LABORATORY ACTIVITY

Resource Person : Julia Hartati/Ratna Damailia

Subject : Identification Bacterial Pathogen and Mycoses in Tropical Diseases
Department : Microbiology
A. Sequent
I Introduction : 30 minutes
II Pretest : 10 minutes
III Lab Activities : 120 minutes
IV Post test : 10 minutes
B. Topic
Identification Bacterial Pathogen and Mycoses in Tropical Diseases : 120 minutes

C. Venue
Biomedical Laboratory, Faculty of Medicine, Unisba, Jl. Tamansari No.22 Bandung 40116
D. Introduction
1. Tropical Diseases
E.

2. Bacterial Pathogens in Tropical Diseases

A. Corynebacterium diphtheriae
Physiology and Structure
C. diphtheriae is an irregularly staining, pleomorphic rod (0.3 to 0.8 × 1.0 to 8.0 μm). After
overnight incubation, large 1- to 3-mm colonies are observed on blood agar medium. More
selective, differential media can be used to recover this pathogen from specimens with
other organisms present, such as pharyngeal specimens. This species is subdivided into four
biotypes based on their colonial morphology and biochemical properties: belfanti, gravis,
intermedius, and mitis, with most disease caused by biotype mitis.
Pathogenesis and Immunity

Diphtheria toxin is the major virulence factor of C. diphtheriae. The tox gene that codes for
the exotoxin is introduced into strains of C. diphtheriae by a lysogenic bacteriophage, β-phage. Two
processing steps are necessary for the active gene product to be secreted: (1) proteolytic cleavage
of the leader sequence from the Tox protein during secretion from the bacterial cell and (2)
cleavage of the toxin molecule into two polypeptides (A and B) that remain attached by a disulfide
bond. This 58,300-Da protein is an example of the classic A-B exotoxin.

Three functional regions exist on the toxin molecule: a catalytic region on the A subunit and
a receptor-binding region and a translocation region on the B subunit. The receptor for the toxin is
heparin-binding epidermal growth factor, which is present on the surface of many eukaryotic cells,
particularly heart and nerve cells; its presence explains the cardiac and neurologic symptoms
observed in patients with severe diphtheria. After the toxin becomes attached to the host cell, the
translocation region is inserted into the endosomal membrane, facilitating the movement of the
catalytic region into the cell cytosol. The A subunit then terminates host cell protein synthesis by
inactivating elongation factor-2 (EF-2), a factor required for the movement of nascent peptide
chains on ribosomes. Because the turnover of EF-2 is very slow and approximately only one
molecule per ribosome is present in a cell, it has been estimated that one exotoxin molecule can
inactivate the entire EF-2 content in a cell, completely terminating host cell protein synthesis. Toxin
synthesis is regulated by a chromosomally encoded element, diphtheria toxin repressor (DTxR).
This protein, activated in the presence of high iron concentrations, can bind to the toxin gene
operator and prevent toxin production.

Culture

Specimens for the recovery of C. diphtheriae should be collected from both the
nasopharynx and throat and should be inoculated onto a nonselective, enriched blood agar plate
and a selective medium (e.g., cysteine-tellurite blood agar [CTBA], Tinsdale medium, colistin-
nalidixic agar [CNA]). Tellurite inhibits the growth of most upper respiratory tract bacteria and
gram-negative rods and is reduced by C. diphtheriae, producing a characteristic gray to black color
on agar. Degradation of cysteine by C. diphtheriae cysteinase activity produces a brown halo
around the colonies. CTBA has a long shelf life (practical for cultures that are infrequently
performed) but inhibits some strains of C. diphtheriae. Tinsdale medium is the best medium for
recovering C. diphtheriae in clinical specimens, but it has a short shelf life and requires addition of
horse serum. Because infections caused by C. diphtheriae are rarely seen or suspected in
nonendemic areas, CTBA and Tinsdale medium are not commonly available in most laboratories.
CNA is commonly used for the selective recovery of gram-positive bacteria; therefore this is a
practical alternative medium. Regardless of the media used, all isolates resembling C. diphtheriae
must be identified by biochemical testing and the presence of the diphtheria exotoxin confirmed
because nontoxigenic strains occur.
 Albert stain is a type of differential stain used for staining high-molecular-weight polymers of
polyphosphate known as metachromatic granules or volutin granules found in C. diphtheriae.
Metachromatic granules are also found in Yersinia pestis and Mycobacterium species. It is named
metachromatic because of its property of changing color i.e when stained with blue stain they appear red
in color. When grown in Loffler’s slopes, C. diphtheriae produces a large number of granules.
Principle of Albert Staining:
Albert stain is basically made up of two stains that are toluidine blue’ O’ and malachite green both of
which are basic dyes with high affinity for acidic tissue components like cytoplasm. The pH of Albert
stain is adjusted to 2.8 by using acetic acid which becomes basic for volutin granules as the pH of
volutin granule is highly acidic. Therefore on applying Albert’s stain to the smear, toluidine blue’ O’
stains volutin granules i. e the most acidic part of cell and malachite green stains the cytoplasm blue-
green. On adding Albert’s iodine due to effect of iodine, the metachromatic property is not observed
and granules appear blue in colour.

Procedure of Albert Staining

1. Prepare a smear on clean grease free slide.
2. Air dry and heat fix the smear.
3. Treat the smear with Albert’s stain and allow it to react for about 7 mins.
4. Drain of the excess stain do not water wash the slide with water.
5. Flood the smear with Albert’s iodine for 2 minutes.
6. Wash the slide with water, air dry and observe under oil immersion lens.
Result : If Corynebacterium diphtheriae is present in the sample it appears green colored rod-shaped
bacteria arranged at an angle to each other, resembling English letter ‘L’, ‘V’, or Chinese letter
pattern along with bluish-black metachromatic granules at the poles.

Test for Toxigenicity

The identification of an isolate as C. diphtheriae does not mean that the patient has
diphtheria. Diagnosis of diphtheria is established by also demonstrating that the isolate produces
diphtheria toxin. The growth medium and conditions markedly affect toxin production. The iron
content of the medium must be growth rate limiting for full toxin production. The addition of iron
to iron-starved cultures quickly inhibits toxin production. In vivo toxin testing is rarely done
because the in vitro methods are reliable, less expensive, and free from animal use.

The in vitro diphtheria toxin detection procedure is an immunodiffusion test first described
by Elek. In the Elek test, organisms (controls and unknowns) are streaked on medium of low iron
content. Each organism is streaked in a single straight line parallel to each other and 10 mm apart.
A filter paper strip impregnated with diphtheria antitoxin is laid along the center of the plate on a
line at right angles to the inoculum lines of control and unknown organisms (Fig. 16.5). The plate is
incubated at 35° C and examined after 18, 24, and 48 hours. Lines of precipitation are best seen by
transmitted light against a dark background. The white precipitin lines start about 4 to 5 mm from
the filter paper strip and are at an angle of about 45 degrees to the line of growth. If an isolate is
positive for toxin production and it is placed next to the positive control, the toxin line of the
positive control should join the toxin line of the positive unknown to form an arch of identity.

The Elek test requires that reagents and antisera be carefully controlled and titrated.
Because of the difficulty of the test, it is performed only in certain reference laboratories. Rapid
enzymelinked immunosorbent assays and immunochromatographic strip assays are also available
for the detection of diphtheria toxin. In addition, procedures for detecting the C. diphtheriae tox
gene by polymerase chain reaction (PCR) have been developed. The PCR assay can also be applied
directly to clinical specimens.
B. Salmonella enterica, serovar. Typhi
Physiology and Structure

Members of the family Enterobacteriaceae are moderate sized (0.3 to 1.0 × 1.0 to 6.0 µm),
non–spore-forming, gram-negative rods that share a common antigen (enterobacterial common
antigen). All members can grow rapidly, aerobically and anaerobically (facultative anaerobes), on a
variety of nonselective (e.g., blood agar) and selective (e.g., MacConkey agar) media. The
Enterobacteriaceae have simple nutritional requirements, ferment glucose, reduce nitrate, and are
catalase positive and oxidase negative.

The appearance of the bacteria on culture media has been used to differentiate common
members of the Enterobacteriaceae. For example, fermentation of lactose (detected by color
changes in lactose-containing media such as the commonly used MacConkey agar) has been used
to differentiate some enteric pathogens that do not ferment lactose or do so slowly (e.g.,
Salmonella, Shigella, and Yersinia spp.—colorless colonies on MacConkey agar) from lactose-
fermenting species (e.g., Escherichia, Klebsiella, Enterobacter, Citrobacter, and Serratia—pink-
purple colonies on MacConkey agar). Resistance to bile salts in some selective media has also been
used to separate enteric pathogens (e.g., Shigella, Salmonella) from commensal organisms that are
inhibited by bile salts (e.g., gram-positive and some gram-negative bacteria present in the
gastrointestinal [GI] tract). In this way, use of culture media that assess lactose fermentation and
resistance to bile salts is a rapid screening test for enteric pathogens that would be otherwise
difficult to detect in diarrheal stool specimens, where many different organisms may be present.

Pathogenesis and Immunity

After ingestion and passage through the stomach, salmonellae attach to the mucosa of the
small intestine and invade into the M (microfold) cells located in Peyer patches, as well as into
enterocytes. The bacteria remain in endocytic vacuoles, where they replicate. The bacteria can also
be transported across the cytoplasm and released into the blood or lymphatic circulation.
Regulation of attachment, engulfment, and replication is controlled primarily by two large clusters
of genes (pathogenicity island I and II) on the bacterial chromosome. Pathogenicity island I encodes
salmonella-secreted invasion proteins (Ssps) and a type III secretion system that injects the proteins
into the host cell. Pathogenicity island II contains genes that allow the bacteria to evade the host’s
 immune response and encodes a second type III secretion system for this function. The
inflammatory response confines the infection to the GI tract, mediates the release of
prostaglandins, and stimulates cAMP and active fluid secretion.

C. Mycobacterium leprae
The nontuberculous mycobacterium M. leprae is a close relative of M. tuberculosis. This
organism causes leprosy (also called Hansen’s disease). Leprosy is a chronic disease of the skin,
mucous membranes, and nerve tissue.

M. leprae has not yet been cultivated in vitro, although it can be cultivated in the armadillo
and in the footpads of mice. Molecular biologic techniques have provided most of the information
about this organism’s genomic structure and its various genes and their products. Although
polymerase chain reaction (PCR) assays have been used to detect and identify M. leprae in infected
tissues, the technique thus far has not proved as effective diagnostically as anticipated in
indeterminate or paucibacillary (few organisms present) disease. Therefore, diagnosis of leprosy is
based on distinct clinical manifestations, such as hypopigmented skin lesions and peripheral nerve
involvement, in conjunction with a skin smear that tests positive for acid-fast bacilli.

Laboratory diagnosis of Hansen disease depends on the microscopic demonstration of AFB

from skin biopsy specimens. M. leprae has not been grown on artificial media. In patients with
tuberculoid leprosy, organisms are extremely rare and may not be detected in skin scrapings or
biopsy specimens. However, AFB are usually abundant in samples from patients with lepromatous
leprosy (Fig. 26.5). M. leprae is not as acid fast or alcohol fast as in the case of other mycobacteria;
as such, a weaker decolorizer consisting of 10% sulfuric acid is recommended instead of the
standard acid-ethanol decolorizer. The bacteria are rod shaped, usually 1 to 7 µm long and 0.3 to
0.5 µm wide. The entire smear should be examined under oil immersion field (×1000) for the
presence of the microorganisms. PCR assays are also available to definitively detect the bacteria
D. Leptospira
General Characteristics

Organisms of the genus Leptospira are tightly coiled, thin, flexible spirochetes, 0.1 µm wide
and 5 to 15 µm long (Fig. 23.1). In contrast to both Treponema and Borrelia organisms, the spirals
are very close together, so the organism may appear to be a chain of cocci. One or both ends of the
organism have hooks rather than tapering off. Their motion is rapid translational (back and forth)
and rotational.
 Laboratory Diagnosis

Microscopy

Because leptospires are thin, they are at the limit of the resolving power of a light microscope and
thus cannot be seen by conventional light microscopy. Neither Gram stain nor silver stain is reliable
in the detection of leptospires. Darkfield microscopy is also relatively insensitive, capable of
yielding nonspecific findings.

Culture

Leptospires can be cultured on specially formulated media (e.g., Fletcher, EMJH [Ellinghausen-
McCullough-JohnsonHarris], Tween 80-albumin). They grow slowly (generation time, 6 to 16 hours),
requiring incubation at 28° C to 30° C for as long as 4 months; however, most cultures are positive
within 2 weeks. Consistent with the two phases of illness, leptospires are present in blood or CSF
during the first 10 days of infection and in urine after the first week and for as long as 3 months.
Because the concentration of organisms in blood, CSF, and urine may be low, several specimens
should be collected if leptospirosis is suspected. In addition, inhibitors present in blood and urine
may delay or prevent recovery of leptospires. Likewise, urine must be treated to neutralize the pH
and concentrated by centrifugation. A few drops of the sediment are then inoculated into the
culture medium. Growth of the bacteria in culture is detected by darkfield microscopy.

3. Mycoses
Mycoses (singular, mycosis) are diseases caused by fungi. Fungal disease is frequently categorized
on the basis of the site of the infection—superficial, cutaneous, subcutaneous, and systemic mycoses.
Fig. 27.10 shows the different layers of tissues in which fungal infections can occur. With the help of
this figure, it becomes easier to classify infections of the skin, depending on where the infection occurs.
Infections not involving the skin or deeper tissues just under the skin are termed systemic. Superficial
and cutaneous mycoses are caused by fungi that can degrade keratin (dematophytes) and fungi that
are not able to degrade keratin (nondermatophytes).

For clinicians, dividing the fungi into four categories of mycoses, according to the type of infection,
is much more useful. The fungi are categorized as follows:

• Superficial (cutaneous) mycoses

• Subcutaneous mycoses

• Systemic mycoses

• Opportunistic mycoses

The superficial, or cutaneous, mycoses are fungal infections that involve the hair, skin, or nails without
direct invasion of the deeper tissue. The fungi in this category include the dermatophytes (agents of
ringworm, athlete’s foot) and agents of infections such as tinea, tinea nigra, and piedra. All of these
infect keratinized tissues. Some fungi cause infections that are confined to the subcutaneous tissue
without dissemination to distant sites. Examples of subcutaneous infections include
chromoblastomycosis, mycetoma, and phaeohyphomycotic cysts.

Systemic, or disseminated, mycoses are infections that affect internal organs or deep tissues of the
body. Frequently, the initial site of infection is the lung, from which the organism disseminates
hematogenously to other organs, including skin. Generalized symptoms include fever and fatigue.
Chronic cough and chest pain might also accompany these infections. Historically, the term systemic
mycoses has been used to describe diseases caused by thermally dimorphic fungi, including
Histoplasma, Coccidioides, and Blastomyces spp. Other fungal agents are capable of causing systemic
disease and include Aspergillus, Fusarium, Scedosporium, and Curvularia spp. as well as some yeasts,
including Candida and Cryptococcus spp. It is important to note that any fungus is capable of
disseminating from the primary site of infection in the immunocompromised host.

Malassezia furfur

Clinical Manifestations.

The Malassezia furfur complex causes tinea versicolor, a disease characterized by patchy lesions or
scaling of varied pigmentation. It is also thought to be a cause of dandruff. Lesions associated with tinea
versicolor typically appear as pale patches in individuals with darkly pigmented skin, but they can also
be described as fawn-colored liver spots in individuals with a fair complexion. Lesions become
especially evident in warm months, when sun exposure is more likely. Tinea versicolor may involve any
area of the body, but the most prevalent sites include the face, chest, trunk, and abdomen. Antifungal
therapy is not typically indicated, but the appearance of lesions may be diminished by treatment with
antidandruff shampoos. Interestingly, M. furfur complex has also been implicated in disseminated
infections in patients receiving lipid replacement therapy (total parenteral nutrition), particularly in
infants. Removal of indwelling feeding lines is usually sufficient to clear infections without using
antifungal therapy.

Laboratory Diagnosis

M. furfur can be identified by visualizing skin scrapings from characteristic lesions in a potassium
hydroxide (KOH) preparation or by observing yellow fluorescence with the Wood lamp on examination
of the infected body site. Microscopic examination of the direct smear in KOH preparations reveals
budding yeasts, approximately 4 to 8 µm, along with septate, sometimes branched, hyphal elements.
This microscopic appearance has gained M. furfur the nickname the spaghetti and meatballs fungus
(Fig. 27.11). M. furfur requires lipids for growth and will not grow on routine fungal media that have
not been supplemented with a lipid source. Because of its special nutritional requirements, routine
fungal cultures are negative for growth. Typical yeastlike colonies may be observed only after the
culture medium has been overlaid with olive oil. Colonies are cream colored, moist, and smooth.
4. Dermatophytes
The dermatophytes produce infections involving the superficial areas of the body, including the hair,
skin, and nails (dermatomycoses). The genera Trichophyton, Microsporum, and Epidermophyton are
the principal etiologic agents of the dermatomycoses

Trichophyton spp.

Members of the genus Trichophyton are widely distributed and are the most important and common
causes of infections of the feet and nails; they may be responsible for tinea corporis, tinea capitis, tinea
unguium, and tinea barbae. They are commonly seen in adult infections, which vary in their clinical
manifestations. Most cosmopolitan species are anthropophilic, or “human loving”; few are zoophilic,
primarily infecting animals. Generally, hairs infected with Trichophyton organisms do not fluoresce
under the ultraviolet (UV) light of a Wood’s lamp. Fungal elements must be demonstrated inside,
surrounding, and penetrating the hair shaft or within a skin scraping to diagnose a dermatophyte
infection by direct examination. Confirmation requires recovery and identification of the causative
organism.

Microscopically, Trichophyton organisms are characterized by smooth, club-shaped, thin-walled

macroconidia with three to eight septa ranging from 4 × 8 µm to 8 × 15 µm. The macroconidia are
borne singly at the terminal ends of hyphae or on short conidiophores; the microconidia (which may
be described as “birds on a fence”) predominate and are usually spherical, pyriform (teardrop shaped),
or clavate (club shaped), and 2 to 4 µm (Figure 60-8).

Only the common Trichophyton species are described here. T. rubrum and T. mentagrophytes are the
most common species recovered in the clinical laboratory. T. rubrum is a slow-growing organism that
produces a flat or heaped-up colony, generally white to reddish, with a cottony or velvety surface. The
characteristic cherry-red color is best observed on the reverse side of the colony; however, this is
produced only after 3 to 4 weeks of incubation.

Microsporum spp. 

Species of the genus Microsporum are immediately recognized by the presence of large (8-15 × 35-150
µm), spindle-shaped, echinulate, rough-walled macroconidia with thick walls (up to 4 µm) containing
four or more septa (Figure 60-14).

The exception is Microsporum nanum, which characteristically produces macroconidia having two
cells. Microconidia, when present, are small (3 to 7 µm) and club shaped and are borne on the hyphae,
either laterally or on short conidiophores. Cultures of Microsporum spp. develop either rapidly or
slowly (5 to 14 days) and produce aerial hyphae that may be velvety, powdery, glabrous, or cottony,
varying in color from whitish, buff, to a cinnamon brown, with varying shades on the reverse side of
the colony.

Epidermophyton sp. 

E.floccosum, the only member of the genus Epidermophyton, is a common cause of tinea cruris and
tinea pedis. Because this organism is susceptible to cold, specimens submitted for dermatophyte
culture should not be refrigerated before culture, and cultures should not be stored at 4°C. In direct
examination of skin scrapings using the calcofluor white or potassium hydroxde preparation, the
fungus is seen as fine branching hyphae. E. floccosum grows slowly; the growth appears olive green to
khaki, with the periphery surrounded by a dull orange-brown. After several weeks, colonies develop a
cottony white aerial mycelium that completely overgrows the colony; the mycelium is sterile and
remains so even after subculture. Microscopically, numerous smooth, thin-walled, club-shaped,
multiseptate (2 to 4 µm) macroconidia are seen (Figure 60-16).
They are rounded at the tip and are borne singly on a conidiophore or in groups of two or three.
Microconidia are absent, spiral hyphae are rare, and chlamydoconidia are usually numerous. The
absence of microconidia is useful for differentiating this organism from Trichophyton spp.; the
morphology of the macroconidia (smooth, thin walled) is useful for differentiating it from Microsporum
spp.
 Note:
If the pre-test score less than 50, the student can’t allow to do lab. Activities
F. Lab Activities
1. The Students were divided into six group
2. Each group perform discussion accompanied by tutor
G. Reference
1. Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition, the McGraw−Hill
Companies, 2002
2. Sherris Medical Microbiology, An Introduction to Infectious Diseases 6th Edition,
Kenneth J. Ryan, Md, C. George Ray, Md, Editors, Mcgraw-Hill Medical Publishing
Division, 2018
3. Bailey and Scott’s Diagnostic Microbiology, 14th edition, Betty A. Forbes, Daniel F. Sahm,
Alice S. Weissfeld, Elsevier, 2017
4. Medical Microbiology, Eight edition, Patrick R. Murray, Ken S. Rosenthal, Michael A.
Pfaller, Editors, Elsevier, 2016

H. HOMEWORK ASSIGNMENT
1. Explain the morfology of bacteria:
a. Corynebacterium diphteriae
b. Leptospira
c. Mycobacterium leprae
d. Salmonella enterica serovar Typhi
2. Explain the pathogenesis infections from bacteria:
a. Corynebacterium diphteriae
b. Leptospira
c. Mycobacterium leprae
d. Salmonella enterica serovar Typhi
3. Define and differentiate superficial, cutaneous, subcutaneous, and systemic mycoses,
including the tissues involved.
4. Define differentiate the identification of yeasts and filamentous fungi (molds).
5. Describe differentiate the sexual and asexual reproduction of fungal