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Dehydrated Culture Media

CLAUSEN MEDIUM

DITHIONITE-THIOGLYCOLLATE (HS-T) BROTH

Code: CM0353

The Nordic Pharmacopoeia Board have recommended this medium, containing neutralising compounds and supplementary minerals, for sterility testing.

Typical Formula*

mg/litre

Tryptone

15.0

Yeast extract

6.0

Soya peptone

3.0

Glucose

6.0

Sodium chloride

2.5

Dipotassium hydrogen phosphate

2.0

Sodium citrate

1.0

L-cystine

0.5

L-asparagine

1.25

Sodium dithionite

0.4

Sodium thioglycollate

0.5

Lecithin

0.3

Magnesium sulphate .7H2O

0.4

Calcium chloride .2H2O

0.004

Cobalt sulphate .7H2O

0.001

Cupric sulphate .5H2O

0.001

Ferrous sulphate .7H2O

0.001

Zinc sulphate .7H2O

0.001

Manganese chloride .4H2O

0.002

Resazurin

0.001

Agar

0.75

pH 7.1 ± 0.2 @ 25°C

 
* Adjusted as required to meet performance standards

Directions
Suspend 40g in a solution composed of Tween 80 (polyethylene sorbitan mono-oleate) 3g: glycerol 5g and distilled water 1 litre. Bring to the boil to dissolve completely.
Distribute into tubes or bottles and sterilise by autoclaving at 121°C for 15 minutes.
THE MEDIUM MUST NOT BE RE-STERILISED.

Description
Dithionite-thioglycollate (HS-T) Broth was developed by Clausen in Oslo University and has been recommended for sterility testing by the Nordic Pharmacopoeia Board. The problems of sterility testing by selecting random samples is recognised by the Board and they refer to the process as the Microbial-Contamination Test. The standard microbial- contamination test is designed solely to establish that the number of non-sterile units, if any, in a batch is below a certain level.

The following description of the Standard Microbial-Contamination Test has been abridged from the detailed description published as an addendum in the Nordiska Farmakopenämnden.

The tests must be performed with all precautions taken to prevent laboratory contamination occurring more than once in every 100 tests. The use of laminar air-flow cabinets is recommended. Tests are to be made by qualified and experienced staff and the efficiency of the methods used must be checked at regular intervals.

A random sample of sufficient quantity to be representative of the whole bulk, should be examined.

Two methods of detecting non-sterile units may be used in the microbial-contamination test.

Membrane Filter Method
The test substance is dissolved or suspended in 200ml of 0.1% w/v sterile (pH 7.0-7.2) Peptone water CM0009 and immediately filtered through one or more membrane filters (average pore diameter 0.45µm or less).

Each filter is then washed three times, by passing 100ml volumes of peptone solution through the membrane.

After filtration the membranes are transferred to tubes of media, containing at least 15ml of Clausen Medium and tubes of Tryptone Soya Broth (soybean-casein digest medium) CM0129. If only one filter is used, this is divided into two and the two halves placed in separate tubes.
Tubes of Clausen Medium are incubated for at least 14 days at 30-32°C.

Tubes of Tryptone Soya Broth (soybean-casein digest medium) are incubated for at least 14 days at 20-25°C.

Dilution Method
From each sample 1.0ml of material or suspension is transferred to each of at least 10 tubes containing a minimum quantity of 15ml of Clausen Medium.

One half of the number of tubes is incubated at 30-32°C for at least 14 days and the other half at 20-25°C for the same time.

If the medium becomes turbid on incubation, subcultures must be taken as soon as possible. Subcultures must also be taken after normal incubation and observed for a further period of 14 days.

Assessment Of The Results
The standard microbial contamination test is passed if growth is not observed in any of the tubes. If growth is observed, the test may be repeated with twice the number of samples. The test is then passed if no growth is observed in any of these tubes. Growth is diagnosed by the appearance of turbidity in fluid or semi-fluid media, by the formation of colonies on solid media, or by microscopy of culture samples.

Controls
Both methods of testing must be controlled for microbial inhibitors by adding a small inoculum of organisms (approximately 10 colony-forming bacteria) either to the tubes prepared in Method II or to peptone diluent, prior to filtration, in Method I.

If no growth occurs in the tubes containing the test organisms then the test must be repeated with the growth-inhibitory effect inactivated.

The test organisms recommended are:
Staphylococcus epidermidis
Clostridium sporogenes
Rhodotorula rubra

They are maintained on agar slants or deep agar stabs and the test inoculum is prepared from 24 hour cultures grown in Clausen Medium at 30-32°C. The Rhodotorula rubra inoculum is prepared from a 48 hour culture grown in the same broth at 20-25°C.

Dithionite-Thioglycollate Broth was formulated by Clausen as a highly nutritious medium containing reducing agents and essential metals for the recovery of anaerobic spore-bearing organisms. It also contains lecithin and Tween 80 to overcome the effects of cationic agents which may show powerful bacteriostatic effects in vitro.

The broth should be prepared as directed and transferred to tubes or bottles in sufficient volumes (at least 15ml) for the standard microbial-contamination test and rapidly cooled to 20°C after sterilisation.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Prepared medium: The medium can be stored in a cool place (above 4°C) away from light, for a maximum period of one month.

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

Quality control

Positive control:

Expected results

Staphylococcus epidermidis ATCC® 14990 *Turbid growth

Negative control:

 

Uninoculated medium

No change

* This organism is available as a Culti-Loop®

Precautions
The medium is yellowish in colour and almost clear. It turns pale-pink under aerobic conditions. The upper third only of the medium should be pink by the time it is to be used.

References
1. Clausen O. G., Aasgaard N. B. and Solberg O. (1973) Ann. Microbiol. (Inst. Pasteur) 124 B. 205.
2. Christensen E. A., Kristensen H. and Jensen K. M. (1969) Arch. Pharm. Chem. 76. 625.
3. Clausen O. G. (1973) `A study of the growth-promoting properties of fluid and solid microbial-contamination test media on small numbers of micro- organisms.’ `Pharmaceutica Acta Helvetiae’’48. 541-548.
4. Clausen O. G. (1973) `An examination of the Bactericidal and Fungicidal Effects of Cetylpyridinium Chloride, separately and in combinations embodying EDTA and Benzyl Alcohol’. Die. Pharm. Ind. 35. Nr. 12 869-874.
5. Mohamed A. and Abdou F. (1974) `Comparative Study of Seven Media for Sterility Testing’. Jnl. of Pharma. Sci. Vol. 63. No.1 Jan.
6. Mohamed A. and Abdou F. (1974) `Sterilitatstest III Vergleichsuntersuchungen von 3 Medien zum Nachweis von Bakterien’. Pharm. Ind. 36. Nr. 5. 337-334.

 
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