Microbiology Lab practical: Identification of Gram Negative organisms

How do the procedures for a BEA slant and a TSI slant differ?

• With the BEA slant you’re only streaking to top of the slant with the culture

• The TSI slant uses a needle to go through to the “Butt” portion of the slant" and then streaks the stop portion

What sugars are in the TSI tube?

1. Glucose

2. Lactose

3. Sucrose

What is the gas that is produced from the TSI test ?

CO2 is produced

What would happen to the TSI slant if CO2 was produced?

There would be a crack, separation, bubble or lifting in the tube

POSITIVE= crack/ bubble

NEGATIVE= no crack or bubble

What would happen to the TSI slant if there’s H2S production?

• H2S production= black coloring in the tube: POSITIVE

• No H2S production= no black coloring: NEGATIVE

What would it mean if the slant results were K/A, A/K, A/A and K/K

1. K/A= Slant portion is Alkaline (red) and Butt portion is acidic (yellow)→ ONLY glucose fermented

2. K/K= BOTH the Butt and slant portion is alkaline (red)→ NO sugar fermented

3. A/A= BOTH the Butt and slant portion are acidic (yellow)→ glucose and lactose fermented

What would this mean?

K/A (Slant= red & Butt= yellow)

CO2= (-)

H2S= Black

The Microgen GNA-ID system is a collection of biochemical tests to identify ___species and some additional ___species.

1. Eneterobacteriaceae

2. Gram-negative

**Contains 12 wells of dehydrated substrates

Which wells are anaerobic and what does that mean we need to add?

1,2,3, and 9

• Mineral oils gets 2-3 drops in each well

Well 1

• Tests for Lysine

• Looking for Lysine Decarboxylase

• pH Indicator: Brothymol Blue

• Results: (+) end product= cadaverine raise pH= blue/green

• Note: Anaerobic process

well 2

• Tests for Ornithine

• Looks for Ornithine decarboxylase

• pH indicator= Brothymol Blue

• Results: (+) end product= putrescine raise pH= BLUE

• Note: Anaerobic process

Well 3

• Tests for H2S

• Looks for thiosulfate reductase

• Results: (+) H2S gas reacts with ferric salts= Black precipitate

• Note: Anaerobic

Well 4,5,6

• Tests for Glucose, Mannitol & Xylose

• Looks for Carbohydrate metabolized?

• Indicator: Brothymol Blue

• Results: (+) End products are acidic and will LOWER pH

Well 7

Tests for Beta-galactosidase and nitrate reductase

• If synthetic lactose (ONPG) is broken down= yellow color change

Then tests for nitrate reductase

• After reagent drops

• Red color= positive= REDUCED NITRITE → NITRATE

• Colorless= add zinc

→ Red= negative

→ Colorless= positive

Well 8

• Tests for Indole production

• Kovac reagent is added

• Red= Positive

Well 9

• Tests for Urease

• (+)= Hot pink

• (-)= light yellow or pink

well 10

VP test

• Can it ferment glucose with ONE end product?

• VP 1&2 react with Acetylmethocarbinol

• (+)= RED

Well 11

• tests for Citrate utilization

• pH indicator brothymol

• (+)= RED

• (-)= yellow

Well 12

tests for Tryptophan Deaminase?

• (+)= RED

• (-)= Brown