Dep.

Medical microbial., college of medicine

Dr.Sawsan Q. Taha

Corynebacteria
They are Gram-positive, non-spore-forming, non-motile, non-capsulated,
catalase positive, rod-shaped bacteria that are straight or slightly curved.
Metachromatic granules are usually representing stored phosphate regions. The
bacteria group together in a characteristic way, which has been described as the
form of a "V", "palisades", or "Chinese letters". They may also appear
elliptical. They are aerobic or facultatively anaerobic, with a 51–65% genomic
G:C content. They are pleomorphic through their life cycle: they come in various
lengths and frequently have thickenings at either end, depending on the
surrounding conditions.

Classification
Some of Corynebacteria are normal flora of respiratory mucous
membrane, skin, and vagina. The most important pathogen is Corynebacterium
diphtheriae that cause diphtheria and produce exotoxin. Commensals or normal
flora are known as Diphtheroids including:
1. C amycolatum
2. C minutissimum
3. C jeikeium
4. C pseudodiphtheriticum
5. C striatum
6. C urealyticum
7. C xerosis

Corynebacterium diphtheriae
C. diphtheriae is a pathogenic bacterium that causes diphtheria. It is also
known as the Klebs-Löffler bacillus, because it was discovered in 1884 by
German bacteriologists Edwin Klebs and Friedrich Löffler. Corynebacteria are
0.5–1µm in diameter and several micrometers long.
Four biotypes of C. diphtheriae have been widely recognized: gravis, mitis,
intermedius, and belfanti. These variants have been classified on the basis of
growth characteristics such as colony morphology, biochemical reactions, and,
severity of disease produced by infection.

Microscopic Characteristics
C. diphtheriae possesses irregular swellings at one end that give them the
"club-shaped" appearance. Irregularly distributed within the rod (often near the
poles) are granules staining deeply with aniline dyes that give the rod a beaded
appearance. Individual corynebacteria in stained smears tend to lie parallel or at
acute angles to one another as the form of a "V","Z", or "Chinese letters".

Cultural Characteristics monstion

C. diphtheriae and other Corynebacteria grow aerobically on most
E
ordinary laboratory media. On blood agar, the C. diphtheriae colonies are small,
granular, and gray, with irregular edges, and may have small zones of hemolysis.
Tellurite blood agar used for isolating, differentiating and identifying
Corynebacterium diphtheriae. Other organisms are generally inhibited. The
I

l B

MK
Blipsbeoffler
Diphtheria e
 medium is enriched by the addition of blood and made selective by the addition
of potassium tellurite. The colonies on this medium are brown to black with a
brown-black halo because the tellurite is reduced intracellularly to tellerium.

pint Black

Blood THEY
Agog
Throat culture on blood agar (left) and tellurite medium (right).
Black colonies on tellurite medium are Corynebacterium diphtheriae

On this medium C. diphtheriae can be differentiated into three variants:

1- Gravis variant: it shows large gray nonhemolytic colonies, these are
known as Daisy head colonies.
2- Mitis variant: it gives small black hemolytic convex colonies, like poached
egg.
3- Intermedius variant: its nonhemolytic small colonies.
Tinsdale agar is semilar to tellurit medium, contaning cystine, used also
as selactive medium for C. diphtheriae and showing black colored colonies
serounded with halo zone.

II

 On Loeffler's serum medium, Corynebacteria grow much more rapidly than
other respiratory organisms; the Loeffler's slant may yield organisms of typical
"diphtheria-like" morphology.

Corynebacterium diphtheriae (ATCC® 13812) colonies growing on Loeffler's Medium . Incubated

aerobically for 72 hours at 35º C
1
Laboratory Diagnostic Tests
Specimens and Smear:
Swabs from the nose, throat, or other suspected lesions must be obtained
before antimicrobial drugs are administered. Swabs should be collected from
beneath any visible membrane. The swab should then be placed in semisolid
transport media. Smears stained with Albert's stain or Gram's stain show
beaded rods in typical arrangement.

swab fromthetonsils
1
Albert's Stain Technique
blue
91 yjnethyline
1- Prepare dry smear from specimen or culture
2- Cover the smear with Alberts reagent-1 and allow for 3-5 minuts
3- Wash with water
4- Cover the smear with Alberts reagent-2 (Alberts Iodine) for 1 minutes
5- Wash with water and blot dry
6- Examine under oil immersion
Observations: granules stain bluish black while the protoplasm seems
green.

III

 Culturing
Inoculate a Loeffler's slant, tellurite blood agar plate, tinsdale
mediumand plate, and incubate all at 37 °C. In 12–18 hours, the Loeffler slant
may yield organisms of typical "diphtheria-like" morphology. In 36–48 hours,
the colonies on tellurite medium are sufficiently definite for recognition of C
diphtheriae.

Toxigenicity Tests
1- Elek's Test: A filter paper disk containing antitoxin is placed on an agar
plate. The cultures to be tested for toxigenicity are spot innoculated 7 to 9
mm away from the disk. After 48 hours of incubation, the antitoxin
diffusing from the paper disk has precipitated the toxin diffusing from
toxigenic cultures and has resulted in precipitate bands between the disk
and the bacterial growth.

pathog

Pathogenic
am non Path
non Path

a.BE Ave

a I
1- Positive control

o
2- Negative control
3- Tested bacteria (-ve)
4- Tested bacteria (+ve)

in General
Diagnosis Stain IV

Mek Test

 2- Polymerase chain reactions (PCR): based methods have been described
for detection of the diphtheria toxin gene (tox). PCR assays for tox gene
can also be used directly on patient specimens before culture results are
available. A positive culture confirms a positive PCR assay. A negative
culture following antibiotic therapy along with a positive PCR assay
suggests that the patient probably has diphtheria.
3- Enzyme-linked immunosorbent assays can be used to detect diphtheria
toxin from clinical C diphtheria isolates.
4- An immunochromographic strip assay allows detection of diphtheria
toxin in a matter of hours. This assay is highly sensitive.

5 9828119
Attenuated 01 WII Mo
The Schick test: Allergic
Immunological Test
I 5 1.10
inject a small amount of specially-prepared diphtheria
Mtoxin beneath the skin. If the person is susceptible to the disease, a red swollen
rash appears around the injection area. A vaccine may then be used to protect
the person from diphtheria.
16mA ve
Treatment summer ve
Neutralization of the toxin, using antitoxins, and administration of
antibiotics, such as erythromycin or penicillin.

Albert stain
Culture
Elek Test
Schik test