Arbaminch University

College of Natural Sciences

Department of Biology
Internship Final Report
1
03/15/2024
 Internship Final Report
Title : Isolation and Identification of Pathogenic Bacteria

By : Hawi Dereje
ID No. : NSR/1233/13
University Adivisor : EtalemG.
Company Advisor : Semira Ibrahim

Submitted to: AMU, College of Natural Sciences, Department of Biology

03/15/2024 2

December 26, 2023

 Outline
 Introduction
 Components of EPHI department of clinical Bacteriology
 Media Preparation
 Sterilization
 Inoculation
 Identification
 Observation and Results
 Internship Discussion
 Conclusion and Recommendation
 References

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 Introduction
 Background of the study of institute

• EPHI was established by the merging of the former National

Research Institute of Health (NRIH), the Ethiopian Nutrition
Institute (ENI) and the Department of Traditional Medicine (DTM)
in April 1995 by the minister of health.
• The institute focused on major health and nutritional problems,
as well as traditional and modern medicine.
• The merger was affirmed by the council of minister’s regulation
No.4/1996, which recognize the institute as an autonomous public
authority having its own legal personality.
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 It has several departments called;
• Department of bacteriology and Mycology
• Department of parasitology, and others like;
Department of molecular genetics and immunology
/especially in TV, HIV and serological analysis/,Department
of chemical analyses, Department of vaccine preparation
and, Department of traditional medicine.
• Clinical Bacteriology and Mycology National Reference
Laboratory isolate and identify bacteria and fungi from patient
samples and recommend appropriate antibiotics.
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 EPHI provides evidence-based information for public health policy
formation and resource allocation through its key functions including
such as disease surveillance, detection, and monitoring; outbreak
investigation and control; health information analysis for policy
development; research; training; health promotion and health
education; and laboratory science.
 The institute also;
contribute to the development of health science and technology
serve as reference, cause prevention and diagnosis of major
diseases of public health importance
establish
and support national laboratory quality assurance
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program and system.

 Vision, Mission and Values of EPHI

 Vision
 To be Center of excellence public health institute in Africa.
 To see healthy, productive and prosperous Ethiopian people.

 Mission
 The mission for Ethiopian Public Health Institute is to improve the health of
the general public through undertaking research on priority health and
nutrition issues for evidence based information utilization and technology
transfer, effective public health emergency management, establishing
quality laboratory system, training public health researchers and
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practitioners for best public health interventions.

 Values

 Accountability - focus on results

 Creativityand innovation - believe in creative and innovative thinking to
address public health problems.
 Collaboration and partnership - are committed for scientific collaboration
and partnership.
 Customer satisfaction - serve to customers to their best satisfaction with
respect and courtesy.
 Evidence based decision - generate high quality research findings for
evidence based decisions.
 Research ethics and professional commitment - work with integrity and do
not harm to patients and keep the maximum benefit of research to them.
 Team work - work as a team in harmony with maximum knowledge sharing
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 Objectives of the Labratory Experiment
 General objective
• to develop skills; how to use laboratory equipment and chemicals and,
• to learn profession in practical biotechnology activities
 Specific Objectives
• To practice the technique of culture media preparation.
• To understand method of sterilization of media and laboratory
equipment.
• To identify disease causing microbes which are bacteria and fungi.
• To find the most reliable antibiotic that can be used kill the infection
causing bacteria. 03/15/2024 9
 Components of EPHI department of clinical
Bacteriology
 I done my internship in Ethiopian public health institute in the department of
national clinical bacteriology and reference laboratory which concerned on
culturing, isolating and identifying of bacteria from patient sample.
 We develop this technique and isolate and identify different bacteria from
sample of different patient sample.
 The department of bacteriology has four different room for characterization of
disease causing bacteria.
 These are;
• Media Preparation Room
• Sterilization Room
• Inoculation Room
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• Identification Room
 Media Preparation Room

 Media preparation room is the basis and backbone of a microbiology

laboratory.
 In this room we were prepare different culture media that used for culture
and characterize different bacteria.
 What is Culture Media?
• A culture media is a biologic product used to grow bacteria and fungi
that is used for isolation, identification, and susceptibility testing.
• It contains the nutrients the microorganisms need for growth, in
particular an energy source, useable sources of the elements
especially nitrogen, sulfur and phosphorous.
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 General guideline for media preparation
• Follow all manufacturers’ instructions to weigh, dilute/suspend the
dehydrated media.
• Clean glassware carefully with detergent and rinse well with distilled water.
• Use distilled or deionized water to prepare solutions unless specified
otherwise.
• Mix the ingredients using a hot plate and magnetic stir bar placed in the
bottom of the flask.
• Avoid excessive heating.
• Use the manufacturer’s recommended sterilization method.
• For quality control of autoclaving, place specialized tape or paper on the
medium flask at the time of autoclaving. 03/15/2024 12
• Cool sterilized agar media to 45 to 500C or recommended temperature
• Dispense correct volume of media into Petri dishes
• After pouring media, leave lids ajar for 20 minutes to avoid excess
moisture on surface of the agar.
• After approximately 20 minutes, place the lids back on.
• Slant tubed media (e.g. Triple Sugar Iron (TSI) agar/Kligler Iron Agar)
to provide a deep butt (2 to 3 cm) and a short slant.
• After media has hardened, invert the plate (agar side up) and allow
media to cool at room temperature before storage.
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 Label with:
 Media name - accepted abbreviations can be used, such as SBA
(Sheep Blood Agar), MAC (MacConkey agar),
 Lot number
 Date of preparation (can be the lot number) and expiration date.
 Storage

 Store media in the dark, at 2 to 8OC (avoid freezing),

 Tighten caps of tubed media before storage to avoid drying,
 Store plated media in sealed plastic bags (or other containers) to
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avoid drying.
 Types of Media
Media in microbiology are classified on the basis of various criteria,
including;
 Based on state  Based on Purpose
 Solid Media  Simple media
 Semisolid Media  Enriched media
 Liquid Media  Differential media
 Selective media
 Based on Composition  Selective-deferential media
 Chemically-defined (Synthetic):  Storage media
 Complex (Undefined):  Transport media
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 Based on State

• Solid Media: - Solid medium is a  Liquid Media: -These media

mixture of agar and other nutrients. contain specific amounts of
 Examples of solid media are: blood nutrients but don’t have a trace of
agar, nutrient agar, MacConkey agar, gelling agents such as gelatin or
and chocolate agar. agar.
 Semisolid Media: - Semisolid media  For example, Broth medium
are prepared with lower agar has no agar at all.
concentrations of 0.2 to 0.5%.  Other examples include:
 Semisolid media can be used to TSB, Selenite broth
distinguish between typhoid and
colon bacilli.
 Examples: SIM, Cary biller, Amines
transport 03/15/2024 16
 Based on Composition
• Chemically-defined (Synthetic): - Synthetic or defined media
such as Davis & Mingioli medium are specially prepared media
for research purposes where the composition of every
component is well known.
• Complex (Undefined): - The exact composition is not known.
Usually contain complex materials of biological origin, such as;
sheep blood, Milk, Yeast extract, Beef extract.

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 Based on Purpose

• Simple media: - These media support growth of most bacteria. They

don’t have inhibitors or pH indicators.
 Examples: TSB, Nutrient broth.
• Enriched media: - Addition of extra nutrients in the form of blood,
serum, egg yolk, etc, to the basal medium makes an enriched medium.
 Blood agar, chocolate agar are a few examples of enriched media.
 Differential media: -More than one type of micro-organisms grow on it.
Separation is based on color and other characteristic differences.
 Example: Blood Agar. 03/15/2024 18
 Selective-deferentialmedia: -Containing inhibitors (Selective)
and pH indicators (Differential).
 Examples: MAC, MSA
 Storage media- are the ones that store organisms alive.
 Examples are Selenite broth, TSB.
 Transport media- these medium is used to transport samples from
certain reception site to examination center. These samples cannot
be transported in free-state.
 Examples are Carry Blare, Amines and SIM.
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 Sterilization Room

 Sterilization room is one of the most important and basic site for
all activity carried out in the bacteriology room.
 Most material used in the laboratory like glass and plastic were
sterilized in this room using Autoclave at different pressure and
time. Also different media was sterilized, boiled and cooled in this
room.
 The most common material used in this room was Autoclave and
Hotplate for sterilization.
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Autoclave
 Many autoclaves are used to
sterilize equipment and supplies
by subjecting them to high-
pressure saturated steam at
121 °C (249 °F) for around 15–
20 minutes.
 Itis used to sterilize materials
like glass, plastic and the
prepared media to kill spore-
forming Microorganism. Autoclave

 Itis also used to sterilize most

agar and fluid culture media.
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The Hotplate
Used to sterilize the media and
to provide equal pressure for the
media and to mix the
ingredients and also to heat
glassware or its contents. Hotplate

Some hot plates also contain a

magnetic stirrer, allowing the
heated liquid to be stirred
automatically. 03/15/2024 22
 Inoculation Room
 Inoculation room is one of the room found in the Clinical
Bacteriology and Mycology room where different samples
are received and cultivated or inoculated.
 There were several samples come from different healthy
center like blood, nail scraping, throat, pus, wound, urine,
stool, sputum and ear swab.
 There are some steps performed before inoculation.
Specimen collection
• Clinical specimens are submitted for bacteriological
investigation in the diagnosis of infectious diseases.
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These specimens should be collected, stabilized, and transported according to
exact specifications to ensure valid results. Poor specimen quality contributes to
misdiagnosis and inappropriate antimicrobial therapy.

Gram staining
The Gram stain is used to classify bacteria on the basis of their forms, sizes and
cellular morphologies and Gram reactions.
The test was originally developed by Christian Gram in 1884.
Bacteria stain either Gram-positive or Gram-negative on the basis of differences in
their cell wall compositions and architectures.
 Gram-positive species have a thick peptidoglycan layer and large amounts of
teichoic acids whereas Gram-negative species have a single peptidoglycan
layer on their cell wall. 03/15/2024 24

Interpretation of Gram-stained smears involves consideration of
staining characteristics, cell size, shape and arrangement.
 These characteristics may be influenced by many factors,
including culture age, medium, incubation, atmosphere, staining
methods and presence of inhibitory substances.

Gram stain area

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Inoculation Area

It is a place where an artificial seeding or introduction of

microorganisms on/in to culture take place in the area of safety
cabinet.
When inoculating culture media, an aseptic technique must be
used to prevent contamination of specimens and culture media, and
laboratory worker and the environment.
 Immediately before inoculating a culture medium, check the
medium for visual contamination

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Sample Media Culture and Gram Incubation Diagnosis Suspected organisms
staining

Blood BAP,CHP,MAC Direct culture and for Bottles are initially incubated Any growth of Staphylococcus aureus, Group
turbid bottles gram for 18-24 hoursSubculture organism is reported as Streptococcus, E.coli,
staining plates (BAP and CAP) pathogen Klebsiellia pneumonia Proteus
incubated for 24-72 hoursMAC mirabilis
in aerobic incubator for 24

Sputum BAP,MAC,CHP Direct culture and Gram Subculture plates(BAP and Pneumonia Streptococcus pneumonia,
staining CAP) Klebsiellia

Urine BAP,MAC Do not gram staining Subculture plates(BAP and Urinary tract infections E.coli, Proteus
MAC) incubated for 2

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 Identification Room
 It is one the important room in clinical bacteriology laboratory;
helps to identify and characterize different pathogenic bacteria.
 Multiple tests are done in this room for getting enough clues to
identify the specific bacteria.
 These can be categorized as;
 Solid media test
 Enzyme test
 Biochemical media test

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Solid media test
 BAP test - Blood agar plate is a type of growth medium often used to
grow fastidious organisms and to differentiate bacteria based on their
hemolytic properties.
The bacteria are classified as;
 α-hemolytic bacteria - partially distract RBCs and greenish to
brownish color colonies are formed.
 β-hemolytic bacteria - completely distract RBCs and whitish or
colorless colonies are formed.
 γ- hemolytic bacteria or non-hemolytic bacteria - which don’t
distract RBCs.
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Growth of bacteria on BAP
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 MAC test - MacConkey agar is selective and differential medium for gram
negative bacteria which identify bacteria based on lactose/non lactose
fermenting nature of bacteria.
 The bacteria are classified as;
 Lactose fermenter; bacteria pinkish colonies are formed.
 Non-lactose fermenter bacteria; whitish or yellowish colonies are formed.

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Lactose fermenter bacteria
 MSA test - Mannitol salt agar is used as selective and differential
medium for the isolation of staphylococcus aureus from clinical
and non-clinical specimens.
 Gram +ve staphylococcus fermenting mannitol indicate by
yellow color.
 Gram +ve staphylococcus which cannot ferment mannitol does
not change color.

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Enzyme test
•Catalase test - This test is used to identify organisms that produce the
enzyme, catalase. This enzyme detoxifies hydrogen peroxide by breaking
it down into water and oxygen gas.
The bubbles resulting from production of oxygen gas clearly indicate a
catalase positive result.
 The catalase test separates staphylococci (positive) from streptococci
and enterococci (negative).
 For spore forming organisms, Bacillus spp. are catalase positive, and
Clostridium spp. are catalase negative.
 Neisseria gonorrhoeae produces an enhanced elaboration of bubbles
not seen with other members of the genus due to superoxol.
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 Coagulase test - Coagulase is an enzyme that clots blood plasma.
Coagulase is a virulence factor of S. aureus. This test
differentiates Staphylococcus aureus from other coagulase
negative Staphylococcus species.
 Oxidasetest - The oxidase test is used to detect the production of
the enzyme cytochrome oxidase by bacteria.
Development of a deep blue to purple color in 10-30 seconds
is a positive reaction.
Negative Oxidase test is no color change in 60 seconds.
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Biochemical media test

Med ia Positive Negative Interpretation for positive result

result result
Urea Pink Yellow Urease enzyme prod ucer
microorganism and acid ic
environment is establishe d .
SIM Black color Yellow color Black Color-Formation of H2S
Pink color before and Tryptophan prod ucer
after Kovax after Kovax’s microorganism after Kovax’s
reagent reagent reagent or Indole positive
Diffusion of Diffusion ind icts motile
suspension microorganism

Citrate Blue Green Microorganism uses sodium

citrate as carbon source
KIA Crack Light red Yellow butt- only glucose is
Yellow butt fermented
Yellow slant Yellow butt and slant- glucose,
and butt lactose and are fermented
Crack- gas formation
LDA Pink or red Purple slant Prod uces lysine deaminase
LIA slant
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LDC Purple butt Yellow butt Prod uces lysine decarboxylase
 Observation and Results
 Results from media preparation room

SDA agar results

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CHP agar results TSB agar results BAP agar results

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Biochemical agar results

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 Result from inoculation process
• The samples used for the experiment/ in our internship period/
was blood. After incubation time different results have been
observed in different media.
Result -Macconkey agar plate
 Bacteria that grew was lactose fermenter
- Gram stain
 Gram stain is necessary to check the bacteria really negative or not,
and also to know its arrangement.
The bacteria was Gram negative diplococcus.
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 - Blood biochemical test
 Oxidase; positive
 Catalase test; positive result
 Acid production in glucose and maltose
- KIA test; positive result
- SIM test; positive result

 The final sum up result was N. meningitides.

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 Internship Discussion
 The practice section in EPHI help me/us develop a technique of media
preparation, sterilization of media and lab equipment, inoculation of bacteria
sample; study characteristics of bacteria and identification of target bacteria
from a given sample.
 Here are some of the things we discussed about;
• General guideline of media preparation
• why some media are selective and have color formation after culture.
Challenges
Ingeneral I/we spent a good time working and studying during my/our internship
and I/we got good experience in media preparing.
But,there was some challenges with timely allocation of money and the air
03/15/2024 41

condition also was also a bit challenging.

 Conclusion and Recommendations
 Conclusion  Recommendation
 Generally, in this internship I have got Here are some of the recommendations
good skills and knowledge about of mine about the training;
bacteriology lab. I learned a lot of  The samples does not come on time.
about laboratory activates and  The training time was short so we
technical method; I performed the have not enough time to gain wide
media preparation by myself that knowledge.
makes me to have a real hand  The molecular room of bacteriology
practice in the laboratory. must be functional for clinical test
 I also learned how the media are and for internship student training
sterilized and inoculate in preferred purpose.
medium and also I learn  The financial issues of our campus
differentiation and identification of sholud be corrected. /Money for the
03/15/2024 42

bacteria. training/
References

 Laboratory manuals (Standard operating procedure(SOP) of

company.
 Manual for the Laboratory Identification and Antimicrobial
Susceptibility Testing of Bacterial Pathogens of Public Health
Importance in the Developing World. U.S. Centers for Disease
Control and Prevention (CDC), Atlanta, Georgia, U.S.A, and World
Health Organization (WHO) Geneva Switzerland. 2003.
 Paul W. (2010). General microbiology manual. 2 nd edition.

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Thank You!

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