MLCB AGAR

Code: CM0783

Mannitol Lysine Crystal Violet Brilliant Green Agar for the isolation of salmonellae (not Salmonella typhi or Salmonella paratyphi A.).

Typical Formula*	gm/litre
Yeast extract	5.0
Peptone	10.0
`Lab-Lemco’ powder	2.0
Sodium chloride	4.0
Mannitol	3.0
L-lysine hydrochloride	5.0
Sodium thiosulphate	4.0
Ammnium iron (III) citrate	1.0
Brilliant green	0.0125
Crystal violet	0.01
Agar	15.0
pH 6.8 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards

Directions
Suspend 49.0g in 1 litre of distilled water. Mix and bring gently to the boil with frequent agitation to dissolve the medium completely. Cool to 50°C and pour approximately 20ml into sterile Petri dishes.
DO NOT AUTOCLAVE OR OVERHEAT.

Description
Mannitol Lysine Crystal Violet Brilliant Green Agar (MLCB Agar) is based on the formula of Inoue et al.¹ for the selective isolation of salmonellae from faeces and foods. Visual detection of very small numbers of hydrogen sulphide- producing strains is easy because of the distinctive colonial appearance.

The concentration of Magnesium ions appears to be critical for maximum growth of salmonellae on MLCB Agar. van Schothorst et al.² showed that Oxoid MLCB Agar did not inhibit any of the salmonellae species investigated.

Salmonellae serotypes that have a high incidence of H₂S-negative strains e.g. Salmonella sendai, Salmonella berta, Salmonella pullorum and Salmonella senftenberg may produce atypical pale colonies. MLCB Agar is not suitable for Salmonella typhi and Salmonella paratyphi A because of the inhibitory concentration of brilliant green.

The medium may be inoculated directly with the specimen or from an enrichment culture. Selectivity is relatively weak and its performance may be adversely effected by heavily contaminated specimens. Because of these limitations MLCB Agar should not be used alone.

MLCB Agar is specified as a plating medium following enrichment in Modified Semi-Solid Rappaport Vassiliadis (MSRV) CM0910 for isolation of Salmonella spp. from human faeces³.

Van Schothorst et al.² reported MLCB Agar to be excellent for the isolation of H₂S-positive salmonellae after enrichment in Rappaport-Vassiliadis (RV) Enrichment Broth CM0669. They found that the selectivity of MLCB Agar was substantially increased after RV Broth enrichment. They suggested Brilliant Green Agar and MLCB Agar should be used when examining heavily contaminated samples.

Salmonellae grow as large purple-black colonies due to hydrogen sulphide production. Mannitol is utilised by the organism and the resultant pH fall initiates lysine decarboxylation which controls further downward pH movement and promotes blackening.

MLCB Agar does not depend on lactose fermentation and is therefore recommended when investigating lactose-fermenting salmonellae (Salmonella arizona).

Atypical Salmonella strains that produce little or no hydrogen sulphide grow as mauve-grey colonies and may develop a central black `bulls eye’.
To assist the detection of these atypical strains Brilliant Green Agar (modified) CM0329 or Bismuth Sulphite Agar CM0201 should also be used.
Gram-positive and most Gram-negative organisms are inhibited although some strains of Citrobacter spp. may grow sufficiently well to mimic the appearance of Salmonella spp. and some Proteus spp. may swarm.

Most contaminating organisms that are able to grow develop as small colourless colonies.

Technique
Dry the surface of the agar before use.

Inoculate the medium heavily with the specimen or enrichment culture and incubate for 18-24 hours at 35°C.

Examine for typical large purple-black colonies of H₂S positive salmonella. Search carefully for H₂S negative strains that atypically grow as large mauve-grey colonies with a cratered centre. A proportion may show a black `bulls eye’.

Pick all colonies presumed to be Salmonella spp. and confirm by biochemical and serological testing.

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 plates of medium at 2-8°C.

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

Quality control

Positive controls:	Expected results
Salmonella typhimurium ATCC® 14028 *	Good growth; mauve coloured colonies with black centres
Negative control:
Escherichia coli ATCC® 25922 *	Inhibited