REPORT ON IDENTIFICATION OF SALMONELLA IN CHICKEN’S

LIVER
DATE : 6 MARCH 2018

BY :
DINESH RADZA KRISHNAN B04148005
KAUSALYAA K GAJAPATHI RAO B04158017
JOEY LW B04158021

DEPARTEMEN ILMU PENYAKIT HEWAN DAN KESMAVET

FAKULTAS KEDOKTERAN HEWAN
INSTITUT PERTANIAN BOGOR
2018
 INTRODUCTION
Salmonella enterica is a gram-negative, rod-shaped, flagellated bacterium that is of interest
due to its ability to cause infectious disease in humans and animals. Human salmonellosis, S.
enterica infection, occurs in about 1.3 million people per year, an estimated 30% of all food
borne illness, causing about 500 deaths and has an estimated cost of $2.4 billion dollars per
year. Salmonella sp. are facultative, being capable of both aerobic respiration for the production
ATP as well as fermentation in the absence of oxygen. Dr. Daniel E. Salmon was the first
person to identify Salmonella species over 100 years ago, currently there are approximately
2,300 serotypes known. S. enterica subspecies enterica serovar thyphi is a subspecies that is
responsible for typhoid fever in humans by invading the gastrointestinal tract. Much of the
current understanding of cellular and molecular functions in a cell has been made possible by
using Salmonella. For example, S. enterica subspecies enterica serovar Typhimurium, was the
first bacteria used to observe transduction by Zinder and Lederberg in 1952. Since then, a vast
number of discoveries have been made using Salmonella ranging from gene regulation
mechanisms, to cell surface antigen interactions with immune cells.
Salmonella species are non-spore-forming, predominantly motile enterobacteria with cell
diameters between about 0.7 and 1.5 µm, lengths from 2 to 5 µm, and peritrichous flagella (all
around the cell body). They are chemotrophs, obtaining their energy from oxidation and
reduction reactions using organic sources. They are also facultative anaerobes, capable of
generating ATP with oxygen ("aerobically") when it is available; or when oxygen is not
available, using other electron acceptors or fermentation ("anaerobically"). S. enterica
subspecies are found worldwide in all warm-blooded animals and in the environment. S.
bongori is restricted to cold-blooded animals, particularly reptiles.
Salmonella is the name of the group of over 2,500 types of bacteria that most commonly
causes food poisoning in humans and animals. Salmonella is spread by ingesting foods that are
contaminated by salmonella such as raw eggs, raw meat, eggs, fruits, vegetables, and
contaminated water. Contamination takes place when these foods come into contact with
animal or human feces and are not cooked properly. Symptoms of Salmonella are diarrhea,
vomiting, fever, cramps, headache, and last around 4-7 days. Symptoms can get more serious
in infants and the elderly but overall will go away by themselves.
 METHOD

Materials and Apparatus

The apparatus that were used in this experiment are clean glass slides, inoculating loop, Bunsen
burner, bibulous paper, microscope, lens cleaner, immersion oil, distilled water, marking pen, petri
dishes, spoid, test tubes, and a lighter. The materials that were used in this experiment were crystal
violet, lugol, acetone alcohol, safranin and chicken’s bile that was infected with salmonella, indole,
urea, citrate, TSIA, glucose broth, phenol red indicator, tetramethyl-p-phenylenediamine
dihydrochloride.

Gram Staining

Aseptic technique was used throughout the experiment. The glass was first cleaned using alcohol
and cotton to sterilize it. Then bile liquid was extracted from the bile and a few drops was placed onto
the glass slide using a syringe. The slide had to be air dried before heat fixing it, so that the Gram
staining may begin. Firstly, the primary stain, Crystal Violet was placed, on the slide where the bacteria
was heat fixed. The dye had to stay on the slide for about one minute before rinsing with distilled water.
Then the gram’s Iodine (which is the Gram stain dye) was placed on the slide for another minute. After
the one minute was up, the slide was rinsed with distilled water. The next step was to use 95% Ethanol
to wash off the dyes from the Gram-negative bacteria. The slide was tilted slightly and the alcohol was
applied drop by drop for 5 to 10 seconds until the alcohol runs almost clear. It was immediately rinsed
with distilled water. Lastly, the saffarin dye was placed on the slide to counterstain the Grampositive
bacteria for one minute, rinsed with distilled water, and blotted dry. The smear was viewed using a light
microscope under oil immersion.

Glucose Fermentation test

A test tube containing glucose broth, phenol red indicator and a durham tube was used in this test. The
durham tube was added to indicate gas production. First, the inoculating loop was fixated and then it
was used to transfer the bacteria filled with the glucose broth. For positive results, the durham tube has
to be checked for bubbles and if the result is negative, there will be no changes in colour and no bubbles.

Oxidase test
A filter paper was taken and soaked with the substrate tetramethyl-p-phenylenediamine dihydrochloride.
The paper was then moistened with sterile distilled water. Then, the colony to be tested was placed on
the filter paper using a sterile inoculating loop. The inoculated area of the paper was observed for colour
change to deep blue or purple within 10 seconds.

Agar test
For the blood agar and MCA agar, the liquid from the bile was extracted using a syringe and placed
onto the agar. Then, using an inoculating loop that was fixated the liquid was spread onto the agar. The
agar was then observed after 24 hours.

Gram Staining
The glass was first cleaned using alcohol and cotton to sterilize it. Then bacteria that was inoculated in
the slanted tube was placed onto the glass slide using an inoculating loop. The slide had to be air dried
before heat fixing it, so that the Gram staining may begin. Firstly, the primary stain, Crystal Violet was
placed, on the slide where the bacteria was heat fixed. The dye had to stay on the slide for about one
minute before rinsing with distilled water. Then the gram’s Iodine (which is the Gram stain dye) was
placed on the slide for another minute. After the one minute was up, the slide was rinsed with distilled
water. The next step was to use 95% Ethanol to wash off the dyes from the Gram-negative bacteria. The
slide was tilted slightly and the alcohol was applied drop by drop for 5 to 10 seconds until the alcohol
runs almost clear. It was immediately rinsed with distilled water. Lastly, the saffarin dye was placed on
the slide to counterstain the Gramnegative bacteria for one minute, rinsed with distilled water, and
blotted dry. The smear was viewed using a light microscope under oil immersion.

Biochemical Test
Citrate test

Bacteria was inoculated in a tube containing citrate medium. It was also possible to streak or
perform a deep inoculation into "Simmons citrate tube". Media was incubated at 30-37ºC for 24-48
hours.

Indole test

Several drops of Spot Indole Reagent were placed on a piece of filter paper. A plastic loop was filled
with bacteria from a colony cultivated for 24-48 hours. It was rubbed onto the reagent saturated
area of the filter paper.

Urease test
The surface area of a urea agar slant with a portion of a well-isolated colony or inoculate slant was
streaked with 1 to 2 drops from an overnight brain-heart infusion broth culture. The cap was left on
loosely and the tube was incubated at 35°-37°C in ambient air for 48 hours to 7 days. The
development of a pink colour was examined over the course of 7 days

TSI Agar

With a sterilized straight inoculation needle, the top of a well-isolated colony was touched. TSI Agar
was inoculated by first stabbing through the center of the medium to the bottom of the tube and
then streaking on the surface of the agar slant. The cap was left on loosely and the tube was
incubated at 35°C in ambient air for 18-24 hours.
 RESULT
Citrate test

Result: positive
Urea test

Result: Negative
Indol test

Result: Negative
Catalase test

Result: positive
Fermentation test

Result: positive
Gram staining test

Result: bacillus negative

MCA agar

Result: negative

Blood agar

Result: positive

TSIA agar

Result: positive
 DISCUSSION
From the gram staining, gram negative bacteria was observed under the microscope. The
gram negative bacteria were seen as red and rod-shaped bacterium with rounded ends. Being
Gram-negative bacteria, have an additional outer membrane that is composed of phospholipids
and lipopolysaccharides. The presence lipopolysaccharide Gram negative bacteria are easily
decolorized because of their thinner peptidoglycan layer and are subsequently dyed red by the
safranin on the outer membrane of bacteria gives it an overall negative charge to the cell wall.
Because of these properties, gram negative bacteria does not retain crystal violet during the
Gram staining process. (Halebian S,1981).

The bacteria was cultured in MacConkey agar, then it was used for the isolation of gram-
negative enteric bacteria and the differentiation of lactose fermenting from lactose non-
fermenting gram-negative bacteria. The selective action of this medium is attributed to crystal
violet and bile salts, which are inhibitory to most species of gram-positive bacteria. Sodium
chloride maintains the osmotic balance in the medium. Neutral red is a pH indicator that turns
red at a pH below 6.8 and is colourless at any pH greater than 6.8. Agar is the solidifying agent.
From the results we can conclude that the bacteria can ferment lactose because pink/red
colonise was found. (Anderson, N.L,1905)

Blood agar is an enriched, bacterial growth medium. Fastidious organisms, such as

streptococci, do not grow well on ordinary growth media. Blood agar is a type of growth
medium containing trypticase soya agar enriched with 5% sheep blood that encourages the
growth of bacteria, such as streptococci, that otherwise wouldn’t grow. Blood contains
inhibitors for certain bacteria such as Neisseria and Haemophilus genera and the blood agar
must be heated to inactivate these inhibitors and to release essential growth factors .Heating of
blood agar converts it into chocolate agar (heated blood turns a chocolate colour) and supports
the growth of these bacteria. From the results, we can see that grey colour of the colony in
blood agar this shows partial lysis of red blood cells. This is because non-haemolytic colonies
of Salmonella enterica cultivated on blood agar. An example, is gamma-haemolysis which
equals to no haemolysis.. Some strains of Salmonella, grows on media with blood surrounded
by a zone of beta-haemolysis’s most probably Salmonella enterica is the bacteria growing on
the media. (Wayne, PA. 2011)

From the second gram staining was conducted, the bacteria that was observed was to be a
gram negative bacteria.

TSI agar is used to determine whether a gram negative rod utilizes glucose and lactose or
sucrose fermentative and forms hydrogen sulphide (H2S). TSI contains 10 parts lactose: 10
parts sucrose: 1 part glucose and peptone. Phenol red and ferrous sulphate serves as indicators
of acidification and H2S formation, respectively. The formation of CO2 and H2 is indicated by
the presence of bubbles or cracks in the agar or by separation of the agar from the sides or
bottom of the tube. The production of H2S requires an acidic environment and is indicated by
blackening of the butt of the medium in the tube. From the results, we could conclude that the
butt is brown and the slant remains yellow with gas production. This shows that glucose,
sucrose and lactose were fermented. ( Bontron S,2016)

The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme
of the bacterial electron transport chain. When present, the cytochrome c oxidase oxidizes the
reagent such as tetramethyl-p-phenylenediamine to indophenols purple colour end product.
When the enzyme is not present, the reagent remains reduced and is colourless. From the
results, we can see that, the bacteria does not produce cytochrome c oxidase so it remains
reduced and colourless.( Lau SH, Reddy S,2016)

Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy. The
medium contains citrate as the sole carbon source and inorganic ammonium salts as the sole
source of nitrogen. Bacteria that can grow on this medium produce an enzyme, citrate-
permease, capable of converting citrate to pyruvate. Pyruvate can then enter the organism’s
metabolic cycle for the production of energy. Growth is indicative of utilization of citrate, an
intermediate metabolite in the Krebs cycle. When the bacteria metabolize citrate, the
ammonium salts are broken down to ammonia, which increases alkalinity. The shift in pH turns
the bromothymol blue indicator in the medium from green to blue above pH 7.6. From the
results, the colour of the agar remains green thus the bacteria is citrate negative and does not
use citrate as a carbon source.(Takeshwar Acharya,2016)

Urea is a diamide of carbonic acid. It is hydrolysed with the release of ammonia and
carbon dioxide.. The ammonia combines with carbon dioxide and water to form ammonium
carbonate which turns the medium alkaline, turning the indicator phenol red from its original
orange yellow colour to bright pink. From the results, we can see that there is no colour change
to pink seen. This shows the bacteria does not produce the enzyme urease.( Sherman
Natalie ,2002)

Indole test is used to determine the ability of an organism to split amino acid tryptophan
to form the compound indole. Tryptophan is hydrolysed by tryptophanase to produce three
possible end products – one of which is indole. Indole production is detected by Ehrlich’s
reagent which contains 4- (p)-dimethylamino benzaldehyde, this reacts with indole to produce
a red coloured compound. From the results, we can see that, the presence of a red or red-violet
colour in the surface which indicates the ability of an organism to split amino acid tryptophan
to form the compound indole.( A. Forbes ,1998)

The carbohydrate fermentation test is used to determine whether or not bacteria can ferment
a specific carbohydrate. Carbohydrate fermentation patterns are useful in differentiating among
bacterial groups or species. It tests for the presence of acid and/or gas produced from
carbohydrate fermentation. Basal medium containing a single carbohydrate source such as
Glucose, Lactose, Sucrose or any other carbohydrate is used for this purpose. An Andrade
solution is used as an indicator. Which will detect the lowering of the pH of the medium due
to acid production. Small inverted tubes called Durham tube is also immersed in the medium
to test for the production of the gas (hydrogen or carbon dioxide). From the results, we observe
that glucose, sucrose, lactose, maltose and mannitol were fermented by the bacteria to produce
acid.( Mizer E. Helen ,1999)
 CONCLUSION
Salmonella is a rod-shaped genus, gram-negative, non-spore forming enterobacteria. It is motile with
a diameter of about 0.7-1.5 μm, a length of from 2 to 5 μm, and a flagellum which projectiles in all
directions. In identifying and isolating the bacteria, many tests were perfomed. For the biochemical
test, the urea test and indole test were negative and the citrate test was positive. For the catalase and
glucose fermentation test, the results were positive but for the gram staining test the result was
negative
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