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Practical medical microbiology
Laboratory Schedule
Exercise
1
2
3
4
Laboratory Topic
A. General Rules, Safety and Review of Microbiology Techniques
B. Gram-positive cocci: I- Staphylococci and Micrococci
Gram-positive cocci: II-Streptococci and Enterococci.
A- Gram-positive bacilli : Aerobic spore forming: Bacillus spp,
B- Gram-positive bacilli : Aerobic non-spore forming: Corynebacterium spp,
A- Gram-positive bacilli : Anaerobic spore forming: Clostridium spp,
B- Acid fast bacteria: Mycobacterium tuberculosis.
A- Gram-negative rods: Oxidase & Oxidative-Fermentative test to distinguished
5
Enterobacteriaceae, Pseudomonas & other Gram negative rods.
B- Enterobacteriaceae: General characters, Selective and differential media
6
Enterobacteriaceae: A- Lactose fermenters
7
Enterobacteriaceae: B- Lactose non fermenters
8
9
10
11
13
14
15
A. Quiz 1
B. First practical Exam
Nonfermentative, Gram negative Bacilli" Pseudomonas aeruginosa"
A. Vibrio cholerae and other Vibrio
B. Haemophilus spp.
Mycobacterium tuberculosis and Acid fast bacteria
A- Antibiotic Susceptibility Testing
B- Medical Mycology: Candida
Microbiology of water, food and milk
A. Quiz 2
B. Final Practical Exam
1
Page
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Practical medical microbiology
Practical Number 1
General Safety Rules for the Microbial Pathogenesis
Laboratory
The microorganisms used for instruction in this course are pathogenic for humans
or animals. The safety of every student depends upon the conscientious observation of
rules that must be followed by all who work in the laboratory. Certain precautions
must be followed to avoid endangering well being, that of neighbors and those who
clean the laboratory. Any student who is in doubt about how to handle infectious
material should consult an instructor. Laboratory attendance is mandatory. There will
be no way to make up missed work. The following rules must be observed at all
times.
1. Always wear a laboratory coat when working in the laboratory classroom.
2. Put nothing in mouth which may have come in contact with infectious
material.
3. Smoking, eating and drinking in the laboratory are not permitted at any time.
4. Mouth pipetting is not permitted under any circumstances. Use the safety
pipetting devices which are provided. Dispose of used pipettes in the
appropriate receptacle. Any infectious material which may accidentally fall
from pipettes to the laboratory bench or floor should be covered with a
disinfectant and reported to any instructor immediately.
5. Any spilled or broken containers of culture material should be thoroughly wet
down with a disinfectant and then brought to the attention of an instructor.
There are no penalties for accidents provided they are reported promptly.
6. Report at once an accident which may lead to a laboratory infection.
7. The microscope issued to you is both an expensive and delicate instrument-treat it accordingly. Always, at the end of each laboratory period, carefully
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Practical medical microbiology
clean oil from the objective and condenser lenses, align the low power dry
objective with the condenser and rack condenser up and body tube down. You
will be held personally responsible for any defect found on microscope when
it is recalled at the semester's end.
8. When finished for the day, dispose of all used glassware and cultures in the
appropriate receptacle, clear workbench and wash the top with a disinfectant.
Wash hands thoroughly with soap and water before leaving the laboratory.
9. Do not throw refuse of any kind into the sink. Use the containers provided.
10. Be sure all burners are turned off at the end of the laboratory period. Double
check to be sure that handles on all gas outlets are in the off position.
11. The inoculating needle should be heated until red hot before and after use.
Always flame needle before you lay it down.
12. Always place culture tubes of broth or slants in an upright position in a rack.
Do not lay them down on the table or lean them on other objects. They may
roll onto the floor and break.
13. All culture containers which are to be incubated should bear the following
notations: 1) initials (or last name of the student), 2) specimen (name of
organism or number of unknown) and 3) date. When using Petri plates, these
notations should be entered on the bottom half, not the lid. Unless otherwise
directed, all plates are to be inverted, all plugged tubes should have the plugs
firmly set into the tubes, and all screw cap tubes should have the caps
loosened one-half turn to permit gas exchange.
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Practical medical microbiology
Practical Medical Microbiology
Infectious
Microorganisms
Bacteria
"Bacteriology"
Fungi
"Mycology"
Virus
"Virology"
Bacteria
I- Gram
positive
II- Gram
negative
III- Acid fast
Mycoplasma
Chalamydia
Rickettsia
Spirochetes
I- Gram Positive bacteria
A- Gram positive cocci
B- Gram positive rods
Non spore-forming
Corynebacterium
Spore-forming
Aerobic
Bacillus anthracis
Anaerobic
Clostridium
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Practical medical microbiology
A- Gram-positive cocci
I- staphylococci
II- streptococci
I- staphylococci
 Objective:
1-
Detect the presence of catalase in staphylococci and to differentiate between
staphylococci (catalase positive) and streptococci (catalase negative)
2-
Learn procedure of Gram stain and to know Gram reaction, shape and
arrangement of staphylococci
3-
Use of mannitol salt agar as selective and differential medium for
staphylococci and to differentiate between mannitol fermenter and mannitol
non fermenter staphylococci
4-
Learn the use of plasma to differentiate coagulase positive and coagulase
negative staphylococci
5-
Detect DNase in staphylococci and to compare activity in S. aureus and S.
epidermidis
 Material requirements (per 20 students):
123456789-
Overnight cultures of S. aureus, S. epidermidis, & S. saprophyticus
Grams stain dyes (Crystal violet and Saffranin) and reagents (Iodine and
alcohol), filter paper, slides, immersion oil.
Nutrient agar plates (20)
Mannitol Salt Agar (MSA) plates (20)
Deoxyribonuclease (DNase) agar plastes (20)
1N HCl (100 ml)
Hydrogen peroxide 3% (10ml)
Rabbit plasma (20 ml)
Novobiocin disk
 Practical protocol:
123456-
Gram stain:
Catalase test
Mannitol fermentation on MSA
Coagulase test
DNase test
Novobiocin susceptibility
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Practical medical microbiology
Characters of the genus Staphylococcus:

Staphylococci are Gram positive cocci, 0.5-1.5 m in diameter, which occurs
in irregular "grape-like" clusters.

3 None: Non motile, Non spore forming and typically Non encapsulated.

Catalase positive.

Facultative anaerobes.

Grow on simple media (i.e. non fastidious).
Three species of staphyloccoci have medical importance:

S. aureus is the most pathogenic and commensally found in nose (nares).

S. epidermidis usually non pathogenic and common commensals in nares& skin

S. saprophyticus is also a common cause of urinary tract infection and
occasionally commensally found skin.
Laboratory diagnosis of Staphylococcus species:

Specimens: Pus, Urine, Blood, Cerebrospinal fluid (CSF).

Stain: Gram positive cocci, arranged in clusters.

Culture:
1- Colonial appearance
A- On nonselective media:

The typical 24 h isolated colonies of staphylococci are 1-3 mm in
diameter. Colonies of coagulase positive staphylococci i.e. S. aureus
are pigmented, smooth, entire, and hemolytic on blood agar.
However, colonies of coagualse negative staphylococci (e.g. S.
epidermidis) are unpigmented, smooth, entire, glistening, slightly
raised to convex, opaque and non hemolytic.
B- On selective medium:

Mannitol salt agar (MSA) is selective differential medium for
staphylococci, which contains mainly on 7.5% NaCl, mannitol and
phenol red. The selectivity of MSA is due to staphylococci are able to
grow in presence of high salt concentration i.e. staphylococci are
halotelorant. However, MSA is differential because contains mannitol
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Practical medical microbiology
(sugar) and phenol red (pH indicators turns yellow in acidic pH and
turns red in alkaline pH). S. auerus ferment mannitol and release acid
which decreased in the pH of medium. The resultant acidic pH is
detected by phenol red and colonies of S. aureus appear yellow. On the
other hand, other staphylococci do not ferment mannitol and
subsequently no changes occur in the pH of medium, therefore
colonies of S. epidermidis appear red.
Mannitol fermentation on MSA
Mannitol nonfermenter
Red colonies:
S. epidermidis& S. saprophyticus
Mannitol fermenter
Yellow colonies:
S. aureus
2- Microscopical examination:

Staphylococci are Gram positive cocci, which occur in irregular
"grape-like" clusters.
3- Catalase test:

The catalase test is important in distinguishing streptococci (catalasenegative) from staphylococci which are catalase positive. The test is
performed by flooding an agar slant or broth culture with several drops
of 3% hydrogen peroxide. Catalase-positive cultures bubble at once.
The test should not be done on blood agar because blood itself will
produce bubbles.
Catalase test
Positive
Microcococcaceae
Staphylococci
Negative
Streptococcaceae
Streptococci
4- Coagaulase production:

The ability to clot plasma continues to be the most widely used and
generally
accepted
criterion
7
for
identification
of
pathogenic
Practical medical microbiology
staphylococci. The enzyme act by converting fibrinogen into fibrin.
Coagulase test used to classify staphylococci into 1- Coagulasepositive staphylococci (e.g. S. aureus) and 2- Coagulase negative
staphylococci (S. epidermidis, S. saprophyticus). It is thought that
coagulase-positive staphylococci may avoid host defense mechanisms
by forming this fibrin clot around them.
Two different coagaulase tests can be performed:

Tube test for free coagulase and a slide test for bound coagulase or
clumping factor. While tube test is definitive and slide test may be
used as a rapid screening technique to identify S. auerus. Variety of
plasma may be used for either test; however dehydrated rabbit plasma
containing EDTA is mostly used.
o The tube coagulase test is performed by mixing 0.1 ml of an
overnight culture with 0.5 ml of plasma, incubating the mixture
at 370C in water bath for 4 h, and observing the tube for clot
formation by slowly tilting it 900 from the vertical. Any degree
of clotting constitutes a positive test.
o The slide coagulase test is performed by making heavy
suspension of growth in distilled water, add one drop heavy
bacterial suspension and one drop of plasma on clean slide,
mixing well and observing for clumping within 10 seconds.
Coagulase test
Coagulase Positive
Staphylococus aureus
Coagulase-Negative
S. epidermidis & S. saprophyticus
4- Deoxyribonuclease (DNase) test:

A heat
stable
staphylococcal
nuclease that
has
endo-
and
exonucleolytic properties that cleave DNA is produced by most strains
of S. aureus. The test is performed by inoculating DNA agar with
tested organism in circular motion to create an area of inoculation.
Incubate at 370C for 24-48h. After incubation, observe DNase activity
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Practical medical microbiology
by adding 1N HCl to the agar surface, a zone of clearing indicates a
positive test. The zone represents the absence of DNA. The medium
around colonies not producing DNase remains opaque, which is a
reflection of the precipitation of DNA by the added acid.
DNase test
Positive
Negative
Staphylococus aureus
S. epidermidis
S. saprophyticus
5- Novobiocin Resistance:

A simple disk diffusion test for estimating novobiocin susceptibility
and distinguishing S. saprophyticus from other clinically important
species can be performed by use novobiocin disk on Mueller-Hinton
agar inoculated with 105 CFU/ml overnight bacterial cultures and
incubated at 370C overnight. Novobiocin resistance is intrinsic to
S. saprophyticus but uncommon in other clinically important species.
Novobiocin test
Sensitive
Resistant
S. aureus
S. epidermidis
S. saprophyticus
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Practical medical microbiology
Gram stain:
Smear preparation:
The preparation of a smear is required for many laboratory procedures,
including the Gram-stain. The purpose of making a smear is to fix the bacteria onto
the slide and to prevent the sample from being lost during a staining procedure. A
smear can be prepared from a solid or broth medium. Below are some guidelines for
preparing a smear for a Gram-stain.
1. Place one needle of solid bacterial growth or two loops of liquid bacterial growth in
the center of a clean slide
2. If working from a solid medium, add one drop of water to your specimen with a
water bottle. If using a broth medium, do not add the water.
3. Now, with your inoculating loop, mix the specimen with the water completely and
spread the mixture out to cover about half of the total slide area.
4. Allow the smear to dry at room temperature. Gently heat-fix by passing the dried
smear through the flame of Bunsen burner. Caution must be exercised not to overheat
the slide. Remember to gently heat-fix and not to cook the bacteria on the slide.
Gram-staining Procedure:
Gram-staining is a four part procedure which uses certain dyes to make a bacterial cell
stand out against its background. The specimen should be mounted and fixed on a
slide before you proceed to stain it. The reagents you will need to successfully
perform this operation are:

Crystal Violet (Primary Stain)

Iodine Solution (Mordant)

Decolorize (Ethanol is a good choice)

Safranin (Counter stain)

Water (preferably in a squirt bottle)
Before starting, make sure that all reagents, as well as the squirt-bottle of water, are
easily accessible because you won't have time to go get them during the staining
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Practical medical microbiology
procedure. Also, make sure you are doing this near a sink because it can get really
messy.
Step 1: Place your slide on a slide holder or a rack. Flood the entire slide with crystal
violet. Let the crystal violet stand for about 60 seconds. When the time has elapsed,
wash your slide for 5 seconds with water. The specimen should appear blue-violet
when observed with the naked eye.
Step 2: Now, flood your slide with the iodine solution. Let it stand about a minute as
well. When time has expired, rinse the slide with water for 5 seconds and immediately
proceed to step three. At this point, the specimen should still be blue-violet.
Step 3: This step involves addition of the decolorize, ethanol. Step 3 is somewhat
subjective because using too much decolorizer could result in a false Gram (-) result.
Likewise, not using enough decolorizer may yield a false Gram (+) results. To be
safe, add the ethanol drop wise until the blue-violet color is no longer emitted from
your specimen. As in the previous steps, rinse with the water for 5 seconds.
Step 4: The final step involves applying the counter stain, safranin. Flood the slide
with the dye as you did in steps 1 and 2. Let this stand for about a minute to allow the
bacteria to incorporate the saffranin. Gram positive cells will incorporate little or no
counter stain and will remain blue-violet in appearance. Gram negative bacteria,
however, take on a pink color and are easily distinguishable from the Gram positives.
Again, rinse with water for 5 seconds to remove any excess of dye.
Step 5: After you have completed steps 1 through 4, you should blot the slide gently
with bibulous paper or allow it to air dry before viewing it under the microscope. DO
NOT RUB THE SMEAR.
Step 6: Apply a drop of immersion oil directly to the smear and place the slide on the
stage of the microscope and examine the film using oil immersion lens (x100).
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Practical medical microbiology
Practical NO 2
II- Streptococci
Objective:
1. Use of blood agar to differentiate between, , and  hemolytic
streptococci.
2. To know Gram reaction, shape and arrangement of streptococci.
3. To differentiate between group A and non group A streptococci using
bacitracin sensitivity
4. To identify Strep. agalactiae by CAMP test.
5. To differentiate between Streptococcus pneumoniae and viridans
streptococci using optochin sensitivity and bile solubility.
Required materials (per 20 students):
1. Overnight cultures of Strep. pyogenes, Strep. agalactiae, Strep.
pneumoniae, Strep. viridans and S. aureus .
2. Gram stain "dyes and reagents", filter paper, slides, immersion oil
3. 20 plates of blood agar
4. Bacitracin disks (20 disks)
5. Optochin disks (20 disks)
6. 40 tubes containing 5 ml of 10% bile salts
Experimental:
123456-
Gram stain of streptococci
Hemolysis on blood agar (, , and  hemolysis)
Bacitracin susceptibility test
CAMP test
Optochin susceptibility test
Bile solubility test
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Practical medical microbiology
Practical NO 3
Gram positive bacilli
Gram-positive bacilli
Anaerobic
Aerobic
Non spore forming
Corynebacterium
Listeria
Spore forming
Clostridium
Spore forming
Bacillus
I- Aerobic Gram positive bacilli:
A- Non spore forming: Corynebacterium
These are group of aerobic, non spore forming, Gram positive bacilli
 General characters:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Gram positive bacilli, with characteristic morphology
Non motile
Non spore forming
non capsulated
Facultative anaerobic
Breakdown glucose by oxidative and fermentative i.e. O+/F+
Fastidious
Catalase positive
Oxidase negative
Corynebacterium
Pathogenic
C. diphtheriae
Commensal "Diphtheriods"
C. hofmannii
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Practical medical microbiology
Corynebacterium diphtheriae
 Diphtheria:
o
o
o
o
o
o
o
o
o
o
o
Diphtheria is an acute, toxin-mediated disease caused by C. diphtheriae.
Diphtheria is a childhood disease affecting the upper respiratory tract and
transmitted by droplet infection from case or carrier.
Incubation period is 2-6 days, with gradual onset.
C. diphtheriae multiply locally on epithelial cells of pharynx or adjacent surfaces.
Early symptoms include malaise, anorexia, headache, sore throat, exudative
pharyngitis and low grade fever.
Within 2-3 days a thick firmly bluish-white adherent pseudo membrane forms.
This membrane composed of bacteria, lymphocytes, fibrin and dead cells that can
cover the tonsils, uvula, and palate and extended up into the nasopharynx or
down into the larynx.
The membrane is adherent to the tissue, and forcible attempts to remove it cause
bleeding.
Extensive membrane formation may result in respiratory obstruction.
The patient may recover at this point; or if enough toxins are absorbed,
develop severe prostration, pallor, rapid pulse, stupor, coma, and may die
within 6 to 10 days.
Complication with sever disease include myocarditis, neuritis and palate
perforation.
Patients with severe disease may develop marked edema of the submandibular
areas and the anterior neck along with lymphadenopathy, giving a characteristic
"bull neck" appearance.
 Laboratory diagnosis
o The laboratory diagnosis serves to confirm the clinical manifestation
but the specific treatment must be never delayed for laboratory results
if the clinical manifestation is strongly suggestive of diphtheria.
 Diagnosis of a case:
o Specimen: A throat swap by gentle touching the membrane.
o Culture: The swap is inoculated on Loeffler's serum medium and/or
on blood tellurite agar aerobically at 370C for 24.
 On Loeffler's serum: the colonies of C. diphtheriae are small,
smooth, and creamy.

There are 3 biotypes of C. diphtheriae (gravis, intermedius & mitis).

The most severe disease is associated with the gravis biotype,
but any strain may produce toxin.
On blood tellurite agar, three biotypes of C. diphtheriae are
characterized.




Colony of gravis biotype is large, non-hemolytic and grey.
Colonies of mitis biotype are small, hemolytic and black
Colonies of intemedius biotype are intermediate in size,
non-hemolytic with black center & grey margin.
o Stain:
 Gram stain: C. diphteriae are gram positive bacilli arranged in
Chinese letters form often club shaped
 Polychrome methylene blue stain: C. diphteriae appears beaded
due to the presence of intercellular “Metachromatic or volutin"
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Practical medical microbiology
granules. By stain, the granules appear red while the rest of
organism appears blue.
o Animal inoculation:
 Toxigenic strains are highly pathogenic for laboratory animals.
When injected into a guinea pig it will die in 2-3 days.
 Diagnosis of carrier:
o Two steps must be done for diagnosis of carrier:
o Isolation of the microorganism:
o Detection of exotoxins produced by isolated microorganism:
 A swap are taken from the nose and throat, then inoculated
onto Loeffler's serum or blood tellurite agar and incubated
aerobically at 370C for 24 h. The suspected colonies are
examined by Gram stain and any diphtheria-like organisms
must be submitted to virulence tests.
o Virulence tests:
 In Vivo: Two guinea pigs are used; one is the test and the other
is the control. The control is injected with diphtheria antitoxin
24 h before the experiment. Then the both guinea pigs, the test
and the control, are injected with the suspension of the isolated
microorganism


If the test animal dies while the control animal survives, it
means the isolated organism is virulent C. diphtheriae.
If both guinea pigs survive, it means the isolated organism is
avirulent strain




In Vitro: "Eleks test"
 A strip of filter paper impregnated with diphtheria
antitoxin is placed on the surface of serum agar. The
tested organism is streaked at right angels to the filter
paper. After 48 hrs incubation, the antitoxin diffusing
from filter paper strip and the toxigenic strains produce
exotoxin, which diffuses and resulted in lines four
precipitation lines radiating from intersection of the
strip and the growth of organism.
Immunization:
o Active immunization:
 Diphtheria toxoid either formol or alum precipitated toxoid
 D.P.T. vaccine used against diphtheria, pertussis and tetanus.
 It is given in three doses two months intervals, at 2, 4
and 6 months after birth.
o Passive immunization:
 Diphtheria antitoxic serum is given to children in contact with a
case as prophylaxis to give rapid protection.
Treatment:
o The important step for treatment of diphtheria is the early
administrated of diphtheria antitoxin for specific neutralization of
exotoxin. Antibiotic therapy such as penicillin and erythromycin has
proved in eliminating C. diphtheriae from patients who have the
disease and also from those who are asymptomatic carrier.
Diphtheroids
Diphtheroids are gram positive bacilli oval short parallel bacilli, not beaded.
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Practical medical microbiology




Most of them are commensals in vagina, on the skin and throat.
Examples of diphtheroids are C. hofmannii, C. xerosis and C. acne.
C. acne may play a role in the pathogenesis of acne vulgaris.
They can grow on ordinary media as nutrient agar.
Practical NO 5
B-Aerobic spore forming: Bacillus
Bacillus
Commensal
Pathogenic
B. anthracis
B. cereus
e.g. B. subtilis
 General characters:
1. Very large Gram positive bacilli, 1-1.2 µm in width x 3-5µm in length,
arranged in long chains
2. Motile except B. anthracis
3. Spore forming (outside the host)
4. Capsulated (inside the host)
5. The bacteria can be cultivated in ordinary nutrient medium i.e. non Fastidious
6. Facultative anaerobic
7. Breakdown glucose by oxidative and fermentative i.e. O+/F+
8. Catalase positive
9. It is found in soil habitats around the world
Bacillus anthracis




Anthrax is an acute infectious disease caused by the spore-forming bacterium
Bacillus anthracis.
Anthrax most commonly occurs in wild and domestic animals (cattle, sheep,
goats, camels, and other herbivores) i.e. zoonotic disease.
Humans infected incidentally when brought into contact with diseased
animals, which includes their flesh, bones, hair i.e. occupational disease to
farmers, slaughters and wool workers.
Direct person-to-person spread of anthrax is extremely unlikely to occur.
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Practical medical microbiology


There are three main types of anthrax.
o Cutaneous anthrax:
 The most common form of the disease to humans
 It is usually acquired when the spores from the soil or
contaminated animal or carcass infect injured skin or mucous
membrane usually in face, neck and arm
 The spores germinate, vegetative cell multiply and a
characteristic gelatinous edema (no pus) develops at the site
 Then develops into papule within 12-36 hrs after infection
 The papule changes rapidly to a vesicle, then a malignant
pustule and finally into necrotic ulcer and then dries to form the
characteristic brown or back and infection may disseminate,
giving rise to septicemia.
 The lesion is painless
 The disease is frequently fatal.
o Pneumonic anthrax (Woolsorter’s disease):
 It is results most commonly from inhalation of spore-containing
dust where animal hair or hides are being handled.
 The disease begins suddenly with high fever and chest pain
 It is progress rapidly to a systemic hemorrhagic pathology
 This disease is often fatal
 B. anthracis spores are used in biological war by spreading in
atmosphere
o Intestinal anthrax:
 It is analogous to cutaneous anthrax but occurs on the intestinal
mucosa
 Intestinal anthrax is rare and occurs accidently among butchers
and in primitive societies eating meat of infected animals.
 Initial signs of nausea, loss of appetite, vomiting, fever are
followed by abdominal pain, vomiting of blood, and severe
diarrhea.
 Intestinal anthrax results in death in 25% to 60% of cases.
Virulence factors:
o B. anthracis owes its pathogensity to two major determinates of
virulence
 Poly-D-glutamyl capsule, which mediates the invasive stage of
the infection
 Anthrax toxin, which mediates the toxigenic stage.
 The toxin consists of three distinct antigenic
components, which is thermolabile protein.
o
o
Edema Factor (EF): necessary for edema production
Protective Antigen (PA): induces protective antitoxic
antibodies in guinea pigs
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Practical medical microbiology
o



Lethal Factor (LF): has a lethal effect of anthrax toxin
Diagnosis of anthrax:
Anthrax is diagnosed by isolating B. anthracis from the blood, skin lesions, or
respiratory secretions or by measuring specific antibodies in the blood of
persons with suspected.
o Specimen:
 Pastular exudates in malignant pustule, sputum in pneumonic
anthrax or stool in intestinal anthrax is collected
 Stool specimen is emulsified and heated to 800C to kill non
spore forming microorganism.
o Stain:
 Direct smear is done from specimen and stained by Gram stain
 The stained smear revealed Gram positive bacilli, found in
chains, capsulated inside the host, sporulated outside the host
(Spore is central oval and non-bulging) and non motile.
 By spore stain, the spore appears green and vegetative cell
appear red when stained with malachite green and safranin.
o Culture:
 On ordinary medium, grow aerobically at 370C with
characteristic mucoid or smooth colonies, which indicates the
pathogensity of organism (presence of capsule). Rough
colonies are relatively avirulent.
 Stab culture on gelatin medium results in inverted fire tree
appearance.
o Biochemical reactions:
 Ferment glucose, galactose, maltose and dextrin with acid
production only.
o Animal inoculation:
 Guinea pig is injected with B. anthracis. After injection
gelatinous edema will appear at the site of injection and dark
watery blood in heart and spleen and death will occur.
o Ascoli test
 Infected tissue is extracted and mixed with specific antibody. A
precipitation reaction is observed.
Immunization of anthrax:
o
o
o
o
o
o
o
Vaccines composed of killed bacilli and/or capsular antigens produce no
significant immunity
Live attenuated vaccine is used for immunization of animals only
The Sterne strain of B. anthracis produces sub-lethal amount of the toxin that
induces formation of PA
The anthrax vaccine for humans is a preparation of PA recovered from
avirulent non-capsulated strain of B. anthracis that produces PA
Immunization consists of 3 SC injection given 2 weeks apart followed by 3
additional SC injections given at 6, 12 and 18 months and annual booster
dose is required to maintain a protective level of immunity.
This vaccine is indicated for individuals who come in contact in workplace
with imported animals hides, furs, bone, meat and animal hair
It has been indicated for military during the era of biological warfare.
Bacillus cereus
18
Practical medical microbiology




B. cereus is a normal inhabitant of soil, also isolated from food such as grains
and spices.
B. cereus causes two types of food poisoning:
Emetic form or short incubation:
o It is caused by heat stable enterotoxin
o It is characterized by nausea, vomiting and abdominal cramps and
has incubation period of 1-6 hrs.
o It resembles S. aureus food poisoning.
Diarrheal form or long incubation:
o It is manifested primarily by abdominal cramps and diarrhea with an
incubation period of 8-16 hrs.
o Diarrhea may be a small volume or profuse and watery
o It resembles food poisoning caused by Clostridium perfringens
o In either type, the illness usually lasts less than 24 hrs after onset.
o It is caused by heat labile enterotoxin.
Differential characteristics of B. anthracis and B. cereus
Characteristic
B. anthracis
Hemolysis on blood agar
Motility
-
19
B. cereus
+
+
Practical medical microbiology
Practical NO 5
Clostridium spp
Clostridia are:








Large Gram positive
Straight or slightly curved rods with slightly rounded ends
Anaerobic bacilli
Spore bearing
Spore do not germinate and growth does not normally proceed unless a
suitably low redox potential Eh exists
Saprophytes
Some are commensals of the animal & human gut which invade the blood and
tissue when host die and initiate the decomposition of the corpse (dead body)
Causes diseases such as gas gangrene, tetanus, botulism & pseudomembranous colitis by producing toxins which attack the neurons pathways
Clostridium
Causing
Tetanus
e.g. Cl. tetani
ِAntibiotic associated
Botulism
e.g. Cl. botulinum diarrhea
Gas gangrene
e.g. Cl. difficille
Saccharolytic
Proteolytic
e.g. Cl. perfringens &Cl. septicum
e.g. Cl. sporogenes
Mixed: Cl. histolyticum
Clostridium-causing tetanus (Clostridium tetani)
a) Description








Gram positive, straight, slender rod with rounded ends
The fully developed spores gives the organism the appearance of a drumstick
with a large round end
Obligate anaerobe
Motile and has numerous peritrichous flagella
Grows well in cooked meat broth and produces a thin spreading film when
grown on enriched blood agar
Spores are highly resistant to adverse conditions
Spores of some strains resist boiling in water for up to 3h. They may resist dry
heat at 160 0C for 1 h and 5% phenol for 2 weeks or more
Iodine (1%) in water is able to kill the spores within a few hours
20
Practical medical microbiology
b) Occurrence of tetanus bacilli



Occurs in the intestine of humans and animals
Derived primarily from animal faeces and indirectly via soil
Especially prevalent in manure soil
c) Tetanus and clinical symptoms









Cl. tetani is the causative agent of tetanus
In developing countries most cases occur in
o Children secondary to wounds
o Neonates with contaminated umbilical stump after labor or abortion
o After surgical procedures done with non-sterilized instruments
o Also due to use of badly sterilized cut gut "surgical ligatures"
The spores vegetate and the organism multiply locally at the site of infection
producing exotoxins (neurotoxins)
Toxins spreads via
o The toxins is absorbed from the site of its production in an infective
focus and is transmitted to the CNS via motor nerves and apparently
specifically by motor fibers.
o Via blood to all nerves & the subsequent transmission to the CNS
The period between injury and the first signs is usually about 10-14 days
Onset of signs and symptoms of tetanus is gradual, usually starting with some
stiffness and perhaps pain or near a recent wound
The toxin act by interfering with synaptic transmission and leads to increased
excitability of motor nerve cells and produces tonic spasms.
The spasm of master muscles (Trismus or Lock Jaw) develops early.
When tetanus occurs naturally, the tetanus bacilli stay at the site of the initial
infection and are not generally invasive, but the toxin diffuses to affect the
relevant level of the spinal cord (local tetanus) and then to affect the entire
system (generalized tetanus).
d) Toxin

Cl. tetani produces two types of toxins
o Tetanolysin, which causes lysis of RBCs
o Tetanospasmin is neurotoxin and essential pathogenic product
 Tetanospasmin is toxic to humans and various animals when
injected parenterally, but it is not toxic by the oral route
 Tetanospasmin which causes increasing excitability of spinal
cord neurons and muscle spasm
e) Laboratory Diagnosis



The diagnosis of tetanus depends primarily upon the clinical manifestation of
tetanus including muscle spasm and rigidity.
Specimen from wound exudates using capillary tube
Culture: On blood agar and incubated anaerobically. Growth on blood agar
often appears as a fine spreading film.
21
Practical medical microbiology

Gram stain is a good method for identifying Clostridium. Cl. tetani is Gram
positive rod motile with a round terminal spore giving a drumstick appearance.
f) Treatment



Large doses of Anti Tetanic Serum (ATS) are given
The patient requires skilled sedation, constant nursing, surgical washing of the wound
If generalized spasms are worrying, the patient is ventilated mechanically until
the toxin that has been taken up has decayed: this may take some weeks
g) Immunization


Passive immunization →ATS
Active immunization
o Toxoid either Formol or Alum precipitated Toxoid
o
DPT vaccine
Clostridia causing gas gangrene
Clostridia causing gas gangrene
Saccharolytic organisms
Proteolytic organisms
Cl. perfringens, Cl. septicum
Cl. sporogenes
Ferment carbohydrates
Acid and gas are produced
Digest proteins with blackening
bad smell production
Mixed saccharolytic & proteolytic
Cl. histolyticum

All of these organisms are gram positive rods, their most characteristics
feature is the formation of spore, which is produced under drastic conditions

Gas gangrene is only likely to occur when:
 There is extensive damage to tissues
 Interruption of blood supply
 Contamination of wound with soil and other foreign material
o Such conditions are particularly associated with
 War wounds
 Accidental fracture
 Industrial injuries
 Uterine infections may occur after septic abortion
 The main source of the organisms is animal and human excreta
22
Practical medical microbiology
Saccharolytic organisms causing Gas gangrene
I- Clostridium perfringens
Cl. perfringens
Causing
Gas gangrene
Food poisoning
a) Description






Large Gram-positive bacilli with stubby ends
Capsulated
Non motile
Anaerobic
Grown quickly on selective media
Can be identified by Nagler reaction (see down)
b) Pathogenesis of gas gangrene









Gas gangrene is life treating infection of skeletal muscles
The organism is a normal constituent of the fecal flora
Impairment of the normal blood supply of tissue with a consequent reduction
in oxygen tension may allow an anaerobic focus to develop
When clostridial infection has been initiated in a focus of devitalized
anaerobic tissue, the organisms multiply and produce a range of toxins.
They spread and attack adjacent viable tissue, particularly muscle fibers, kill it
& render it anaerobic, causing fermentation of muscle glycogen with
production of acid and gas
The gas separates the muscle fibers from its sheath, cutting of blood supply
leading to necrosis.
The organism is further colonized with the production of more toxins.
The incubation period is 1-4 days and earliest symptom is severe pain at the
site of wounds.
In puerperal infections or in cases of septic abortion, the organisms may gain
access from faeces-contaminated perineal skin or contaminated instruments to
necrotic or devitalized tissues in the uterus. Here they set up a dangerous &
often fulminating pelvic infection, possibly with prompt invasion of the bloodstream


Cl. perfringens may also participate in peritoneal infections occurring as a
result of extension of pathogens from the alimentary or intestinal obstruction
or mesenteric thrombosis
The toxins of Cl. perfringens :
o  toxin (phospholipase C, lecithinase) is the most important toxin
 Lyses of RBCs, platelets, leucocytes and endothelial cells
23
Practical medical microbiology

o
o
Increased vascular permeability with massive hemolysis and
bleeding tissue destruction
 Hepatic toxicity and myocardial dysfunction
-toxin is responsible for necrotic lesions in necrotizing enterocolitis
Enterotoxin is heat labile toxin produced in colon → food poisoning
c) Laboratory Diagnosis

Specimen: Histological specimen or wound exudates
o Histological specimen transferred aseptically into a sterile screwcapped bottle & used immediately for microscopical examination & culture
o



Specimens of exudates should be taken from the deeper areas of the
wound where the infection seems to be most pronounced
Gram stain:
o Gram-positive bacilli, non motile, capsulated & sporulated. The spore
is oval, sub-terminal & non bulging. Spores are rarely observed.
Culture: Anaerobically at 370C
o On Robertson's cooked meat medium → blackening of meat will
observed with the production of H2S and NH3
o On blood agar → -hemolytic colonies
Biochemical reactions
o Cl. perfringens ferment many carbohydrates with acid and gas.
o Cl. perfringens acidified litmus milk with stormy clot production
o Nagler's reaction:
 This test is done to detect the lecithinase activity
 The M.O is inoculated on the medium containing human serum
or egg yolk (contains lecithin), colonies of Cl. perfringens are
surrounded by zones of turbidity due to lecithinase activity and
the effect is specifically inhibited if Cl. perfringens antiserum
containing  antitoxin is present on the medium
d) Treatment




Prompt and adequate surgical attention to the wound is of the utmost importance
Sutures are removed, necrotic & devitalized tissue is excised with careful
debridement; facial compartments are incised to release tension; any foreign
body is found and removed
Antibiotic therapy is started immediately with very high doses. Penicillin,
metronidazole and an aminoglycoside may be given in combination
Alternatively, clindamycin or a extended-spectrum -lactam antibiotic
Cl. perfringens food poisoning



Cl. perfringens is normally present in considerable numbers in human faces
These bacteria also occur in animals; thus, meat is often contaminated with
their heat-resistant spores
When meat is cooked in bulk, heat penetration is slow and subsequent cooling
is slow unless special precautions are taken. The heat-resistant spores may
survive and during the cooling period, they will germinate in the anaerobic
environment produced by the cooked meat and multiply. Thus, anyone who
24
Practical medical microbiology


eats this will consume the equivalent of a cooked meat broth culture of the
organism. The organisms are protected from the gastric acid by the protein in
the meal and pass in large numbers into the intestine where they sporulate
Enterotoxin released in the gut will cause abdominal cramps about 8-12 h
after ingestion, followed by diarrhea. Fever and vomiting are not typically
encountered and symptoms generally subside within a day or two.
Symptoms are similar to food intoxication with B. cereus "Diarrheal form"
Laboratory Diagnosis

This depends upon the isolation of Cl. perfringens from the faces of patients &
from those at risk who have eaten the suspected food, and from the food itself.
Prevention: Prevent mishandling of foods
II Clostridium septicum
a) Description







Gram positive bacilli
Shorter, longer and filamentous forms
Spores are readily formed
Actively motile
Numerous peritrichous flagella
It is one of the less exacting anaerobes and grow well at 37 0C on ordinary media
Surface colonies are irregular, transparent and droplet-like, later becoming greyish
and opaque
b) Pathogenesis




One of the gas gangrene group of clostridia
Also occurs in the human intestine where it is usually harmless
Intramuscular injection of cultures in laboratory animals produces a spreading
inflammatory edema, with slight gas formation in the tissues.
The organisms invade the blood and the animal dies within a day or two.
Proteolytic organisms


The proteolytic organisms attack the necrotic muscle fibers leading to
blackening and foul smell of the gangrene
They also enhanced the action of saccharolytic organisms
Clostridium sporogenes





Gram positive motile bacillus
Widely distributed in nature and also in the intestines of animals
Harmless saprophyte
Spores are oval, central or sub-terminal
Spores may survive boiling for periods ranging from 15 min up to 6h
25
Practical medical microbiology
Clostridium-causing botulism (Clostridium botulinum)
a) Pathogenicity

Botulism (food poisoning)
b) Rout of infection
 Ingestion of food containing botulinal toxin, often from eating of canned food
c) Description








Strict anaerobic Gram positive bacillus
Motile with peritrichous flagella
Spores are oval and sub-terminal
It is widely distributed saprophyte occurring in soil, vegetables, fruits, leaves,
silage, manure, the mud of lakes and sea mud
Its optimum growth temperature is about 35 degrees
The widespread occurrence of C. botulinum in nature
Produce a potent neurotoxin in food and resistance of its spores to inactivation
combine to make it a formidable pathogen of humans & a range of animals
Insufficient heating in the process of preserving foods is an important factor in
the causation of botulism and great care must be taken in canning factories to
ensure that adequate heating is achieved in all parts of the can contents
d) Toxins of Cl. botulinum



Botulinal toxins are among the most poisonous natural substances known
During the growth of the microorganism, toxin is liberated into the food
Classified into seven antigenic types (A-G) with types A, B and E most
frequently associated with human disease
e) Human Botulism




Botulism is a severe, often fatal, form of food poisoning characterized by
pronounced neurotoxin effects
The disease has been caused by a wide range of foods, usually preserved hams
(preserved meat of upper parts of pig's leg), large sausages, home-preserved
meats and vegetables, canned products such as fish, liver paste
Foods responsible for botulism may not exhibit signs of spoilage
The preformed toxin in the food is absorbed from the intestinal tract. Although
it is protein, it's not inactivated by the intestinal proteolytic enzymes. The
toxin primarily affects the cholinergic system and seems to block release of
acetylcholine, chiefly at points in the peripheral nervous system
f) Clinical Features


The period between ingestion of the toxin and the appearance of signs and
symptoms is usually 1-2 days, but it may be much longer
They may be initial nausea and vomiting
26
Practical medical microbiology


The oculomotor muscles are affected and the patient may have diplopia and
drooping eyelids with a squint. There may be vertigo and blurred vision
There is progressive descending motor loss with flaccid paralysis but no loss
of consciousness or sensation, though weakness & sleepiness are often described




The patient is thirsty, with a dry mouth and tongue
Difficulties in speech and swallowing, with later problems of breathing
There may be abdominal pain and restlessness
Death is due to respiratory or cardiac failure
g) Laboratory Diagnosis






The diagnosis must be suspected on clinical manifestation
The diagnosis may be confirmed by demonstration of
o Organism and/or its toxin may be detected in the patient's stool or gastric
contents
o Organism and/or its toxin may be detected in the suspected food
o Toxin may be demonstrated in the patient's blood
Samples of vomit or faces may also yield such evidence
Food or stool specimens are emulsified, heated at 800C & inoculated on blood agar
Gram stain of the suspected colonies revealed that the organism is gram
positive bacilli, motile, and sporulated. The spores are oval and sub-terminal
Toxin is detected in either food or blood by toxin-antitoxin neutralization test in mice
h) Treatment

The priorities are
o To remove unabsorbed toxin from the stomach and intestinal tract
o To neutralize unfixed toxin by giving polyvalent antitoxin and
o To give relevant intensive care and support
i) Control




Home canning of foodstuffs should be avoided and commercial canning must
be strictly controlled
Acid fruits must be bottled safely in the home with heating at 100 0C, since
low pH is inhibitory to its growth
A prophylactic dose of polyvalent antitoxin should be given intramuscularly to
all persons who have eaten food suspected of causing botulism
Active immunization should be considered for laboratory staff who might
have to handle the organism or who might have to handle specimens
containing the organism or its toxin
Clostridium difficile and antibiotic associated diarrhea



Cl. difficile is part of the normal intestinal flora in a small proportion of
healthy persons and hospitalized patients
Exposure to antibiotics alerts the normal enteric flora, permeating overgrowth
of Cl. difficile or making the patient more susceptible to exogenous
acquisition of Cl. difficile.
Proliferation of Cl. difficile with localized production of their toxins in the
colon leads to disease
27
Practical medical microbiology
a) Description




Motile Gram positive rod
Oval spores
Quite commonly in the faces of neonates, nut is not generally regarded as a
normal commensals of adults
Produces an enterotoxin (toxin A) and a cytotoxin (toxin B)
b) Pathogenesis

This organism has a direct relationship with pseudomembranous colitis
c) Laboratory Diagnosis


Can be isolated from the faces by enrichment and selective culture procedures
Toxin can be detected in the patient's faces by testing extracts against cell
monolayer of human embryo fibroblasts or other susceptible cells or by
immunological methods such as ELISA
d) Treatment

It is essential to discontinue the antibiotic that is presumed to have precipitated the
disease and to suppress the growth and toxin production by giving oral vancomycin
or metronidazole
e) Prevention



Clinical awareness is the keynote
If a patient develops unexplained diarrhea, especially if this is antibioticassociated, the possibility of Cl. difficile and pseudomembranous colitis
should be borne in mind
If several cases occur in a hospital unit, the possibility of cross-infection
should be considered and the existing antibiotic policy of the unit should be
reviewed
28
Practical medical microbiology
Practical NO 6
Gram negative rods
Objective:
1-
To identify Enterobacteriaceae from other Gram negative rods and
to differentiate between lactose fermenter and non lactose
fermenter by:
A. Detection of the presence of cytochrome oxidase in Gram-negative rods.
B. To determine the ability of bacteria to metabolize glucose oxidatively or
fermentatively.
C. To know Gram reaction of Enterobacteriaceae.
D. Use of MacConkey agar to differentiate between Lactose fermenter and
non lactose fermenter Enterobacteriaceae.
Required materials:
1- Overnight cultures of E.coli, K. pneumoniae, Shigella, Salmonella,
Pseudomons.
2- Gram stains dyes and reagents, filter paper, slides.
3- 80 plates of MacConkey agar
4- 160 tube containing 5ml OF media
5- 10 ml of oxidase reagent
Experimental:
1- Gram stain of Enterobacteriaceae.
2- Oxidase teat.
3- OF test.
4- Inoculation of MacConkey agar with lactose fermenter and non lactose
fermenter Enterobacteriaceae.
 Oxidase and Oxidative-Fermentative (OF) tests can be used in
differentiation Enterobacteriaceae from other Gram negative rods
as showed in the table:
29
Practical medical microbiology
I-
Microorganism
Oxidase test
OF test
Enterobacteriaceae
Negative
Fermentative
Pseudomonads
Positive
Oxidative
Vibrio
Positive
Fermentative
Oxidase Test:
1. Principle - To determine the presence of cytochrome oxidase in bacteria.
Recall that in aerobic bacteria the cytochrome serve as electron carriers during
aerobic respiration. The detection of cytochrome is extremely beneficial in
differentiating many groups of bacteria. All members of Enterobacteriaceae
are oxidase negative, while other Gram-negative rods, such as Pseudomonas,
are oxidase positive. The oxidase test is based on the ability of bacteria to turn
(oxidize) a reagent (tetramethyl-p-phenylenediamine), which serves as an
alternate substrate for the cytochrome oxidase, to a purple color.
2. Materials:
o
Overnight cultures of E. coli and Pseudomonas aeruginosa.
o
Oxidase reagent "tetramethyl-p-phenylenediamine".
o
Pieces of filter paper.
3. Method - hold a piece of the oxidase test paper with forceps and touch onto an
area of heavy growth.
4. Results - Color change to purple within:
o
10 seconds = positive
o
10 - 60 seconds = delayed positive
o
>60 seconds = negative
5. Special Features
o
Useful in differentiation of Enterobacteriaceae (-) and Pseudomonas (+).
o
An oxidase positive organism will be catalase positive.
o
Strict anaerobe organisms are oxidase negative.
6. Precautions in interpretation - The test is only reliable as long as the time
limit for a positive result is adhered to (up to 60 seconds maximum)
30
Practical medical microbiology
 Oxidation-Fermentation test:
1. Principle - To determine the ability of bacteria to breakdown glucose
oxidatively or fermentatively. The OF test is used to determine whether a
bacterium has the enzymes necessary for the aerobic breakdown of glucose
(i.e. oxidation) and/or for the fermentation of glucose.
2. Materials:
a- Overnight cultures of E. coli and Pseudomonas aeruginosa.
b- Four tubes of OF medium/each student.
c- Sterile liquid paraffin.
3. Method
a- Inoculate two tubes of OF medium for each organism being tested.
Inoculation is carried out as a stab to within 1 cm of the bottom of the
tube.
b- Overlay one tube (covered) only with sterile paraffin oil to exclude all
oxygen. Not overlay the second tube (open).
c- Incubate at 37°C for 24 hours.
4. Results
Open tube
Covered tube
Result
Yellow
Green
oxidation (O)
Yellow
Yellow
fermentation (F)
Green
Green
No action on glucose
5. Special Features - The test differentiates Enterobacteriaceae (F) from the
Pseudomonas sp. (O), and Micrococcus sp. (O) from Staphylococcus sp. (F).
6. Precautions in Interpretation - Some organisms require prolonged
incubation before acid production is visible.
31
Practical medical microbiology

Members of Enterobacteriaceae are characterized by:
Small Gram-negative non-spore-forming enteric bacilli
All Enterobacteriaciae:
1. ferment glucose with acid production
2. reduce nitrates into nitrites (NO3 to NO2 or all the way to N2)
3. are oxidase negative
All are aerobic but can be facultative anaerobic
Motile via peritrichous flagella except Shigella and Klebsiella which are non-motile
Non-capsulated except Klebsiella
Grow on ordinary medium i.e. non-fastidious as well as grow on bile containing
media as MacConkey's medium, which used in primary classification depends on
lactose fermentation on MacConkey's agar medium
Some members of the Enterobacteriaceae are true pathogens such as:
Salmonella spp.
Shigella spp.
Yersinia spp.
Certain strains of Escherichia coli
ETEC = enterotoxigenic E. coli
EIEC = enteroinvasive E. coli
EPEC = enteropathogenic E. coli
EHEC = enterohemorrhagic E. coli
EaggEC = enteroaggregative E. coli
UPEC = uropathogenic E. coli
32
Practical medical microbiology
Most members of the Enterobacteriaceae are opportunistic or cause secondary
infections of wounds, the urinary and respiratory tracts, and the circulatory system
e.g. E. coli.

Primary classification of Enterobacteriaceae depends on lactose fermentation:
Enterobacteriaceae
Lactose fermenter
Lactose non fermenters
e.g. E. coli, Klebsiella,
Enterobacter, Citrobacter
e.g. Salmonella, Shigella,
Proteus
Laboratory Identification:
Specimens whether pus, sputum, urine, feces, CSF should be cultured immediately
or placed on special media to prevent overgrowth
Culture:
Colony morphology: moist, gray (except Serratia marcescans which appears red)
smooth colonies on non-selective media
Special differential and selective media used for separation of genera and species
I- MacConkey agar medium:
 MacConkey agar is selective and differential medium for isolation of
Enterobacteriaceae. The nutritive base includes variety of peptones. The
medium is made selective by the incorporation of bile (although at levels
less than those used in other enteric medium) and crystal violet, which
inhibit gram-positive bacteria, especially staphylococci and enterococci.
The medium is differential by use of combination of neutral red (in acidic
pH the color turns red) and lactose. When an organism ferment lactose, the
drop in pH (due to acid formation as end product of fermentation of
33
Practical medical microbiology
lactose) causes the colony to take on a pink-red appearance whereas
organism which unable to ferment lactose appears as pale yellow colonies.
II Eosin Methylene Blue (EMB) agar medium:

EMB agar is a selective and differential medium used for the isolation and
differentiation of enteric pathogens from contaminated clinical specimens.
Pancreatic digest makes up the nutritive base. Eosin and methylene blue
are the selective agents and inhibit gram-positive organisms. EMB
contains lactose. Organisms that ferment lactose binds to dyes under acidic
conditions and appear as blue-black colonies with a metallic sheen. Under
less acidic conditions, other coliforms, such as Klebsiella, Enterobacter,
and Citrobacter, appear as brown-pink "dark" colonies. Nonfermenters,
such as Salmonella, Shigella, and Proteus, appear as the color of medium
(amber) or transparent and colorless.
III. Salmonella-Shigella (SS) agar:

SS agar is a selective and differential medium used for isolation and
differentiation of Salmonella and Shigella from clinical specimens. The
nutritive base contains animal and casein peptones and beef extract. The
selective agents are bile salts, and brilliant green dye, which inhibit
gram-positive organisms. The high degree of selectivity of the medium
results in the inhibition of some strains of Shigella, and the medium is not
recommended as primary medium for isolation of this species. The
medium contains only lactose and thus differentiates organisms on the
basis of lactose fermentation. The formation of acid on fermentation of
lactose causes the neutral red indicator to make red colonies. Non lactose
fermenting organisms are clear on the medium. SS agar contains sodium
thiosulfate and ferric ammonium citrate allows the differentiation of
organisms that produce H2S. Lactose fermenters, such as E. coli, have
colonies which are pink with a precipitate, Shigella appears transparent or
amber, and Salmonella appears transparent or amber with black centers.
34
Practical medical microbiology
IV. Xylose-Lysine-Desoxycholate (XLD) agar:

XLD is a selective and differential medium used for the isolation and
differentiation of enteric pathogens from clinical specimens. The nutritive
base includes carbohydrates and yeast extract. This medium is supportive
of fastidious enteric organisms such as Shigella. The selective agent is
desoxycholate, which inhibit gram-positive organisms. Phenol red is the
color indicator. As with SS agar, ferric ammonium citrate (indicator) and
sodium thiosulfate (sulfur source) allow identification of organisms that
produce H2S with appearance of colonies with black center. The medium
contains xylose, which most of enteric organisms ferment. The most
important exception is Shigella, the colonies appear to be transparent or
the color of red medium. The lysine in the medium is utilized by the
enteric organisms that contains lysine decarboxylase enzyme. For
Salmonella, which contains lysine decarboxylase enzyme, this reaction
converts the pH to an alkaline state and the colonies appear transparent or
red with black center. The lactose and sucrose in the medium help to
differentiate other enteric organisms. When other enteric organisms
ferment these sugars, they maintain the pH at an acidic condition and the
colonies appear yellow or yellow red.
V. Selenite broth:

Selenite broth is an enrichment broth medium used for isolation of
Salmonella and Shigella species from clinical specimens. Casein and meat
peptones provide nutrients. Selenite inhibits enterococci and coliforms that
are part of the normal flora if they subcultured within 12 to 18 h. However,
reduction of selenite produces an alkali condition that may inhibit the
recovery of Salmonella. Lactose and phosphate buffers are added to allow
the stability of the pH. When fermentating organisms produce acid, the
acid neutralizes the effect of selenite reduction and subsequent
alkalinization. Cystein is added to selenite broth to enhance the recovery of
Salmonella.
35
Practical medical microbiology
Practical NO 4
Enterobacteriaceae: I- Lactose Fermenters
Biochemical tests for identification of Enterobacteriaceae:
 As mentioned in the previous lesson, all members of
Enterobacteriaceae are ferment glucose, oxidase negative, and
reduce nitrate into nitrites.
 Nitrate Reduction:
1. Principle - To determine the ability of an organism to reduce nitrate to nitrites
or free nitrogen gas
2. Method - Inoculate a nitrate broth and incubate for 5 days at 37°C.
3. Result
1. To each nitrate broth culture add 1 ml of sulphanilic acid and 1 ml of
-naphtylamine. The production of a red color occurs in the presence
of nitrite indicates the ability of the organism to reduce nitrate to
nitrite.
2. To broths showing a negative reaction add a few particles of zinc. The
appearance of a red color indicates that nitrate is still present and hence
has not been reduced by the organism. If the solution does not change
color the organism has reduced the nitrate through nitrite to nitrogen
gas.
4. Special Features - Used in identification of Enterobacteriaceae (usually +).
5. Precautions in Interpretation - Interpret results immediately as the color
produced in a positive reaction may fade quickly.
 Triple Sugar Iron Agar (TSI) and H2S production:
1. Principle - To determine the ability of an organism to attack a specific
carbohydrate incorporated into a basal growth medium, with or without the
36
Practical medical microbiology
production of gas, along with the determination of possible hydrogen sulphide
(H2S) production.
2. Method - Inoculate TSI medium with an inoculating needle by stabbing the
butt and streaking the slant. Incubate at 37°C for 24 hours.
3. Results
Butt
Slant
color
color
Yellow
Red
Yellow
Red
Yellow
Yellow
Red
Red
H2S
Negative
Result
Glucose only fermented
Example
Lactose non fermenter
e.g. Shigella
Positive/blackening
Glucose only fermented
Lactose non fermenter
in butt
with H2S production
e.g. Salmonella & Proteus
Negative
Glucose fermented, also
Lactose fermenter
lactose and/or sucrose
e.g. E. coli
No action on glucose,
Non fermenter organisms
lactose or sucrose
e.g. Pseudomonas
Negative
37
Practical medical microbiology
4. H2S production is indicated by the presence of a black precipitate
5. Special Features - H2S production and carbohydrate fermentation patterns are
generally characteristic for specific bacterial groups, especially the
Enterobacteriaceae.
6. Precautions in Interpretation - An H2S organism may produce so much of
the black precipitate (ferrous sulphide) that the acidity produced in the butt is
completely masked. However, if H2S is produced, an acid condition does exist
in the butt even if it is not observable.
Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests:
o The following four tests comprise a series of important determinations
that are collectively called the IMViC series of reactions (I= indole;
M=methyl red; V=Voges Proskauer; and C=citrate). The IMViC series
of reactions allows for the differentiation of the various members of
Enterobacteriaceae.
 Indole "Tryptophan Hydrolysis" Test:
1. Principle – Certain microorganisms can metabolize the amino acid tryptophan
through the action of the enzyme tryptophanase. Once again, the activity of the
enzyme is not measured directly, but rather, one of the end products (Indole) is
detected. The enzymatic degradation leads to the formation of pyruvic acid,
indole and ammonia. The presence of indole is detected by addition of Kovac's
reagent.
2. Materials (per 20 students)
1. Tube of tryptone water (20)
2. Tested microorganism (such as E. coli (+) and Klebsiella (-)
3. Kovac's reagent (20 ml)
3. Method
1. Inoculate tryptone water with the tested microorganism
2. Incubate at 37°C for 48 hours
3. After incubation interval, add 1 ml Kovacs reagent, shake the tube
gently and read immediately.
38
Practical medical microbiology
Kovacs reagent
p-dimethylaminobenzaldehyde 50 g
amyl or butyl alcohol
750 ml
HCl (conc.)
250 ml
4. Result - a bright pink color in the top layer indicates the presence of indole.
The absence of color means that indole was not produced, and that the
organism does not possess the tryptophanase enzyme i.e. indole is negative
5. Special Features - Used in the differentiation of genera and species. e.g. E.
coli (+) from Enterobacter (-).
6. Precautions in Interpretation
o
Cultures to be tested for indole production must be incubated
aerobically
o
The optimum pH for tryptophanase activity is one that is slightly
alkaline (ph 7.4 - 7.8); a decrease in pH results in decreased indole
production and a possible false negative.
 Methyl Red "Mixed acid Fermentation" Test:
1. Principle – All enteric bacteria can utilize sugar for their energy demands.
Mixed acid fermenters such as E. coli ferment glucose to produce large
amounts of acetic, formic and succinic acids as end products. The large
amount of acid produced lowers the pH of the medium to below 5.0. By using
the indicator methyl red (MR), the production of these acids as the end product
of fermentation can be monitored. If pH drops below 4.5, the color of MR
indicator will be red. If the pH is above 6.0, the color will be yellow/orange.
2. Material (per 20 students)
1. 20 tubes of MRVP medium (Phosphate buffered glucose peptone water)
2. Tested microorganism
3. Methyl red indicator (20 ml)
3. Method
1. Inoculate the tested organism into a tube of MRVP broth.
2. Incubate the tube at 37°C for 24 hours.
39
Practical medical microbiology
3. After incubation, add a few drops of MR solution to the culture. Read
immediately.
4. Result
Red
MR positive
Yellow/orange
MR negative
5. Special features - The test is used to differentiate between genera. e.g. E. coli
and Citrobacter (+) from Klebsiela and Enterobacter (-)
6. Precautions in Interpretation - If the MR test is performed too early, the
results may be a false positive since MR negative organisms may not have had
time to completely metabolize the initial acid products that accumulated from
the glucose fermentation
 Voges Proskauer "Butanediol Fermentation" Test:
1. Principle – Some bacteria, rather than producing abundant acid in the
fermentation of glucose, produce other products such as alcohols. All species
of Enterobacter and Klebsiella will form products such as alcohol and 2,3butanediol rather than the large amount of acid, as does E. coli and
Citrobacter. A test for acetylmethylcarbinol (a precursor of 2-3, butanediol
that appear in the growth medium) is performed. If Barrit's reagent is added, a
pink color developing after few minutes indicates the presence of
acetylmethylcarbinol and the test is positive.
2. Materials (per 20 students)
1. Tubes of MRVP (20)
2. Tested microorganism
3. Barrit's solution A "40%KOH" (20 ml)
4. Barrit's solution B "-Naphthol" (20 ml)
3. Method
1. Inoculate buffered glucose broth with the organism
2. Incubate the inoculated tube at 37°C for 24 h.
40
Practical medical microbiology
3. After it has become apparent that growth has occurred, add
approximately 0.5 ml of -naphthol, followed by 0.5 ml of 40% KOH.
4. Shake vigorously and let the tube stand for 1 to 2 h.
5. Results
Pink
VP(+)
No change
VP(-)
6. Special Features - The test is used to differentiate between genera.
e.g. K. pneumoniae (+) and Enterobacter (+) from E. coli (-) and
Citrobacter.
7. Precautions in Interpretation - After exposure to the reagents for
over 2 hour, a negative VP culture may show a copper-like color due to
the action of the KOH on the alpha naphthol. This is not a positive
reaction.
 Citrate Utilization Test
1. Objective – determine whether microorganism can utilize citrate as a sole
source of carbon.
2. Principle – Some microorganisms can metabolize citrate as a sole of carbon
source. Like many of the nutrients already used, citrate needs to be transported
into the bacterial cells before it can be metabolized. This transport depends
upon the enzyme citrate permease. Once inside the cell, the citrate is broken
down to pyurvate and carbon dioxide. The carbon dioxide produced combines
with sodium in the medium and water to form sodium carbonate, an alkaline
product. The rise in pH is detected by the color change in bromothymol blue
indicator present in the medium from green to deep blue.
Citrate →Pyruvate →CO2 + Na + H2O →Na2CO3 →↑ pH
3. Method - Streak a Simmon's Citrate agar slant with the organism and incubate
at 37°C for 24 hours.
41
Practical medical microbiology
4. Result - Examine for growth (+). Growth on the medium is accompanied by a
rise in pH to change the medium from its initial green color to deep blue.
5. Special Features - Aids in the differentiation of genera and species.
6. Precautions in Interpretation - The medium must be lightly inoculated (from
plate cultures, not from a broth) to avoid a carry over of nutrients, which may
lead to a false positive result.
 Urea Hydrolysis (Urease):
1. Principle – Urease is an enzyme that catalyzes the conversion of urea to CO2
and NH3. The urease test is particularly useful in identifying Proteus,
Klebsiella and Enterobacter. The Christensen's urea agar used for the test
contains yeast extract, urea and the indicator phenol red. In the presence of
urease, urea split and ammonia and carbon dioxide are produced. Ammonia
combines with water to produce ammonium hydroxide, a strong base which
raises the pH of the medium. This rise in the pH causes the phenol red
indicator to turn a deep pink. This is indicative of a positive reaction for
urease.
2. Method - Streak a urea agar tube with the organism and incubate at 37°C for
24 h
3. Result –If color of medium turns from yellow to pink indicates positive test.
Proteus give positive reaction after 4 h while Kelebsiella and Enterobacter
gave positive results after 24 h...
4. Special Features - Aids in the differentiation of members of Proteus also
differentiates between E. coli (-) from Klebsiella (+) and Enterobacter (+).
5. Precautions in Interpretation - The test must be read after 4 h and 24 h
Item
Result
Example
Non-urease producer
Negative
E. coli
Week urease producer
Positive after 18 hrs
Klebsiella
Strong urease producer
Positive after 2-6 hrs
Proteus
The differences between Lactose fermenter are summarized in the following table:
42
Practical medical microbiology
Items
E. coli
MacConkey's Rose pink colonies
Citrobacter
Klebsiella
Enterobacter
Rose pink colonies
Rose pink colonies
Rose pink colonies
EMB
Metallic sheen
Dark colonies
Dark colonies
Dark colonies
Indole
+
+
-
-
MR
+
+
-
-
VP
-
-
+
+
CIT
-
+
+
+
Urease
-
+
+
+
Motility
Motile
Motile
Non motile
Motile
43
Practical medical microbiology
Practical NO 7
Enterobacteriaceae: I- Non Lactose Fermenters
Enterobacteriaceae
Lactose fermenter
Lactose non fermenters
e.g. E. coli, Klebsiella,
Enterobacter, Citrobacter
e.g. Salmonella, Shigella,
Proteus, Yersinia
I-
Salmonella species:
 General characters:

Salmonella species are:
o Gram-negative rods
o Oxidase negative
o Ferment glucose
o They do not ferment lactose and sucrose
o Motile
o Non capsulated
o Non-spore forming
o Facultative anaerobic
o Characterized by O, H, and Vi antigens.
o There are over 1800 known serovars which current classification
considers being separate species.

Two important members of Salmonella causing diseases:
1- Salmonella causing enteric fever which caused by either Salmonella typhi
or Salmonella paratyphi
2- Salmonella causing food poisoning which is caused by either Salmonella
enteritidis or Salmonella typhimurium.
44
Practical medical microbiology

Enteric fever " Typhoid"

Enteric fevers are severe systemic forms of salmonellosis.

The best studied enteric fever is typhoid fever, the form caused by S
typhi, but any species of Salmonella may cause this type of disease.

The symptoms begin after an incubation period of 10 to 14 days.

Enteric fevers may be preceded by gastroenteritis, which usually
resolves before the onset of systemic disease.

The symptoms of enteric fevers are nonspecific and include fever,
anorexia, headache, myalgias, and constipation.

Enteric fevers are severe infections and may be fatal if antibiotics are
not promptly administered.

Transmission via fecal-oral route = person-to-person spread by
chronic carrier through fecally-contaminated food or water

10-14 day incubation to signs of sepsis; sustained fever (delirium) for
one to several weeks before abdominal pain and gastrointestinal
symptoms

Laboratory diagnosis of typhoid:
A- Direct diagnosis:
1- Specimen: Blood during 1st week, urine during 2nd week and stool during 3rd week
2-Isolation of microorganism:
o From blood using blood culture. Five to 10 ml of blood is taken aseptically
from patient during the 1st week of infection, add to 50-100 ml sterile nutrient
broth and incubate at 370C for 24 hrs. Subculture is done on MacConkey's
agar which shows pale yellow colonies in positive case i.e. non lactose
fermenter.
o From stool by culture on enrichment medium such as selenite F or
tetrathionate broth which inhibits the growth of coliform and allow the growth
of Salmonella and Shigella. Subculture is made on selective medium such as
Salmonella-Shigella (SS) or deoxycholate citrate (DCA) agar which support
the growth of Salmonella and Shigella. The suspected colonies are picked and
identified by Gram stain and biochemical reactions.
45
Practical medical microbiology
3- Biochemical identification of Salmonella:

Salmonella is oxidase negative, ferment glucose, non ferment lactose
and sucrose., on MacConkey's agar they give yellow colonies, on SS
agar they give pale yellow colonies with black edges due to H2S
production and on EMB they appear as colorless colonies. The
biochemical reactions of Salmonella are outlined in the following
Table.
Biochemical Test
Indole
MR
VP
CIT
Urease
TSI/H2S
Motility
Salmonella typhi
-
+
-
-
-
A/Alk/+
Motile
B- Serological diagnosis:

In the 2nd week of the disease, antibodies against Salmonella are present in the
patient's serum and can be detected by:

Widal test is positive and valid during 2nd week. This is an agglutination test
used to detect antibodies which appear in the serum of patient during 2nd week.
Serial dilutions of patient's serum are added to an equal volume of common O
and specific H antigens. Agglutination of O- antigen and one only of the Hantigens at a titer 1/80 or above is diagnostic.
II-
Shigella species
The members of this genus are:

Gram negative rods

Non motile

Non spore-forming

Non capsulated

Oxidase negative

Ferment glucose

Non lactose fermentating organism

They are the causative agent of bacillary dysentery
Four species:
1. Shigella dysenteriae: causes most serious form of bacillary dysentery
46
Practical medical microbiology
2. Shigella
flexneri:
most
common
cause
of
shigellosis
in
underdeveloped countries
3. Shigella sonnei: most common cause of shigellosis in developed
countries
4. Shigella boydii : causes mild intestinal upset
Classification:
Shigella
Non Mannitol
Fermenters
Mannitol fermenter
Subgroup B: S. flexneri
Subgroup A: Shigella dysenteriae
Dysentery
Subgroup C: S. boydii
Subgroup D: S. sonnei
Clinical Syndromes (shigellosis):
Ranges from asymptomatic infection to severe bacillary dysentery
Two-stage disease: watery diarrhea changing to dysentery with frequent small
stools with blood and mucus, tenesmus, cramps, fever
Early stage:
Watery diarrhea attributed to the enterotoxic activity of Shiga toxin
Fever attributed to neurotoxic activity of toxin
Process involves:
1. Ingestion
2. Noninvasive colonization and cell multiplication
3. Production of the enterotoxin by the pathogenic bacteria in the small intestine;
Second stage:
Adherence to and tissue invasion of large intestine
Typical symptoms of dysentery
Cytotoxic activity of Shiga toxin increases severity
47
Practical medical microbiology
Laboratory diagnosis:
Specimen: Stool or rectal swap
Stool culture is done from mucous bloody part of stool on enrichment media such
as Selenite broth at 370C for 24 h then subculture on SS or DCA agar. The suspected
pale yellow colonies is picked up and examined by:
Gram stain: Gram negative bacilli, non motile, non spore forming and non
capsulated.
Biochemical reactions:

Shigella is oxidase negative, ferment glucose, non ferment lactose and,
on MacConkey agar they give yellow colonies, on SS agar they give
pale yellow colonies and on EMB they appear as colorless colonies.
The biochemical reactions of Shigella are outlined in the following
Table.
Biochemical Test
Indole
MR
VP
CIT
Urease
TSI/H2S
Motility
Shigella
-
+
-
-
-
A/Alk/-
Non motile
48
Practical medical microbiology
III-
Proteus species
General characters of Proteus:
Gram negative rods
Oxidase negative
Ferment glucose
Not ferment lactose
Facultative anaerobes
Non spore forming
Actively motile:
a. Flagella (H antigen)
b. Swarms on lab media, producing spreading colonies
Urease positive after 2-6 hrs (urea → NH3+ CO2)
Grows well at alkaline pH
Major pathogens are Proteus mirabilis and Proteus vulgaris
P. mirabilis causes urinary tract infections (UTIs): urease alkalinizes urine
→precipitation of calcium and magnesium salts → stone formation →renal
epithelium damage
.P. vulgaris causes nosocomial infections (pneumonia, bacteremia) and UTIs.
Laboratory diagnosis:
Specimen: Urine or Stool.
Culture:
 On MacConkey agar →non lactose fermenters→ pale yellow colonies
 On EMB agar → non lactose fermenters →colorless colonies
 On SS agar →non lactose fermenters→ pale yellow colonies with black center
 On ordinary media, such as nutrient agar, blood agar, show swarming
(successive waves on the surface) due to high motility of Proteus.
 The suspected is picked up and examined by:
Gram stain: Gram negative bacilli, motile, non spore forming and non
capsulated.
49
Practical medical microbiology
Biochemical reactions:

Proteus is oxidase negative, ferment glucose, non ferment lactose. The
biochemical reactions of Proteus are outlined in the following Table.
Biochemical Test
Indole
MR
VP
CIT
Urease
TSI/H2S
Motility
Proteus mirabilis
-
+
-
-
+ (2-6 h)
A/Alk/-
Non motile
The differences between non lactose fermenter are summarized
in the following table:
Items
Salmonella
Shigella
Proteus mirabilis
MacConkey
Pale yellow colonies
Pale yellow colonies
Pale yellow colonies
SS agar
Pale yellow colonies
Pale yellow colonies
Pale yellow colonies with black
with black center
center
EMB
Colorless colonies
Colorless colonies
Colorless colonies
Swarming
No swarming
No swarming
Swarm on non inhibitory medium
Indole
-
-
-
MR
+
+
+
VP
-
-
-
CIT
-
-
+
Urease
-
-
+ (2hrs)
TSI/H2S
A/Alk/+
A/AlK/-
A/AlK/+
Motility
Motile
Non-motile
Motile
50
Practical medical microbiology
Practical NO 8
First Practical Exam from section No1 to No7
Pseudomonas aeruginosa
Characteristics:












It is a Gram-negative, aerobic rod belonging to the family
Pseudomonadaceae.
These bacteria are common inhabitants of soil and water.
Motile by means of a single polar flagellum.
Non spore forming
Capsulated "Polysaccharide capsule"
Aerobic
Breakdown glucose by oxidation
Oxidase and catalase positive
It has very simple nutritional requirements (able to grow on commercial
distilled water).
Its optimum temperature for growth is 37 degrees, and it is able to grow at
temperatures as high as 42 degrees.
It is tolerant to a wide variety of physical conditions, including temperature. It
is resistant to high concentrations of salts and dyes, weak antiseptics, and
many commonly used antibiotics.
Pseudomonas aeruginosa is opportunistic pathogen and associated with a
variety of infections including:
o Urinary tract infections
o Wound and burn with blue green pus
o Respiratory system infections
o Eye infection and may lead to blindness
o Ear infection (external ear or otitis media)
o A variety of systemic infections
Pseudomonas aeruginosa colonies on agar
51
Practical medical microbiology


P. aeruginosa strains produce two types of soluble pigments:
o Pyoverdin or fluorscein: It is yellow-green pigment and fluorescent.
o Pyocyanin: It is a blue-green pigment and non-fluoroscent.
Laboratory diagnosis
o Specimen: Urine, pus, sputum, or blood
o Stain: Gram-negative rods and motile
o Culture:
 On Nutrient agar→ Colonies are surrounded by bluish green
coloration
 On Blood agar → -hemolytic colonies
 On MacConkey agar → pale yellow colonies i.e. non lactose
fermenters
 On selective media "Cetermide" → pigments are more obvious
 Pseudomonas aeruginosa able to grow at temperatures as high
as 42 degrees.
o Biochemical reactions:
 Oxidase positive (See details of test under practical No3).
 Breakdown glucose oxidatively i.e. OF test is open type turned
from green to yellow and the covered tube with oil still green
(See details of test under practical No3).
 Gelatin liquefaction test:
 Principle – Upon boiling collagen, a major connective tissue,
the product gelatin is formed. Certain bacteria are capable of
producing a proteolytic exoenzyme called gelatinase, which
hydrolyze the protein to amino acids. These amino acids can
then be transported into the cell for further metabolism. At
temperature below 25°C, gelatin will remain a gel, but if the
temperature rises about 25°C, the gelatin will be liquid. If you
cool the liquefied gelatin, it will resolidify. Gelatin hydrolysis
has been correlated with pathogenicity of some
microorganisms. It is thought that pathogenic bacteria may
breakdown tissue and spread to adjacent tissues.
 Method - Streak a slope of nutrient agar tube with heavy
inoculums of tested organism. By using a sterile forceps
transfer aseptically gelatin tablet containing charcoal to
inoculated nutrient agar tube and incubate at 37°C for 24 h
 Result –If a tablet of gelatin dissolve and turns to liquid with
black coloration indicates positive test (Pseudomonas
aeruginosa). If the tablet remains solid indicates negative test
(E. coli).
52
Practical medical microbiology
Practical NO 9
Vibrionaceae: Vibrios
 General characters:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Gram negative, curved (comma shaped) bacilli
Motile by single polar flagella
Non spore forming
Non capsulated
Most vibrios have relatively simple growth factor requirements and grow well
in alkaline pH
Facultative anaerobes
Vibrios are capable of both respiratory & fermentative metabolism i.e. O+/F+.
Oxidase and catalase positive
Natural inhabitants of aquatic environment
 Species of Vibrio:
Vibrios
Vibrio cholerae
Cholera
V. parahaemolyticus
Gastroenteritis
Classical type
V. cholera






Allied vibrios
Saprophytic
El-Tor-type
V. El-Tor
V. cholerae and V. parahaemolyticus are pathogens of humans.
Vibrio cholera is the cause of cholera
Vibrio parahaemolyticus is the cause of acute gastroenteritis following
ingestion of contaminated sea-food such as raw fish.
Allied vibrios: are a large group of organisms; some of them are saprophytic
while others cause disease in animals.
V. cholerae and V. parahaemolyticus produce diarrhea, but in ways those are
entirely different.
o V. parahaemolyticus is an invasive organism affecting the colon
o V. cholerae is noninvasive, affecting the small intestine through
secretion of an enterotoxin.
o V. cholerae can be divided serologically into 6 groups based on
somatic O-antigens
o Most of pathogens belong to the O1 group
o V. cholerae can be subdivided into two biotypes, V. cholerae and V.
El-Tor
El-Tor Vibrio can be differentiated from classical V. cholera by the following
characters:
53
Practical medical microbiology
Item
Hemolysis on BA
Voges-Proskauer test
Resistance to polymyxin B
V. cholera
Non hemolytic
Negative
Sensitive
Vibrio El-Tor
Hemolytic
positive
Resistant
Cholera:







Cholera is a severe diarrheal disease caused by Vibrio cholerae O1 seotype.
It is endemic in southern Asia (India, Pakistan, and Bangladesh).
Transmission is by contaminated water or food through oral-fecal routs.
Incubation period of the disease is 1-4 days.
It characterized by sudden onset of intense vomiting and diarrhea (rice water
stool) with rapid dehydration.
The disease progresses from the first liquid stool to shock in 4-12 hours, with
death following in 18 hours to several days.
V. cholerae produces cholera toxin "enterotoxins", whose action on the
intestinal mucosal epithelium is responsible for the characteristic diarrhea of
the disease cholera through stimulation of cAMP, resulting in active secretion
of chloride and secondary loss of sodium and water.
 Diagnosis of cholera:
o Primary case: A suspected case of cholera appearing in a nonendemic area (primary case) must be fully identified before being
considered as cholera and the identification is done by:
 Specimen: rice watery stool
 Culture:
 Inoculation of rice water stool in alkaline peptone
water, pH9 in which the organisms multiply rapidly and
tend to form pellicle at the surface of the medium after
6-8 h at 370C.
 Subculture is made into Thiosulphate Citrate Bile
Sucrose (TCBS) agar.
 TCBS is selective and differential medium for
vibrios
o This medium is selective because:
 It contains bile salt
 pH of the medium is alkaline (pH9)
o This medium is differential because:
 It contains sucrose, some of Vibrio spp
ferment sucrose and form acids as end
product of fermentation, the acid is
detected by pH indicator, bromothymol
blue, which turns from deep blue
(alkaline pH) to yellow (acidic pH).
54
Practical medical microbiology
Practical NO 10
Haemophilus
 The term Haemophilus represents a large group of Gram-negative
rods that like to grow on blood agar and restricted to organisms that
are dependent on one or both of certain growth promoting
substances termed X (haematin) and V (NAD) which present in
blood; therefore these organisms grow only on blood agar or
chocolate agar.
 General characters:
1.
2.
3.
4.
5.
6.
7.
Non intestinal pleomorphic Gram negative coccobacilli
Grown under aerobic conditions or under slight CO2 tension (5% CO2)
Non motile
Non spore forming
Usually capsulated
Fastidious
Oxidase and catalase positive
 Species of Haemophilus:




H. influenzae
H. parainfluenzae
H. aegypticus
H. ducreyi
 H. influenzae:



Naturally-acquired disease caused by H. influenzae seems to occur in
humans only (5 months-5 years).
Non capsulated (nontyable) strains colonize the nasopharynx in up
to 80% of healthy individuals in the upper respiratory tract and become
a secondary pathogen on respiratory mucosa that has become
susceptible to bacterial attack after primary influenzae virus infection.
 Non capsulated strains are less invasive, but they are apparently
able to induce an inflammatory response that causes disease.
 It may spread locally and cause pneumonia, otitis media, and
sinusitis
Encapsulated strains, especially type b (Hib), are a very minor
colonized in 5% of healthy individuals in the upper respiratory tract.
 Capsule (type b polysaccharide capsule) is known to be the
major factor in virulence.
 All strains are serotyped into 6 different types (a-f) based on
their biochemically different capsules and the most
pathogenic one is type b
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Practical medical microbiology
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Encapsulated organisms can penetrate the epithelium of the
nasopharynx and invade the blood capillaries directly.
Their capsule allows them to resist phagocytosis and
complement-mediated lysis in the non-immune host.
It cause pediatric meningitis, otitis media & epiglottitis
(obstructive laryngitis)
H. influenzae requires both X and V factors
 H. parainfluenzae:
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Similar to non capsulated strain of H. influenzae
This organism needs only V factor
 H. aegypticus:
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It is cause of acute mucopurulent conjunctivitis (Pinkeye)
The disease is common in Egypt
It is spread very easily, especially among children.
Pinkeye is transmitted mechanically by common towels or by flies
This organism requires both X and V factors
The diagnosis is made from the conjunctival discharge.
 H. ducreyi
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This is the cause of chancroid (Soft chancre) which is venereal disease
The disease is characterized by painful genital ulcers
This organism needs only X factor.
The diagnosis is made from the discharge of the ulcer.
 Diagnosis of Haemophilus:
o Your child's physician may diagnose the illness based on clinical
examination and a medical history. It can be confirmed by detecting
the bacteria in blood, spinal fluid, or other body fluid.
o Specimen:
 according to site of infection; swap, sputum, CSF, discharge
o Stain:
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Gram negative coccobacilli
o Culture:
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H. influenzae and H. aegypticus grow on blood agar or
chocolate agar as it requires X factor and V factor that found on
blood
 On Blood agar: A 24 h colony of H. influenzae on
blood agar is very small usually non hemolytic
 On chocolate agar: A 24 h colony of H. influenzae on
chocolate agar is larger than that observed on blood
agar
H. duceryi requires only X factor
H. parainfluenzae requires only V factor
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Practical medical microbiology
o Satellitism:
 Blood agar contains much X factor and little V factor and so
the presence of another microorganism such as Staph. aureus
which produces V factor will support the growth of H.
influenzae i.e. larger colonies of H. influenzae are observed
near Staph. aureus colonies.
o Capsule swelling:
 Specific antiserum added to a slide of the organism allows
swelling of the bacterial capsule thus permitting rapid diagnosis
of H. influenzae in sputum.
 Prevention of H. influenzae:
o In 1985, the first Hib polysaccharide vaccines were licensed for use in
the United States.
o These vaccines contained purified polyribosylribitol phosphate (PRP)
capsular material from the type b serovar.
o There are several types of Hib conjugate vaccines available for use.
o Immunization against type b is routinely administered in a three- or
four-part series.
 The first vaccine is received at 2 months of age and the two
subsequent doses are given at about 4 months and 6 months. A
booster is then given between 12 and 15 months of age.
 If a child did not receive the vaccine and is older than 5 years,
it may not be necessary for them to be immunized.
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Practical medical microbiology
Practical NO 11
Acid-fast bacteria
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Acid fast bacteria include the Mycobacterium and few species of Nocardia.
The Mycobacterium include 2 species:
o Mycobacterium tuberculosis, which causes tuberculosis
o Mycobacterium leprae, which causes leprosy
Mycobacterium tuberculosis, and M. bovis all cause the disease known as
tuberculosis (TB) and are members of the tuberculosis species complex.
Each member of the TB complex is pathogenic, but M. tuberculosis is
pathogenic for humans while M. bovis is usually pathogenic for animal
Mycobacterium contains 40% lipid content in their cell envelop.
High lipid content difficult to stain by ordinary dye but requires special dye
as CF and heating, and once stained are difficult to decolorize with acidalcohol mixture.
Acid fastness is due to high lipid content of cell envelop
Morphology, metabolism and characters:
o Pleomorphic rods, slender (thin) straight or slightly curved rods, 2-4 
long x 0.2-0.5  in diameter
o Acid-alcohol fast
o Mycobacteria are Gram-positive (no outer cell membrane)
o Non-motile
o Non-spore forming
o Non-capsulated
o Obligate aerobic
o Catalase positive
o Most Mycobacteria are found in habitats such as water or soil.
However, a few are intracellular pathogens of animals and humans.
Cultural and Physiological characteristics:
o TB is obligate aerobes→ infect lung → rich in oxygen
o They can grow on simple liquid medium
o They are grow on complex enriched & selective media e.g.
Lowenstein-Jensen medium which is made by addition of malachite
green & some antibiotics which inhibit the growth of fungi and other
bacteria.
o Slow growing organism generation time each 20 h need 4 weeks
for growth.
o Presence of CO2 enhances the growth.
o Optimum growth temp is 370C.
Biochemical reactions: not routinely used. TB are catalase positive
Antigenic structure:
o Lipoid fraction or Cord factor Trehalose dimycolate
o Lipid fraction
 Long chain fatty acids called Mycolic acidsAcid fastness and
slow growth
 Wax Denhance the immune response
 Phsphatides  play role in caseation
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Practical medical microbiology
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o Protein fraction which when combined with wax  elicits delayed
hypersensitivity.
 These proteins are antigens in the purified protein derivative
used in skin test.
Epidemiology of Mycobacterium:
o Source of infection:
 Human with active pulmonary disease
 Milk of infected animals
o Portal of entry:
 Lung is the major portal of entry by inhalation of infected
droplets and 70% of tuberculous patients have pulmonary
disease.
 Oral rout by ingestion of infected milk extra-pulmonary
disease.
 TB can persist in dried sputum and dust for long periods.
 TB is resistant for heating at 600C for 1 h. They killed at 1000C
for 10 min.
 TB is sensitive to Phenol and formalin disinfectants.
 Acquiring infection is proportional to the rate of active
infection the community, crowding, socioeconomic status and
inadequacy of medical care.
 Development of clinical disease occur only in a small number
of infected persons and is affected by:
 Age (16-21 y)
 Malnutrition
 Immunological status
Pathogenesis of TB:
o I- Primary tuberculosis:
 The first exposure to M. tuberculosis is called primary
tuberculosis
 TB is a highly communicable disease that is transmitted mainly
by droplets due to M. tuberculosis causing pulmonary
infection or by ingestion of cow milk infected with M. bovis
resulting in intestinal infection
 Primary tuberculosis may occur at any epithelial site but
common in lung
o II- Secondary or Reactivation infection:
 This is usually caused by endogenous TB that have survived in
the primary lesion in less than 5% of those infected and rarely
due to exogenous infection
 Reactivation is seen primarily in immuno-compromised
patients
 It is characterized by chronic tissue lesions, formation of
tubercles, caseation and fibrosis
 Reactivation lesions occur at apex of lung and other well
oxygenated organs such as brain, kidney
Diagnosis:
A. Clinical manifestations:
o Fever, fatigue, night sweats, and weight loss are common.
o Cough and bloody sputum are common in pulmonary TB
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Practical medical microbiology
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o Meningitis or UTI can occur
B. Laboratory Diagnosis:
Direct methods:
o Microscopical examination:Acid-fast bacteria
o Rapid culture:
 Bactec radiometric culture: a liquid broth in bottle , with
radioactive palmitate as a carbon source
 Mycobacteria grow and use the carbon, allowing early
detection (1-2 weeks) even before colonies can be seen
Indirect methods: used
o When direct method give negative result or
o When there is no available material for direct examination
PCR and DNA probes
Chest X-ray
Tuberculin test (PPD test):
o This is a skin test which is useful in diagnosis of TB specially during
the primary infection where the majority of cases are symptom less
o During primary infection the host acquires hypersensitivity to TB and
becomes positive to tuberculin
o It is carried out by intradermal injection of 0.1ml of purified protein
derivative (tuberculin) in the forearm
o Red indurations are observed in positive test, measuring not less than
10 mm in diameter appears after 2-3 days after injection. The best time
for reading is 72 hours after injection
Significance of tuberculin test:
o A positive tuberculin test:
 Means that the person has got a tuberculosis focus in his body
but does not usually tell whether is active or latent
 A positive test in adults is of no much significance because
80% of the adults become infected with TB but the disease is
manifested only in much lower percentage
 A positive test in children below 5 years is more significant
because any tuberculosis focus is seriously considered
o A negative tuberculin test:
 Means that there is no infection at all or very old healed one
o Uses of tuberculin test:
 To determine the incidence of TB infection in community
 Before B.C.G vaccination to minimize the tuberculin negative
individual only
o Immunity against tuberculosis:
 TB stimulates
 Cellular immunity by CD4 T cell (protective)retard
the multiplication
 Humoral immunity (non protective)
 Delayed hypersensitivity (harmful): related to pathology
of disease. It is due to protein fraction
o Prevention and control:
 Eradication of infected animals
 Pasteurization of milk and better nutrition
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Practical medical microbiology
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Vaccination of children during the first month of age, otherwise
vaccinated person should be tuberculin negative using BCG
vaccine: live attenuated of mycobacterium bovis by subculturing 250 times
 INH prophylaxis for immunosuppressed patients
o Treatment:
 Isoniazide
(Isonicotinc
Hydrzide,
INH),
rifampicin,
pyrazinamide, ethambutol and streptomycin
 Anti TB are used in combination to overcome resistance e.g.
INH + rifampicin (Rimectazide).
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