Selective and Differential Media for Gram-Negative Bacteria

Amino Acid Structure

3 parts: carboxyl group, amino group, and 'R' side chain

Decarboxylation

Removal of the COOH group (carboxyl group) which exists as COO- in a pH of 7.2; therefore decarboxylation releases carbon dioxide.

Deamination

Removal of the NH3+ group (amino group).

Lysine

An essential amino acid.

Agar slants

Agar which has hardened on a slant; inoculated by a stab and streak method to allow aerobic growth at the top and anaerobic growth at bottom of tube (known as the butt).

Sensitivity

Ability to detect small amounts of the chemical or organism (i.e., a test's ability to find true positive results); failure yields false negatives.

Specificity

Ability to discriminate between the chemical or organism in question and similar chemicals or organisms; failure yields false positives.

Selective Media

Suppresses growth of unwanted bacteria and encourages growth of desired microbes.

Differential Media

Facilitates distinguishing the desired microbe from others in a mixed sample.

Enterobacteriaceae

Family name; includes gram (-) organisms found in human GI tract.

Enterics

A general term for the above family members.

Fecal Coliforms

Commonly used bacterial indicator of sanitary quality of foods and water.

Lactose

A disaccharide composed of glucose + galactose.

Eosin Methylene Blue (EMB) Agar

Both selective and differential; selects for gram (-) and differentiates between vigorous lactose fermenters, moderate lactose fermenters, and non-lactose fermenters.

Hektoen Enteric (HE/SS) Agar

Agar used to identify gram (-) microbes.

Vigorous lactose fermenters

Produce enough acidity to produce a dark purple complex with a green metallic sheen under acidic conditions.

Moderate lactose fermenters

Ferment lactose but do not produce as much acidity as vigorous fermenters.

Non-lactose fermenters (NLF)

More pathogenic organisms such as Salmonella, Shigella, and Proteus.

Gram (+)

Will not grow on Eosin Methylene Blue Agar.

Acidity of media

Increased by end products of lactose fermentation.

Metallic sheen

Produced by vigorous lactose fermentation under acidic conditions.

Inhibitory dyes

Eosin and methylene blue dyes inhibit growth of gram (+) bacteria.

Aerobic growth

Occurs at the top of an agar slant.

Anaerobic growth

Occurs at the bottom of an agar slant (known as the butt).

HEKTOEN ENTERIC AGAR

Both selective and differential media that selects for gram (-) bacteria and differentiates among enterics that can reduce sulfur to hydrogen sulfide gas (H2S).

H2S Producers

Bacteria that can reduce sulfur to hydrogen sulfide gas, resulting in black colonies on HEKTOEN ENTERIC AGAR.

Lactose Fermenters

Bacteria that can ferment lactose, resulting in pink or yellow colonies on HEKTOEN ENTERIC AGAR.

Non-Lactose Fermenters

Bacteria that do not ferment lactose, resulting in colorless colonies on HEKTOEN ENTERIC AGAR.

Lysine Iron Agar (LIA)

Differential media that separates Gram (-) enteric bacteria based on their ability to decarboxylate or deaminate lysine.

Ferric Ammonium Citrate

A component in media that reacts with H2S to form a black precipitate.

Bromcresol Purple

A pH indicator used in LIA that turns purple in alkaline conditions and yellow in acidic conditions.

Lysine Decarboxylase Positive (K/K)

Indicates a purple slant and purple butt in LIA, showing lysine decarboxylation.

Lysine Deaminase Positive (R/A)

Indicates a red slant and yellow butt in LIA, showing lysine deamination.

Black Precipitate

Indicates H2S production in LIA, which must be deaminated to observe.

Gram (+) Growth on Media

Gram (+) bacteria will not grow on HEKTOEN ENTERIC AGAR.

Salmonella

A non-lactose fermenter that produces H2S, resulting in black colonies on HEKTOEN ENTERIC AGAR.

Shigella

A non-lactose fermenter that does not produce H2S, resulting in colorless colonies on HEKTOEN ENTERIC AGAR.

E. coli

A lactose fermenter that produces pink colonies on HEKTOEN ENTERIC AGAR.

Klebsiella pneumoniae

A lactose fermenter that produces yellow to salmon pink colonies on HEKTOEN ENTERIC AGAR.

Sodium Thiosulfate

A sulfur source in HEKTOEN ENTERIC AGAR that is reduced to H2S.

0.1% Glucose

A fermentable carbohydrate in LIA that induces acid production.

Acid Production in LIA

Acid production from glucose fermentation turns the medium yellow.

Proteus vulgaris

is a gram (-) bacillus, highly motile, facultative anaerobe normally present in intestinal tract of humans.

Proteus mirabilis

is a gram (-) bacillus, highly motile, facultative anaerobe commonly found in intestinal tract of humans.

Morganella morganii

is a gram (-) bacillus, motile, facultative anaerobe normally found in environment and in intestinal tract of humans.

Shigella spp

is a gram (-) bacillus, non-motile, facultative anaerobe, non-lactose fermenter, responsible for causing shigellosis in humans.

Salmonella spp

is a gram (-) bacillus, motile, facultative anaerobe, non-lactose fermenter, responsible for causing salmonellosis in humans.

Lactose Fermenter

grow pink / red colonies.

Non-Lactose Fermenter

grow colorless colonies.

Vigorous Lactose Fermentation

grow dark purple with a green metallic sheen.

Lysine Iron Agar

Gram (+) org. Inhibited by bile salts.

H2S Production

Hydrogen sulfide gas (H2S) producer.

Reiter's triad

inflammatory arthritis, conjunctivitis, and urethritis.

Symptoms of Shigellosis

diarrhea, fever, nausea, vomiting, stomach cramps, tenesmus, and flatulence.

Transmission of Shigella

by fecal-oral route which results in destruction of epithelial cells of intestinal mucosa in cecum and rectum.

Symptoms of Salmonellosis

diarrhea, fever, vomiting, and abdominal cramps 12 to 72 hours after infection.

Salmonella Infection Statistics

About 142,000 (reported) Americans are infected each year with Salmonella enteritidis from chicken eggs and about 30 die.

Infection Duration

In most cases, the illness lasts four to seven days, and most people recover without treatment.

Proteus vulgaris Characteristics

ferments glucose (with gas production), but does not ferment mannitol or lactose.

Proteus mirabilis Characteristics

urease positive and a non-lactose fermenter.

Klebsiella pneumoniae Infections

causes pneumonia with destructive changes to human lungs, inflammation and hemorrhage with cell death (necrosis) that sometimes produces a thick, bloody, mucoid sputum (currant jelly sputum).

Morganella morganii Infections

is an uncommon cause of community-acquired infection and is most often encountered in postoperative and other nosocomial settings.