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PEPTONE WATER (ANDRADE)

Code: CM0061

A nutrient base containing Andrade’s indicator, to which carbohydrates and other organic compounds may be added for use in catabolic studies.

Typical Formula*	gm/litre
Peptone	10.0
Sodium chloride	5.0
Andrade’s Indicator (acid-Fuchsin)	0.1
pH 7.4 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards

Directions
Suspend 15 g in 1 litre of distilled water. Mix well to dissolve, distribute into tubes or bottles, containing Durham tubes and sterilize by autoclaving at 121°C for 15 minutes.

Ensure that each individual bottle of peptone water sugar is correctly coded for the contained sugar. Some sugar solutions may affect the pH of the peptone water, check and correct if necessary.

Description
Andrade’s indicator is a solution of acid-Fuchsin that when added to peptone water is colourless or slightly pink at pH 7.4. It becomes pink at acid pH levels and yellow at alkaline pH levels (working over a pH range of 5-8). Filter-sterilised carbohydrate solutions are added to the base medium post-sterilisation. These solutions are usually 10% w/v concentrations and it is important to allow for the dilution of the peptone water when making up the initial volume of medium. A final concentration of 1% w/v carbohydrate in peptone water is normally used.

The peptone used in this medium is free from fermentable carbohydrates and also free from nitrates which may interfere with gas production. The biochemical identification of organisms capable of growing in this medium is made by observing the catabolism of the various carbohydrates added to separate tubes of peptone water. To detect fermentation, which is the production of acid and gas a small, inverted tube (Durham fermentation tube) is inserted into the peptone water to trap any gas produced by the organism. A single bubble in the tube is sufficient to label the organism positive. Some organisms will utilise the sugar to produce acid only, without gas formation.

Technique
The bottles should be inspected before inoculation to confirm that they are clear, that there is no colour or only a slight pinkness in the medium (pH 7.4) and that the glucose tube contains no air trapped in the Durham tube.
1. Aseptically inoculate each bottle with a single, well-separated colony or with a colony from a purity plate.
2. Incubate the bottles at 35-37°C for the required period of time. Incubation is normally carried out aerobically but if anaerobic incubation is required then fresh indicator may have to be added to the bottles after incubation at the time of examination.
3. Note which sugars give an acid reaction and look for bubbles of gas in the Durham tube of the glucose bottle.
Although most reactions are complete after 18-24 hours, it may be necessary to look for late reactions after prolonged incubation.

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 in the dark at room temperature.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: reddish-pink solution when hot. A colourless to a slight pink coloured solution at room temperature

Quality control

Positive control:	Expected result
Salmonella typhimurium ATCC® 9642 *	See table.

* This organism is available as a Culti-Loop®

Table

Carbohydrate	Growth	Acid	Gas
Glucose	Good growth	+	+
Mannitol	Good growth	+	+
Lactose	Growth	No reaction	No gas
Sucrose	Growth	No reaction	No gas
Control (no carbohydrate)	Good growth	No reaction	No gas

One or two drops of  1N hydrochloric acid added to a control bottle will demonstrate the acid-reaction colour and prove that the indicator is active.