrate reduction test is used for identifying microorganisms that is capable of
reducing nitrate (NO3) to nitrite (NO2) using the enzyme nitratase (also known as nitrate
reductase). This enzyme catalyzes reaction involving conversion of the starting nitrate into
nitrogen gas as an end product. Addition of Nitrogen reagent 1 (sulfanilic acid) and Nitrogen
reagent 2 (dimethyl-alpha-naphthylamine) are done after incubation.
III. Voges-Proskauer Test
The unknown microorganism showed a dark brown pigment after 10 minutes of addition
of the VP Reagent 1 (40% KOH) and VP Reagent 2 (alpha-naphtol). The Voges-Proskauer test is
used to determine if an organism produces acetyl methyl carbinol from glucose fermentation. If
acetyl methyl carbinol is present, it will be converted to a diacetyl in the presence of alphanaphthol which is a color intensifier. If the media turned color red, then acetyl methyl carbinol is
produced and is a positive test result.
IV. NAG (N-Acetylglucosamine) Fermentation test
The unknown organism showed a yellow color after incubation. Acidification of NAcetylglucosamine which has a positive result of the media turning yellow and a negative result
where the media turn color red.
V. MDG (mthyl-alpha-D-glucopyranoside) Test
The solution did not change initial color; it retained the color red. Acidification of
Mthyl-alpha-D-glucopyranoside which has a positive result of the media turning yellow and a
negative result where the media turn color red.
VI. ADH decarboxylation of the amino acid arginine by arginine dihydrolase
The solution showed a yellow color after incubation. Arginine is an amino acid that some
bacteria can use because of an enzyme known as arginine dihydrolase. Arginine dihydrolase is
used to determine whether the microorganism can use the amino acid arginine for carbon and
energy. For a positive result the organism must first use the glucose present to make the pH of
the media drop which is indicated by a purple to yellow color change. Once the pH of the media

is acidic the enzyme arginine dihydrolase is activated and degrades the amino acid arginine. The
final results are observed after 48 hours where a positive result is indicated by the media turning
purple from yellow.
VII. Urease test
The solution turned red after incubation. Urease test produces water, carbon dioxide, and
ammonia as product from urea. If a result turns pink or red, the test is positive since it produces
acid. Some species of Staphylococcus are positive like S. aureus, S. epidermidis, and S.
saprophyticus. However, if the result is yellow it means it is alkaline, or orange if neutral which
are negative result.
Table 2 shows the scores of the API Staph results, wherein positive results scores are
added to form a 7-digit code to enter onto the API Catalog for verification.
Table 2. Summation of scores of API Staph Results
Tube

11

0
Rx

20

Digital code: 661 215 3

The digital code was entered onto the API Catalog where possibilities of what the
unknown organism is are listed. Figure 1 shows the results.
Figure 1. Results from the API Catalog

The group garnered a result of 67% with Staphylococcus hominis, 15.1% Staphylococcus
saprophyticus, 10.1% Staphylococcus simulans, 3.4% Staphylococcus haemolyticus, and 1.7%
Staphylococcus lugdunensis. Identification of the organism cannot be considered valid as the
percentage is below 90%.
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