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ADDIS ABABA UNIVERSITY

COLLEGE OF HEALTH SCIENCES
DEPARTMENT OF MEDICINE

MICROBIOLOGY LABORATORY REPORT ON GRAM STAINING

AND CULTURE MEDIA PREPARATION
COURSE: BCDT (MODULE 4)

Group ID NO.

1. ABREHAM GETYE DEJENE-----------------------------UGR/4588/16

2. BIRUK DEGNEH JABIR-----------------------------------UGR/1619/16

3. EBASA WAKJIRA KEBEDE------------------------------UGR/3420/16

4. EYOEL YONAS TERFASA--------------------------------UGR/9961/16

5. FANA GEBRETSADIK GEBREHIWOT--------------UGR/0084/16

6. KALID MOHAMMEDNUR IBRAHIM------------------UGR/0607/16

SUBMITTED TO:AAU CHS medical microbiology department

SUBMITTED DATE: 21/3/2025 G.C.
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Section one: laboratory safety

laboratory safety practices include appropriate facilities and equipment, adequate training, personal
protective equipment, chemical management, standard operating procedures, waste handling, signage,
proper laboratory practices and safe working conditions.

Technical Procedures

Controlling risk through safe conduct of laboratory techniques.

 Avoiding inhalation of biological agents

 Avoiding ingestion of biological agents and contact with skin and eyes

 Avoiding injection of biological agent.

Personal Protection

1. Laboratory coats must be worn at all times for work in the laboratory.

When putting on lab coats, care should be taken to minimize contact with the outer/exposed side of the
material in case the material is contaminated from the previous use.

2. Appropriate gloves must be worn for all procedures.

3. Eye protection

Lab glasses should be put on with clean hands (for example, not after handling microorganisms) to avoid
contamination of the face and the eye protection itself.

Eye protection should be removed after taking off your lab coat with clean hands to avoid contamination
of the head.

4. Hand washing

Personnel must wash their hands after handling infectious materials and animals, and before they leave
the laboratory working areas.

5. It is prohibited to wear protective laboratory clothing outside the laboratory, e.g. in the hallway or
washrooms.

You could contaminate non-lab environments with microorganisms.

Even if your coat is sterile, your appearance will scare others in the college!
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6. Protective laboratory clothing that has been used in the laboratory must be stored in the plastic bag
when not in use.

Section two
Title: Gram staining technique

Purpose
To differentiate Gram-positive from Gram-negative bacteria depending on the structure of their
cell walls.

Principle
Gram Staining is the common, important, and most used differential staining techniques in
microbiology, which was introduced by Danish Bacteriologist Hans Christian Gram in 1884.
This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps
in the classification and differentiations of microorganisms.

Gram positive and Gram negative are two broad classes of bacteria. The classification comes
from the results of the Gram stain test, which in turn, depends on the nature of the bacterial cell
wall. Gram positive bacteria have a thick coating of peptidoglycan and stain purple with crystal
violet. Gram negative bacteria lack this thick coating. They don’t retain crystal violet, so are
stained red or pink with carbol fuchsin or safranin.

Method
A clean glass slide was prepared by adding a loopful of water using a sterilized inoculating loop.
A bacterial sample was then transferred onto the slide using the same sterilized loop and mixed
with the water. The smear was allowed to air dry and then heat-fixed by passing it through a
Bunsen burner three times.

The slide was first stained with crystal violet for one minute and then rinsed with water. Iodine
solution was added as a mordant and left for one minute before washing with water. Acetone was
then applied for 20 seconds to decolorize Gram-negative bacteria, followed by another water
rinse. The smear was then counterstained with safranin for one minute and washed again with
water.
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Finally, the slide was air-dried and examined under a microscope using the oil immersion lens
(100x magnification) to observe Gram-positive and Gram-negative bacteria.

Material required
 Microscopic slide
 Inoculating loop
 Microscope
 Bunsen Burner
 Personal protective equipment
 Petridish
 Dropper
Reagents used

 Stains (crystal violate, safranin),

 alcohol,
 iodine,
 distilled water,
 immersion oil

Procedure
1. A loop full of water drop was added on clean slide using inoculating loop.
2. The loop was sterilized with heat and the sample was added on the slide using the
sterilized inoculating loop
3. The smear was air dried
4. The smear was fixed by passing the smear 3 times on the Bunsen burner and the smear
was made ready for staining
5. The slide was first stained with crystal violet, and then rinsed with water after waiting for
one minute.
6. Iodine was added, and after waiting for one minute, it was washed with water.
7. Acetone was then added (this will decolorize gram-negative bacteria), and after waiting
for 20 seconds, it was washed with water.
8. The slide was counter-stained with safranin for about one minute and then washed with
water.
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9. The sample was air-dried and observed under the oil immersion lens (100x) of a
microscope.

Result
we observe six samples under microscope.

Sample 1- Gram negative, Cocci, staphyl(chain)

Sample 2- Gram positive, Rod, Random

Sample 3- Gram positive, Bacilli, Chain, has some Epithelial cells

Sample 4- Gram positive, Cocci, Cluster, has some Yeasts

Sample 5- Gram positive, Cocci, Cluster

Sample 6- Gram negative, Coccobacilli, Cluster

Sample 7- Gram positive, Bacilli, Chain, has some Epithelial cells

From the observation on the bacterial samples , regardless of their morphology, the gram positive
bacteria retained purple color, while the gram negative bacteria retained pink color, because of
the difference in their cell wall composition.

Interpretation
Gram-positive bacteria have a thick peptidoglycan layer in the cell wall. The primary stain
Crystal violet is fixed in a thick peptidoglycan layer by the Mordant Grams iodine. Crystal
Violet-Iodine complex is insoluble and remains trapped inside the thick peptidoglycan layer. In
the decolorization step alcohol doesn’t wash out this complex. In the next step counterstain,
safranin doesn’t stain because the Crystal Violet-Iodine complex is already occupied the space in
the peptidoglycan layer. So the cell shows the color of crystal violet i.e. Purple. That’s why
gram-positive bacteria stain purple in grams staining.

The cell wall of gram-negative bacteria is complex. The thin peptidoglycan layer is surrounded
by the additional membrane known as the outer membrane. This outer membrane contains more
lipid. Most of the lipids get dissolved by alcohol during decolorization step. Alcohol easily
washes out Crystal Violet-Iodine complex from thinner peptidoglycan layer. Such cells appear
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colorless. In next step counterstain safranin stain such colorless cells to pink-red color. That’s
why gram-negative bacteria stain pink-red in grams staining.

Draw backs
 Due to procedural errors and certain specimen characteristics, there may be false
positives and negatives.
 Some gram-positive organisms may appear to be gram-negative when the primary stain is
lost due to over decolorization.
 Mixed or insufficient cultures may have been used.
 Some bacteria cannot be stained using the Gram-method, such as Chlamydia,
Mycoplasmas and Spirochetes.

Recommendations
In order to achieve a more convenient outcome and more accurate measurement, we would
recommend using a fresh sample. Implement rigorous procedural checks and consider specimen-
specific protocols. Strictly adhere to decolorization time limits; practice controlled application.
Ensure pure, viable cultures are used; re-culture if necessary. Employ alternative staining or
identification methods for suspected non-Gram stainable organisms.

References for section 1 and 2

 sciencenotes.org / https://sciencenotes.org/gram-positive-vs-gram-negative-bacteria/
 microbiologyinfo.com / https://microbiologyinfo.com/gram-staining-principle-procedure-
interpretation-examples-and-animation/
 rbrlifescience.com / https://rbrlifescience.com/category/microbiology/staining/
 bio.libretexts.org/
https://bio.libretexts.org/Bookshelves/Microbiology/Introductory_Bacteriology_Lab_Manual_(T
urnbull)/01%3A_Labs/1.01%3A_Microbiology_Lab_Safety_and_General_Procedures /

SECTION THREE: Sterilization and Disinfection

Sterilization and disinfection are essential aspects of infection control in healthcare settings,
ensuring patient safety and preventing the spread of harmful microorganisms.
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 Sterilization:-Sterilization refers to the process of completely eradicating all

microorganisms, including highly resistant endospores, from a surface or instrument. It
can be classified into three main types:

1. Physical Sterilization

Heat Sterilization:

Moist Heat: Works by denaturing and coagulating proteins, making it more effective than dry
heat. Common methods include pasteurization, autoclaving, tyndallization, and boiling.

Dry Heat: Also denatures proteins but requires higher temperatures and longer exposure times.
Examples include incineration, flaming, and red heat.

Radiation Sterilization:

Ionizing Radiation: Includes gamma rays and X-rays, which penetrate packaging to sterilize
objects.

Non-Ionizing Radiation: UV light is used for surface sterilization but does not penetrate through
solid materials.

2. Chemical Sterilization

Utilizes strong chemical agents such as hydrogen peroxide, heavy metals, ethylene oxide, and
formaldehyde to eliminate microbes.

3. Mechanical Sterilization

Uses high-pressure steam or filtration techniques to remove microorganisms.

 Disinfection

Disinfection is used for instruments and materials that cannot withstand sterilization methods. It
significantly reduces microbial load but does not completely eliminate spores. Disinfectants are
categorized based on their potency:

High-level disinfectants:- eliminate most pathogens, including some spores.

Intermediate-level disinfectants:- are effective against bacteria, fungi, and most viruses.
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Low-level disinfectants:- target common bacteria and some viruses but may not eliminate all
pathogens.

With the rising number of invasive procedures in medical practice, the risk of infections due to
contaminated instruments has increased. Any medical device that comes into contact with sterile
tissues or mucous membranes can introduce infections if not properly sterilized or disinfected.
The consequences of using improperly cleaned medical tools include cross-contamination
between patients, transmission to healthcare personnel, and environmental spread of harmful
pathogens. Several outbreaks have been traced back to insufficient decontamination of medical
equipment, emphasizing the need for stringent infection control measures.

In many parts of the world, inadequate hygiene practices exacerbate these risks. However, this is
not an isolated issue—numerous healthcare facilities globally have reported infections linked to
improperly disinfected instruments, such as endoscopes and surgical tools. To prevent such
occurrences, hospitals and clinics must implement strict protocols for cleaning, disinfecting, and
sterilizing medical devices. Proper training of healthcare workers, routine monitoring, and
adherence to established guidelines are crucial to ensuring patient safety and maintaining high
standards of infection control.

SECTION FOUR
Title: CULTURE MEDIA PREPARATION AND INVITRO DRUG SENSITIVITY
TESTING

Purpose
The purpose of this introductory lab course was to introduce the students to the basic ideas of
culture media. Understanding the meaning of culture media, investigating the several kinds of
media that are available, and discovering their particular applications in microbiology were all
part of this. This lab report tries to illustrate how various culture media offer the required
nutrients and conditions for microbe development by preparing and observing them.
Additionally, this experiment introduced fundamental procedures that are necessary to avoid
contamination and guarantee precise findings in microbiological investigations.
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Principles
A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the
growth of a population of microorganisms. These media contain essential nutrients such as
amino acids, vitamins, carbohydrates and minerals, which provide environment for the
organisms to thrive. In applications like hygiene monitoring, sterilization process validation, and
assessing the efficacy of preservatives and antimicrobial agents, culture media are utilized for
microbial contamination (sterility) tests as well as quality control tests of non-sterile raw
materials and completed goods.

Culture media can be classified based on physical state as

 Solid media

 Semi solid medium and

 Liquid media.

Solid media

It contain agar at a concentration of 1.5-2.0% or some other primarily inert solidifying

agent. Solid medium has a physical structure and allows bacteria to grow in physically
informative or useful ways (e.g., as colonies or in streaks). MacConkey agar, chocolate
agar, nutrient agar, blood agar, etc., are some examples of solid culture media.

Semi solid media

This type of culture media are prepared with agar at 0.5% or less concentrations. Semisolid
medium has a soft custard-like consistency and is helpful for the cultivation of microaerophilic
bacteria or for determining bacterial motility. Motility test medium, Stuart’s and Amies
transport media, etc., are semisolid media.

Liquid media

These media contain specific amounts of nutrients but don’t have a trace of gelling agents such
as gelatin or agar. Commonly used liquid media in the lab are; nutrient broth, glucose broth,
brain-heart infusion (BHI) broth, alkaline peptone water (APW), tryptic soy broth (TSB), and
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selenite F broth. Broth medium serves various purposes such as propagation of many organisms,
fermentation studies, and various other tests.

Based on chemical composition:

 routine laboratory media and

 synthetic media

Routine Laboratory Media

These are classified into six types:

 Basal media,
 Enriched media,
 Selective
 Indicator media,
 Transport media, and
 Storage media.

Basal media

These are simple media that will support the growth of microorganism that do not have special
nutritional requirements. Example – Nutrient agar ,Nutrient broth

Enriched media

These media are required for growth of organism with extra nutritional requirement such as
H.Influenza , Neisseria spp and some streptococcus species.

Selective media

These are media which contain substances that prevent or slow down growth of microorganisms
other than pathogen for which the media are intended
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Differential media

There are media to which dyes or other substances are added to differentiate microorganism

Transport media

These are mostly semisolid media that contain ingredients to prevent the overgrowth of
commensals and ensure the survival of aerobic and aneorobic pathogens when specimens cannot
be cultured immediately after collection

Methods
 Precise media preparation using measured ingredients and sterile techniques.

 Sterilization to prevent contamination.

 Controlled inoculation and incubation for optimal microbial growth.

 Antibiotic disc diffusion for susceptibility testing.

 Quantitative measurement of inhibition zones to assess antibiotic efficacy.

Materials required

 A balance
 Agar (Meuller-Hinton)
 Petri dishes
 Distilled water
 Aluminum foil
 Test tube
 Bunsen burner
 Antibiotics
 Incubator
 Inoculating tube
 Measuring cylinder
 Beaker
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Procedure
1. A dry, dehydrated version of a commercially produced media was prepared.
2. A desired weight of powder medium was weighted.
3. Deionized and distilled water was measured.
4. Using a flask, the powder and the measured water were combined and carefully swirled.
5. Heat sensitive ingredients were added in to the flask.
6. The flask mouth was covered using aluminum foil.
7. Using flaming (dry heat) method, the mouth of the flask was passed through Bunsen burner.
8. The inoculating loop was sterilized using Bunsen burner until it became red.
9. The desired micro-organism was inoculated systematically using inoculating loop on the petri dish
lid.
10. The lid was filled with 15 milliliters of the liquid medium.
11. Antibiotics were added to the petri dish using dispenser disks.
12. The Petri dish was placed in the incubator at the ideal temperature of 35 to 37 degrees Celsius.
Additionally, the diameter of the additional antibiotics' zone of inhibition was noted and valued.

Results
The Petri dish will establish the culture media. The media will be tested for viability using
microorganisms that have previously grown in different culture media. If viable, it is prepared
for microbial growth.
And from the anti-biotics testing, three results can be observed
 Resistant: A pathogen will not respond to treatment with the drug to which it is resistant
irrespective of dose or site of infection.
 Intermediate: A pathogen is likely to respond to treatment when the drug is used in larger
doses than normal or when the drug is concentrated at the site of infection.
 Sensitive :-A pathogen is likely to respond to treatment when the drug is used in normal
recommended doses.

Interpretation

Viability of Microorganisms: The successful growth in Petri dishes confirms that the basal media
provided essential nutrients and optimal conditions for the inoculated microorganisms.
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Growth Characteristics: Observations of colony morphology can help identify different microbial
species, demonstrating the media's versatility.

Antibiotic Efficacy: The zones of inhibition around antibiotic discs indicate the susceptibility of
microorganisms, with larger diameters reflecting higher efficacy.

Validation of Conditions: Comparing results with expected outcomes confirms that the media
and incubation conditions were appropriate.

Quality Control: Successful microbial growth serves as a quality assurance check, emphasizing
the importance of proper media preparation and sterilization.

Drawback
 The disk diffusion method is not a complete indication of how a drug will perform in vivo
(inside the body) due to several reasons. It provides qualitative results rather than a
quantitative measure of minimum inhibitory concentration.
 The results can be affected by several factors including inoculum density (number of
bacteria), agar quality and thickness, incubation time and temperature, and antibiotic disk
quality and storage. These factors can lead to inconsistent results specially if they are not
carefully controlled.
 The size of inhibition zone which indicates effectiveness of the antibiotic is measured
visually which can be subjective and prone to human error leading to variation s in results.

Recommendation
 Antibacterial dilution tests can be used to determine a particular drug’s minimal inhibitory
concentration (MIC), the lowest concentration of drugs that inhibits visible bacterial growth
and, and minimal bactericidal concentration (MBC), lowest drug concentration that kills
≥99.9% of the starting inoculum.
 Consider using alternative methods or calibrating the disk diffusion methods for specific
bacteria/ antibiotic combinations.
 Some antimicrobials and anaerobic bacteria are not well suited for testing with disks.
Consider alternative methods or specialized techniques for these agents.
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Reference for section 3 and 4

 Wikipedia/ https://en.wikipedia.org/wiki/Growth_medium# /2025
 labtestsguide / https://www.labtestsguide.com/culture-media-2
 microbeonline.com / https://microbeonline.com/types-of-bacteriological-culture-medium/