BIOL 362 Unknown Project
ge210058
DifferentiationofGramnegativerods2020.pptDifferentiation of Gram (-) rods
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E. coli
E. coli
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
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Carbohydrate broths
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Phenol Red Carbohydrate Broths
- Contains Phenol Red as a pH indicator
- pH < 6.8 = yellow
- pH > 7.4 = pinky red
- Fermentation = acid production = yellow
- Degradation of the amino acids in peptone = NH3 (ammonia)(basic) = pinky red
- Inverted tubes (Durham) check for gas production
- Test should not be read after 48 hours
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Phenol Red Carbohydrate Broths
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Eosin Methylene Blue (EMB) agar
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Protection of the water supply
- We need clean water for bathing, drinking, and cooking.
- How can we adequately screen for pathogens in the water?
- Screen for an organism that indicates fecal contamination (coliforms)
- E coli is a good indicator organism.
- Sole habitat is the intestine of warm blooded animals
- Easy to detect
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How can we test for coliforms?
1) Multiple Fermentation Test
A) Add the water to media containing lactose and test for growth and lactose fermentation.
B) If positive, look for growth under selective conditions for coliforms.
C) Streak onto EMB agar.
2) Filter Test
A) Filter 100 mls of water.
B) Place the filter directly on selective media.
C) Look for growth of coliforms.
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EMB agar
- Eosin Methylene Blue Agar
- Eosin
- Methylene Blue
- Used to isolate coliforms in a sample; often used in checking water supplies
- Selective
- Aniline dyes inhibit growth of Gram positives
- Differential
- Lactose fermenters produce acid
- Dark Colonies
- Green metallic sheen due to precipitation of methylene blue
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EMB agar
Lac (-)
Lac (+)
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Citrate Utilization
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Citrate Utilization
- Measures whether an organism can utilize citrate as a nutrient (sole carbon source)
- Inoculate the top of the slant with the organism
- After 48 hours, look for growth on the slant, and a shift in color of the agar from green to blue
(-)
+
+
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TSI Agar
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TSI (Triple Sugar Iron Agar)
- Differentiates bacteria on their ability to:
- Ferment glucose, lactose, and/ or sucrose to acid and gas products
- Reduce sulfur to hydrogen sulfide
- Medium also includes:
- 0.1% glucose, 1% lactose, and 1% sucrose
- 2% polypeptone
- Phenol red as a pH indicator
- pH < 6.8 = yellow
- pH > 6.8 = red
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TSI (Triple Sugar Iron Agar)
- Medium also includes:
- Thiosulfate as an electron acceptor for sulfur reduction and ferric ammonium citrate as a H2S indicator
If H2S is present = black precipitate
If the color of the butt is not observable due to a lot of H2S, the test is recorded as acid positive.
- If gas is produced, the agar will be cracked (fissures) or lifted off the bottom of the tube.
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TSI (Triple Sugar Iron Agar)
- Glucose fermentation occurs first, turning the butt yellow.
- If the organism cannot ferment lactose or sucrose, it will break down the amino acids producing NH3 and raising the pH = acid (yellow) butt and alkaline (red) slant
- If the organism can ferment lactose or sucrose, fermentation continues = acid (yellow) butt, acid (yellow) slant
- If the organism cannot ferment any of the sugars, it will utilize the peptone turning the medium red.
- Catabolizes the peptone aerobically and anaerobically = red butt, red slant
- Catabolizes the peptone aerobically = red slant
- Catabolizes the peptone anaerobically = red butt
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(-)
A
B
C
D
E
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Hektoen Enteric Agar
- Main goal is to identify Salmonella and Shigella from other enterics
- Selective for Gram negative organisms due to presence of bile salts
- Differential for fermentation of lactose, sucrose or salicin
- Differential for reduction of sodium thiosulfate (sulfur source) to H2S
H2S + ferric ammonium citrate → black precipitate
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Lactose
fermentation
No Growth
No fermentation, H2S production
http://textbookofbacteriology.net/salmonella_3.html
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