EXERCISE 10

ENTERIC GRAM-NEGATIVE SHORT RODS

I. INTRODUCTION

Enteric organisms are a large, heterogeneous group of Gram-negative non-sporeforming rods whose natural habitat is the intestinal tract of humans and animals. This group also contains species that are regularly pathogenic. These pathogens are frequently isolated in the hospital diagnostic laboratory. The purpose of this exercise is to acquaint you with tests and procedures used in their identification, and the biochemical basis behind these reactions.

It is convenient to begin the differentiation of enteric bacteria with carbohydrate fermentation patterns. Special plating media used for this purpose include MacConkey's agar and Hektoen-Enteric agar.

MacConkey's agar (MAC) contains lactose as the sole carbohydrate. Bacteria that ferment lactose appear as bright pink colonies on this agar, due to acid production. Those enterics that do not ferment lactose appear as colorless colonies.

Hektoen-Enteric agar (HE) contains lactose, sucrose and salicin for determining sugar fermentation; bile salts to inhibit Gram-positive organisms; and ferric salts to detect H2S production. Organisms that ferment the carbohydrates produce yellow colonies on this agar, and non-fermenters appear as translucent light green. H2S production by bacteria on this agar results in dark brown or black colonies.

A variety of biochemical reactions are used to identify enteric bacteria to the species level. Commercially produced kits, each containing a battery of tests, are available for this purpose. The Enterotube IIŪ does not require an understanding of the biochemical reactions contained within it. To more clearly demonstrate these biochemical reactions, several individual tests will also be performed

II. LAB WORK

• 2 BAP, each with a numbered unknown enteric organism (A)
• 2 BAP, each with a numbered unknown enteric organism (B)
• 4 MacConkey agar plates (MAC) (one green stripe)
• 4 Hektoen Enteric plates (HE) (printed label)
• 2 Enterotube IIŪ, for unknown A
• 2 'classic' sets of biochemical tests for unknown B, including:
• 2 Triple Sugar Iron (TSI) slants (large green cap)
• 2 Tryptophan broths (red cap)
• 2 Urea broths (dark blue cap)
• 2 Citrate slants (pink cap)
• 2 Motility agar gel tubes (black cap)

1. Examine your unknown BAP plates, A and B, and describe the colonial morphology. Gram stain both unknowns.
2. Streak each unknown to a set of MAC and HE plates. Label the plates and the Report Sheet with the unknown identification numbers and letters written on the back of the BAPs.
3. Incubate the plates (inverted) at 37°C until the next lab period.
4. Use unknown A to inoculate the Enterotube IIŪ according to the directions provided by the manufacturer (handed out in lab). The instructor will demonstrate the procedure. Incubate the Enterotube IIŪ at 37°C until the next lab period.
5. Follow the instructions below to inoculate the "classic" identification set with your OTHER (B) unknown. Label this set of tubes with your name and unknown number. Incubate them at 37°C until the next lab period.

Tryptophan - inoculate with a colony

Urea - inoculate with a colony

Citrate - use your needle to streak the slant (DO NOT STAB!)

Motility - use your needle to CAREFULLY stab the agar (DO NOT MIX!)

NEXT LAB PERIOD

1. Examine the colonies on all agar plates and circle the correct results for the MAC and HE agar plates on the Report Sheet.
2. Read and record the reactions obtained with the Enterotube IIŪ, before adding the Indole reagent. (The indole reagent changes the carbohydrate results, so be sure to record these first, and don't change your answer sheet afterwards.)
3. Complete the Indole test as described in the Enterotube IIŪ booklet, with the reagent provided.
4. On the basis of the information obtained from the agar plate cultures and the Enterotube IIŪ tests, use the Enterotube IIŪ Computer Coding Manual and your ID Value to complete the identification of your (A) organism.
5. Use the information below and the chart to determine your 'classic' tube results and the identity of your second (B) unknown.

TSI:

Lactose fermentation = Acid (yellow) slant, Acid (yellow)butt = (A/A)

Dextrose fermentation = Basic (red) slant, Acid (yellow) butt = (K/A)

Sucrose fermentation = Basic (red) slant, No Change butt = (K/NC)

H2S production = Blackening of the slant; ą bubbles in the agar

CO2 production = Bubbles or splits in the agar butt

INDOLE:

Add Kovak's reagent:

a bright red layer = positive for indole

yellow or no change = negative for indole

CITRATE:

Blue slant = positive for citrate as sole source of carbon

Green slant = negative for citrate utilization

UREA:

Bright pink broth = positive for urease production

No change = negative for urease production

MOTILITY:

Diffuse red precipitate = motile bacteria

Compact red stab line = non=motile bacteria