Plot No. C40, Road No. 21Y, MIDC, Wagle Industrial Estate,
Thane(W)-400604, MH, India.
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Intended Use
Recommended for selective isolation and differentiation of Corynebacterium diphtheriae.
Composition**
| Ingredients | g/L |
|---|---|
| HiVeg™ peptone | 20.000 |
| Sodium chloride | 5.000 |
| L-Cystine | 0.240 |
| Sodium thiosulphate | 0.430 |
| Agar | 15.000 |
| Final pH (at 25°C) | 7.4±0.2 |
**Formula adjusted, standardized to suit performance parameters
Directions
Suspend 40.67 grams 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. Cool to 45-50°C and aseptically addTinsdale Selective Supplement (FD073, Part A and Part B). Mix well and pour into sterile Petri plates.
Principle And Interpretation
The Corynebacteria are gram-positive, non-sporulating, non-motile rods. They are often club-shaped and frequently banded or beaded with irregularly stained granules. These bacteria are generally aerobic or facultative, but microaerophilic species do occur. Corynebacterium diphtheriae produces a powerful exotoxin that causes diphtheria in humans. In nature, C. diphtheriae occurs in nasopharyngeal area of infected persons or healthy carriers.
The three biotypes of C. diphtheriae are mitis, intermedius and gravis (1). The signs and symptoms of diphtheria are sore throat, malaise, headache and nausea (2). Tinsdale Agar Base Medium was developed by Tinsdale (3,4) for the selective isolation and differentiation of C. diphtheriae from diphtheroids. This medium was modified by Billings (2), which improved the recovery and differential qualities of C. diphtheriae. The present medium is according to the modified Billings Medium. Moore and Parsons (3) confirmed the halo formation as a characteristic property of C. diphtheria with the exception of C. ulcerans, which forms colony with similar features as C. diphtheriae.
Tinsdale HiVeg™ Agar Base is prepared by using vegetable peptones in place of animal based peptones which make the media free of BSE/TSE risks. HiVeg™ peptone provides nitrogenous compounds. L-cystine and sodium thiosulphate form the H2S indicator system. Potassium tellurite from the supplement 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 do not form such colonies. Dark brown halo around the colony is due to H2S production from cystine combining with the tellurite salt. Moore and Parsons (3) found Tinsdale Medium as an ideal medium for the routine cultivation and isolation of C. diphtheriae. They also confirmed the stability of halo formation on clear medium and its specificity for C. diphtheriae and C. ulcerans. C. ulcerans found in nasopharynx form colonies same as C. diphtheriae and require further biochemical confirmation (5).
Do not incubate the plates in 5-10% CO2 as it retards the development of characteristic halos (6). Tinsdale Agar is not suitable as a primary plating medium, since it may not support the growth of some strains of C. diphtheriae (1). C. ulcerans, C. pseudotuberculosis and (rarely) Staphylococcus species may produce a characteristic halo on Tinsdale Agar (1). Several organisms may exhibit slight browning on Tinsdale Agar in 18 hours; therefore the plates should be read after complete incubation period (48 hours) (1).
Type of specimen
Clinical samples - Throat swab
Specimen Collection and Handling:
After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions :
Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations :
- Do not incubate the plates in 5-10% CO2 as it retards the development of characteristic halos (6)
- Further biochemical and serological tests need to be carried out for confirmation.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.
Quality Control
Appearance
Cream to yellow homogeneous free flowing powder
Gelling
Firm, comparable with 1.5% Agar gel
Colour and Clarity of prepared medium
Light amber coloured clear to slightly opalescent gel forms in Petri plates
Reaction
Reaction of 4.07% w/v aqueous solution at 25°C. pH: 7.4±0.2
pH
7.20-7.60
Cultural Response
Cultural characteristics observed after an incubation at 35-37°C for 40-48 hours with added Diptheria Virulence Supplement (FD073, Part A and Part B).
| Organism | Inoculum (CFU) | Growth | Recovery | Colony characteristics |
|---|---|---|---|---|
| Corynebacterium diphtheriae type gravis | 50-100 | good-luxuriant | >=50% | brown-black with halo |
| Corynebacterium diphtheriae type intermedius | 50-100 | good-luxuriant | >=50% | brown-black with halo |
| Corynebacterium diphtheriae type mitis | 50-100 | good-luxuriant | >=50% | brown-black with halo |
| Klebsiella pneumoniae ATCC 13883 (00097*) | >=104 | inhibited | 0% | |
| Streptococcus pyogenes ATCC 19615 | 50-100 | good | 40-50% | black pin point, without halo |
Key: *Corresponding WDCM numbers.
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques (7,8).
| Product Name | Tinsdale HiVeg™ Agar Base |
|---|---|
| SKU | MV314 |
| Product Type | HiVeg™ |
| Physical Form | Powder |
| Origin | Animal Free (Veg) |
| Packaging type | HDPE |
| References | 1. Tinsdale G. F. W., 1947, J. Pathol. Bacteriol., 59:461. 2.Billings E., 1956, An investigation of Tinsdale Tellurite Medium: its usefulness and mechanisms of halo-formation, M.S.thesis, University of Michigan, Ann Arbor, Mich. 3.Moore M. S. and Parsons E. I., 1958, J. Infect. Dis., 102:88. 4.MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. I, Williamsand Wilkins, Baltimore. 5.Murray P. R., Baron E. J., Jorgensen J. H., Pfaller M. A., Yolken R. H., (Eds.), 8th Ed., 2003, Manual of ClinicalMicrobiology, ASM, Washington, D.C 6.Isenberg, (Eds.), 1992, Clinical Microbiology Procedures Handbook, Vol. 1, American Society for Microbiology,Washington, D.C. |
| Customized Product Available | No |
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