Gram negative bacilli comprise a vast array of bacteria, yet most clinically significant Gram-negative bacilli can be divided into two groups the Enterobacteriaceae and the Non-Enterobacteriaceae.

Gram‑negative bacilli in the family Enterobacteriaceae are the most frequently encountered microbes in the clinical microbiology laboratory. They are often referred to as “enterics” since since they are normally found in the intestinal tract of humans and other animals. The presence of Escherichia coli was used by public health officials for many years to indicate fecal contamination of food and water supplies. Enterobacteriaceae include many genera of bacteria such as E. coli, Klebsiella, Salmonella, Shigella, and Proteus.

Escherichia coli, Klebsiella, Enterobacter and Proteus as part of the normal intestinal microbiome and are non‑pathogens in that environment. Of course, these bacteria can cause illness under other circumstances. For example, E. coli is a common cause of urinary tract infections and Proteus has been isolated from patients with kidney stones, gangrenous wounds, and otitis media.

The truly enteric pathogens in the Enterobacteriaceae family include Salmonella, which causes typhoid fever and mild to severe gastroenteritis (inflammation of the gastrointestinal tract) due to “food poisoning” and Shigella, which causes bacterial dysentery (bloody diarrhea with fever) due to “food poisoning”.

The Enterobacteriaceae have four common characteristics:

• They are all Gram-negative bacilli
• They all ferment glucose
• They do not produce oxidase
• They all reduce nitrate to nitrite

The Gram-negative bacilli that are not in the Enterobacteriaceae family are composed of several families but are collectively called Non-Enterobacteriaceae or nonfermenters (of glucose) to differentiate them from the Enterobacteriaceae. Non-Enterobacteriaceae include bacteria such as Pseudomonas aeruginosa and Alcaligenes faecalis. These bacteria are mostly found in the environment, yet some of these organisms can cause wound infections and serious life-threatening infections in immunocompromised patients.

The Non-Enterobacteriaceae also have three common characteristics:

• They are all Gram-negative
• They do not ferment glucose
• Many (but not all) are oxidase positive

To identify the Gram negative bacilli, we will perform biochemical tests to detect the presence or absence of metabolic enzymes. Substrates upon which these enzymes act are incorporated into the culture medium along with an indicator system to allow detection of specific metabolic products.

PRE-ASSESSMENT

PROCEDURE

For this exercise:  Work in groups of 3 at Pecos groups of 4 at Williams.  Perform the following tests on each color dot culture.

KLIGLER IRON AGAR (KIA)

KIA media contains two carbohydrates, glucose and lactose. It also has a phenol red pH indicator, ferrous sulfate, and peptones in the agar.

KIA media is used to determine if the organisms can ferment glucose with or without gas production. If the organism can ferment glucose, then KIA will also demonstrate if it can ferment lactose. The media can also determine if the organism can produce hydrogen sulfide.

If bacteria cannot ferment glucose, they are called “nonfermenters” and will not be able to ferment any other carbohydrates.

If bacteria can ferment glucose, they are called “fermenters” and can be differentiated by determining if it can also ferment lactose or produce hydrogen sulfide.

1.  Obtain a KIA tube and a straight inoculating wire.

Use a permanent marker to write the color dot, your name (or initials), and the name of the media (KIA) on the side of the tube.

2.  Use the sterile straight inoculating wire to take an inoculum of the bacteria.

3.  Begin by stabbing in the middle of the KIA slant to just above (3-5mm or 0.25 inch) the bottom of the tube. As the needle is withdrawn from the agar, streak the agar slant.

4.  Incubate the KIA tube in the class test tube rack.

AFTER INCUBATION –Observe the reactions in the KIA tube and record the reactions/results. Record the reactions and results on the worksheet. Use the growth on the KIA slant to perform the oxidase test.

Butt of KIA tube:

Yellow butt = Positive glucose fermentation (A black precipitate may mask the yellow color)

Red butt = Negative glucose fermentation

Gas production from glucose fermentation:

Bubbles or splitting of the agar = Positive for gas (If bacteria cannot ferment glucose, they will not produce gas)

Slant of the KIA tube:

Yellow slant = Positive lactose fermentation

Red slant = Negative lactose fermentation

Hydrogen sulfide production:

Black precipitate in the media = Positive Hydrogen Sulfide Production