Tinsdale Agar: Introduction, Principle, Composition, Preparation, Procedur

Tinsdale Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

Introduction of Tinsdale Agar

Tinsdale Agar is used for selective isolation and differentiation of Corynebacterium diphtheriae and it is Gram-positive, facultatively anaerobic, non-motile bacteria that exhibit a fermentative metabolism (carbohydrates to lactic acid) under certain conditions. Most species of genus Corynebacterium are normal flora of humans present virtually at all anatomic sites. Corynebacterium diptheriae is the causative agent of Diphtheria. Tinsdale Agar was developed by Tinsdale for the selective isolation and differentiation of Corynebacterium diphtheriae from diphtheroids and further modified by Billings, which improved the recovery and differential qualities of C. diphtheriae. The present formulation of the medium is according to the modified Billings Medium. Moore and Parsons confirmed the halo formation as a remarkable property of C. diphtheriae with the exception of Corynebacterium ulcerans, which forms colonies with the similar property as C. diphtheriae.

Principle of Tinsdale Agar

Peptic digest of animal tissue (PDAT) provides nitrogenous compounds. L-cystine and sodium thiosulphate form the hydrogen sulfide (H₂S) indicator system. Potassium tellurite of the medium inhibits all gram-negative bacteria and most of the upper respiratory tract normal flora. C. diphtheriae forms grayish-black colonies surrounded by a dark brown halo while diphtheroids commonly found in the upper respiratory tract (UPRT) do not form such colonies. The dark brown halo around the colony is because of H₂S production from cystine combining with the tellurite salt.

Composition of Tinsdale Agar

Ingredients Gms / Litre

Peptic digest of animal tissue (PDAT): 20.0
Sodium chloride: 5.0
L-Cystine: 0.24
Sodium thiosulphate: 0.43
Agar 15.0
Final pH (at 25°C) 7.4±0.2

Additional Ingredient/s

Part A Diphtheria Virulence Supplement
Part B Diphtheria Virulence Supplement

Preparation of Tinsdale Agar

Suspend 40.67 grams of the dehydrated medium in 1000 ml purified/distilled water.
Heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes i.e. validated cycle.
Cool to 50°C.
Aseptically add extra ingredients provided by the vendor i.e. Part A Diphtheria Virulence Supplement and Part B Diphtheria Virulence Supplement.
Mix well before pouring into sterile Petri plates.
Leave for drying.

Storage and Shelf life of Tinsdale Agar

Store at 2-8ºC and away from direct light.
Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements for Tinsdale Agar

Test specimen
Tinsdale Agar
Inoculating loop/ dropper
Bunsen burner
Incubator
Control strains
Waste bin

Procedure of Tinsdale Agar

Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
Inoculate and streak the specimen as soon as possible after collection.
If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
Streak for isolation with a sterile loop.
Incubate plates aerobically at 35-37°C for 48 hours (or more).
Examine colonial characteristics.

Colony Morphology of Tinsdale Agar

Organism Growth and Colony characteristics

Corynebacterium diphtheriae type gravis: Good-luxuriant with brown-black with halo

Corynebacterium diphtheriae type intermedius: Good-luxuriant with brown-black with halo

Corynebacterium diphtheriae type mitis: Good-luxuriant with brown-black with halo

Klebsiella pneumoniae: No growth

Streptococcus pyogenes: Good with black pinpoint without halo

Uses of Tinsdale Agar

Tinsdale agar is an ideal medium for the routine cultivation and isolation of C. diphtheriae.
It also supports the growth of C. ulcerans.

Limitations

Colony characteristics only provide presumptive identification and thus biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for final identification.
Tinsdale Agar is not favorable as a primary plating medium, because it may not support the growth of some strains of C. diphtheriae.
Some species of Genus Corynebacterium like C. ulcerans and C. pseudotuberculosis, and Staphylococcus species (rarely) may produce a characteristic halo on Tinsdale Agar.
Several organisms may also exhibit slight browning on Tinsdale Agar in 18 hours; hence, the plates should be read after the complete incubation period (48 hours).

Keynotes on Tinsdale Agar

Do not incubate the Tinsdale agar plates in 5-10% CO₂ as it retards the development of characteristic halos.
Corynebacterium diphtheriae occurs in the nasopharyngeal area of infected persons or healthy carriers.
The four biotypes of C. diphtheriae are gravis, mitis, belfanti, and intermedius and the signs and symptoms of diphtheria are sore throats, malaise, headache, and nausea.
C. ulcerans found in the nasopharynx form colonies same as C. diphtheriae and hence it requires a further biochemical test for confirmation.
Loffler serum slope restores virulence and other identifying properties (microscopic and colonial) after they have been lost due to prolonged incubation or repeated subculturing and it is also used for the demonstration of pigmentation and ascospores.
Downie’s blood tellurite agar (BTA), Hoyle medium (modification of Neill’s medium), Tinsdale, and Loeffler’s serum slope are the most common media used for the cultural isolation and differentiation of Corynebacterium diphtheriae.

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