MICR LAB

1. Mannitol salt agar or MSA is a commonly used selective and differential growth medium in microbiology. It
encourages the growth of a group of certain bacteria while inhibiting the growth of others. This medium is
important in medical laboratories by distinguishing pathogenic microbes in a short period of time. [1] It contains
a high concentration (~7.5%-10%) of salt (NaCl), making it selective for gram positive bacterium Staphylococci
(and Micrococcaceae) since this level of NaCl is inhibitory to most other bacteria

* selective for: gram-positive Staphylococci bacteria -differential for: mannitol fermentation

Type: Selective and differential

Purpose: Selects for Staphylococci, which grow at high salt concentrations; differentiates
Staphylococcus aureus from other Staphylococci
Interpretation: Staphylococcus aureus is yellow (ferments mannitol), other staphylococci are white

SA - Gram + Staphylococcus : fermenting mannitol: Media turns yellow (eg. S. aureus)]

Enterococcus - Gram - : inhibited growth

2. EMB ( EOSIN-METHYLENE BLUE AGAR)

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Type: Differential (lactose) and selective (dye inhibition and precipitation at acid pH)

Purpose: Differentiates lactose fermenters (E. coli) from non-fermenters (Salmonella, Shigella)
Interpretation: Lactose fermenters blue/black; non-fermenters colorless or light purple

Expected colony characteristics of organisms in EMB agar (Quality control)

Escherichia coli ATCC 25922: Blue-black bulls eye; may have green metallic sheen

Pseudomonas aeruginosa ATCC 27853: Colorless

3. MAC (MacConkey (lactose) Agar)

Type: Selective and differential

Purpose: Contains bile salts and crystal violet which selects for gram-negative enterics, differentiates lactose-
fermenters from non-fermenters. Can include sugars other than lactose for further differentiation (for example,
to differentiate enterohemorrhagic E. coli (EHEC), which does not ferment sorbitol, from other E. coli types
which do.)
Interpretation: Selects for non-fastidious gram-negatives; red colonies indicate fermentation of lactose, white
indicates no fermentation of lactose

E. COLI PINK
PSEUDOMONAS - WHITE

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4. TSIA ( TRIPLE SUGAR IRON AGAR )
Escherichia coli in a triple sugar iron (TSI) slant. This medium contains glucose, lactose,
and sucrose. Escherichia coli ferments glucose and lactose producing acid and carbon
dioxide. Acid causes the phenol red indicator in the agar to turn yellow. Carbon dioxide
is observed as bubbles or cracks in the agar. There is no hydrogen sulfide production,
as indicated by the lack of black precipitate in the agar.

Result (slant/butt) Symbol Interpretation

Glucose fermentation
1 Red/Yellow K/A only, peptone
catabolized.
Glucose and lactose
2 Yellow/Yellow A/A and/or sucrose
fermentation.
No fermentation,
3 Red/Red K/K
Peptone catabolized.
Glucose and lactose
Yellow/Yellow with and/or sucrose
4 A/A,G
bubbles fermentation, Gas
produced.
Red/Yellow with Glucose fermentation
5 K/A,G
bubbles only, Gas produced.
Red/Yellow with Glucose fermentation
6 bubbles and black K/A,G,H2S only, Gas produced,
precipitate H2S produced.
Glucose and lactose
Yellow/Yellow with and/or sucrose
7 bubbles and black A/A,G,H2S fermentation, Gas
precipitate produced, H2S
produced.
Red/Yellow with black Glucose fermentation
8 K/A,H2S
precipitate only, H2S produced.
Glucose and lactose
Yellow/Yellow with
9 A/A,H2S and/or sucrose
black precipitate
fermentation, H2S

Pseudomonas - Growth; red slant, red butt, no gas, no H 2 S

produced

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5. Lysine iron agar slant or LIA slant test is used to distinguish bacteria which are able to
decarboxylate lysine and/or produce hydrogen sulfide from those that cannot. This test is particularly useful for
distinguishing different Gram-negative bacilliespecially among the Enterobacteriaceae.

Tube 1: Positive decarboxylation (butt), negative deamination (slant)

Tube 2: Negative decarboxylation (butt), positive deamination (slant)

PSEUDOMONAS

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6. INDOLE TRYPTONE TEST

This test determines whether the microbe produces indole from the amino acid tryptophan.

If indole is produced, it will react with a chemical reagent added after incubation to produce a color change.

A positive and a negative result from an indole test done in tryptone broth. The positive result given
by Escherichia coli in the positive control (tube 1) and the test (tube 2) is indicated by the red layer at the top of
the tube after the addition of Kovcs reagent. The negative result given by Klebsiella pneumoniae in the negative
control is indicated by the lack of color change in the top of the tube after the addition of Kovcs reagent.

7. MR AND VP ( Nutrient Broth with Glucose )

Voges-Proskauer (VP) Test

Certain bacteria produce neutral-reacting end products when cultivated in specific media. Particular enteric
bacteria that ferment glucose, further metabolize pyruvic acid to form acetyl-methyl carbinol (acetoin). This end
product, in the presence of atmospheric oxygen and 40% potassium hydroxide is converted to diacetyl. Diacetyl,
under the catalytic action of alpha-naphthol and creatine, is converted into a red complex. (5) This is a positive
Voges-Proskauer (VP) test reaction. The VP test is used primarily to separate Escherichia coli (VP-negative) from
the Klebsiella -Enterobacter groups (VP-positive).

Methyl Red (MR) Test

Clark and Lubs, in 1915, discovered the ability of coliforms to produce and maintain acid products when
cultivated in specific media. (9) They found that cultures of Escherichia coli produced a red color upon addition
of methyl red. This color development was a result of strong acid production from dextrose-fermentation.

The Methyl Red (MR) test is based on the use of methyl red, a pH indicator, to determine acidity when an
organism ferments glucose. (10) Because all Enterobacteriaceae ferment glucose, acidic metabolic by-products
are initially formed. However, with further incubation (2-5 days), MR-positive organisms continue to produce

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more acids. The increased acidity overcomes the phosphate buffer, thus resulting in a low pH and development
of a red color.

MR-negative organisms, such as Klebsiella pneumoniae and Enterobacter aerogenes , further metabolize the
fermentation products by decarboxylation. The result is an alkaline reaction (no red color) due to the production
of acetyl-methyl carbinol, a neutral-reacting end product.

Clark and Lubs developed MR-VP Broth which allowed both the MR and VP tests to be performed from the same
inoculated medium by aliquoting portions to different tubes.

Voges-Proskauer Test
A positive VP test is demonstrated by the development of a pink-red color on the surface of the medium 15
minutes to one hour after the addition of the reagents.

A negative VP test is demonstrated by the appearance of a yellow color on the surface of the medium.
Development of a copper-like color is also interpreted as negative.

Methyl Red Test

A positive MR test is demonstrated by the development of a stable red color on the surface of the medium after
the addition of methyl red indicator.

A negative MR test is demonstrated by the development of a yellow color on the surface of the medium.

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8.) SCA (Simmons Citrate Agar)

- Simmons' citrate agar is used for differentiating gram-negative bacteria on the basis of citrate utilization.[1] It
is useful for selecting for organisms that use citrate as it's main carbon and energy source. that It is a defined,
enrichment medium that tests for an organism's ability to use citrate as a sole carbon source and ammonium
ions as the sole nitrogen source.

The medium contains citrate, ammonium ions, and other inorganic ions needed for growth.

It also contains bromothymol blue, a pH indicator. Bromothymol blue is green at pH below 6.9, and then turns
blue at a pH of 7.6 or greater.

Organisms growing on Simmons' citrate agar are capable of using citrate as the sole carbon source and they can
metabolize the ammonium salt in the medium (serving as a sole nitrogen source for growth [2]).

Use of citrate results in the creation of carbonates and bicarbonates as byproducts. Organisms degrading citrate
must also use the ammonium salts, producing ammonia,[3] thus increasing the pH of the medium.[4] The increase
in pH then causes color change in the bromothymol blue indicator, turning it blue. Under neutral conditions the
medium remains a green color. The color change to blue is useful because growth on Simmons' citrate agar is
often limited and would be hard to observe if it were not for the color change.

Sometimes, it is possible to detect growth on the Simmons' citrate agar without the accompanying color change
to blue. This is most likely due to insufficient incubation. Either a combination of blue color and growth or growth
alone without the blue color should be scored as a positive for the citrate use test. Bacteria such as Salmonella
and Providencia develop on such mediums.

E. coli (Gram negative)

Klebsiella oxytoca (Gram positive)

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9.) SA (Starch Agar Plate)

Starch agar is a differential medium that tests the ability of an organism to produce certain exoenzymes,
including a-amylase and oligo-1,6-glucosidase, that hydrolyze starch. Starch molecules are too large to enter
the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized
by the organism.

Starch agar is a simple nutritive medium with starch added. Since no color change occurs in the medium when
organisms hydrolyze starch, we add iodine to the plate after incubation. Iodine turns blue, purple, or black
(depending on the concentration of iodine) in the presence of starch. A clearing around the bacterial growth
indicates that the organism has hydrolyzed starch.

Growth of Bacillus subtilis on a starch agar plate before

the addition of iodine solution (A) and after the addition
of iodine solution (B). After the addition of iodine, the
clearing surrounding the bacterial growth indicates
starch hydrolysis.

Growth of Staphylococcus aureus on a starch agar plate

before the addition of iodine solution (A) and after the
addition of iodine solution (B). After the addition of
iodine, the absence of a clearing surrounding the
bacterial growth indicates no starch hydrolysis.

10.) Nutrient Gelatin deep

Nutrient gelatin is a differential medium that tests the ability of an organism to produce an exoenzyme, called
gelatinase, that hydrolyzes gelatin.

Gelatin is commonly known as a component of gelled salads and some desserts, but it's actually a protein
derived from connective tissue. When gelatin is at a temperature below 32C (or within a few degrees thereof),
it is a semisolid material. At temperatures above 32C, it is a viscous liquid.

Gelatinase allows the organisms that produce it to break down gelatin into smaller polypeptides, peptides, and
amino acids that can cross the cell membrane and be utilized by the organism.

When gelatin is broken down, it can no longer solidify. If an organism can break down gelatin, the areas where
the organism has grown will remain liquid even if the gelatin is refrigerated.

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 Gelatin hydrolysis test. After 30 minutes in an ice bath, the uninoculated control remained solid.

Gelatin hydrolysis test. After 30 minutes in an ice bath, the nutrient gelatin tube inoculated with
Bacillus subtilis exhibited positive gelatin hydrolysis as shown by medium liquefaction.

Gelatin hydrolysis test. After 30 minutes in an ice bath, the nutrient gelatin tube inoculated with
Staphylococcus aureus, exhibited positive gelatin hydrolysis as shown by medium liquefaction

11.) Nitrate Broth

Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the
enzyme nitrate reductase. It also tests the ability of organisms to perform nitrification on nitrate and nitrite to
produce molecular nitrogen.

Nitrate broth contains nutrients and potassium nitrate as a source of nitrate.

After incubating the nitrate broth, add a dropperful of sulfanilic acid and -naphthylamine. If the organism has
reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid. When sulfanilic acid is added, it will
react with the nitrous acid to produce diazotized sulfanilic acid. This reacts with the -naphthylamine to form
a red-colored compound. Therefore, if the medium turns red after the addition of the nitrate reagents, it is
considered a positive result for nitrate reduction.

If the medium does not turn red after the addition of the reagents, it can mean that the organism was unable
to reduce the nitrate, or it could mean that the organism was able to denitrify the nitrate or nitrite to produce
ammonia or molecular nitrogen. Therefore, another step is needed in the test.

If the medium does not turn red after the addition of the nitrate reagents, add a small amount of powdered
zinc. Be careful, as powdered zinc is hazardous! If the tube turns red after the addition of the zinc, it means
that unreduced nitrate was present. Therefore, a red color on the second step is a negative result. The addition
of the zinc reduced the nitrate to nitrite, and the nitrite in the medium formed nitrous acid, which reacted with
sulfanilic acid. The diazotized sulfanilic acid that was thereby produced reacted with the -naphthylamine to
create the red complex.

If the medium does not turn red after the addition of the zinc powder, then the result is called a positive
complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite present in the medium,
either, that means that denitrification took place and ammonia or molecular nitrogen were formed.

Important: We do not use Durham tubes in our nitrate broth.

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 Nitrate reduction test results following growth of
Escherichia coli, Alcaligenes faecalis, and Pseudomonas
aeruginosa in nitrate broth. Escherichia coli reduces nitrate
to nitrite, as evidenced by the red color after addition of
nitrate reagents A (sulfanilic acid) and B (dimethyl--
naphthylamine) to the culture. Alcaligenes faecalis is
negative for nitrate reduction as the culture turns red only
after addition of reagents A, B, and zinc powder.
Pseudomonas aeruginosa denitrifies nitrate to nitrogen gas.
Note the gas bubbles captured by the Durham tube (arrow)
and visible at the top of the broth. An uninoculated nitrate broth is shown for comparison.

12.) MIO (Motility Indole Ornithine - deep)

Hardy Diagnostics MIO (Motility, Indole, Ornithine) Medium is recommended for use in testing motility, indole
production, and ornithine-decarboxylase activity of enteric bacilli.

Ederer and Clark, et al., developed MIO (Motility Indole Ornithine) Medium to be used as an aid in the
identification of members of the Enterobacteriaceae family. (7) Motility, indole production, and ornithine-
decarboxylation are the three differentiating tests that are provided in the one culture tube.

The addition of agar to the medium allows for the detection of motility along the stab line of inoculation. Motile
organisms extend from the stab line and produce turbidity or cloudiness throughout the medium. Non-motile
organisms grow only along the stab line and leave the surrounding medium clear.

Tryptophan, present in the basal medium, is degraded by organisms that possess the enzyme tryptophanase.
Degradation of tryptophan produces indole which is detected upon addition of Kovacs Reagent (Cat. no. Z67) to
the surface of the medium. Indole combines with p-dimethylaminobenzaldehyde and produces a red band at
the top of the medium. A negative indole test results in no color change upon addition of Kovacs Reagent.

Bromcresol purple serves as the pH indicator which allows for detection of decarboxylase activity. Organisms
that ferment dextrose will produce acids, thereby lowering the pH. A decreased pH results in the indicator
changing from purple to yellow. The presence of acid also results in stimulation of enzyme activity. Once the
enzyme has decarboxylated ornithine, the by-product diamine putrescine is produced. Production of putrescine
causes an alkaline shift which turns the medium a dark purple. Organisms which do not produce decarboxylase
remain acidic due to dextrose-fermentation, and the medium retains a yellow (acidic) color throughout or
yellow with a purple band near the top of the tube.

Ingredients per liter of deionized water:*

Pancreatic Digest of Gelatin 10.0gm

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 Pancreatic Digest of Casein 10.0gm

L-Ornithine 5.0gm

Yeast Extract 3.0gm

Dextrose 1.0gm

Bromcresol Purple 0.02gm

Agar 2.0gm

Final pH 6.5 +/- 0.2 at 25C.

* Adjusted and/or supplemented as required to meet performance criteria.

PROCEDURE

Specimen Collection: This medium is not intended for use as a primary isolation medium. It is used in
characterizing pure cultures. Consult listed references for information regarding the processing and inoculation
of specimens. (2-5)

Method of Use: Allow medium to warm to room temperature prior to inoculation. Using a straight needle, select
isolated colonies from a pure 18-24 hour culture and stab the center of the medium to about one-half its length.
Incubate the tubes aerobically at 35 degrees C. for 18-24 hours. Caps should be loose during incubation. Examine
for motility and ornithine production. Record the above reactions then add a few drops of Kovacs Reagent (Cat.
no. Z67) and observe for indole production.

INTERPRETATION OF RESULTS

Positive motility is denoted when turbidity or cloudy growth extends from the line of inoculation. Growth only
along the stab line is indicative of a negative motility test.

A positive test for indole is denoted when a pink to red color band is formed at the top of the medium after
addition of Kovacs Reagent. A yellow color denotes a negative indole test after addition of Kovacs Reagent.

A positive test for ornithine is denoted by a dark, turbid purple color in the medium. A yellow color throughout
the medium denotes a negative ornithine result.

The reactions in this medium are observed as follows:

Motility. Observe for cloudiness in the medium (growth away from the stab line). For a non-motile
organism, growth may be seen along cracks in the medium caused by gas production, but there will be
clear pockets of no growth.
Ornithine Decarboxylation. Observe the lower three-quarters (anaerobic region) of the medium for
change in color of the pH indicator; growth must be present in this part of the tube for correct analysis
of result:
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 Gray, blue or purple color: Positive reaction for ornithine decarboxylation formation of a highly alkaline
product, over-neutralizing the acid produced from glucose fermentation.

Yellow color: Negative reaction. Yellow color is due to the "default" acid production from glucose
fermentation.

Indole Production. About one-half dropperful of Kovacs reagent is added to the medium. A red ring
indicates production of indole from the breakdown of tryptophan.

corresponding tube no. above (tubes arranged in

pairs with Kovacs reagent added to right tube in each 1 2 3
pair)
deamination of amino acids (aerobic alkaline rx.) + + +
motility (cloudiness disregarding stab line) + + +
decarboxylation of ornithine (anaerobic alkaline rx.) + + -
indole production (red color with Kovacs reagent) + + -
glucose fermentation (acid rx.) + + + -
Klebsiella Enterobacter Escherichia Pseudomonas
typical examples
oxytoca aerogenes coli aeruginosa

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13.) Litmus Milk

Litmus Milk is differential media containing milk, litmus (a pH and oxidation-reduction indicator), lactose, and
casein (a milk protein) all of which can be metabolized by some bacteria. Litmus milk also contains sulfur and
many nutrients that are found in milk making it is an undefined media that is used to help differentiate and
identify Enterobacteria, Clostridium, and lactic acid bacteria.

A number of results may be obtained in the Litmus Milk test, making it a rather complex test. There are four
main reactions: lactose fermentation, litmus reduction, casein coagulation, and protein hydrolysis. Some or all
of these reactions may occur at the same time, and many of them can have more than one reaction (for example,
casein coagulation can be from a solid clot, as in an acid clot, or from the casein being converted to a solid curd
in the bottom below a liquid whey (as in curd formation).

Four reactions can occur in Litmus Milk: Litmus Color pH Notes

4.5 and below
1. Lactose fermentation (without gas) pink acid
(4.5 and
(fermentation with gas production) (pink) (acid with bubbles or fissures)
below)
2. Reduction of litmus white any white starts in tube bottom
solid in lower tube (acid clot or
3. Casein protein coagulation any any
curd)
4. Casein and protein hydrolysis (partial) blue
8.3 & above basic (especially at tube top)
(Digestion or nearly complete (clear &
(8.3 & above) (basic and media is thin)
hydrolysis) dark)
pH uncertain
Any combination of the above reactions other a combination of reactions
or difficult
purple
There can also be no reaction 4.6 to 8.2 near neutral pH, check for growth
shades

As you might have noticed, it is a rather complex test. Note that reactions in Litmus Milk tend to vary a little,
probably because milk is a complex substance and its components vary by lot and by what the cows were
eating. Therefore, the results of this test should not be used to solely identify a species, other tests must be
used along with a close fitting but not necessarily an exact match to the expected Litmus results to confirm your
identification.

Purpose: To test for lactose fermentation, the reduction of litmus, protein coagulation, and protein hydrolysis
to mainly aid in the identification and differentiation of Enterococcus, Clostridium, and Lactic acid bacteria. The
media may also be used to grow lactic acid bacteria.

Results:

Obtain your tubes after one week of incubation, checking for gas bubbles before you handle the tubes as these
disappear after handling. Obtaining results for such a complex test can be tricky if several reactions occur
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together, but if looked at in a certain order, it is easier. First, determine if reduction has occurred, second,
determine the color of the media using your observations on day 2 or 3 if needed, third determine if the media
has solidified, and lastly look for specific characteristics of each reaction. Follow this guide for assistance:

First, determine if there is white in the tube, especially at the bottom (check that is not sedimented
milk).
o If there is no white in the tube, there is no reduction of litmus. (Result so far is No
Reduction which is usually not even recorded.)
o If there is white in the tube, you have reduction of litmus unless there was a problem with the
making of the media and the uninocculated controls have the same amount of white in the
bottom, then it is sedimented milk. If in doubt, use a marker to mark the area of white and
reincubate the tube. If the area of white expands with time, you have litmus reduction,
otherwise it is sedimented milk.
o If the litmus has been totally reduced and you have no color other than white, and you did not
make an observation on day 2 or 3, then it will be difficult to obtain further results for this
test. For the below steps, ignore the color white and use the other colors in the tube to base
your remaining results on. (Result so far is Reduction which may occur with any of the below
results.)
Second, determine if the tube is (or in the case of reduction was on day 2) pink.
o If the tube is pink, it means that lactose fermentation has occurred resulting in the production of
acid end products (result, an Acid reaction).
o If the tube is pink, check for bubbles or check for fissures or breaks in any clotted milk. The
bubbles usually rise to the surface and pop at the first disturbance, so they are difficult to see. If
any of these are present, there is fermentation with the production of gas (result, Acid and Gas).
o If the tube is pink, check to see if any of the tube contents are solid, especially in the tube
bottom. The acids that accumulate from fermentation may cause casein to clot. Usually, one
can tip the tube and immediately tell if the tube is solid or not. If needed, you can sterilize a loop
and gently stick it into the milk to test if any of it has solidified. Keep the tube sterile in case it
needs to incubated further, and be careful not to damage the loop! If there is a solid pink clot,
coagulation has occurred (result, is an Acid Clot, with or without gas). (Note, an acid clot only
occurs if an acid reaction has occurred.)
o If there is an acid clot, it is easier to observe gas production from fermentation; look for the
presence of fissures in the clot. If studying Clostridium, note if there is a lot of gas that has broken
up an acid clot, it is called stormy fermentation.
Third, determine if the tube is or on day 2 was blue.
o If the tube is blue (especially at the top), the media has become basic from the partial hydrolysis
and degradation of proteins, especially casein, which tends to release ammonia. (Result is a Basic
reaction.)
o If the tube is blue, look to see if there is any solid media especially in the bottom of the tube (see
the discussion above for an Acid Clot). If there is a blue solid, it may only be in the bottom with
a fluid whey on top, then there is coagulation of casein (result, a Basic reaction and a Curd).
o If the tube is or was blue, check to see if there is is any clarification of the media, where the milk
has become thin, as if watered down. The top may appear as a transparent fluid that is dark, or
brownish (or grey if there is reduction). The curd appears to be partially or fully dissolved. If so,
this is digestion of protein that is nearing complete hydrolysis. (Result is Digestion or
Peptonization which always starts as a Basic reaction, with or without curd.)
Fourth, determine if the tube is (or in the case of reduction, was on day 2) purple.
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 o If the tube is purple, then the pH of the media is near neutral.
o If the tube is purple, check to see if there is a solid, especially in the bottom of the tube (see the
discussion above for an Acid Clot). If there is a solid, then there is coagulation of casein by
enzymes released by the bacteria without an acid or a basic reaction (result is a Curd).
o If the tube is purple and if none of the above have occurred, then there is no change in the tube. If
there is no change, sterilize a loop, cool it, stick it in the litmus milk, and streak it across a general
purpose agar plate to ensure that there is bacterial growth in the tube.
If few colonies grow on the plate, then the strain of bacteria did not grow in the litmus
milk (result is Little or No Growth). If there is little or no growth, the results to the litmus
test are not really valid and cannot be recorded as no change.
If there are many colonies, the organism grew, so the results of the experiment are valid
(result is No Change).

14.) Glucose Fermentation Broth (GFB)

This is a differential medium. It tests an organism's ability to ferment the sugar glucose as well as its ability to
convert the end product of glycolysis, pyruvic acid into gaseous byproducts. This is a test commonly used when
trying to identify Gram-negative enteric bacteria, all of which are glucose fermenters but only some of which
produce gas.

Like MSA, this medium also contains the pH indicator, phenol red. If an organism is capable of fermenting the
sugar glucose, then acidic byproducts are formed and the pH indicator turns yellow.

The end product of glycolysis is pyruvate. Organisms that are capable of converting pyruvate to formic acid and
formic acid to H2 (g) and CO2 (g), via the action of the enzyme formic hydrogen lyase, emit gas. This gas is
trapped in the Durham tube and appears as a bubble at the top of the tube.

*Note - broth tubes can be made containing sugars other than glucose (e.g. lactose and mannitol). Because the
same pH indicator (phenol red) is also used in these fermentation tubes, the same results are considered
15
positive (e.g. a lactose broth tube that turns yellow after incubation has been inoculated with an organism that
can ferment lactose).

Tube 1: No fermentation. The pH indicator remains purple. There can still be growth due to the use of amino
acids as sources of energy (usually by respiration).

Tubes 2A and 2B: Fermentation with the production of acid (yellow color) but no gas. A slight amount of acid is
seen in tube 2A, but fermentation is still recorded for this tube.

Tubes 3A and 3B: Fermentation with the production of acid (yellow color) and insoluble gas (bubble in Durham
tube). Tube 3B shows an alkaline reaction on top; this is simply due to deamination of amino acids whose
alkaline reaction has not been over-neutralized by the acid diffusing through the tube from fermentation.

Escherichia coli demonstrates a positive fermentation reaction (A or acid conditions) as indicated by the yellow
broth color. When Escherichia coli produced organic acids from the initial carbohydrate, the pH of the medium
fell causing the indicator, phenol red, to turn the broth yellow. Gas production (G) is also detected by observing
displacement of the liquid in the Durham tube by a gas bubble.

Pseudomonas aeruginosa produces a negative reaction (K or alkaline conditions) as indicated by the broths red
color. At a neutral or alkaline pH (7.3 0.2), the indicator, phenol red, remains red.
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