Microbiology lab 3&4

A.L. Ayat Majed

Corynebacterium diphtheriae

• Gram – positive , non- acid fast , nonmotile rods

with irregularly stained segments , and sometimes

granules. They frequently shows club shaped

swelling, so the names corynebacterial ( from

coryne, meaning club).

Corynebacterium diphtheriae

• Reservoir: throat and nasopharynx

• Transmission: bacterium or phage via respiratory droplets

• Toxin-producing strains have β-prophage carrying genes for the toxin (lysogeny, β-

corynephage). The phage from one person with diphtheria can infect the normal

nontoxigenic diphtheroid of another, and thus cause diphtheria

• Pathogenesis: Organism not invasive; colonizes epithelium of oropharynx or skin.

• Diphtheria toxin (A-B component)—inhibits protein synthesis by adding ADPribose to eEF-

• Effect on oropharynx: Dirty gray pseudomembrane (made up of dead cells and fibrin

exudate, bacterial pigment)

• Extension into larynx/trachea → obstruction

• Effect of systemic circulation → heart and nerve damage

 Disease
symptoms

1-diphtheria (sore throat with pseudomembrane,

bull neck) potential respiratory obstruction

2-myocarditis, cardiac dysfunction,

3-recurrent laryngeal nerve palsy and lower limb

polyneuritis)

4- renal failure
• Diagnosis

1- Elek test to document toxin production (ELISA for toxin is now

gold standard) toxin produced by Elek test toxin-producing

strains diffuses away from growth Antitoxin diffuses away from

strip of filter paper Precipitin lines form at zone of equivalence

2-  Direct smear microscopy:

Gram stain: Corynebacterium diphtheria appears as irregularly stained club shaped

gram-positive bacilli arranged in Chinese letter or cuneiform arrangement (V or L

shaped). It is difficult to differentiate them from other commensal coryneforms found

in the respiratory tract.

Albert’s stain is more specific for C.diphtheriae, where they appear as green bacilli with

bluish black metachromatic granules

2- culture: in addition to blood agar using specialized media

A-Tellurite: Black colonies not diagnostically significant, tellurite inhibits many organisms

but not C. diphtheria. Selective & differential medium because Corynebacteria are resistant

to tellurite – Reduced to tellurium and Forms deposit in colonies – Colonies appear dark,

Gray-to-black colonies

• B-Loeffler :On Loeffler serum medium, corynebacteria grow much more readily than

other respiratory organisms,., and Granules (volutin) produced on Loeffler coagulated

serum medium stain metachromatically

 Albert stain

Gram stain

Blood agar
Treatment

• Erythromycin and antitoxin

• For endocarditis, intravenous penicillin and aminoglycosides

for 4–6 weeks

 GENUS: MYCOBACTERIUM
• Acid fast rods with waxy cell wall, Obligate aerobe

• Cell wall

-Unique: high concentration of lipids containing long chain fatty acids called mycolic

acids

-Wall makes mycobacteria highly resistant to desiccation and many chemicals

(including NaOH used to kill other bacteria in sputa before neutralizing and culturing)
• Sensitive to UV
• Species of Medical Importance
• M. tuberculosis
• M. leprae
• M. avium-intracellulare
• M. kansasii
• M. marinum
• M. ulcerans
 Mycobacterium tuberculosis
• Acid fast, Aerobic, slow growing on Lowenstein-Jensen medium; new culture systems

(broths with palmitic acid) faster

• Produces niacin, Produces a heat-sensitive catalase: catalase-negative at 68.0°C (154.4 F)

(standard catalase test); catalase active at body temperature.

• Reservoir: human lungs
• Transmission: respiratory droplets
 Diseases
• Primary pulmonary TB

• Symptoms can include — fever, dry cough Organisms replicate in naive alveolar

macrophages, killing the macrophages until CMI is set up (Ghon focus)

 Diagnosis
• Microscopy of sputum: acid fast stain

• PPD skin test (Mantoux): measure zone of induration at 48–72 hours, Positive skin test indicates

only exposure but not necessarily active disease.

• Quantiferon-TB Gold Test: measures interferon-gamma production when leukocytes exposed to TB

antigens

• Culture:Slow-growing (3–6 weeks) colonies on Lowenstein-Jensen medium (faster new systems)

Treatment
• Multiple drugs critical to treat infection
• First 2 months: rifampin + isoniazid + pyrazinamide + ethambutol (RIPE)
• Next 4 months: rifampin and isoniazid
• Streptomycin added for possible drug-resistant cases until susceptibility tests are
• back (if area acquired has >4% drug-resistant mycobacteria)