III. epidermis was identified to be catalase positive which

III. Significance of Staphylococcus epidermis

Staphylococcus epidermis is a gram-positive coccus and an
aerobe of the genus Staphylococcus.

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It is also non-motile and nonsporing. Species of the genus Staphylococcus are common bacterial invaders of the skin and mucous
membranes in humans and other mammals. S.

epidermis, does cause any life-threatening disease but it does thrive in infectious
areas of the body and the treatment is complex. In the tests conducted in
class, Staphylococcus epidermis was identified
to be catalase positive which indicates that the organism is able to flourish
in aerobic conditions and is also able reduce nitrate to nitrite. However, this
organism is unable to tolerate salt-rich environments and it does not produce
enzymes that aid in the process of DNA hydrolysis. S. epidermis also exhibited gamma hemolysis on the Blood Agar plate
and sensitivity to the antimicrobials, Bacitracin and Novobiocin. It also
showed negative growth on the MSA plate and responded negative to the Bile Esculin
test.

Staphylococcus epidermis is significant because it is a
prevalent cause of many health care-related infections. S. epidermis is most commonly transferred by any type of indwelling
medical devise such as catheters. The bacteria are first introduced from the
skin of patients or the health care professional in the process of inserting
the indwelling medical device. Thus, it is known as the “accidental pathogen.” Fortunately,
S. epidermis is not virulent for its
avirulent strains exceeds its virulent strains.  However, it is the main cause of
catheter-related bloodstream infections, neonatal sepsis, and other biomedical
device- related infections such as prosthetic joint infections. Compared to Staphylococcus aureus, information about
Staphylococcus epidermis is limited and
it may be due its avirulent impact on individuals.  

Unfortunately,
there is no vaccine for S. epidermis-
related infections for it has been proven that the eradication of this organism
may cause more harm than good. Staphylococcus
epidermis is a common part of the human microflora, thus if it is removed
more pathogenic microorganisms might take its place. Therefore, prevention is
key in dealing with S. epidermis infections.

Prevention tips includes the thorough sterilization of medical equipment and of
both patient and health care professionals in possible contact with contaminated
medical devices.

 

Materials and Methods

Streak
Plate

The streak
plate test is used to isolate the unknown bacteria from its contaminant. During
the streaking process, the decrease in cell density allows individual cells to
form colonies on the agar side of the plate. There were two plates that was
used to achieve the isolation of
colonies from the unknown bacteria.

 

The TSA Plates are non- selective nutrient
medium that does not specifically inhibit any microorganism but is utilized to count the amount of aerobics that it
develops.2

 

MSA
Plate

The
Mannitol Salt Agar is a selective and differential media that consists of mannitol
carbohydrate and 7.5% of sodium chloride, and phenol red— a pH indicator.

Phenol red turns yellow if the pH is below 6, remains red at the pH between 7.4
to 8.4, and turns pink at the pH 8.4 and above. The manner in which the
mannitol supplies the substrate for fermentation makes the MSA a differential
medium. Yellow halos form when the mannitol fermentation process produces acids
that lowers the pH of the medium.

 

Gram
Stain

The gram
stain test is used to determine the gram reaction of the species. The gram
stain should show two types of results: positive (purple) reaction or negative
(pink) reaction. There are several dyes involved in the gram staining method.2

 

The
primary stain in the gram stain process is crystal
violet. The crystal violet stains both the Gram- positive and Gram-
negative cells purple as it enters the cytoplasm of the cells. After the
crystal violet is washed off with distilled water, the iodine is the mordant that fixes the dye to the cell. The decolorizer that contains either
alcohol or acetone removes the crystal violet dye from Gram-negative cells
leaving it with no color. Safranin
is used to counterstain the Gram- negative cells with a pinkish hue. 2

 

Oxygen
Requirement Tests

The Oxygen
Requirement Tests is consisted of three different tests to determine if the
unknown bacterium is an obligate aerobe
or facultative anaerobe. It also
indicates the fermentation and/or oxidization of glucose.

 

Brewer’s Plate Test: The purpose of the Brewer’s Plate
in the Gas Pak is to identify if the unknown bacterium is a strict aerobe or a facultative anaerobe. The Brewer’s plate has reazurin
which is the indicator for O2 depletion and whether or not if the
organism is a strict aerobe or facultative anaerobe. The chemical pack produces
H2 gas that reacts with O2
to produce H2O which as a result depletes the oxygen inside. The
reazurin turns pink in the presence of oxygen and turns colorless without
oxygen. A chunk of a colony is streaked onto a plate and placed into the Gas
pack for a couple of days. If growth occurs, then it is a facultative anaerobe.

If there is no growth, then it is a strict aerobe.2

 

Oxidation- Fermentation (O-F) Test: This test is designed to
distinguish organisms on its ability to ferment
and oxidize glucose.2 There
are two different test tubes used for the test. The first test tube contains
just the original medium while an oil layer is added to the second test tube.

If the tube with the oil is yellow, it is positive for the fermentation of
glucose.2 If the tube without the oil is yellow, then it is positive
for the oxidation of glucose.2

 

The FTM Test is another media to determine
if the unknown is a strict aerobe or
a facultative anaerobe. The key
constituents of this medium are sodium thioglycollate, L-cystine and reazurin.

The sodium thioglycollate and L-cystine reduce oxygen into water and the reazurin indicates the presence of O2.2
If the organism is a strict aerobe then growth will occur only at the top. If
it is a facultative anaerobe, then the growth is equally dispersed throughout
the tube. 2

 

Catalase

This test
is to determine whether or not the organism is able to break down hydrogen peroxide into water and oxygen. If the species is catalase positive, bubbling should occur
immediately after the hydrogen peroxide is added. If no bubbling occurs, then
it is catalase negative.2

 

Oxidase

This test
is to determine if there is a presence of cytochrome
oxidase in the electron transport chain of the organism. A positive result
will show immediate color change to a dark blue/purple in less than a minute.

No color change is a negative result.2

 

Nitrate
Reduction Test

The first
part of the Nitrate Reduction Test is used to determine if the organism can
reduce nitrate to nitrite. The tube
is first examined for a gas bubble within the test tube which is from N2 gas. If there is a
bubble, the organism is a dentrifyer. This examination is just simply to check
for a presence of gas. The reagents, Nitrate A and B, are added directly into
the tube to test for the nitrate reductase activity. If a reddish color
develops, the organism is positive for nitrate reduction. If no color develops,
there is one last test that can help detect nitrate reduction. Zinc is used as
another reagent. If red develops at the presence of zinc, it indicates that the
organism is negative and does not reduce nitrate. If no red color develops, it
indicates that the organisms is positive for nitrate reduction for some other
nongaseous nitrogenous compound but not nitrite.2

 

Vogues-
Proskauer

The
Vogues- Proskauer test detects the production of acetoin, a byproduct of glucose fermentation, in the 2, 3 butandiol fermentation path. Like
the methyl red test, the cells are inoculated into MR-VP medium and are
incubated. After the incubation period, two VP reagents, Barrits A and Barrits
B, are added directly to the test tube. Since the results are not immediate
like the MR test, it takes time for a reaction. A positive result will display
that the indicators detect the presence of acetoin by generating a pinkish/red
color.2

 

Coagulase
Test

The
Coagulase test is made up of rehydrated Rabbit plasma and it detects the
production of the exoenzyme coagulase which causes clots in the fibrin of blood
plasma. The formed fibrin barriers make the bacterial cells resistant to
phagocytosis and other types of immune responses. There are two types of the
coagulase enzymes— the free coagulase
and the bound coagulase. The free
coagulase reacts with the plasma (also known as the coagulase- clotting factor) which forces the formation of the
fibrin. The bound coagulase (also known as the clumping factor) directly reacts with the fibrin which causes the
cells to clump together. A negative coagulase test will show no change or
cloudiness. A positive coagulase test will display visible clumping at end of
the test tube.

 

Blood Agar
Plate

The Blood
Agar Plate contains 5% sheep’s blood in a tryptic soy agar base which is used
to identify the production of exotoxins called hemolysins in Gram-positive cocci species. Hemolysins destroy the
red blood cells (RBCs) and hemoglobin. There are three types of hemolysis
results: alpha, beta, and gamma. The alpha hemolysis occurs when there is partial
lysis of RBCs and hemoglobin. The green zones around the colonies indicates the
partial lysis. The beta hemolysis is the complete lysis of the RBCs and the
hemoglobin and should have clear zones around the colonies. The gamma hemolysis
indicates no hemolysis.

 

Antimicrobial
Disks

Bacitracin
is an antibiotic produced by Bacillus licheniformis that interferes
with the transport of peptidoglycan subunits during bacterial cell wall
synthesis. This antibiotic is only effective on bacteria that have cell walls
and that is able to grow. An organism sensitive to Bacitracin will display a
clearing zone around the disk at least 10mm or greater.

 

Novobiocin
is an antibiotic that is produced by Streptomyces
niveus that interferes with the ATPase activity linked with DNA gyrase, an
essential enzyme used in DNA replication. An organism sensitive to Novobiocin
will produce a clearing zone around the disk of 16mm or greater.

 

Bile
Esculin

The Bile
Esculin Agar (BEA) is selective and differential medium that contains bile,
Esculin, gelatin, and ferric acid. The beef extract and gelatin provides nutrient
and energy while the bile acts as a selective agent to detach the Group D Streptococci from non- Group D Streptococci. During the process in which
the Esculin is hydrolyzed by esculinase, it produces D-glucose and esculetin. Ferric
acid is used as the indicator for oxidized iron that turns the media dark
brown/ black when it reacts with the esculetin. No dark color change signifies
a negative for Bile Esculin.

 

6.5%
Salt Broth

The 6.5%
NaCl test is a selective medium that tests for salt tolerance when sodium
chloride is added to the Tryptic Soy Broth. Furthermore, this test examines
that organism’s survival ability in a salt-rich setting. A positive result will
show growth in the inoculated tube broth. A negative result will show no
growth.

 

DNAse
Test

The DNAse
Agar test is a differential medium that examines an organism’s ability to
produce an exoenzyme, deoxyribonuclease, which aids in the process of DNA
hydrolysis. Methyl green is the indicator for this test. A positive DNAse test
will show a clear zone around the inoculated line where the DNAse- producing is
growing. A negative result will show no growth or clear zone around the
inoculated line.

 

Immunochromatography

The Immunochromatography
test is used to detect particular antigens or antibodies within a given sample.

This test consists of a single-use test strip that is placed into a sample
fluid and it generates a positive or negative test by the formation of color on
the lines of the strip. A positive test strip indicates that an antigen or
antibody is captured by a second antibody (usually IgG) within the test area. A
negative test (or no color change) indicates that no antigens or antibodies
were detected.

 

Mannitol
Fermentation Test

The Mannitol
Fermentation test is to detect the organism’s ability to ferment mannitol as a
carbon source. This test also shows gas production which is indicated by the gas
trapped in the Durham tube. The organism is inoculated into a phenol red broth with
a mannitol medium. The acid production from fermentation lowers the pH level
turning the medium to a yellow color. A positive fermentation will turn the
inoculated broth to yellow. This means that there was acid fermentation.

 

 

 

 

Part Three:
Streptococcus/ Enterococcus/ Staphylococcus Comparison

Staphylococcus is gram-positive coccus that is commonly
found on the skin and/ or mucous membranes of humans. The genus Staphylococcus is non-motile, nonsporing,
and are facultative anaerobes. This organism tests positive for catalase and
negative for oxidase. There are many types of Staphylococcus, however, the two most common ones are Staphylococcus epidermis and Staphylococcus aureus. S. aureus resides in nasal passageways and
is known to cause a variety of diseases and symptoms. This type of species of Staphylococcus is said to be virulent
compared to S. epidermis for its
virulent strains surpasses its avirulent strains. S. aureus is known to cause boils, styes, and other external skin
infections. In extreme cases of S. aureus infection, it could cause meningitis,
food poisoning. S. epidermis, on the
other hand, is known to cause pretty benign bacterial infections that is usually
transmitted accidently by indwelling medical devices.

Streptococcus is a gram- positive coccus that is
the most similar to the genus Enterococcus.

The major distinguishing factor between all three organisms is the catalase test.

Unlike Staphylococcus, Streptococcus, and Enterococcus tests negative for catalase. However, like the genus
Staphylococcus, it is non-motile, nonsporing, and are facultative anaerobes.