Question

How would you determine whether a colorless colony on MacConkey agar is Salmonella or Shigella? Why would you want to identify a colorless colony? Briefly explain

Answer 1

• Salmonella has Lysine DeCarboxylase, whereas Shigella does not.
• Shigella is a gram negative rod found in natural habitat of intestinal tract of humans and other primates. The major pathogen is Shigella dysenteriae, but Shigella sonnei is more common in the U.S. It causes dysentery, which is an invasive bloody diarrhea.
• Shigella is facultative anaerobe but grows best aerobically and is a non-lactose fermenter so will have colorless colonies on MacConkey agar. Hektoen enteric agar: green colonies.
• Hektoen enteric agar:

Selective medium: selective growth of Salmonella/Shigella (contains deoxycholate which suppresses other Enterobacteriaceae and Gram positive bacteria).
Differential medium: distinguishes Shigella (green colonies since no H₂S production) from Salmonella (black colonies since have H₂S production).

• Shigella is non-motile and Produces heat-labile exotoxin called Shiga toxin whereas salmonella is motile.
• Shigella acts as enterotoxin and produces bloody diarrhea, it is also associated with haemolytic uremic syndrome. Toxin is similar to Shiga toxin of Shiga toxin-producing E. coli (STEC), but is not encoded by a bacteriophage.
• Salmonella is gram negative bacilli and most common cause of bacterial foodborne infection in U.S. Major pathogen is Salmonella Typhi, which causes enteric fever (typhoid fever)
• Never ferment lactose (colorless colonies on MacConkey agar).
• Hektoen enteric agar is a differential medium to distinguish Salmonella from Shigella.
• Salmonella will look black due to the production of H₂S.
• Different strains possess different somatic O antigens and flagellar H antigens.
• Loss of O antigen causes a change from smooth to rough colonies.
• Bacteria, known as “lactose fermenters”, eat the sugar lactose that is part of the Mac medium, and, in the process, create an acidic by-product that causes the pH indicator in the medium, neutral red, to turn pink.
• With MacConkey’s, it is not the media that changes color, but rather the actual colonies of lactose fermenting bacteria that appear pink.
• Non-lactose fermenting bacteria will be colorless (or, if the colonies have any color, it will be their natural color rather than pink). Whenever bacteria are growing on MacConkey’s Agar, they are Gram-negative (since Gram+ do not grow on this type of medium). This is the selective aspect of this specialized medium.
• If the bacterial colonies growing on MacConkey's are pink, they are Gram- lactose fermenting bacteria. This is the differential aspect of this specialized media.
• These pink colonies are typically coliform bacteria in the family Enterobacteriaceae, including, but not limited to, the genera Escherichia, Klebsiella, Enterobacter, Hafnia and Citrobacter. The presence of coliform bacteria is commonly used as an indicator of unsanitary food and water.
• Non-lactose fermenting, non-coliform bacteria growing on MAC are not pink (typically colorless), and include members of Enterboacteriaceae such as the genera Proteus, Morganella, Providencia, Edwardsiella, Salmonella, Shigella and Yersenia (plague bacteria).
• MacConkey Agar is a modification of Neutral Red Bile Salt Agar developed by MacConkey. It was one of the earliest culture media for the cultivation and identification of enteric organisms.
• It has also been used in the isolation of pathogens from foods and coliforms in water samples.
• The MacConkey Agar formulation presently in use is a modification of the original. In addition to containing sodium chloride, the modified formula has a lowered agar content and an adjusted concentration of bile salts and neutral red. Differentiation of enteric microorganisms is achieved by the combination of the neutral red indicator and lactose. Lactose-fermenting organisms form pink colonies surrounded by a zone of bile salt precipitation.
• Color change is due to the production of acid which changes the neutral red pH indicator from colorless to red. Acid production is also responsible for the formation of bile salt precipitation.
• Non-lactose-fermenters (Salmonella spp. and Shigella spp.) develop into transparent, colorless colonies with no precipitated zone.
• Peptones are incorporated into MacConkey Agar to provide amino acids and nitrogenous compounds. Sodium chloride is present to maintain osmotic equilibrium. Lactose is added as a possible carbon source for energy, and the acids produced from this activity precipitate out the bile salts. Bile salts and crystal violet are added to inhibit the growth of most gram-positive organisms.
• Bile salts inhibit gram positive basteria, which allows for the isolation of gram negative bacteria. Neutral red and crystal violetfurther inhibit the gram positive bacteria. Lactose is the onlycarbohydrate source. Neutral red indicator is brown in pH 6.8 to 8.0and pink-red at pH less than 6.8.