Bio and Emerging technologies institute

Biotechnology department

Internship laboratory report presentation on screening of

bacteriocin producing lactic acid bacteria and isolation and
identification of salmonella

Date: October 10, 2022 G.C

• Lab mentor :- Tigest
• Interns :-
• Bethelhem Mandefro
• Etsubdink Ephrem
• Hanna yoseph
• Mariamawit Yimer
• Natnael Tamirat
• Salem Sebsebe
• Thomas Masresha
• Zetseat Admasu

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 Activity One
Screening of bacteriocin producing lactic acid bacteria from
milk, yogurt and bacterial isolate
1. Sample collection and isolation.
- By Bethelehem Mandefro

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2. Agar well diffusion assay – Part One
- By Zetseat Admasu

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Objective
• To separate the product (supernatant) of lactic acid bacteria from it
• To neutralize the acidity of the supernatant

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 procedure
• MRS broth media prepared (for to 22 samples)
• The bacteria inoculated in the MRS broth from the MRS media and
incubated
• After incubation the broth mixed well and transferred 2ml of
bacterial broth to eppendorf tube
• Centrifuged for 10 min in 10000 rev/min in centrifuge
• The PH meter placed in the PH value of 7 and 4 buffers and
stabilized
• The supernatant dispensed on the beaker and measured its PH and
adjusted the PH to be neutral (PH= 7) by adding a drop of
base(NaOH) or acid(HCl)
• Then the neutralized supernatant moved to new eppendorf tubes

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 Result
• When the PH value of the whole supernatant measured the value
was in the range of 4.4 up to 4.7

• After the addition of a drop of base(NaOH) the PH value range

became from 6.75 up to 7.00

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 Discussion
• If we want the product that produced by lactic acid bacteria we
should remove the bacteria and take the supernatant and check the
anti bacterial effect of the product.
• Most of the time lactic acid bacteria are acidic or there PH is low .
if we apply on other bacteria the acidic effect will not make them
grow .So we can’t know whether the lactic acid bacteria produce
anti bacterial product(bacteriocin) or not. But if it is neutralized
and not allowed bacterial growth we sure that something in it other
than the acidity.

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3. Agar well diffusion assay – Part Two
- By Etsubdink Ephrem

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Chemicals and materials
• Mullen Helton agar • Flask
• H2so4 • Tips
• BCl • Pipettes
• Distill water • Petridis
• E.coli • Beaker
• P.mirab • Magnetic stirrer
• protases • Ependorph tube
• L.p • Sensitive balance
• S. Aeurase • Laminar airflow hood
• E fecales • Auto clave

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 Agar well diffusion assay
Introduction
• The agar diffusion assay is one method for quantifying
the ability of antibiotic to inhibit bacterial growth
• Agar well diffusion method is widely used to evaluate
the antimicrobial activity of plats or microbial extract
• Agar diffusion test is qualitative, easy to perform and
simple

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 Cot’d
• First we prepared 200ml of Mullen henton agar for 10 plate
• Why we used Mullen Helton agar ?
1000ml=38g of MHA
200ml=7.6g
• Stare with magnetic stirrer after that autoclaved for 15 min
by 121oc

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cont’d
• After 1 hr dispensed on 10 plate and we waited until the agar
will be solidify
• Stare with magnetic stirrer after that autoclaved for 15 min
by 121oc

• Then prepared McFarland solution

HOW TO PREPAR MACFERLAND SOLUTION
• We took 1 % of BCl =0.005 and 1% OF H2so4=9.995ml
• We prepared 10 ml =5% of McFarland solution =1.2*10^-18
number of living thing

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Cont’d
• Then we took 1ml water and 0.0025ml supernatant of from 7
bacteria for each ependroph tube
• such as : E.coli
S.auerus
P mirab
E.feacales
L-P
Protease

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 Cont’d
• Then compared with McFarland.
• If the bacteria has less denser than McFarland solution we
added some water if the bacteria has more denser than
McFarland solution we added some bacteria until the
turbidity become the same.
• After that we took the plate that carry Mullen Helton agar
• Than we took 100ml from bacteria then swab on Mullen
Helton agar.
• Then we created 5 holes on Mullen Helton agar
• We used 2 controls and 5 treatments.

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Cont’d
• For the first hole we added negative control we used distill
water.
• For the second hole we added positive control we used anti
bacteria which is gentamicin.
• For the 3 reaming hole we used treatment that took from 7
bacteria.
• After that putted on incubator for 2 day.

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 Cont’d
• After 2 day we saw the result the result was from 10
sample 2 samples was potential 3/3t E.coli and 36/t p.
mirab was in habited.
• It has anti-bacterial property which means it produce
lactic acid bacteria and both have bactrocin so we needed
lactic acid bacteria that produce bacteriocin.
• We took both 3/3t e.coli and 36/t p. mirab

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Cont’d

• Then we measured the diameter the first length was 12 the

second 13 the third was 16
• 12+13+16%3
• After that Re swab the reaming un potential, not in habited
result on Mullen Helton agar the result was from 4 sample 1
sample was potential and in habited.
• In general from 10 samples 5 samples was potential, in
habited and produce lactic acid that produce bacteriocin the
other remaining sample was not potential.

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 Activity - Two
Isolation and identification of salmonella from bacterial isolate
4. Isolation of salmonella
- By Salem Sebsebe

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5. Gram staining and catalase test
- By Thomas Masresha

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Test - 1
Gram staining
Objective
• To identify salmonella by its color and shape after gram staining.

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Procedure
1. Smear preparation
2. Addition of methylene blue (crystal violate)
3. Addition of iodin
4. Addition of acetone
5. Addition of safranin
6. Microscope adjustion and oil
immersion

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Result

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Discussion
• Given procedures were followed precisely.
• For all the smears results were obtained interpretations were made.
• For salmonella our staining showed pink / safranin colored and rod
shaped samples assuring for gram negative interpretations.

Conclusion
• We have successfully identified potential salmonella species.

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Test - 2
Catalase test
Objective
• To identify potential salmonella species using catalase test.

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Procedure
1. 3% H2O2 preparation
2. Smear preparation (no heat fixing)
3. Adding a drop of H2O2

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Results

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Discussion
• According to references salmonella can produce catalase
enzyme.
• For a potential salmonella species next to the gram stain our
samples are all catalase producers.

Conclusion
• All potential salmonella species are catalase producers / Positive.

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6. TSI (Triple sugar iron) test
- By Hanna Yoseph

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Objective
To determine the ability of Salmonella bacteria to ferment glucose, lactose, and sucrose,
and their ability to produce hydrogen sulfide as well as produce gas.

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 Introduction

● The triple sugar- iron agar test employing Triple Sugar Iron Agar is designed to differentiate
among organisms based on the differences in carbohydrate fermentation patterns and
hydrogen sulfide production. Carbohydrate fermentation is indicated by the production of
gas and a change in the color of the pH indicator from red to yellow.
● To facilitate the observation of carbohydrate utilization patterns, TSI Agar contains three
fermentative sugars, lactose and sucrose in 1% concentrations and glucose in 0.1%
concentration. Due to the building of acid during fermentation, the pH falls. The acid base
indicator Phenol red is incorporated for detecting carbohydrate fermentation that is
indicated by the change in color of the carbohydrate medium from orange red to yellow in
the presence of acids.

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 Cont’d
● In case of oxidative decarboxylation of peptone, alkaline products are built and the pH rises. This
is indicated by the change in color of the medium from orange red to deep red. Sodium thiosulfate
and ferrous ammonium sulfate present in the medium detects the production of hydrogen sulfide
and is indicated by the black color in the butt of the tube.

● To facilitate the detection of organisms that only ferment glucose, the glucose concentration is
one-tenth the concentration of lactose or sucrose. The meagre amount of acid production in the
slant of the tube during glucose fermentation oxidizes rapidly, causing the medium to remain
orange red or revert to an alkaline pH. In contrast, the acid reaction (yellow) is maintained in the
butt of the tube since it is under lower oxygen tension.After depletion of the limited glucose,
organisms able to do so will begin to utilize the lactose or sucrose. To enhance the alkaline
condition of the slant, free exchange of air must be permitted by closing the tube cap loosely.
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Materials Used
● TSI Agar, straight inoculation needle, well-isolated colony, balance, culture tube, Incubator

● Media of TSI agar Contains:-Enzymatic digest of casein (5 g), enzymatic digest of animal
tissue (5 g), yeast enriched peptone (10 g), dextrose (1 g), lactose (10 g) sucrose (10 g),
ferric ammonium citrate (0.2 g), NaCl (5 g), sodium thiosulfate (0.3 g), phenol red (0.025
g), agar (13.5 g), per 1000 mL, pH 7.3.

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Method
● With a straight inoculation needle, touch the top of a well-isolated colony.
● Inoculate TSI by first stabbing through the center of the medium to the bottom
of the tube and then streaking the surface of the agar slant.
● Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air for 18
to 24 hours.
● Examine the reaction of medium.

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Expected results
● An alkaline/acid (red slant/yellow butt) reaction: It is indicative of dextrose
fermentation only.
● An acid/acid (yellow slant/yellow butt) reaction: It indicates the fermentation
of dextrose, lactose and/or sucrose.
● An alkaline/alkaline (red slant, red butt) reaction: Absence of carbohydrate
fermentation results.
● Blackening of the medium: Occurs in the presence of H2
● Gas production: Bubbles or cracks in the agar indicate the production of gas (
formation of CO2and H2)

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Results observed

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Discussion
It is recommended that biochemical, immunological, molecular, or mass
spectrometry testing be performed on colonies from pure culture for complete
identification.
● It is important to stab the butt of the medium. Failure to stab the butt
invalidates this test. The integrity of the agar must be maintained when
stabbing. Caps must be loosened during this test or erroneous results
will occur.
● TSI Agar must be read within the 18-24 hour stated incubation period. A
false-positive reaction may be observed if read too early. A false-
negative reaction may be observed if read later than 24 hours.
● An organism that produces hydrogen sulfide may mask acid production
in the butt of the medium. However, hydrogen sulfide production
requires an acid environment, thus the butt portion should be
considered acid.
● TSI is not as sensitive in detecting hydrogen sulfide in comparison to
other iron containing mediums, such as Sulfide Indole Motility (SIM)
Medium.
● Certain species or strains may give delayed reactions or completely fail
to ferment the carbohydrate in the stated manner.
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 Conclusion
● TSI test determines the ability of Salmonella bacteria to ferment glucose,
lactose, and sucrose, and their ability to produce hydrogen sulfide as well as
produce gas.The test is used primarily to differentiate members of the
Enterobacteriaceae family from other gram-negative rods. It is also used in
the differentiation among Enterobacteriaceae on the basis of their sugar
fermentation patterns.
● In this experiment non lactose fermenter plus gas producers and hydrogen
sulphide producers were considered as potential and were to be further
examined.

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7. Motility and citrate test
- By Mariamawit Yimer

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8. Urease test
- By Natnael Tamirat

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 INTRODUCTION

• Urease test helps for the identification of urease positive & to

differentiate it from other non-lactose fermenting Enterobacteriaceae
family.
• Urease test is used for the presumptive evidence of the presence
of Helicobacter pylori in tissue biopsy material.
• the test also uses to know the utilization of ammoniam by help of urase
enzyme.
• Stuart’s Urea broth media It was commonly used to get rapid results of the
urease test,
• Urea Agar (Christensen) was developed by Christensen in 1946 for the
differentiation of enteric bacilli.
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Principle of Urease Test
• Urea is the product of decarboxylation of amino acids.
• Hydrolysis of urea produces ammonia and CO2

• 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 color to bright
pink.

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Media used in Urease Test
• Christensen’s Urea Agar
• Preparation
• 1.Dissolve the ingredients in 100 ml of distilled water and filter sterilize
(0.45-mm pore size).
• 2.Suspend the agar in 900 ml of distilled water, boil to dissolve
completely.
• 3.Autoclave at 121 degree C and 15 psi for 15 minutes.
• 4.Cool the agar to 50 to 55 degree C.
• 5.Aseptically add 100 ml of filter-sterilized urea base to the cooled agar
solution and mix thoroughly.
• 6.Distribute 4 to 5 ml per sterile tube (13 x 100 mm) and slant the tubes
during cooling until solidified.

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Procedure of Urease Test

• 1.Streak the surface of a urea agar slant with a portion of a

well-isolated colony or inoculate slant with 1 to 2 drops from
an overnight brain-heart infusion broth culture.
• 2.Leave the cap on loosely and incubate the tube at 35°-37°C
in ambient air for 6 hours to 7 days.
• 3.Examine for the development of a pink color for as long as u
see change.

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Result
• The result after 24 hours of
incubation in 35-36°c is shown
below.
• The result is delayed +ve,
• The limitations might be because
of less
• incubation time, if we have given
it more
• Incubation time it will turn to
• full bright pink.

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DISCUSSION
• Positive Reaction: Development of an intense magenta to bright pink
color in 15 min to 24 h.
• Examples: Proteus spp, Cryptococcus spp, Corynebacterium spp, Helicob
acter pylori, Yersinia spp, Brucella spp, etc.
• Negative Reaction: No color change.
• Examples: Escherichia, Shigella, Salmonella, etc.
• If organism produces urease enzyme, the color of the slant changes from
light orange to magenta.
• If organism does not produce urease the agar slant and butt remain
light orange (medium retains original color).
• The pH at 6.8 and between 7.5-8.1, the colour of the phenol red
indicator
appears yellow-orange and red, respectively.
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