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Dehydrated Culture Media

Sector: Clinical

SIM MEDIUM

Code: CM0435

A medium for the differentiation of enteric bacteria on the basis of sulphide production, indole production and motility.

Typical Formula *

gm/litre

Tryptone

20.0

Peptone

6.1

Ferrous ammonium sulphate

0.2

Sodium thiosulphate

0.2

Agar

3.5

pH 7.3 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards 

Directions
Suspend 30g in 1 litre of distilled water and boil to dissolve the medium completely. Dispense into final containers and sterilise by autoclaving for 15 minutes at 121°C.

Description
A motility-indole medium has been found to be helpful in the identification of the Enterobacteriaceae; e.g. in the differentiation of Klebsiella from Enterobacter and Serratia species1. For convenience, these two important tests have been combined with sulphide-production in one tube. The production of hydrogen sulphide is a useful diagnostic test in the identification of enteric bacteria and is helpful in the differentiation between Salmonella and Shigella. The sulphate-reducing bacteria will produce hydrogen sulphide and further chemical substitution results in ferrous sulphide being formed along the line of inoculation.

The presence of fermentable sugars may suppress the enzyme mechanism which forms hydrogen sulphide, as a result of the acid products formed (Bulmash and Fulton2 ) and therefore sugars are not included in the medium. Oxoid SIM Medium can be used in conjunction with Triple Sugar Iron Agar CM0277 to assess the ability of the culture to ferment lactose, sucrose and glucose.

The production of indole from tryptophan is one of the diagnostic tests used in identifying enteric bacteria. For example, unless it is an unusual form, a Salmonella culture never produces indole from tryptophan in amounts detectable in usual tests. Tryptone is incorporated into the medium since it is a tryptophan-rich peptone, and after incubation, indole can be identified by a red dye complex reaction with one of several reagents, e.g. Kovac’s Reagent which consists of amyl alcohol, para-dimethylaminobenzaldehyde and concentrated hydrochloric acid3.

The presence of glucose in the medium is avoided as recommended4. False negative reactions have been recorded when fermentation has occurred5.

The use of only 0.35% agar in the medium results in the production of a semi-solid medium, ideal for the examination of motility. Non-motile organisms will grow along the line of inoculation only, whereas motile species will grow away from it.

SIM Medium is therefore designed to determine three characteristics: hydrogen sulphide production, indole production and motility.

Technique
The medium should be dispensed in tubes or bottles and when cool, inoculated once with a pure culture, by inserting a straight wire to about one third of the depth of the medium. If papers are used for the detection of indole, then these are wedged between the cotton wool plug or cap, and side of the container.

The inoculated medium is incubated at 35°C for 18 hours or longer, if necessary, and examined for motility, hydrogen sulphide production and finally indole production from tryptophan.

To test for indole production:
1. Add 0.2ml of Kovac’s Reagent to the tube and allow to stand for 10 minutes. A dark red colour in the reagent constitutes a positive indole test. No change in the original colour of the reagent constitutes a negative test.
Or
2. Suspend a strip of filter paper, soaked in a solution of saturated oxalic acid and dried, over the medium4. Indole formed by positive organisms is volatile and causes the test paper to turn pink.

Colonial appearances
Non-motile organisms grow only along the line of inoculation, whereas motile species show either a diffuse even growth spreading from the inoculum, turbidity of the whole medium, or more rarely, localised outgrowths which are usually fan-shaped or occasionally nodular.
Hydrogen sulphide production is shown by blackening of the line of inoculation.

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.

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

Quality control

Organism

Motility

H2S

Indole

Escherichia coli ATCC® 25922 *

V

-

+

Proteus hauseri ATCC® 13315 *

+

+

+

Shigella sonnei ATCC® 25931 *

-

-

-

* This organism is available as a Culti-Loop®

Precautions
To avoid delay in initiating growth always sub-culture from solid media. The reactions given by SIM Medium are not sufficient to speciate organisms. Additional biochemical and serological tests are required for confirmation.

References
1. Blazevic D. J. (1968) Appl. Microbiol. 16. 668.
2. Bulmash J. M. and Fulton M. (1964) J. Bact. 88. 1813.
3. Harrigan W. F. and McCance M. E. (1966) `Laboratory Methods in Microbiology’ Academic Press. 53.
4. Wilson G. S. and Miles A. A. (1964) Topley and Wilson’s `Principles of Bacteriology and Immunity’ 5th ed., Arnold, 1. 490.
5. Giles R. R. (1956) J. Clin. Path. 9. 368-371.

 
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