COLLAGE OF HEALTH SCIENCE

DEPARTMENT OF ANESTHESIA
DIAGNOSTIC MEDICAL MICROBIOLOGY LAB
REPORT
GROUP 1
Group Members
NO NAME ID
1. GEBIRU MITIKU UGR/0390/13
2. HABTAMUA ESSAYAS UGR/8364/13
3. IRSHAD YUSUF UGR/0378/13
4. KUMALA FUFA UGR/2101/13
5. RUZIYA DESTA UGR/6728/13
6. SENA GUTA UGR/4368/13
Submitted to Mr. Shemse S.
May 3, 2022
 Morphological test
Morphological tests are tests used to investigate the physical structure of microbes.
Here we see gram stain and acid fast stain
1.Gram Stain
The Gram stain is a differential stain commonly used to differentiates bacteria on the
basis of their cell wall structure.This test differentiate the bacteria into Gram positive
and negative. It is also used for fungal staining.
Principle
When the bacteria is stained with primary stain Crystal Violet and fixed by the
mordant, some of the bacteria are able to retain the primary stain and some are
decolorized by alcohol. The cell walls of gram positive bacteria have a thick layer of
protein-sugar complexes called peptidoglycan and lipid content is low. Decolorizing
the cell causes this thick cell wall to shrink, which closes the pores in the cell wall and
prevents the stain from exiting the cell. So the ethanol cannot remove the Crystal
Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram
positive bacteria and appears blue or purple in colour.
In case of gram negative bacteria, cell wall also takes up the crystal violet-Iodine
complex but due to the thin layer of peptidoglycan and thick outer layer which is
formed of lipids,crystal violet-Iodine complex gets washed off. When they are
exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the
crystal violet-iodine complex to leach out of the cells. Then when again stained with
safranin, they take the stain and appears red in color.
Materials
1. Light Microscope
2. Glass Slide
3. water
4. Immersion Oil
5. Wire Loop or use inoculating loop
6. Gram stain reagent
i. Primary stain (Crystal Violet)
ii. Mordant (Gram’s Iodine)
iii. Decolourizer (Acetone Alcohol)
iv. Counterstain (Safranin)
Procedure
1. Using a sterile inoculating loop, add 1 drop of sterile water to the slide. Prepare a
mixed smear of the specimen
2. Air dry and Heat fix.
3. Cover the smear with Crystal Violet (primary stain) for 1 min and wash off the
slide with water
4. Add Gram’s Iodine (mordant) for 1 min and wash with water
5. Decolorize with 95% ethanol(acetone alcohol). Stop decolorizing with alcohol as
soon as the purple color has stopped leaching off the slide (time will vary depending
on thickness of smear). immediately wash with water.
6. Cover the smear with Safranin for 30 seconds.
7. Wash both the top & the bottom of the slide with water.
8. Blot the slide dry with bibulous paper.
9.Examine your slide with the microscope, starting out with the low power (10X)
objective and working your way up to the oil immersion lens (100X objective) of the
microscope.

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10.Under the 100x objective, note whether the bacteria are Gram positive or negative,
based on their color.
Result and interpretation
Gram positive bacteria appear in purple and the negative are pink/red. In addition to
the cell wall structure, gram stain aids in the shape and arrangement of the bacterium.
For example,During lab demonstration, we observed the Gram positive spherical
bacteria arranged in cluster (staphylococcus) under microscope.
If the organism under investigation is fungi, they take the color of gram positive
bacteria but they are different by their size (about 2-3x larger)

gram positive(left) and negative bacteria (right)

2.Acid fast stain
The acid-fast stain is a differential stain that separates bacteria on the basis of the
lipid content of their cell walls.The main aim of this staining is to differentiate
bacteria into acid fast group and non-acid fast groups. This method is used for those
microorganisms which are not stained by simple or Gram staining method,
particularly the member of genus Mycobacterium, are resistant and can only be
visualized by acid-fast staining.

Principle

When the smear is stained with carbolfuchsin, it solubilizes the lipid material present
in the Mycobacterial cell wall but by the application of heat, carbolfuchsin further
penetrates through the lipid wall and enters into cytoplasm. Then after all cell appears
red. Then the smear is decolorized with decolorizing agent (3% HCL in 95% alcohol)
but the acid fast cells are resistant due to the presence of large amount of lipoidal
material in their cell wall which prevents the penetration of decolorizing solution. The
non-acid fast organism lack the lipid material in their cell wall due to which they are
easily decolorized, leaving the cells colorless. Then the smear is stained with
counterstain, methylene blue. Only decolorized cells absorb the counter stain and take
its color and appears blue while acid-fast cells retain the red color.

Materials/ Equipments;
1. Microscope
2. wire loop
3. Immersion oil

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 4. Sterile water
5. Microscopic slide
6. Acid-Fast reagent

i. Basic dye (Carbolfuchsin)

ii. Decolourizer ( 3% of Acid Alcohol)
iii. Counterstain (Methylene Blue)

Methods;

1. Ziehl-Neelsen stain: uses steam heat (hot method) and the most
common one
2. Kinyoun stain: don’t use heat (cold method)

Procedure of Ziehl-Neelsen staining

1. Add one loopful of sterile water to a microscope slide.
2. Make a heavy smear of Mycobacterium tuberclosis obtained from the patient’s
sputum. Mix thoroughly with your loop.
3. Air dry and heat fix well.
4. Cover the smear with carbolfuchsin.
5. Place the slide on the rack over dry heat for 2 minutes.
6. Cool and rinse with water.
7. Decolorize by placing a drop of acid alcohol on the slide and allow to sit for 15
seconds and wash the slide with water
8. Counterstain with Methylene Blue for 30 seconds to 1 minute.
9. Wash and blot the slide dry with bibulous paper.
10. Focus 10X - then use oil immersion
Result and its interpretation
During lab demonstration, we observed red curved rod mycobacterium tuberclosis
(acid fast bacilli) under light microscope. So,it is reported as Acid fast bacilli positive

Acid fast bacilli (+)

Biochemical tests

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Biochemical tests are tests used to identify bacteria based on their activity toward
different biochemical compounds. They are used as diagnostic tool for certain
bacterial infections because of their ability to differentiate morphologically similar
species. Here we will look catalase and coagulase test.
1. Catalase test
Catalase test is test used to distinguish between Staphylococci and Streptococci ( or
more generally aerobic and anaerobic bacteria).Catalase is a peroxidase enzyme that
converts hydrogen peroxide into water and oxygen.
Principle
Catalase test normally uses the ability of the enzyme to convert hydrogen peroxide
(H2O2) into H2O and O2. If the bacterium under investigation is catalase positive, the
following reaction will take place
2H2O2(l) → 2H2O(l)+O2(g)
This reaction is confirmed by rapid formation of gas bubbles on the test slide (the
tube) which is the oxygen being released. To confirm positive result, the bubble
formation must be rapid because catalase is fast acting. If there is bubble formation
but slow, that may be due to another factors like light scattering rather than the
catalase.
Materials required
 Test slide (tube)
 Hydrogen peroxide
 Applicator stick (inoculating loop)
 A 24-hr bacterial culture
Method
Catalase test can be done either by slide test using 3% H2O2 or tube method using
15% H2O2
Procedure
A. Slide test
Prepare 3% hydrogen peroxide by adding 3 ml of the regent and filling the tube until
it reaches 100ml. Then,
a) First, sterilize glass slides by either using an autoclave or a hot air oven.
b) Then, take 24 hours old bacterial inoculum via a sterilized inoculating loop or
applicator stick under sterile conditions.
c) Prepare a bacterial smear over the glass slide.
d) Add 1-2 drops of H2O2 to the top of the bacterial smear.
e) Observe the glass slide for the formation of bubbles.
B. Tube test
f) Prepare the 15% of the regent using the above principle. Then,
g) First, sterilize the test tubes by either using an autoclave or a hot air oven.
h) Then, pour 1-3 ml of hydrogen peroxide into the tubes under sterile conditions.
i) After that, take 24 hours of bacterial inoculum via a sterilized inoculating loop.
j) Dip the inoculating loop straight into the test tube containing H2O2
k) Observe the tubes for instant bubbling.
Result and interpretation
Positive result-if there is rapid formation of bubble, the culture contains a catalase
positive bacterium like Staphylococcus ( or generally aerobic bacterium).
Negative result- if there is no formation of bubble the culture is catalase negative
bacteria like Streptococci.

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Limitations of Catalase Test
 To perform a catalase test, a test organism incubated for 18 to 24 hours should be
used which makes the procedure a little bit delayed.
 Hydrogen peroxide should be freshly prepared to perform the experiment, as it is
a volatile compound.
 Test organism inoculated from the blood agar media frequently gives false-
positive results, as the RBCs are catalase-positive cells
2. Coagulase test
Coagulase test is a test used to differentiate Staphylococcus aures and clinically
significant coagulase negative Staphylococci(CONS) such as S. epidermidis and S.
saprophyticus.
Principle
Coagulase test examines whether the suspected bacterium produces the enzyme
coagulase. This enzyme degrades fibrinogen into fibrin. If the bacterium is S. aureus,
which is coagulase positive, there is conversion of fibrinogen to fibrin and this is
confirmed by formation of agglutinate on the test slide (or in the tube).
S.aureus produces two types of coagulase:bound and free. The bound
coagulase(clumping factor) cross-links the α and β chain of fibrinogen in plasma to
form fibrin clot that deposits on the cell wall. As a result, individual coccus stick to
each other and clumping is observed. The free coagulase secreted by S.aureus reacts
with coagulase reacting factor (CRF) in plasma to form a complex, which is thrombin.
This converts fibrinogen to fibrin resulting in clotting of plasma.
Materials required
 Glass slide or tube
 Applicator stick
 Fresh culture
 Plasma- preferably rabbit plasma because it gives better clotting and is free of
inhibitors. Human plasma contains anticoagulants like sodium citrate that
inhibits the action of the enzyme, hence false result.
Methods
 The slide coagulase method for bound coagulase
 The tube coagulase method for free coagulase
Procedure
A. Slide test
a) Take a clean, grease-free glass slide.
b) Pour one drop of undiluted blood plasma towards the centre of the glass slide.
c) Sterilize an inoculating loop and carefully take the bacterial suspension.
d) After that, mix the bacterial suspension with the drop of blood plasma until the
formation of milky suspension.
e) Allow the glass slide to stand for a few seconds, and then note down the
results based on the agglutination reaction.
B. Tube test

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 a) Perform serial dilution of the blood plasma in a series of sterile test tubes.
b) Pour 1 ml of diluted plasma into the sterile tube.
c) Sterilize an inoculating loop under the flame and carefully take the bacterial
suspension.
d) After that, emulsify the bacterial suspension with plasma until the formation of
a milky suspension.
e) Allow the test tube to stand for 4 hours in a hot water bath.
f) At last, note down the results based on the clot formation by slightly tilting the
tube.
Result and interpretation
Positive result- if there is formation of fibrin clot confirmed by clumping or
agglutination, the bacterium is S.aureus
Negative result- if there is no formation of fibrin clot the bacterium is one of the
CONS.

Serological Tests
Serological test, also called serology test or antibody test, is any of several laboratory
procedures carried out on a sample of blood serum (the clear liquid that separates
from the blood when it’s allowed to clot) for the purpose of detecting antibodies or
antibody-like substances that appear specifically in association with certain disease.
By the following discussions, we are going to see Widal test, Weil felix test, and tests
for HIV and HBV viruses.
1. Widal test
Widal test is an agglutination test which detects the presence of serum agglutinins (H
and O) in patients serum with typhoid and paratyphoid fever which are the result of
the genus Salmonella.The patient’s serum is tested for O and H antibodies
(agglutinins) against antigen suspensions (usually stained suspensions)
Principle
The main principle of widal test is that if homologous antibody is present in patients
serum, it will react with respective antigen in the reagent and gives visible clumping
on the test card and agglutination in the tube. The antigens used in the test are “H”
and “O” antigens of Salmonella typhi and “H” antigen of S.paratyphi. The
paratyphoid “O” antigen are not employed as they cross react with typhoid “O”
antigen due to the sharing of factor 12. “O” antigen is a somatic antigen and “H”
antigen is flagellar antigen.
It is preferable to test two specimens of sera at an interval of 7 to 10 days to
demonstrate a rising antibody titre. The antibody stays long in the serum even after
clearance of the bacterium and therefore, the test is done twice at acute and
convalescence stage to avoid false positive results.
Materials
 Antigens

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  0 ( taken from cell wall)
 H –from flagella
 Vr-from virulence factor
 Test card
 Micro pipette(used to measure small amount of fluid)
Procedure
 Put 40micro litre serum on the test card
 Add one drop of antigen suspension (O,H ) on each serum(test card)
 Mix and rotate for 1-2 minute
Result and interpretation
We saw agglutination (antigen antibody reactive reaction). It indicates presence of
clinically significant levels of the corresponding antibody in the patient serum.
Agglutination indicates presence of antibody in the patient serum and No
agglutination is a negative test result and indicates absence of clinically significant
levels of the corresponding antibody in the patient serum.
2. Weil felix Test
The Weil-Felix test detects typhus and specific rickettsial infections. Rickettsia is
bacteria transmitted by ticks, fleas, lice and is the root of diseases in human beings.
Principle
Weil-Felix reaction is a serological test for detecting Rickettsial antibody in serum of
patient using heterophile antigen. This is a non specific agglutination reaction.
Antibodies produced against Rickettsial antigen cross reacts with OX-19 and OX-2
strain of Proteus vulgaris and OXK strains of Proteus mirabilis. This cross reactivity
between some strains of Rickettsiae.
Patients infected with R. rickettsiae develops antibody which is reactive to Proteus
vulgairis OX19 and OX2 while patients infected to R. prowazekii develops antibody
which is reactive to Proteus vulgaris OX-19.
Anti-Rickettsial antibodies develops within 5-7 days of infection and titer reaches
peak within 2 weeks. Serum of typhus patient agglutinate in presence of non motile
Proteus vulgaris OX strain. Somatic antigen (O) of Proteus vulgaris reacts with anti-
Rickettsial antibody giving agglutination reaction.
Material
 Antigens suspension
 OX -19(Commonly used)
 OX-2
 OX-K
 Test card
 Pippete
PROCEDURE
• Put 40micro litre serum on the test card
• Add one drop of antigen suspension (OX 19, OX 2) on each serum(test
card)
• Mix and rotate for 1-2 minute
Result and interpretation
If we see OX 19 and OX 12 reaction, which indicates positive result (the presence of
antibodies in the patients serum. The Weil Felix test can be an important diagnostic
tool in screening for typhus and various other Rickettsial infections. The Weil Felix
test positive treatment would depend on the type of infection.
Antibiotics Susceptibility Test(AST)

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Antibiotics susceptibility(sensitivity) test are tests used to examine the potency and
effectiveness of a given antibiotics against a suspected bacteria for effective
treatment. It is a procedure done to identify the susceptibility of a microbe to a
suitable antibiotic.
Principle
AST uses the ability of the antibiotics to inhibit the growth of the bacterium under
investigation. Certain culture is used and the formation of clear zone around the anti-
microbe drop ensures its ability to inhibit the growth of the bacterium (this is what
Alexander Fleming used for his sudden discover of penicillin, the first antibiotics).
Materials
Suitable culture media
Sample of the bacterium
(these can be avoided if there is already prepared culture)
Filter papers
Incubator
Methods
1) Agar Plate method
2) Dilution method
Procedure
1. Agar plated method
 Here, the nutrient agar is used as a substrate for the growth of microbes
involved in the test for antibiotic sensitivity.
 Spread the microbe culture uniformly on the surface of nutrient agar.
 Dip filter paper disks of 1 cm radius in different antibiotic solutions and place
over the surface of inoculated agar in the plate.
 Incubate the plate at a suitable temperature for a week and check for the growth
of microbe around the paper disks.
2. Dilution method
 Here liquid nutrient media is used to dilute the microbial culture in suitable
cylindrical tubes like test tubes.
 Add the same dilution of microbes in all tubes.
 Then add fixed concentrations of different antibiotics to each tube with microbial
culture by labeling them.
 After a week of incubation, measure the growth in each tube in terms of the
turbidity of the solution.
Result and interpretation
For the agar plate method, the antibiotics around which more clear zone is formed is
more effective against the bacterium
For the dilution method, the antibiotics in the tube with less turbidity is more effective.
 Concerning the result of AST, there are three terms worth of noting, susceptible,
intermediate and resistant. They are defined as follows
 Susceptible — likely, but not guaranteed to inhibit the pathogenic microbe;
may be an appropriate choice for treatment
 Intermediate — may be effective at a higher dosage, or more frequent dosage,
or effective only in specific body sites where the antibiotic penetrates to
provide adequate concentrations
 Resistant — not effective at inhibiting the growth of the organism in a
laboratory test; may not be an appropriate choice for treatment
When we use the agar plated test, there are standard distances to be cleared set by
WHO, CDC or other authorized body to say a bacterium is susceptible intermediate or

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resistant against the required antibiotics. Using these standards one can select the right
antibiotics for the right bacterium.
1.HBV Test
HBV test is test used to test whether serum under investigation contains HBV virus.
Principle
This test uses a test kit that contains cotton like absorbent and two distinct zones
called C-zone and T-zone. The C-zone is control zone and the T-zone is the test zone
that contains known viral antigen. When serum drop is add, the absorbent takes it and
the serum moves by diffusion toward the zones. There, if there is antibody in the
serum there will be reaction at the T-zone and the reaction appear at both the T-zone
and the C- zone.
Materials
Test kit
Micro-pippet
Serum
Procedure
1. Measure small amount of serum with the pippet
2. Carefully drop to the test kit
3. Look for the T-zone
Result and interpretation
If result appears at the T-zone on the test card, the serum contain the antibody against
the virus and thus the suspected person is positive. If it appears at C-zone only, the
test is negative and the person is free.

2.HIV Test
HIV can be diagnosed either by the detection of HIV-specific antibodies in serum or
by assessing the presence of the virus by nucleic acid detection using polymerase
chain reaction (PCR). Antibody testing is the method most commonly used to
diagnose HIV infection.

Principles
If antibodies to HIV-1 are present in the sample,they combine with an
HIV-1 antigen and this complex binds to the immobilized antigens in the test region
of the device forming a visible pink/red band. The control line should always appear
as a visible pink/red band in the control region of the device to indicate that the test is
undergoing correctly. A positive result is visualized by a pink/red band in the test
region of the device. A negative reaction occurs in the absence of detectable levels of
human immunoglobulin antibodies to HIV-1 in the specimen; consequently no
visually detectable band appear in the test region of the device.
Materials
1. Test kit
2. Pipette
3. Serum/oral fluid/saliva
Procedure of HIV blood test
1. Your finger is cleaned with an antiseptic solution.

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 2. Using a spring-loaded tool called a lancet, the health professional pricks your
finger to take a drop of blood (serum).
3. The blood is drawn into a tiny glass tube called a pipette, which is then placed
in a reagent called a buffer.
4. The buffer and two other chemicals (a dying agent and clearing solution) are
poured into a test device.
5. After 15–20 minutes, the device is checked. If no visual band is appeared in
the test region, the test is nonreactive (negative). If pink/red band is appeared,
the test is reactive (preliminary positive).
6. A confirmatory blood test is then performed. Results are available a few days
later.

Result and its interpretation

 If the preliminary test is nonreactive, you are HIV-negative, that means you
do not have HIV, but you need to be tested soon.
 If the preliminary test is reactive, the result is considered a preliminary
positive.
 If both your preliminary and confirmatory tests are reactive, you are HIV-
positive. Otherwise, you are HIV-negative.

Culture
A microbiological culture, or microbial culture, is a method of multiplying microbial
organisms by letting them reproduce in predetermined culture medium under
controlled laboratory conditions. Microbial cultures are foundational and basic
diagnostic methods used as a research tool in molecular biology.
Classification of culture media
Culture can be classified based on different criteria. Here we see classification of
microbial culture medias based on their consistency( physical state) and purpose.
1. Classification on consistency
Culture media can be solid, liquid or semi solid
 Solid media is used for the isolation of bacteria as pure culture. 'Agar' is most
commonly used to prepare solid media. Agar is polysaccharide extract obtained
from seaweed. Agar is an ideal solidifying agent as it is.solid media are solid at
low temperature and liquid at higher ones.
 Semi Solid Media are a type of culture media that contain agar at 0.5 %
concentration. They are used for the determination of bacterial motility.
 Liquid Media. They are generally termed as broth. It is used for profuse growth
and dilution techniques.
2. Classification based on purpose
A. Basic media- culture media that contain necessary nutrients the microbe need for
normal growing. They are used to grow any MOs
B. Enriched media- media that contain additional nutrient than the basic media. They
are used to grow fastidious microbes or when fast growth is required. E.g penitol
E.g blood agar contain blood as additive
Chocolate agar- contain fragments of hemoglobin as additive
C. Selective/differential media- media that selectively grows specific organism ( as
opposing to basic media). E.g -McConky- for gram negative bacteria

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 -Manitol salt agar (MSA) for Staphylococci
D. Transport media- used to transport microbes to required place. E.g Staurt media
E. Indicator media- these are media used to see the presence of microbes. They
change color whenever there is growth.
F. Enrichment media- liquid enriched media.
preparing media
Materials
 Powder of the regent
 Balance
 Volume flask
 Clean, deionized water)
 Petridish
 Clean paper
Procedure
The methods for preparation of culture media come with the powder of the media and
can vary accordingly. Here we are going to see the general procedures used with all
medias.
1. Read the manual carefully to guide you through the preparation and know the
effective concentration.
2. Calculate the amount of the main ingredient for your process
3. Weigh an empty paper on high accuracy balance and then weigh the ingredient
by adding on the paper
4. Add the powder to the flask
5. Add deionized water up to 80% of the required volume and mix well
6. Top up the water
7. Sterilize the culture with autoclave
8. Label and store properly
Adult parasites in the lab
We have seen the adults of
 Ascaris lumbercoides (Ascaris)
 Taina saginata (tape worm)
 Biomphlaria

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