Microbiology

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  • Topic: Escherichia coli, Gram-negative bacteria, Enterobacteriaceae
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Microbiology Laboratory 7

MacConkey Agar

PURPOSE: MacConkey agar selects for gram negative bacteria and also differentiates lactose fermenters (pink-red) from non-lactose fermenters(colorless).

PRINCIPLE: Bile salts inhibit gram positive basteria, which allows for the isolation of gram negative bacteria. Neutral red and crystal violet further inhibit the gram positive bacteria. Lactose is the only carbohydrate source. Neutral red indicator is brown in pH 6.8 to 8.0 and pink-red at pH less than 6.8

PROCEDURE:
1. Streak agar for isolation
2. Incubate at 35-37 °C for 18-24 hours and observe for growth and color. 3.
INTERPRETATION: If lactose is fermented, the medium is acidified, and bile salts are precipitated. The precipitated dye is absorbed, resulting in a pink-to-red complex.

MacConkey Agar – pink colonies (Enterobacter cloacae – rapid lactose fermenter)

MacConkey agar – pink colonies (Escherichia coli – rapid lactose fermenter)

MacConkey agar – colorless colonies (Salmonella typhimurium – nonlactose fermenter)

MacConkey Agar Classification according to use: Differential medium for gram negative bacilli

INDICATOR: Neutral red

A. MAC showing LFO with pink or colored colonies
Organism used : E. coli

B. MAC showing LFO with mucoid colines
Organism used : Klebsiella pneumoniae

C. MAC showing NLFO with colorless transluscent colonies
Organism used: Proteus vulgaris

D. MAC
Organism used: Enterobacter

Salmonella-Shigella Agar

PURPOSE:
Salmonella-Shigella medium provides for inhibition of normal flora coliforms and differentiation of stool pathogens

PRINCIPLE:
Bile salts inhibit gram positive bacteria, and brilliant green agar and bile salts inhibit the gram negative coliforms. Lacotse is the sole carbohydrate source. Neutral red indicator is red in acidic conditions. Lactose fermenters appear pink-red, whereas non-lactose fermenters appear clear. To detect H2s production, sodium thiosulfate serves as sulfur source. When H2S is formed, it combines with ferric ammonium citrate to form ferric sulfide (FeS), which is represented by black centered colonies.

PROCEDURE:
1. Streak agar for isolation.
2. Incubate at 35-37 °C for 18-24 hours and observe for growth and color.

INTERPRETATION:
Normal flora coliforms = pink to red colonies.
Shigella = colorless colonies without balck centers.
Salmonella = colorless colonies with black centers

A. Salmonella-Shigella Agar (SSA)
B. SSA – Salmonella typhii
C. SSA – E. coli

Xylose Lysine Deoxycholate (XLD) Agar

PURPOSE:
The XLD is used for the isolation and differentiation of stool pathogens and inhibition of normal flora coliforms.

PRINCIPLE:
Sodium deoxycholate inhibits gram positive bacteria, partially inhibits the growth of E. coli, and inhibits the swarming of Proteus. Phenol red indicator becomes yellow in acidic environments. Fermentation of xylose results in yellow colonies. Most members of Enterobacteriacae are xylose positive, except Shigella. Most strains of Shigella cannot ferment lactose and thus produce red colonies. Lysine positive bacteria first produce yellow coloniesas xylose is fermented, followed by red colonies, indicating lysine decarboxylation. H2S positive colonis have black centers due to reaction of H2S with ferric ammonium citrate.

PROCEDURE:
1. Streak agar for isolation.
2. Incubate at 35-37 °C for 18-24 hours and observe for growth and color.

INTERPRETATION:
Salmonella = red colonies with black centers
Citrobacter and Proteus = yellow colonies with black centers

Differential capabilities of XLD agar for lactose-fermenting, gram negative bacilli (e.g E. coli, arrow A), nonlactose fermenters (e.g Shigella spp., arrow B) and H2S producers (e.g Salmonella spp., arrow C)

Hektoen Enteric Agar

PURPOSE: Hektoen enteric medium selects for stool pathogens by inhibiting the normal flora of the lower GI tract.

PRINCIPLE:
A high...
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