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MicroBio 225 (Lab Test 3) Selective and Differential Media, IMViC, BioChem Tests (with some bacterial samples)

Mrs. (Thomas) Wick's BIO 225 CCTC Spring 2011
Phenylethyl Alcohol agar (PEA/PAA)
Selects for: Gram (+)
Phenylethyl alcohol
(used for isolation of Gram + from mixed cultures)

Plate A is a PEA plate with a Gram positive organism on the left and a Gram negative organism on the right. Plate B is a nutrient agar plate illustrating normal growth of each of the same bacteria. Notice that the Gram-negative organism shows inhibited growth on the PEA plate.
PEA/PAA agar
Phenylethanol Agar (PEA) Phenylethanol inhibits many Gram negative bacteria and its inclusion in PEA renders the medium selective for Gram positives.
Mannitol Salt Agar (MSA)
7.5% NaCl; Mannitol; Phenol Red pH Indicator
*Selective for:*
7.5% NaCl growth ( Staphylococcus sp)
*Differential for:*
Mannitol fermentation
phenol red turns yellow
Growth: Staph sp and specific other sp.
If bacteria ferments mannitol the phenol red indicator turns yellow
MSA Positive and Negative Results
orange arrow points to positive mannitol fermentation and positive growth
Blue arrow points to negative fermentation and positive growth
MSA S. epidermidis and S. aureus comparison
S. aureus is positive for fermentation
S. epidermidis is negative for fermentation
MSA Sample Results
o Staphylococcus aureus ferments mannitol
o S. aureus changes the color of the medium from pink to yellow due to acid by-products of mannitol fermentation
o Staphylococcus epidermidis grows on MSA, but does not ferment mannitol (media remains light pink in color & colonies are colorles
MSA agar
Mannitol Salt Agar (MSA)
This is a selective medium that favors the growth of staphylococci and for differentiation between pathogenic and non-pathogenic types.
MSA contains 7.5% NaCl and staphylococci are characteristically able to tolerate this high salt concentration. MSA also contains the sugar-alcohol mannitol and the pH
indicator phenol red.
Generally pathogenic staphylococci are able to ferment the mannitol, lowering the pH, and thereby turning the indicator yellow The non-pathogenic staphylococci do not ferment mannitol and the medium remains pink in their vicinity.
MacConkey's Agar Plate (MAC)
Selective for: Gram (-)
Crystal Violet; Bile salts; lactose; neutral red pH indicator
between lactose and non lactose fermentors:
- Lactose (fermentation)
- Coliforms (neutral red -> pink color)
- ( Ferment = pink/red )
- ( No acid = colorless )
*pH indicator *
Neutral red indicator
- Tan = alkaline; rose = neutral pH; hot pink = acidic pH
MAC plate results
MAC Sample Results
o gram-negative Enterobacteria Escherichia coli and Enterobacter aerogenes ferment lactose
o E. coli produces pink to red colonies often with a reddish bile precipitate surrounding colonies on MacConkey's agar
o E. aerogenes produces pink to red mucoid colonies on MacConkey's agar
o gram-negative bacteria Proteus vulgaris and Salmonella typhimurium grow on MacConkey's agar, but do not ferment lactose (media appears yellow to light pink in color & colonies are colorless; swarming of Proteus is inhibited
MAC agar
MacConkey Agar (MAC)
This is a differential plating medium, selective for Gram negative organisms, used primarily for detection and isolation of enteric bacteria.
Gram positives are generally inhibited by crystal violet in the medium. Isolated colonies of lactose-fermenting bacteria are brick red in color and may be surrounded by a zone of precipitated bile.
This reaction is due to the action of the acids, produced by fermentation of lactose, upon bile salts present in the medium, and subsequent absorption of neutral red. Non-lactose fermenters are colorless and transparent.
Eosin-Methylene Blue Agar (EMB)
Selective for: Gram (-)
Eosin dye/MB dye; Lactose
between lactose and non lactose fermentors:
- ( Some acid = pink )
- ( Large acid = metallic green )
- ( No acid = colorless )
(E. aerogenes = Pink, E. coli = Green (Lactose fermentation), Pseudomonas sp = Clear.)
EMB plate results
far left: negative for lactose fermentation (P. aeruginosa)
middle: some acid production (E. aerogenes)
right: heavy acid production (E. coli)
EMB Sample Results
o gram-negative Enterobacteria Escherichia coli and Enterobacter aerogenes ferment lactose
o E. coli produces colonies with a characteristic green metallic sheen on EMB agar
o E. aerogenes produces pink colonies often with a central dark purple dot (fish eye colonies) on EMB agar
o gram-negative bacteria Proteus vulgaris and Salmonella typhimurium grow on EMB agar, but do not ferment lactose

*K. pneumo may show green metallic sheen initially on EMB and it will later turn to dark purple/black along the edges.*
EMB agar
Eosin Methylene Blue Agar (EMB)
This agar medium is both selective and differential. The combination of the two dyes eosin and methylene blue inhibits most Gram positive bacteria but allows many Gram negative organisms to grow.
In addition to peptones, EMB contains lactose (it may also contain sucrose). Gram negative bacteria that ferment the lactose produce acid which turns the colonies dark purple as the acid acts upon the dyes.
In addition, certain lactose-fermenting bacteria produce flat, dark colonies with a green metallic sheen. Other lactose fermenters produce larger, mucoid colonies, often purple only in their center. Lactose non-fermenters are either colorless or light lavender.
Indole Test (IMViC)
The amino acid tryptophan can be broken down by enzyme tryptophanase to form indole, pyruvic acid and ammonia as end products. Tryptophanase differentiates indole-positive enterics, such as E. coli and P.vulgaris from indole-negative enterics, such as S. marcescens.

*Media and Reagents:*
SIM with tryptophan and Kovac's reagent.

*Expected Results:*
Positive test : Kovac's reagent combines with indole and turns the surface red.
Negative test: No red color or copper color development
Hydrogen Sulfide Test (H2S) (IMViC)
(left is negative for H2S and right is positive for H2s)

Bacteria use enzyme cysteine desulfurase to hydrolyze the amino acid cysteine, forming hydrogen sulfide as end-product.

*Media and Reagent:*
SIM with cysteine and ferrous sulfate (detects H2S)

*Expected Results: *
Positive Test: H2S production = Black
Negative Test: No H2S production = No blackening of medium
This is not a biochemical test, but it can distinguish bacteria. It determines presence of flagella.

*Media and reagent:*
Deep agar

*Expected results:*
Positive test: Growth spread away from the line of inoculation = motile
Negative test: Growth only occurred at the line of inoculation = Non-motile
SIM Tube (Sulfur Reduction/Indole Prod/Motility)
A. Indole positive and hydrogen sulfide positive
B. Hydrogen sulfide positive
C. Indole positive and motility positive (note fuzzy growth away from stab line)
D. Negative Control

To differentiate between bacteria based on three tests: sulfur reduction (cysteine desulfurase), indole production (tryptophanase), and motility.

If a bacterium can produce the enzyme tryptohanase then it can use the amino acid tryptophan as a carbon and energy source (as pyruvate). One of the biproducts of this conversion is indole, which is detected with Kovac's reagent. If the bacterium posseses the enzyme cysteine desulfurase, sulfur containing amino acids will be broken down into pyruvate, ammonia and hydrogen sulfide.
Iron in the medium reacts with hydrogen sulfide producing the characteristic black percipitate. Motility is observed as growth away from the stab line
Methyl Red Test (IMViC)
*Methyl red test*
is used to identify enteric bacteria based on their pattern of glucose metabolism. If they use mixed acid pathway and produce acidic products, then they are called methyl-red-positive. If they use butylene glycol pathway and produce neutral end products, then they are called methyl-red-negative.

*Media and reagents:*
MR-VP medium and methyl red indicator

*Expected results:*
Positive test: acids + methyl red = red solution
Negative test: neutral end products + methyl red = yellow color
Voges-Proskauer Test (IMViC)
It is used to identify enteric bacteria based on their pattern of glucose metabolism. The enterics that produce neutral end-products, such as acetoin are detected by VP test.

*Media and Reagent:*
MR-VP medium and Barritt's Reagent A (contains alpha-naphthol) and Barritt's Reagent B (contains KOH).

*Expected results:*
Positive test: acetoin + alpha-naphthol + KOH = red color
Negative test: alpha-naphthol +KOH = copper color
Citrate Utilization (IMViC)
Citrate is an organic molecule that can be utilized by bacteria that produce the enzyme citrase. Citrase is produced by some bacteria such as E. aerogenes but not by others like E. Coli

*Media and Reagent:*
Simmon's Citrate Agar. It has citrase as the only carbon source and PH indicator bromothymol blue

*Expected results:*
Positive test: Growth and color changes to blue
Negative test: No growth and color remains green
Urease Test (BioChem)
Some bacteria produce urease, an enzyme capable of breaking down urea and produce alkaline end products. This distinguishes Proteus from other bacteria

Urea may be hydrolyzed by some bacteria so that the ammonia that is liberated can be used as a nitrogen source. Phenol red indicator is added to the broth so that when the pH reaches 8.4 (due to ammonia production) the tube will turn from an orange color to a pink color.

*Media and Reagent:*
Urea Broth with phenol red

*Expected Results:*
Positive test: production of alkaline end products = pinkish red color
Negative test: No color change
Nitrate Reduction Test (BioChem)
The identification of some bacteria is aided by determining if the organism can reduce nitrate (NO3) to nitrite (NO2)or another nitrogenous compound such as ammonia (NH3) or nitrogen gas (N2).

The test organism is inoculated into nitrate reduction broth, an undefined medium that contains large amounts of nitrate (KNO3). After incubation, alpha-napthylamine and sulfanilic acid are added . These two compounds react with nitrite and turn red in color, indicating a positive nitrate reduction test. (Tubes 2 and 3 in image above.)

If there is no color change at this step, nitrite is absent. If the nitrate is unreduced and still in its original form, this would be a negative nitrate reduction result. However, it is possible that the nitrate was reduced to nitrite but has been further reduced to ammonia or nitrogen gas. This would be recorded as a positive nitrate reduction result.

To distinguish between these two reactions, zinc dust must be added. Zinc reduces nitrate to nitrite. If the test organism did not reduce the nitrate to nitrite, the zinc will change the nitrate to nitrite. The tube will turn red because alpha-napthylamine and sulfanilic acid are already present in the tube. (Tube 1 above.) Therefor a red color shows after the zinc is added indicates the zinc found the nitrate unchanged. The bacteria was unable to reduce nitrate. This is recorded as a negative nitrate reduction test.

If however, the tube does not change color upon the addition of zinc, then the zinc did not find any nitrate in the tube. (Tube 4 above.) That means the test organism converted the nitrate to nitrite and then converted the nitrite to ammonia and/or nitrogen gas. Thus no color change upon the addition of zinc is recorded as a positive nitrate reduction test.
Catalase Test (BioChem)
*Catalase-Positive will form gas bubbles rapidly
*Catalase-Negative will have no production of gas bubbles

Catalase is an enzyme that decomposes hydrogen peroxide into oxygen and water. Excluding the Streptococci, most aerobic and facultative anaerobic bacteria possess catalytic activity.

Hydrogen peroxide forms as one of the oxidative end products of aerobic carbohydrate metabolism. Catalase converts hydrogen peroxide into water and oxygen. The catalase test is commonly used to differentiate streptococci (negative) for staphylococci (positive).

3% Hydrogen peroxide

*Expected Results:*
Positive: rapid , appearance of sustained gas bubbles
Negative: No gas bubble production
Oxidase Test (BioChem)
This test is used to identify microorganisms containing the enzyme cytochrome oxidase (important in the electron transport chain).
It is commonly used to distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae.

Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final electron acceptor) and reduces it to water. In the oxidase test, artificial electron donors and acceptors are provided. When the electron donor is oxidized by cytochrome oxidase it turns a dark purple.

*Expected Results:*
This is considered a positive result. In the picture above the organism on the right (Pseudomonas aeruginosa) is oxidase positive.
Bacterial Sample: Enterobacter aerogenes
Bacterial Sample: Escherichia coli
Bacterial Sample: Micrococcus luteus
Bacterial Sample: Proteus vulgaris
Bacterial Sample: Pseudomonas aeruginosa
Bacterial Sample: Staphylococcus aureus
Bacterial Sample: Staphylococcus epidermidis
Bacterial Sample: Streptococcus pyogenes