Mycobacteria

.Very thin rod shaped and non-motile bacilli

.cell wall : N-glycolylmuramicacid, cord factor
wax D and mycolicacid.
.Mycobacteriaare difficult to stain however
they resist decolorizationhence they are
called ACID FAST BACILLI
.Can be divided into 2 major groups based on
the fundamental differences in epidemiology
and association with disease
.Mycobacterium TB complex
.M. tuberculosis, M.bovis, M. Africanum
.Mycobacteriathat causes tbin human was first isolated
by ROBERT KOCH in 1886.
.Non tuberculousMycobacteriaor MOTT

.
.MYCOBACTERIUM
TUBERCULOSIS COMPLEX.
1. Mycobacterium tuberculosis
Human Tubercle Bacilli

Koch bacillus

or

.cause of most cases of tuberculosis

.captain of all diseases
.found only in humans
.Colonies: non-pigmented, dirty warty and granular
.niacin positive, nitrate negative
.Reservoir: human lungs
.Transmitted via: respiratory droplets
.PATHOGENESIS:
.Facultative intracellular organism
.SULFATIDES(sulfolipidin cell envelope):
.this inhibits the phagosomelysosomalfusion allowing intracellular survival.
.If fusion occurs, waxy nature of cell envelope reduces the killing effect

.PATHOGENESIS
.CORD FACTOR (trehalosedi-mycolate)
.This causes serpentine growth in vitro
.This inhibit WBC migration
.Disrupt mitochondrial respiration and oxidative
phosphorylation
.TUBERCULIN (surface protein) with mycolicacid
.Can cause delayed hypersensitivity and mediate cell
mediated immunity
.Can mediate damage by immune system

2. Mycobacterium bovis
.Causative agent of tuberculosis in cattle, dogs, cats,
swine and rabbits
.Can cause human tuberculosis after ingestion of
milk from infected cow.
.After ingestion it can penetrate the GIT and invade
the lymphatic of oropharynx
.Niacin and nitrate negative
.It resembles water droplets on middle brook media
3.Mycobacterium africanum

DISEASE:
Tuberculosis
. consumption disease
.Koch disease
.One of the oldest disease known to man since STONE
AGE
.Philippines ranks 9thamong the 22 countries still battling
the disease (before 7th)
.250,000 Filipinos are being infected with Tuberculosis
annually and 75 patients are dying every day.
.40% of Philippine population is infected
.Global emergency because of rapid development of
MDRTB
.Major public health ranking as 6thleading cause of
mortality and morbidity
.Chronic infection that frequently involves the lungs,
although other organ may be infected
.It can be disseminated miliary

.PRIMARY INFECTION: it begins in the lower lung or in the middle

lung.
.POST PRIMARY (REACTIVATION TUBERCULOSIS):
.Occurs in those who have had primary tuberculosis
.Occurs in later life under conditions of decreased T cell immunity
.TREATMENT: Long term multidrug therapy is needed 6 months
treatment: the 1stand 2ndmonth: isoniazidand rifampicinplus pyrazinamide
.The next 4 months: isoniazidand rifampicin
.If the organism appears to be resistant to isoniazid, ethambutolor
streptomycin can be added.
.DIRECTLY OBSERVED TREATMENT SHORT COURSE (DOTS)
.This is a strategy for control of Tuberculosis recommended by
WHO which is currently employed in our country
.AIM: to contribute to the reduction of the prevalence of TB in
developing countries by determining and treating
.The success of this strategy lies in part to correct diagnosis and
treatment of TB thus removing source of infection from the
community and reducing transmission.

MOTT
.Non contagious
.Can be found in surface waters. Soils and cigarettes .
.Runyon s Classification:
1. PHOTOCHROMOGENS
.Mycobacteriathat develops pigment when exposed to
light
.Develop yellow pigment when exposed to constant light
source and they are non pigmented in the dark

a. M. kansasi

yellow bacillus

b.M. marinum(of the sea)

.associated with skin infections occurring as red to blue lesions.
.Infection may result from contact with poorly chlorinated or un
chlorinated fresh or salt water
. swimming pool granuloma is a more serious form of the infection

c. M. simiae

recovered from Macacarhesus monkey

d. M. asiaticumsimilar to M. simiae

.SCOTOCHROMOGENS
.Mycobacteriathat are pigmented in the light and in the dark
.Produces yellow to orange pigment in the dark and
when exposed to light the pigment intensifies to red to
orange pigment

a. M. scrofulaceum
b. M. szulgai: both photochromogenand scotochromogen
c. M. xenopi: produces colonies with branching hyphaeon CMA. Describe as birds
st.
d. M. gordonae: tap water bacillus
e. M. flavescence
f. M. thermoresistable: able to grow at 52C.
.NONPHOTOCHROMOGENS
.Mycobacteriathat do not develop pigment

a. M. malmoense
b. M. haemophilum
c. M. terrae-trivalecomplex
d. Mycobacterium avium-intracellulare(MAI): Batty
bacillus; most frequently found in AIDS patients and the
disease usually involves the GIT

ne

.RAPID GROWERS
.Not classified according to pigment
.They are able to grow in 3-5 days in
culture media;
.M.
.M.
.M.
.M.

fortitum
chelonei
phlei
smegmatis

.LABORATORY SAFETY MEASURES

.All procedures should be performed using a class I or IIA biological safety hoo
d
.Wear gloves, face mask, gown and shoe covers for all procedures
.Wash hands before and after working with infectious materials
.Disinfect instruments immediately after use
.A germicide, such as phenol with soap can be use to decontaminate the work stat
ion
.Report appropriate personnel incidents or exposure to infectious agent.
.TYPES OF SPECIMENS
.Tuberculosis most frequently involves the lung, but other organs may be infecte
d.
.Urine,CSF, Synovial fluid, Blood, Tissue specimens, Fecal specimens,
.Pulmonary specimens includes sputum, transtrachealaspirate, laryngeal
swabs, bronchial washings.
.SPUTUM (spontaneous or induced): sputum should represent lung
secretions collected from a deep cough. This is the most common form of
specimen collected for culture of Mycobacteria.
.GASTRIC LAVAGE: may be collected for those who cannot provide sputum
.For DOTS, Early morning specimens collected on 3 consecutive days are
recommended for increased isolation of Mycobacteria.
.If the result of sputum examination of a symptomatic or a suspected patient are
negative, repeat the sputum examination after 2 weeks or after one month.

.CHARACTERISTICS OF SPUTUM:
.It should be mucous fluid with air bubbles, containing solid or purulent partic
les and
occasionally browning streaks of blood
.Should be 3-5 mL
.In gram stain it should have at least 25 WBC/LPO or 5WBC/OIO
.If the sample has > 25 epithelial cells/LPO, it is considered saliva.
.Salivary specimen should not be rejected in the laboratory because highly posit
ive
patients may give positive results even in salivary specimen.

LABORATORY DIAGNOSIS
I . PREPARATION OF SMEAR
DIRECT SMEAR
.Using a stick or a wire loop fish out enough yellowish mucopurulentor cheesy pa
rticle in the sputum
.Spread the sputum in a small circular pattern on a clean slide approximately 2
x 3 in cm in sizeymoving it up and the sand.
.Before flame sterilization of wire loop dip first the wire loop in a washing bo
ttle (with sand and alcohol) and remove the excess sputum from the
wire loop . (WASHING BOTTLE: may contain and or glass beads with 90-95% ethanol
or 5% cresol)
.Fixation of the smear:
.Air dry
.By passing through the flame 2-3 times

SMEAR FROM DIGESTION DECONTAMINATION

DIGESTION and DECONTAMINATION:
.NaOH(4-2%) : acts as mucolyticand antibacterial;exposuretime must be limited up
to 15 minutes only
.N-Acetyl-L-cystine: mucolytic
.Dithiotreitol: mucolytic
.4% oxalic acid: for specimens known to harbor Pseudomonas and Proteus
.ZephiranTrisodiumPhosphate (TSP) : use by labs that cannot control the time of
exposure to decontamination solution.
.4% H2SO4
.20% chlorox
.1% Cetyl-pyridumchloride

ACID FAST STAINS:

.Smears should be examined carefully by scanning at least 300

OIF before reporting a negative smear. When an AFO is observed
on a smear, results must be quantified. The recommended
interpretations and ways of reporting smear results are given in
the table:
.Note: a control slide should be included with each run of stains.
.Positive control: M. tuberculosis H37Ra ATCC 25177
.Negative control: NocardiaasteroidesATCC 3308 or E. coli
Number of AFB
REPORT
0
No AFB seen in 300 OIF
1-9/100 OIF
+n AFB/100 OIF (n-write the
actual number of AFO)
10-99/100 OIF
1+
1-10 /OIF in at least 50 fields
2+
More than 10 / OIF in at
least 20 fields
3+

Main Causes of False reading in smear

preparation
False negative:
.Size of smear is too big or small
.Uneven distribution of sample in slide
.If the smear is either too thick or too thin
.Under decolorization
.Insufficient heating
.Over heating
.Over decolorization
False positive
.If the slide is dirty
.Over heating
.Insufficient heating

II. Culture of Mycobacteria

A.Solid media
Agar base
.Middlebrook7H10 and Middle Brook 7H10
.Middlebrook7H11 and Middle Brook 7H11
.MiddleBrookbiplate

Egg Based
.Lowenstein-Jensen
.L-J Gruft
.L-J with pyruvicacid
.L-J with Iron
.Petragani
.American thoracic Society

B. Liquid media
.Bactec12 B medium
.MiddleBrook7H9 broth
.SepticheckAFB

Note: cultures are incubated at 35C in the dark in an atmosphere of 5-10 %

CO2 and with high humidity

III.BIOCHEMICAL TEST:
1.Niacin test(Nicotinic acid)
Principle: Niacin + aniline dye + cyanobromide= (+)
yellow
.This test is useful particularly in ID of M. tuberculosis.
.M.tb, M. simiae, M. chelonae(+)

2. Catalasetest
.Reagent: 30% H2O2
.Semi-Quantitative test: determined by measuring the
height of the column bubbles when H2O2 is added
.Catalaseheat stability: determined by heating the
specimen to 68 Celsiusfor 20 minsand then by
adding hydrogen peroxide.
.Calataseof M. tb, M. bovis, M. gastriand M. haemophilumis
becomes deactivated.
.POSITIVE RESULT: bubble formation or effervescence

3.Nitrate reduction test

.Detected by: sulfanilicacid and napthylamide
.Positive result: pink color
.M. tb, M. kansasi, M. szulgai, and M. fortitum

4.Tween80(polyoxyethelenesorbitanmono-oleate)
hydrolysis
.Tween80 in the presence of Tween80 lipase it will
produce oleic acid. Upon the release of oleic acid red
color will be formed.
.Positive result: red color/pink color

5.TelluriteReduction Test
.Determine the ability of an organism to reduce
potassium tellurite
.This test is use to differentiate other M. Aviumfrom
other nonscotochromogens
.All rapid growers reduce telluritein 3 days.

6. Arylsulfatase
.Arylsulfataseis an enzyme produced by M. fortitum-chelonei.
.Arylsulfatasesplits the sulfur group from TRIPOTASSIUM
PHENOLPHTALEIN forming free phenolphthalein. This free
phenolphthalein is detected by sodium bicarbonate
.POSITIVE: formation of pink color after the addition of sodium
carbonate.

7. TCH (thiopene-2-carboxylic acid hydrazide) susceptibility test

.This is use to differentiate M. tband M. bovis. M. bovisis unable
to grow in the presence of TCH.

8. Iron uptake
.Determine the ability of organism to grow in the presence of 20%
ferric citrate.
.Only rapid growers are able to grow in the presence of iron
.This is use to differentiate M. fortitumand M. chelonei
.M. fortitum(+)
.M. chelonei(-)

IV. Serodiagnosisis not used.

NONCULTIVABLE MOTT:
Mycobacterium leprae Hansen s bacillus
.Agent of Hansen s disease or leprosy
.Acid fast rods (seen in punch biopsy),
in AFB, cigarette-packet or in picket-fence
.Obligate intracellular parasites that cannot
be cultured in vitro (not cultivable in agar)
.Grown in mouse foot pad and armadillo
.Optimal growth at less than body temperature
.Can hydrolyze 3,4-dihydroxy-phenylalanine(DOPA)
.Exhibits tropism to peripheral nerves
.RESERVOIR: human mucosa, skin and peripheral nerves are
the only significant reservoir;Some infected armadillos in
Texas and Louisiana
.TRANSMISSION: nasal discharge from untreated lepromatousleprosy patient.

Two extreme type of Leprosy

LABORATORY DIAGNOSIS:
.punch biopsy or nasal scrapings ; acid fast
stains
.LEPROMIN skin test
.Cannot grown in artificial culture media
.Can be grown in armadillo and foot pads of
mice
TREATMENT:
.Multidrug Theraphy:
.Rifampicin
.Clofazimine
.Dapsone

Bacillus
Bacillus
.large gram (+) rods, aerobic or facultative
anerobes
.All sp. are motile except B. anthracis
.3 species:
1.Bacillus anthracis
2.Bacillus cereus
3.Bacillus subtilis

1.Bacillus anthracis
.Gram (+) rod in long chain
.Bamboo shaped in
apperance
.Center piece for counter terrorism
.Zoonotic

.Three types of anthrax:

1.Cutaneousanthrax
.The most common, less severe
.Acquired when the organism enters the body
through small cut
2. Pulmonary (inhalation) anthrax
.Acquired through inhalation of spores
3. Gastrointestinal Anthrax
.Most severe
.Results from ingestion of bacilli or spores.

Important Virulence Factors:

.CAPSULE
.EXOTOXINS(lethal toxin and edema toxin)
Specimen of Choice:
Sputum, skin biopsy and stool
LABORATORY DIAGNOSIS
Media
1.PLET (polymixinlysozymeEDTA thallousacetate)
2.Bicarbonate agar

Cultural characteristics
1.In BAP:
.non hemolytic
.describe as medusa head or lion head
colonies
.beaten egg white consistency

Test for Identificaion:

1. Penicillin susceptibility test-it produces a String of pearls when streaked t
o
MHA/BAP and 10units penicillin disk is added
and cover slip is applied.
2. Gamma Phage Susceptibility Test3. Animal Pathogenicity4. Ascoli test-diagnost
ic test for ID of B. anthracis5. Direct fluorescence Antibody Technique6. Gelati
nasepositive

Bacillus Cereus
.fried rice bacillus
.associated with food poisoning from eating
contaminated rice
Two Possible toxins
1.Emetic toxin (heat stable)
.With vomiting within 6hrs after ingestion of
contaminated food.
.Most patient recover without medical
intervention
2. Diarrheal Type (heat labile toxin)
.With diarrheal effects seen within 12 hours
after ingestion of contaminated foods

Colonial Morphology:
.Small shiny colonies to large
spreading ones
.May produce lavander color
colonies in bap and
produces wide zone of
hemolysis.

Differential test
Bacillus anthracis
Bacilluscereus
Motility
Non-motile
motile
Capsule
Present
Absent
Hemolysis
Non hemolytic
.-hemolysison
BAP
Growth on 45C
Not able
Able
Salicilinpenetration
Negative
Positive
PenicilinG
susceptibility
Susceptible
resistant

Bacillus subtillis
.Most common laboratory contaminant
.the spore is centrally located
.Causes opportunistic infection
.Associated in eye infection in heroin
addicts
.COLONIES:
Large, flat, dull -hemolytic producing a
ground glass appearance.

Clostridium
.Anaerobic or aerotolerantgram positive
bacilli that forms spores.
.Soil and environment inhabitants
.Not zoonotic
.Medically important species:
1. C. perfringens
2. C. botulinum
3. C. tetani
4. C. difficle

General Characteristic of Clostridium

.All motile except:
.C. perfringens
.C. ranosum
.C. innoculum

.All have swollen sporangia except

.C. perfringens
.C. bifermentans

.All non-encapsulated except

.C. perfringens

.All has single hemolytic pattern except

.C. perfringens

.All are able to ferment CHO except

.C. histolyticum
.C. tetani

5 Groups Of Clostridia
1.Gas Gangrene Group
-includes: C. perfringens, C. novyi, C.
bifermentans, C. septicum, C. sordelii
2. Tetanus
3.Botulism
4.Antibiotic Associated PseudomembranousColitis
5.Miscellaneous Group

Clostridium perfringens
.C. welchii, Bacillus capsulatus, Gangrene
bacillus
.Myonecrosisand food poisoning
Disease:
1.Gas Gangrene life threatening toxin
mediated destruction of muscles and other
tissues
2.Food Poisoning due to atoxin
3.Necrotic Enteritis / Fire in the Bowel type C
toxin

Laboratory Diagnosis:
1.Abundant growth + gas in chopped meat
medium
2.NaglerReaction or Lecithovitallinreaction
.Due to atoxin, lecithinaseC and
phospholipaseC.
.Mc Clung or NeyomycinEYA
.+ : opalescence in agar without antitoxin
.-: no opalescence in agar with antitoxin

3. Reverse CAMP test

.This test is used to demonstrate the synergistic action of C. perfringens and S.
agalactiae.
.+: arrowhead formation at the intersection of
2 streaks

4. Stormy fermentation of milk

5. BAP (+) double zone of hemolysis
.Theta toxin : inner zone of hemolysis
..toxin and lecithinase activity : outer zone
6. Lecithinase positive and ferments glucose
lactose, maltose and fructose
.Lecithinase produces an opaque yellow halo
on CYA.

Clostridium tetani
.Tack head bacillus, Drum stick, Lollipop
bacillus, Tennis racket bacillus
.Sporangia are terminally located
.Iron loving organism
.Tetanus or lock jaw
.Acquired by deep puncture wound
.Exhibit swarming phenomenon
.Produces exotoxin (tetanolysin,
tetanospasmin)

Disease
Tetanus ( lock jaw) characterized by
prolonged muscle contraction

1. Generalized tetanus-most common form.

The fist sign: trismusor lockjaw and
the facial spasm called risussardonicus(facial spasm that produce
grinningSpasm may occur
frequently, body
may shaped
into characteristic
form OPISTHOTONUS

2. Neonatal tetanus
.Generalized tetanus in new born
.Use of unsterile instrument in cutting the
umbilical cord.
3. Localized tetany -uncommon form
.Spasm at same anatomic area
4. Cranial tetany

rare form

.Occur after head injury

Prevention:
Vaccination:
.Infants : Tetanous toxoid vaccination
.2,4,6 months with booster at 12-15months
.Children: 4-6 yrs DPT

Laboratory Diagnosis
.Gelatinase and indole (+)
.Does not usually cultured from clinical specimens
.Dx is more on clinical findings and history taking

Clostridium botulinum
.Canned good bacillus; Bacillus botulinum
.Agent botulism
.BOTULISM results from the liberation of
botulinumtoxin which binds to the synapse of
the cholinergic nerve fibers resulting to acute
and flaccid paralysis
DISEASES
1.Food botulism: free toxin in canned foods
2.Wound botulism: improperly sterilized surgical
dressing and contaminated cuts
3.Infant botulism: most common type; ingestion
of spores from contaminated honey

Diagnosis
.Based on clinical findings, symptoms and
history taking.
.Confirmatory Test: Mouse Neutralization Test
Treatment:
.Anti-toxin

Clostridium difficile
.Normal flora of the GIT, urethra of small % of
normal healthy adults
.Hospital acquired diarrhea
.Triggered by broad-spectrum antibiotics
(clindamycinand ampicillin)
.Antibiotic associated pseudomembranouscolitis
.Toxins:
.Toxin A (enterotoxin)-hemorrhagic secretion of fluid
in GE
.Toxin B (cytotoxin)
.Diagnosis:
.CytotoxinAssay: confirmatory test
.Tissue Culture: gold standard for the Toxin ID

NON-SPOREFORMERS
Corynebactrium
Mycobacterium
Listeria
Nocardia

GENUS: LISTERIA
.Gram-positive, nonspore-forming rods
.Facultative intracellular
.Tumbling motility

Listeria monocytogenes
.Distinguishing Characteristics
.Small Gram-positive rods
.Beta hemolytic, non spore-forming rod on BA
.Tumbling motility in broth; actin jet motility in cells
.Facultative intracellular parasite
.Cold growth
.Reservoir
.Widespread: animals (gastrointestinal and genital tracts),
unpasteurized milk products, plants, and soil
.Cold growth: soft cheeses, deli meats, cabbages
(coleslaw)

Listeria monocytogenes
.Transmission
.Foodborne, across the placenta, or by contact during
delivery
.Pathogenesis
.Listeriolysin O, a -hemolysin: facilitates rapid egress
from phagosome into cytoplasm, thus evading killing
when lysosomal contents are dumped into phagosome;
jets directly (by actin filament formation) from
cytoplasm to another cell.
.Immunologic immaturity predisposes to serious
infection.

Listeria monocytogenes
.Diseases
.Listeriosis (human, peaks in summer)
.Healthy adults and children: generally asymptomatic or diarrhea with low
% carriage
.Pregnant women: symptomatic carriage, septicemia characterized by
fever and chills; can cross the placenta in septicemia.
.Neonatal Disease
.Early-onset: (granulomatosis infantiseptica) utero transmission; sepsis
with high mortality; disseminated granulomas with central necrosis.
.Late-onset: 2-3 weeks after birth rom fecal exposure; meningitis with
septicemia.
.Immunocompromised Patients (IC pts)
.Septicemia and meningitis (most common clinical presentation)
.Listeria meningitis is the most common cause of meningitis in renal
transplant patients and adults with cancer.

Listeria monocytogenes
.Treatment
.Ampicillin with gentamicin added for IC patients

.Prevention
.Precautions with food may reduce incidence

Actinomyces israelii
.Distinguishing Characteristics
.Anaerobic, Gram-positive branching rods
.Reservoir
.Human; normal flora of gingival crevices andfemale
genital tract
.Transmission
.Endogenous

Actinomyces israelii
.Pathogenesis
.Invasive growth in tissues with compromised oxygen supply; anaerobic
growth

.Disease
.Actinomycosis
.Generally not painful but very invasive,penetrating all tissues including bone
.Tissue swelling .draining abscesses(sinus tracts) with sulfur granules(hard
yellow microcolonies) in exudates that can be used for microscopy or culture
.Only in tissues with low oxygenation (Eh)
.Forms
.Crvicofacial (lumpy jaw): dental trauma or poor oral hygiene
.Pelvic: from thoracic or sometimes IUD s
.Thoracic: aspiration with contiguous spread
.Abdominal: surgery or bowel trauma
.CNS: solitary brain abscessesmost common

Actinomyces israelii
.Treatment
.Ampicillinor penicillin Gand surgical drainage

GENUS: NOCARDIA
.BACTERIA
.Gram-positive filaments breaking up into rods
.Aerobic
.Partially acid-fast (some areas of smear will be
blue and some red)

Nocardia asteroides
.Distinguishing Characteristics
.Aerobic
.Gram-positive branching rods
.Partially acid-fast
.Reservoir
.Soil, dust
.Transmission
.Airborne or traumatic implantation
.Pathogenesis
.No toxins or virulence factors known
.Immunosuppression and cancer predisposeto
pulmonary infection.

Nocardia asteroides
.Diseases
.Nocardiosis
.Cavitary bronchopulmonary nocardiosis
.Sx: cough, fever, dyspnea
.May spread hematogenously to brain (brain abscesses)
.Cutaneous/subcutaneous nocardiosis
.Starts with traumatic implantation
.Sx: cellulitiswith swelling .draining subcutaneous abscesses
withgranules (mycetoma)

.Treatment
.Sulfonamides (high dose) or
trimethoprim/sulfamethoxazole

Corynebacterium
.Slender, pleomorphicgram
positive bacilli, catalasenegative,
oxidasepositive
Corynebacteriumdiptheriae
.Kleb-Loefflersbacillus
.Most virulent specie of Corynebacterium
.Nasopharynxbut not Normal flora
.Agent of diptheria( associated with pseudomembraneformation, toxemia and
no bacterimia)
Two forms of diptheria
1. Respiratory diptheriae
.Diptheriatoxin mucous membrane of RT inflammation and
pseudomembraneformation of oropharynxrespiratory obstruction.
.Sorethroatwith pseudomembranebullneckand with potential respiratory
obstruction.
.MOT: inhalation of contaminated respiratory droplets
2. cutaneousdiptheriae
.Characterized by non healing ulcers and membrane formation
.MOT: contact with cutaneouslesions

Pathogenesis:
.Toxin producing strains have -prophagecarrying genes for
toxin.(lysogeny, corynephage)
.Most important virulence factor: tox-gene
.DiptheriaToxin (A-B component)
.Inhibits the protein synthesis
.Effects on oropharynx:
.Dirty gray pseudomembrane( made up of dead cells and fibrin exudate)
.Extension of infection into larynx, trachea ------obstruction

.Non invasive and it colonizes the epithelium of the oropharynxand the

skin
Specimen of choice:
.Nasopharyngeal swab
.wound/skin c/s
Laboratory Diagnosis:
1.Stain Smears: gram stain, metachromaticstains.
.Cells are arranged singly in pallisadesor parallel cells or in pairs
forming V or L shapes
.Resembles Chinese letters or Chinese characters.

Culture:
1.BAP
2.Modified TindalesAgar
.Contains tellurite
.Important selective media and differentiating media for C. diptheriae
3.LoefflersSerum Media
.This enhances the formation of metachromaticgranules of C.
diptheriae
4.Potassium TelluriteAgar
.Important primary isolation media
1.PAI Coagulated serum
2.CystineTellutiteBlood Agar
.Selective and differential medium for C. diptheriae.
3 colonial Type of C. diptheriae
1.Gravis type : largest colonial type
2.MitisType: has fried egg appearance on BAP; producing a bleach like
odor on telluriteagar
3.Intermediustype: smallest colonies

Definitive test for ID of C. diptheriaeisolate as a true pathogen requires the

demonstration of toxin production.
Methods
1.In vivo: Direct animal inoculation into guinea pigs
2. In vitro :
.PCR to detect the toxic gene
.Tissue culture cell test
.Modified Elektest
.Sterile filter paper impregnated with diphtheria antitoxin is imbedded in agar
culture medium.
.Isolates ofC diphtheriaeare

then streaked across the plate

.ToxigenicC diphtheriaeis

detected because secreted toxin diffuses

from the area of growth and reacts with
antitoxin to form lines of precipitin

Other species of Corynebacterium

1.Corynebacteriumminutissimum
.Agent of erythrasma
.Produces color red fluorescence on woods light
.Because of the presence of porphyrin
2.C. pseudodiptheriticum
.This is a normal flora of the oropharynxand rare cause of endocarditis
3. C. jeikeium
.Associated with peritonitis, endocarditisand pneumoniae
4.C. ulceral
.Produces diptherialike toxin and cause diptheriaelike infection
.Mastitis in cattle
.In humans associated with exposure to cattle or ingestion of
contaminated milk
5.C. Pseudotuberculosis
.Formerly C. ovix
.Rare cause of lymphadenitis
.Ingestion of milk