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Dehydrated Culture Media

STAPHYLOCOCCUS MEDIUM NO.110

Code: CM0145

A selective medium for the isolation and differentiation of pathogenic staphylococci based on salt tolerance, pigmentation, mannitol fermentation and gelatin liquefaction.

Formula

g/litre

Yeast extract

2.5

Tryptone

10.0

Lactose

2.0

Mannitol

10.0

Sodium chloride

75.0

Dipotassium hydrogen phosphate

5.0

Gelatin

30.0

Agar

15.0

pH 7.1 ± 0.2

 


Directions
Suspend 150g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilize by autoclaving at 121°C for 15 minutes. Disperse the precipitate by gentle agitation before pouring.

Description
Staphylococcus Medium No.110 is a selective medium for isolation and differentiation of pathogenic staphylococci (Chapman1,2 ) on a basis of salt tolerance, pigmentation, mannitol fermentation, and gelatin liquefaction. Pathogenic staphylococci (coagulase-positive) are able to grow on the high-salt mannitol medium to form orange colonies which give positive reactions for acid production and gelatin liquefaction.

Stone3 suggested that gelatinase activity was indicative of food poisoning strains but Chapman et al.4 reported that typical food poisoning staphylococci should also produce an orange pigment, be haemolytic, be coagulase-positive, and ferment mannitol. Chapman5 showed that incubation at 30°C produced deeper pigmentation and no interference with the Stone reaction or with acid production from mannitol - both of the latter being about as intense as at 35°C.

Smuckler & Appleman6 made Staphylococcus Medium No.110 selective, for the determination of coagulase-positive staphylococci in meat pies containing large numbers of Bacillus spp., by the addition of sodium azide 0.75 mM (4.875g/L). Staphylococcus Medium No.110 is formulated according to the APHA7 and AOAC8 specifications. Carter9 modified the medium by adding egg yolk (5% v/v SR0047) so that the characteristic egg yolk reactions of staphylococci can be seen.

Technique
Streak or smear the Staphylococcus Medium No.110 plate with the specimen and incubate for 43 hours at 35°C or for 48 hours at 30°C. Pigmented colonies are a deep orange colour, whilst non-pigmented colonies are white. Acid production from mannitol is best demonstrated by adding a drop of 0.04% bromothymol blue indicator to the sites of the individual colonies; yellow indicates acid production.

Gelatin hydrolysis may be demonstrated by adding a drop of a saturated aqueous solution of ammonium sulphate or, preferably, of a 20% aqueous solution of sulphosalicylic acid to an individual colony (’Stone reaction’). A positive `Stone reaction’ is denoted by the presence of a clear zone round gelatinase-producing colonies after 10 minutes’ contact with the reagent.

The above reactions may be conveniently performed using short sleeves, 5mm long and 10mm diameter, cut from polythene tubing. The sleeves act as receptacles for the reagents when placed over discrete colonies, and may be stored in 70% alcohol prior to use. Coagulase tests should not be carried out without first sub-culturing in Nutrient Broth No.2 (CM0067) or on Blood Agar Base (CM0055).

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: Light straw coloured, free-flowing powder.
Prepared medium: Straw coloured gel.
Due to the high slat content of this medium a slight haze may be visable in the prepared medium. This in no way affects the performance.

Quality Control

Positive controls:

Expected results

Staphylococcus aureus ATCC® 25923 *Good growth; cream coloured colonies.
Gelatinase positive; mannitol positive

Negative control:

Escherichia coli ATCC® 25922 *

No growth

* This organism is available as a Culti-Loop®

Precautions
Enterococcus faecalis may grow on this medium as tiny colonies with slight mannitol fermentation.
The high salt content in Staphylococcus Medium No.110 may interfere with the coagulase reaction. Always sub-culture to a non-inhibitory medium before testing.

References
1. Chapman G. H. (1946) J. Bact. 51. 409-410
2. Chapman G. H. (1952) J. Bact. 63. 147-150
3. Stone R. V. (1935) Proc. Soc. Exper. Biol. & Med. 33. 185-187
4. Chapman G. H., Lieb C. W. and Cumco L. G. (1937) Food Research 2. 349-367
5. Chapman G. H. (1947) J. Bact. 53. 367-368
6. Smuckler S. A. and Appleman M. D. (1964) Appl. Microbiol. 12. 335-339
7. American Public Health Association (1978) Compendium of Methods for the Microbiological Examination of Foods. APHA Inc. Washington DC
8. Association of Official Analytical Chemists (1992) Bacteriological Analytical Manual. 7th Edn. AOAC. Washington DC
9. Carter C. H. (1960) J. Bact. 79. 753-756

 
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