VPE 511: Environment and Environmental Hygiene (2+1)

CONFIRMATORY TEST

Inoculation in Eosin methylene blue agar  

• The Gram negative lactose fermenters are confirmed by inoculating the water sample into Eosin methylene blue agar.
• Methylene blue inhibits growth of Gram positive organisms.
•  Coliforms grow in EMB agar and produce dark centered colonies with greenish metallic sheen.
• Enterobacter does not produce metallic sheen. 

Enumeration by pour plate method

Materials Required

• Petridish 
• 10 ml and 1 ml pipette
• Deoxycholate lactose agar.
• Water sample
• Incubator

Deoxycholate lactose Agar

Ingredient	g/L
Peptone	10
Lactose	10
Sodium chloride	5
Sodium citrate	2
Sodium deoxycholate	0.5
Neutral red	0.03
Agar agar	15.0
pH	7.1 ± 0.2

Procedure

• Place 1 ml of water sample using pipette in a petridish.
• Pour 5-8 ml of DCA agar at a temperature of 44-460C into the petridish.
• Thoroughly mix the sample and medium and allow to solidify.
• Incubate at 370C for 18-24 hours.

Note: Entire work is to be carried out under sterile conditions. 

Interpretation

• In positive samples E.coli produces pink colonies and Salmonella produces white colonies.
• As per WHO recommendation drinking water should not have any coliforms per 100 ml of water.
• Environmental Protection Agency (EPA) says that a maximum of 200 colonies per 100 ml water is allowed.