York College - CUNY Bio 265L Lab Notes on Gram Negative Bacteria

York College - CUNY Bio 265L Lab Week #8: Gram Negative Enteric/Non-Enteric Species - Metabolic Activities and Biochemical Tests

Purpose of the Lab Session

• Familiarize students with biochemical tests necessary for differentiating or identifying bacterial species.

• This lab session prepares students for an upcoming session utilizing the API 20E strip for identification of an unknown organism (Lab Week #9).

Main Focus

• The Enterobacteriaceae family.

Biochemical Tests Overview

1. Tests Conducted by Students

   • Gram Stain Reaction

   • Catalase Test

   • Oxidase Test

   • Main inquiries:
     a. Can we distinguish species of the Enterobacteriaceae family using the Gram stain, catalase, and oxidase tests?
     b. What is the purpose of conducting biochemical tests?
     c. What is the significance of each biochemical test shown below?
     d. Are pure or mixed cultures used for biochemical tests?
     e. What is the reason for adding reagents to specific biochemical test tubes after incubation?
     f. Which reagents are used and to which test tubes are they added?

2. Analysis and Discussion of Demonstration Tests

   • Key biochemical tests analyzed:

     • TSI (Triple Sugar Iron)

     • IMViC Tests (Indole, Methyl Red, Voges-Proskauer, Citrate)

     • Motility Test

     • Nitrate Reduction Test

   • Summary of results from demonstration tests:

     Species	TSI	IND	MR	VP	CIT	Mot	NO3
     E. coli	+/+	+	+	-	-	+	NO2
     Enterobacter	+/+	-	-	+	+	+	K. pneumoniae
     K. pneumoniae	+/+	-	-	+	+	-	Salmonella sp.
     Salmonella sp.	+/-	-	+	-	+	+	Shigella
     Shigella	+/-	+/-	+	-	-	-	P. aeruginosa
     P. aeruginosa	N2

   • Note: Pseudomonas aeruginosa is not part of the Enterobacteriaceae family but included for comparison concerning nitrate reduction test.

Reagents for Biochemical Tests

1. IND (Indole Production from Tryptophan Breakdown)

   • Reagent: Kovac’s reagent

   • Positive Test Indicator: Pink ring.

2. MR-VP Tests

   • MR (Mixed Acid Fermentation)

     • Reagent: Methyl red

     • Positive Test Indicator: Red color.

   • VP (Butanediol Fermentation)

     • Reagents: α-naphthol/KOH (Barritt’s solution A/B)

     • Positive Test Indicator: Red-brown color.

3. Nitrate Reduction Test (NIT)

   • Reagents: A, B, and C (zinc)

   • Interpretation of Results:

     • Presence of gas in the test tube may indicate N2.

     • Addition of reagents A/B: Red color indicates a positive result for reduction of NO3 to NO2; a colorless reaction indicates a negative result for NO2 presence, necessitating further analysis with zinc powder.

     • Addition of reagent C (zinc): Colorless reaction is positive for NO3 reduction beyond NO2, while a red color signifies no NO3 reduction.

Control and Organisms Used

• Positive Control: Pseudomonas fluorescens used for the oxidase tests.

• Organisms in Biochemical Tests:

  • Klebsiella pneumoniae

  • Salmonella

  • Shigella

  • Pseudomonas aeruginosa

• Biosafety Level: All tested organisms are categorized under BSL-2.