Lecture Notes on TSI, IMViC, Selective and Differential Media, Enterobacteriacea, MIO, LIA, Oxidase Test

Many intestinal bacteria are classified in the tribe Enterobacteriaceae. These are gram negative rods which are able to ferment glucose.

Enterobacteriacieae which are also capable of fermenting lactose are called coliforms. Coliforms are generally considered to be opportunistic pathogens and non-pathogens, however there are many instances of severe, life-threatening infections caused by coliforms. Coliforms are very important as INDICATORS OF FECAL CONTAMINATION. Some common coliforms are:

• Escherichia coli
• Klebsiella pneumoniae
• Enterobacter aerogenes

• ETEC causes the watery diarrhea commonly known as "Traveler's diarrhea."
• EIEC invades the intestinal epithelium and causes dysentery.
• EPEC causes a watery diarrhea with fever that commonly strikes infants and young children.
• EHEC causes hemorrhagic diarrhea.
HEMOLYTIC UREMIC SYNDROME (HUS) is a life threatening complication of EHEC infections in children under 5 years old. EHEC strains such as O157:H7 are fairly common in cattle. Humans are frequently infected through meat and raw milk, however, it is important to note that recent U.S. outbreaks have been traced to produce such as lettuce and apple juice. The massive 1996 school lunch epidemic in Japan has yet to be conclusively attributed to any particular food.
 

 Hear a special National Public Radio report on food safety.  E. coli O157:H7 is highlighted in this presentation.

• Salmonella typhi causes TYPHOID FEVER, a severe systemic infection which begins several weeks after ingesting the organisms. The disease is characterized by high fever, headache, a rash on the abdomen (rose spots), constipation and/or diarrhea and organisms in the stool, blood and urine.
• Other Salmonellae cause a less severe gastroenteritis commonly called Salmonella "food poisoning" although this is more accurately called a "food infection."
• Shigella dysentariae causes bacillary dysentery. In this disease, frequent bloody, mucoid watery stools are passed. The organism invades and kills the intesinal epithelium. This causes white cells to accumulate and these can be seen in the stool upon microscopic examination.
• Yersinia enterocolitica causes diarrheal disease similar to Shigella. This organism is frequently associated with pigs and therefore undercooked pork is associated with infections.
• Other lactose negative Enterobacteriaceae which are not classified as pathogens include Proteus sp. and Serratia marcescens.

There are a number of non-Enterobacteriaceae which are important intestinal pathogens.

• Vibrio cholerae causes cholera.
• Vibrio parahemolyticus is a marine organism which frequntly contaminates seafood. It causes an acute diarrheal disease 24-48 hours after ingestion of raw or incompletely cooked shellfish and less frequently, fish.
• Partially cooked poultry can be the source of Campylobacter jejuni. This organism can cause diarrhea, often with blood and pus, 1 to 7 days after infection.
• Helicobacter pylori, an organism believed to be associated only with humans, is now known to be the cause of gastric ulcers

A number of selective isolation techniques are used for fecal organisms.

SELENITE BROTH is used mainly to enrich for Salmonella and to some extent Shigella. This broth is called an enrichment broth because it suppresses the growth of most intestinal organisms while allowing the growth of the pathogens thereby increasing the chances of finding the pathogens.

 

Plating media such as:

• Salmonella-Shigella (SS) agar,
• Eosin Methylene Blue (EMB) agar
• MacConkey (MAC) agar

contain a variety of dyes and bile salts to allow the selective growth of the gram negative enteric organisms. These media also contain sugars (such as lactose) and pH indicators to allow one to distinguish between different organisms based on their metabolic abilities. Hence lactose fermentors will appear different from other organisms which are unable to ferment lactose.

 

• On EMB, Lactose (+) organisms appear purple red, sometimes with a metalic green sheen. Lactose (-) organisms appear light colored and transparent.
• On MAC, Lactose (+) organisms are purple-red to brick-red often with a zone of precipitate surrounding the colonies. Lactose (-) organisms can be light pink to white and they are often transparent.
• SS agar is highly selective for Salmonella and Shigella and these form colorless colonies often with black centers indicative of H2S production. Most other organisms don't grow on this medium. If they do they will be white-pink.

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• 0.1% glucose
• 1% lactose
• 1% sucrose
• phenol red

An organism capable of fermenting only glucose and neither of the other two sugars would form only a very small amount of acid (why?).

Whereas lactose and / or sucrose fermenters will form relatively large amounts of acid.

There are three possible sugar reaction interpretations possible on this agar.:

• Alkaline / Alkaline (Red/Red) = No sugars fermented
• Alkaline / Acid (Red/Yellow) = Only glucose fermented
• Acid / Acid (Yellow/Yellow) = Lactose and/or sucrose fermentation occured in addition to glucose fermentation.
 

Notice that an Acid / Alkaline reaction is not an option. (Why?)

The INDOLE test measures the ability of the microorganism to degrade tryptophan into indole, ammonia and pyruvic acid:

The METHYL RED - VOGUES PROSKAUER test measures the types of products produced in the fermentation of glucose.

• The MR test determines if acid products such as lactic and formic acids are formed.
• The VP test determines if neutral products such as ethyl alcohol, acetoin and butanediol are formed.
• The MR test
• uses methyl red as a pH indicator which turns red at acidic pH, hence red is a positive MR test.
• Acetoin reacts with alpha-napthol under alkaline conditions to form a pink or red product which takes several minutes to form.

The CITRATE test determines whether an organism is capable of using citrate as a sole carbon and energy source. Such organisms will produce alkaline end products in the presence of oxygen and these can be measured with a pH indicator. Bromthymol blue is green at neutral pH and blue at alkaline pH, therefore blue represents a positive test.

This medium allows you to test

• (1) if an organism can decarboxylate lysine;
• (2) if an organism can deaminate lysine; and
• (3) if an organism can produce H₂S.

• Alkaline /Alkaline (purple/purple) = lysine decarboxylase positive
• Alkaline / Acid (purple/yellow) = lysine not broken down = negative test
• Red pigment / Acid (red/yellow) = lysine deaminase positive

This medium allows you to

• (1) determine if the organism is motile;
• (2) see if the organism can decarboxylate ornithine; and
• (3) see if the organism can break down tryptophan to produce indole.

The decarboxyation of ornithine will cause the medium to become alkaline, therefore:

• purple color throughout the tube = positive for ornithine decarboxylase
• yellow color in the bottom or throughout = negative for ornithine decarboxylase

OXIDASE TEST

The oxidase test will determine if the organism possesses cytochrome c, one of the electron carrier proteins found in the electron transport chain. (Even though many bacteria are capable of respiration and therefore have an electron transport chain, not all bacteria have the same cytochrome proteins in the chain. For instance, E. coli and the other Enterobacteriaceae lack cytochrome c but have cytochrome o and cytochrome d instead. On the other hand, Pseudomonas aeruginosa, a number of other organisms as well as our mitochondria use cytochrome c in aerobic respiration.)

The test uses a compound called tetramethyl-p-phenylenediamine which is clear in the reduced (electron rich) state and purple in the oxidized (electron poor) state. In the presence of an organism with cytochrome c, the reduced tetramethyl-p-phenylenediamine will donate its electrons to the cytochrome c and become purple colored.

How to do the test:
1. Wet a small piece of white paper towel or filter paper with the tetramethyl-p-phenylenediamine reagent
2. Use a wooden applicator stick to take some growth from TSA or blood agar and then smear this onto the wetted paper.
3. An immediate purple reaction is positive for oxidase