Isolating Staphylococcus sp.

Using Mannitol Salt Agar (MSA) and Escherichia coli using Eosin Methylene
Blue (EMB) Agar

the isolation of specific microbiota were conducted using two kinds of selective media, Mannitol Salt
Agar (MSA) and Eosin Methylene Blue Agar (EMB).

Within 24 hours, colonies were formed on 4 prepared plates with 2 plates contain MSA and the other
two contains emb.

4 plates were prepared for the isolation of specific microbiota using 2 kinds of selective media,2 plates
for mannitol salt agar and the remaining for eosin methylene blue agar.

After 24 hours of incubation, colonies were formed on the prepared plates.

Insert picture.

Figures 1 and 2 are the culture media showing presence of Staphlococcus sp. on Mannitol Salt Agar.
Each plate is divided into two sides left and right for nasal cultures from left and right nostril. Both
plates show higher number of white colony formation on the left side of the plate. Also, there is no
significant change in color of the agar which implies absence of the species Staphylococcus aureus and
other mannitol fermenters

The two EMB plates were used for the isolation of gram negative enteric bacteria, specifically E.Coli.
Each plate contains fecal matter taken from Arlu Bautista’s pet dog. Out of the two, only one plate
obtained a green metallic color which indicates presence of the species E.coli (Figure 3).

Discussion

Selective medium types are formulated to support the growth of one group of organisms, but inhibit the growth of
another. Mannitol Salt Agar is used as a selective media for the isolation of Staphylococcus sp. while Eosin
Methylene Blue agar is for the isolation of gram negative enteric bacteria.

Presence of colonies on msa media indicates positive result of the genus staphylococcus.The high
concentration of salt (7.5%) selects for members of the genus Staphylococcus, since they can
tolerate high saline levels.

Also, the unchanged color of the agar implies absence of the species staphylococcus aureus.
MSA also contains the sugar mannitol and the pH indicator phenol red. If an organism can ferment
mannitol, an acidic byproduct is formed that will cause the phenol red in the agar to turn yellow. Most
pathogenic staphylococci, such as Staphylococcus aureus, will ferment mannitol. On the other hand
non pathogernic Staphylococcus epidermidis does not ferment mannitol. When it grows on MSA, the
natural orange-red color of the msa will remain unchanged (Prescott et al, 2016)
From these data, it can be deducted that s. aureus is absent on the nasal cultures taken from the
nostril of the both subjects and the presence of S.epidermis.

Eosin methylene blue agar (EMB) is a selective and differential medium used to isolate fecal
coliforms. Eosin Y and methylene blue are pH indicator dyes which combine to form a dark purple
precipitate at low pH; they also serve to inhibit the growth of most Gram positive organisms.
(SOURCE)

Vigorous fermenters of lactose or sucrose will produce quantities of acid sufficient to form the dark
purple dye complex. The growth of these organisms will appear dark purple to black as shown in
figure 3. Escherichia coli, a vigorous fermenter, often produces a green metallic sheen.

Only one of the two plates shows green metallic color which indicates presence of E.coli on the
dog’s fecal matter. It ca

Conclusion:

Based on the results acquired. The colony growth on msa agar indicates presence of microorganism
of genus staphylococcus. There is higher number of bacteria on the right nostrils of both subjects
because of higher number of colony formation on it. Also, Staphylococcus epidermidis is present
and isolated in the nasal cultures of the subjects nasal cultures on both left and right nostrils
because of the unchanged color of the msa.

E.coli is present in the fecal matter of arlu bautista’s dog because of the presence of green metallic
sheen on the emb media. Although, in fewer amount
Gram staining:

Three bacteria were observed under the microscope with the aid of gram staining technique.

The species staphylococcus aureus obtained from the msa nasal culture. Figure 2 shows that s.
epidermidis is a gram positive bacteria because it absorbed the crystal violet stain which turned it a
dark shade of violet. Its morphology is round (cocci) with cluster arrangement.

Figure 3 shows stained E.coli taken from emb facal medium with green metallic sheen. Escherichia coli
is a bacillus shaped bacterium that does not form colonies or has a single cell arrangement. It is gram
negative due to reddish coloration.

Figure 1 shows the stained bacteria taken from the laboratory gown of Hanipha Hairulla. It shows that the
isolated bacteria (unknown) is gram-positive due to violet/bluish coloration. It has round shape (coccus)
and have grape-like cluster arrangement (staphylococcus)

Staphylococcus epidermidis is a gram-positive and coagulase-negative staphylococci (4). It typically lives on

the human skin and mucosa and the most common infections on catheters and implants (5). S. epidermidis is
one of five most common organisms that cause noscomial infections due to the increase in usage of
biomaterials in the clinical environment (8). The nosocomial pathogen causes infections on prosthetic valves,
cerebrospinal fluid shunts, joint prosthesis vascular prostheses, valves, and in postoperative wounds and the
urinary tract. It is also the most frequent organism found in the blood of bone marrow transplant patients and on
central venous catheters for patients of total parenteral nutrition (4).

the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories
according to their cell wall.
Gram-positive bacteria take up the crystal violet stain used in the test, and then appear to be purple-
coloured when seen through a microscope. This is because the thick peptidoglycan layer in the
bacterial cell wall retains the stain after it is washed away from the rest of the sample, in the
decolorization stage of the test.
Gram-negative bacteria cannot retain the violet stain after the decolorization step; alcohol used in
this stage degrades the outer membrane of Gram-negative cells, making the cell wall more porous
and incapable of retaining the crystal violet stain. Their peptidoglycan layer is much thinner and
sandwiched between an inner cell membrane and a bacterial outer membrane, causing them to take
up the counterstain (safranin or fuchsine) and appear red or pink.
Despite their thicker peptidoglycan layer, Gram-positive bacteria are more receptive
to antibiotics than Gram-negative, due to the absence of the outer membrane.

Escherichia coli (commonly abbreviated E. coli) is a Gram-negative, rod-shaped bacterium

that is commonly found in the lower intestine of warm-blooded organisms (endotherms).
Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in
humans. The harmless strains are part of the normal flora of the gut, and can benefit
their hosts by producing vitamin K2, and by preventing the establishment of pathogenic
bacteria within the intestine. E. coli and related bacteria constitute about 0.1% of gut flora,
and fecal-oral transmission is the major route through which pathogenic strains of the
bacterium cause disease.
Escherichia coli (/ˌɛʃɪˈrɪkiə ˈkoʊlaɪ/;[1] also known as E. coli) is a Gram-negative, facultatively
anaerobic, rod-shaped, coliform bacterium of the genus Escherichiathat is commonly found in the
lower intestine of warm-blooded organisms (endotherms).[2][3] Most E. coli strains are harmless, but
some serotypes can cause serious food poisoning in their hosts, and are occasionally responsible
for product recalls due to food contamination.[4][5] The harmless strains are part of the normal flora of
the gut, and can benefit their hosts by producing vitamin K2,[6]and preventing colonization of the
intestine with pathogenic bacteria, having a symbiotic relationship.[7][8] E. coli is expelled into the
environment within fecal matter. The bacterium grows massively in fresh fecal matter under aerobic
conditions for 3 days, but its numbers decline slowly afterwards

Escherichia coli
Bacteria Shape
Escherichia coli is a bacillus shaped bacterium that does not form colonies.

Gram Stain
Escherichia coli is a gram negative bacterium. Gram negative means that it has a two layer cell wall
composed of a thin, inner layer of peptidoglycan and an outer membrane of phospholipids,
lipopolysaccharide, and lipoprotein.

https://modmedmicrobes.wikispaces.com/E+coli
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http://www.medicalnewstoday.com/articles/68511.php

isolation in bacteria
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Medical Microbiology. 2001. Volume 50. p. 582-587.

Queck SY & Otto M (2008). "Staphylococcus epidermidis and other Coagulase-Negative

Staphylococci". Staphylococcus: Molecular Genetics. Caister Academic Press. ISBN 978-1-904455-29-5.