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Material Safety Data Sheet

Required Products

Organisms

Organisms in the Clinical sector this product works with:

Dehydrated Culture Media

Sector: Clinical

TINSDALE AGAR BASE

Code: CM0487

A medium for the isolation and identification of Corynebacterium diphtheriae.

Typical Formula*

gm/litre

Proteose peptone

20.0

Yeast extract

5.0

Sodium chloride

5.0

L-cystine

0.24

Agar
15.0

pH 7.9 ± 0.2 @ 25°C

 
* Adjusted as required to meet performance standards 

Directions
Suspend 9g of agar base in 200ml of distilled water. Bring to the boil and dissolve completely. DO NOT AUTOCLAVE. Allow to cool to approximately 50°C and add the contents of one vial of Oxoid Tinsdale Supplement SR0065 reconstiuted as directed. Mix throughly and pour into sterile Petri dishes.

TINSDALE SUPPLEMENT

Code: SR0065

Vial contents (each vial is sufficient for 200 ml of medium)

per vial
per litre

Serum equiv.

20.0ml

100.0ml

Potassium tellurite

0.069g

0.3445g

Sodium thiosulphate

0.085g

0.425g

Description
Oxoid Tinsdale Base is used with Oxoid Tinsdale Supplement SR0065 for the primary isolation and identification of Corynebacterium diptheriae.
Tinsdale’s original agar medium1 containing serum, tellurite, cystine and formolised blood was formulated to differentiate between Corynebacterium diphtheriae and the diphtheroids found in the upper respiratory tract. This differentiation was based on the ability of Corynebacterium diphtheriae to produce black colonies, surrounded by a brown/black halo, after incubation at 35°C for 48 hours. Diphtheroids do not have this ability. The dark halo is due to the production of H2S from cystine, interacting with the tellurite salt.

Oxoid Tinsdale Base and Supplement are based on Billings’2 modification of Tinsdale’s Medium, which improved the differential qualities as well as the reproducibility of the medium. Moore and Parsons3 using Billings’ modification confirmed the stability of halo formation on the clear medium and its specificity for Corynebacterium diphtheriae and Corynebacterium ulcerans.

They considered that as the incidence of diphtheria gets smaller, it becomes more essential to have a medium which gives a distinctive, characteristic colony.

Technique
Inoculate the medium to obtain well separated colonies. Stab deep into the agar at intervals in order to initiate browning at an early stage (10-12 hours incubation).

Plates are incubated at 35°C and examined after 24 hours and 49 hours incubation. Growth of Corynebacterium diphtheriae may be inhibited if Tinsdale Agar is incubated in carbon dioxide-enriched air e.g. in a CO2 incubator.

Browning may be regarded as presumptive evidence of the presence of Corynebacterium diphtheriae although 48 hours incubation may be necessary for the recognition of characteristic colonies.

Colonial characteristics

Corynebacterium diphtheriae biotype gravis

Small, shiny black

Corynebacterium diphtheriae biotype mitis
Corynebacterium ulcerans

Convex colonies with dark brown halos
after 24 hours incubation

Corynebacterium diphtheriae biotype intermedius

Pale brown colonies which form halos after
36 hours incubation

Diphtheroids (Corynebacterium pseudodiphtheriticum) Haemophilus, Klebsiella, Neisseria, Staphylococcus and Streptococcus species

Dark, brown colonies without halos. minute colonies showing no discoloration of the medium

Proteus species

Brown-black colonies showing characteristic odour and morphology


Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C for not more than 4 days.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

Quality control

Positive control:

Expected results

Corynebacterium diphtheriae gravis ATCC® 19409

Good growth, black colonies, black halos

Negative control:

 

Staphylococcus aureus ATCC 25923 *

Inhibited, black colonies, no halos

* This organism is available as a Culti-Loop®

Precautions
Further tests must be carried out on colonies suspected as Corynebacterium diphtheriae, including cell morphology after sub-culture to Loeffler’s Medium and examination for toxin production.
Do not incubate Tinsdale’s Agar plates in enhanced CO2 atmosphere (5-10% v/v).

References
1. Tinsdale G. F. (1947) J. Path. Bact. 59. 461-464.
2. Billings E. (1956) `An Investigation of Tinsdale’s Tellurite Medium, its Usefullness and Mechanism of Halo Formation’. Thesis Univ. Michigan.
3. Moore Mary S. and Parsons Eliz. I. (1958) J. Infect. Dis. 102. 88-91.

 
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