The half MIO is a biochemical test used to aid in the identification of species of bacteria belonging to the Enterobacteriaceae family. It is quite nutritious and is composed of glucose, yeast extract, peptone, triptein, L-ornithine hydrochloride, bromocresol purple and agar.
The meaning of its acronym (MIO) describes each of the parameters that can be observed in this medium; motility, indole and ornithine. Motility is the ability of the microorganism to move due to the presence of flagella. For this property to be observed, the consistency of the medium must be semi-solid, so the preparation contains less agar..
The production of indole shows the presence of the enzyme tryptophanase that acts on the amino acid tryptophan, requiring the use of a revealing reagent to make the production of indole visible..
Finally, ornithine determines if the bacterium is able to decarboxylate the amino acid, that is, if it has the enzyme orinithine decarboxylase.
Article index
These elements contribute to the nutritional power of this medium. They serve as a source of nutrients and essential amino acids for bacterial development.
In addition, triptein is a source of tryptophan to show the presence of the enzyme tryptophanase, which degrades tryptophan by reductive deamination, releasing indole, pyruvic acid, ammonia and energy..
Indole is colorless, therefore its presence is revealed by adding five drops of the Ehrlich or Kovacs reagent, both with p-dimethylaminobenzaldehyde..
The aldehyde group of this compound reacts with indole, generating a fuchsia red product in the shape of a ring on the surface of the agar..
Any trace of color should be considered a positive test. The proof should be read immediately, as over time the color degrades.
Furthermore, this test should be revealed after the motility and decarboxylation results of ornithine have been noted..
Positive test: formation of a red fuchsia ring when adding drops of Kovacs reagent.
Negative test: no ring formation.
The ability of the bacteria to move will be evidenced if a cloudy medium is observed or if there is a thick growth line expanding around the initial inoculation..
A negative motility test will be evidenced by observing a thin line of growth, and everything around it will be without growth..
It is important that the motility is read before the indole is revealed, as the addition of the reagent clouds the entire medium..
In mobile but slow-growing bacteria it is difficult to demonstrate their motility with this medium. In this case, it is recommended to use other tests or methods, such as the medium motility or the drop-pending method..
Glucose is the fermentable carbohydrate that, in addition to providing energy, acidifies the environment, a necessary condition for the decarboxylation of the amino acid ornithine to occur..
Glucose fermentation must always occur, starting from the principle that all bacteria belonging to the Enterobacteriaceae family ferment glucose..
In the event that the bacteria produce the enzyme ornithine decarboxylase, it may act once the medium has been acidified by the fermentation of glucose..
The enzyme ornithine decarboxylase acts on the carboxyl group of the amino acid producing an amine called putresine that again alkalizes the medium.
This test should be read after 24 hours of incubation, because if you try to read before you can misinterpret the test with a false negative.
It should be remembered that the first reaction that occurs is the fermentation of glucose, which is why the medium turns yellow in an initial phase (first 10 to 12 hours). If ornithine decarboxylation subsequently occurs, the medium will turn purple.
It is important to interpret the ornithine decarboxylation test before revealing indole, as the addition of Kovacs' reagent modifies the color of the medium..
Negative test: yellow medium or yellow background.
Positive test: half completely purple.
In this case, bromocresol purple is used; the one in charge of revealing when there is a change in pH in the medium. When acidified, the indicator turns yellow, and when alkalized it turns purple.
To sow the MIO medium, a straight loop or needle is used and with it a portion of the colony to be studied is collected..
A deep puncture is made in the middle of MIO in a straight line. It is not advisable to perform a double puncture, since it can give a false image of motility if the punctures are not carried out in the same place..
Incubate for 24 to 48 hours at 37 ° C in aerobiosis. Observe the results in this order: motility, decarboxylation of the ornithine and finally reveal the indole.
It is advisable to aseptically remove 2 ml of the medium, transfer it to a sterile tube and perform the indole test there, so that if it is negative, the rest of the original tube can be incubated for a further 24 hours, to reveal the indole again..
The development of the indole is carried out as follows: 3 to 5 drops of Kovacs's reagent are added to the MIO medium and it is stirred vigorously. It is observed whether or not a red-fuchsia ring appears.
Weigh out 31 g of the MIO medium and dissolve in a liter of distilled water..
Heat until the mixture boils for one minute, shaking frequently until the agar is completely dissolved. Distribute 4 ml of the medium into 13/100 test tubes with cotton caps.
Sterilize in autoclave at 121 ° C for 15 minutes. Remove from the autoclave and let stand straight in a rack, in such a way that a semi-solid block is formed.
Store in a refrigerator 2-8 ° C. Allow to warm before sowing the bacterial strain.
The color of the dehydrated medium is beige and that of the prepared medium slightly opalescent purple..
The final pH of the prepared medium is 6.5 ± 0.2
The medium turns yellow at acidic pH and is purple at alkaline pH.
This reagent is prepared as follows:
150 ml of amyl, isoamyl or butyl alcohol (any of the three) are measured. 10 g of p-dimethylaminobenzaldehyde are dissolved in it. Subsequently, 50 ml of concentrated hydrochloric acid are slowly added..
The reagent prepared is colorless or light yellow. It should be kept in an amber bottle and kept in a refrigerator. A dark brown color shows its deterioration.
Also the Kovacs reagent can be substituted for the Ehrlich reagent. The latter, being more sensitive, is preferred to reveal indole in bacteria that produce it in minute quantities, such as in some non-fermenting Gram negative rods and certain anaerobes..
This medium is a test that complements a battery of biochemical tests for the identification of bacteria belonging to the Enterobacteriaceae family..
The data of the decarboxylation of ornithine serves to differentiate Shigella sonnei, that gives positive, of Shigella boydii, Shigella flexneri, and S. dysenterieae, that give negatives.
It also differentiates the genus Klebsiella, which tests negative, from the genus Enterobacter, where most of its species test positive..
Each time a batch of MIO medium is prepared, a control test can be performed. For this, known or certified strains are used to observe the behavior of the medium..
The strains that can be used are Escherichia coli, Morganella morganii, Klebsiella pneumoniae, Enterobacter aerogenes Y Proteus mirabilis.
The expected results are E. coli and M. morganii. Dan M: +, I: + and O: +.
Klebsiella pneumoniae gives all negative (M: -, I: -, O :-). Proteus mirabilis Y Enterobacter aerogenes give M: + I: - and O: +.
Yet No Comments