DVC Microbiology 146 Fall 11 (Gard): Lab 40: The Confirmed Test

#40 The Confirmed test

Introduction

1. Confirmed test= in water testing, is a test done on positive and uncertain presumptive test tubes to confirm the presence of coliform bacteria ex. E. coli

-this test allows you to culture and visualize coliforms

2. In this lab-

-we use streak plate technique to culture bacteria from presumptive test tubes on Eosin Methylene Blue (EMB) agar plates

-Eosin Methylene Blue (EMB) agar plates=

a. redish-purple agar that contains:

1. lactose

2. two dyes:

a. Eosin= red

b. Methylene blue

b. is a differential medium- bc it differentiates coliforms from non-coliforms by dyeing coliform colonies

c. is a selective medium- bc it selects FOR Gram negative bacteria

- the two dyes inhibit Gram positive bacteria

3. After incubation, you should be able to identify two specific coliform groups and differentiate them from non coliforms

1. E. coli group= typical coliform group 1= Purple with Green metallic sheen

a. these bacteria form colonies that ferment lactose, producing MANY organic acids

b. the acid causes precipitation of a dye complex (methylene blue eosinate) onto the colonies, forming dark purple colonies with a green metallic sheen

(precipitation= formation of a solid)

2. Enterobacter aerogenes= typical coliform group 2= Pink/purple colonies

a. these bacteria form colonies that ferment lactose, and produce FEWER organic acids

b. less of the dye complex is precipitated, forming pink/purple colonies

3. NON-coliforms= colorless colonies

- NON lactose fermenters

- may include fecal bacteria ex. Salmonella and Shigella

-have colorless colonies (which may transmit the reddish purple color of the medium)

Positive test= you have E. coli (purple w. green sheen) or Enterobacter aerogenes (just pink/purple)

Negative test= you don’t and cultures are colorless

E. coli group =purple with green metallic sheen