Microbiology Laboratory Faculty of Medicine Brawijaya University

MICROBIOLOGY LABORATORY
FACULTY OF MEDICINE
BRAWIJAYA UNIVERSITY
 Infection of the upper respiratory tract  are the most
common type of infection
 Anatomical defenses against airborne pathogens:
 Coarse hairs in the nose
 Mucous membrane contains mucous secreting cells;
cilia
 Lymphoid tissue
 Some potentially pathogens are part of the normal
flora
Microbial diseases of the ENT
 Pharyngitis
 Laryngitis
 Tonsillitis
 Sinusitis
 Epiglotitis
 Otitis media
Bacterial diseases
 Strep throat (streptococcal pharyngitis)
 Diphtheria
 Otitis media
Bacteria:
 Streptococci
 Corynebacterium diphtheriae
 Hemophilus influenzae
Strep throat
 Caused by group A β-hemolytic streptococci –
Streptococcus pyogenes
 The pathogenicity is enhanced by the resistance to
phagocytosis
 Produce streptokinase and streptolysin
 Complications: quinsy (peritonsillar abscess), scarlet
fever, rheumatic fever, acute glomerulonephritis
 Laboratory diagnosis
 Culture & identification: using Blood Agar
 β hemolysis, presumptive
identification  bacitracin test (+)
 Serologic test  anti streptolysin O titer
 Treatment:
 Penicilllin (drug of choice)
Diphtheria
 Corynebacterium is pleiomorphic, non-sporeforming
bacteria that are ubiquitous in plants, animals, and
humans.
 They possess storage granules called metachromatic
granules, and an unique side-by-side palisade
arrangement
 C. diphtheriae is the most important member of the
group, as it can produce powerful exotoxin that causes
diphtheria in humans
 Lysogenic Corynebacterium diphtheriae,
transmitted via respiratory droplets, contains
bacteriophage that codes for diphtheria exotoxin,
which cause the symptoms of potentially fatal
diphtheria. The bacteria can colonize the pharynx
(especially tonsillar regions), the larynx, nose,
genital organ, and skin.
 C. diphtheriae is an aerobic and facultatively
anaerobic organism but grows best under
aerobic conditions
 Complex media are requires for primary

isolation and characterization
 Löeffler’s coagulated serum or Pai coagulated
egg medium  for the primary isolation
 Löeffler’s medium is useful because it does not

support the growth of streptococci and
pneumococci
 The addition of tellurite salts to media used for

primary isolation also reduces the number of
contaminants
RESISTANCE
 C. diphtheriae is more resistant to the action of
light, desiccation, and freezing than are most non
sporeforming bacilli
 They are readily killed, by 1 minute exposure to
1000C or 10 minute exposure to 580C
ANTIGENIC STRUCTURE
 K antigen: responsible for the type specificity of C.
diphtheriae strains, are heat-labile proteins
 Play an important role in antibacterial immunity
 K antigen on the surface, together with glycolipid cord
factor are major determinants of invasiveness and
virulence in diphtheria bacilli
 The heat-stable O antigen of C. diphtheriae is a group
antigen
 It is a polysaccharide containing arabinogalactans
 Is the antigen responsible for the cross reactivity with
mycobacteria and nocardia
 The cord factor, a toxic glycolipid, is 6-6’ diester of
trehalose containing the mycolic acids
characteristic of C. diphtheriae, corynemycolic acid
(C32H62O3) and corynemycolenic acid (C32H64O3)
 The pharmacologic activity of the cord factor of C.
diphtheriae is similar to that of the cord factor
from Mycobacterium tuberculosis
 Other factors contribute are neuraminidase and N-
acetylneuraminate lyase
EXOTOXIN
 Secreted as a single polypeptide chain with a
molecular weight of approximately 58 kD
 When released, the native toxin molecule is
nontoxic until exposure of the active enzymatic
site by mild trypsin treatment
 The biologically active molecule consist of two
functionally distinct polypeptide chains, fragments
A and B, linked by a disulfide bridge
 Both fragments are essential for cytotoxicity
 The amino terminal portion of the toxin, fragment
A (± 21 kD) contains the enzymatically active site
of the toxin. Fragment B (± 37 kD), from the
carboxyterminal of the toxin, contains the
eucaryotic cell receptor binding domain
IMMUNITY
 Depends on the presence in the blood of antitoxin
 < 6 months, infants are passively protected from
diphtheria by transplacental passage of antitoxin
from immune mother
 The widespread immunization of infants and
preschool children with toxoid had reduced the
incidence of diphtheria in children
 The immune status of a person may be assessed by
determination of serum antitoxin levels or by the
Schick test
SCHICK TEST
 Performed by injecting into the skin of the forearm
0,1 ml of highly purified toxin
 A positive reaction indicating the absence of

immunity to diphtheria characterized by local
inflammatory reaction.
 A negative reaction  shows that antitoxin level is

> 0,03 U/ml, and person is immune.
 Based on toxin-antitoxin neutralization

PATHOGENESIS
 After exposure to C. diphtheriae, there is an
incubation period of 1 – 7 days
 The initial lesion usually occurs on the tonsils and
oropharynx, then spread to the nasopharynx,
larynx, and trachea
 The organisms multiply rapidly on epithelial cells
in the local lesion, producing an exotoxin that
causes necrosis of cells in the area
 An inflammatory reaction results, accompanied by
the outpouring of a fibrinous exudate
 At first patchy in appearance, as the local
exudative lesions coalesce a very tough adherent
pseudomembrane forms  that composed of
fibrine, necrotic epithelial cells, lymphocytes,
polymorphonuclear leukocytes, erythrocytes, and
diphtheria bacilli
 In tonsillar diphtheria, onset characterized by low-
grade fever, malaise, and a mild sore throat
 The cervical lymph nodes become edematous and
tender, especially when there is involvement of the
nasopharynx
 Swelling may be so pronounced that the classic
bull neck appearance results
LABORATORY DIAGNOSIS
 Culture: Löffler or Pai slant, and tellurite blood
agar plate should be inoculated immediately
 Invivo test: either guinea pig or rabbit may be
used. The test is performed by injecting 0,2 ml of a
48 hours infusion broth culture of the test
organism intracutaneously into the shaved animal
 Invitro test: Elek test (precipitation test)
Corynebacterium diphtheriae ELEK test
Blood tellurite agar
TREATMENT
 Antibacterial agent: penicillin or erythromycin
 Antitoxin should be given as soon as a presumptive
diagnosis of diphtheria is made clinically, without
waiting for laboratory confirmation, because toxin
binds rapidly to susceptible tissue cells;
given intramuscularly or intravenously in a single
dose
PREVENTION
 Active immunization  key to control and
prevention of diphtheria
 The primary course of immunization should be
started during the first 6 – 8 weeks of life
 Given as toxoid (DPT or Dt only)
Hemophilus influenzae
 Morphology: coccobacilli in pairs or short chains,
pleiomorphic
 Needs X factor and V factor for growth
 Possess capsular polysaccharides
 Somatic antigen: lipooligosaccharides (acts as
bacterial endotoxin)
Pathogenesis
 The non-encapsulated bacteria  normal flora of
respiratory tract
 Type b  causes epiglotitis, meningitis,
pneumonia, and other invasive infections
 Nontypeable H. influenzae tends to cause chronic
bronchitis, otitis media, sinusitis, and
conjunctivitis
Laboratory diagnosis
 Culture and identification: IsoVitaleX medium
(contains X & V factor), capsular swelling test
using specific antiserum
Treatment
 Ampicillin, cephalosporin
Prevention
 Vaccination using Hemophilus influenzae b
conjugate vaccine
VIRAL DISEASES - Common cold
 About 200 different viruses can cause the common
cold, rhinovirus cause ± 50% of all colds, coronavirus
15-20%
 Symptoms: sneezing, nasal secretions, and congestion
 Mostly transmitted by indirect contact
 The incidence increase during cold weather
 Host’s receptor protein for rhinovirus is ICAM-1 
attach and affect the cell lining the nasal passage
 Treatment: symptomatic (untreated cold: recover
in 1 week, w/ drugs: 7 days)
 Prevention: hand washing, using masker
THANK YOU

Microbiology Laboratory Faculty of Medicine Brawijaya University

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MICROBIOLOGY LABORATORY

 Complex media are requires for primary

 Löeffler’s medium is useful because it does not

 The addition of tellurite salts to media used for

 A positive reaction indicating the absence of

 A negative reaction  shows that antitoxin level is

 Based on toxin-antitoxin neutralization

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