MICROBIOLOGY

Biochemical Test in Microbiology

Submitted by
Abhijit padhi
 Biochemical test
Biochemical tests are laboratory procedures that use specific chemical reactions to identify and
characterize microorganisms, such as bacteria. These tests are often used to identify the presence of
specific enzymes or metabolic pathways in a microorganism, which can help to distinguish it from
other microorganisms.
• Some common biochemical tests used in bacteriology include:
1.Oxidase test: This test is used to detect the presence of the enzyme cytochrome oxidase, which is
involved in the metabolism of oxygen.
2.Catalase test: This test is used to detect the presence of the enzyme catalase, which helps to break
down hydrogen peroxide.
3.Indole test: This test is used to detect the presence of the enzyme tryptophanase, which breaks down
the amino acid tryptophan.
4.Nitrate reduction test: This test is used to detect the ability of a microorganism to reduce nitrate to
nitrite.
5.Gelatinase test: This test is used to detect the ability of a microorganism to produce the enzyme
MacConkey Agar
Composition • MacConkey agar is used for the
isolation of gram-negative
Peptone 20gm/1ltr. enteric bacteria.
Lactose monohydrate 10gm/1ltr.
Escherichia coli red/pink non-mucoid
Bile salts 1.5gm/1ltr.
Aerobacter
Sodium chloride 5gm/1ltr. pink mucoid
aerogenes
Neutral red 0.03gm/1ltr. Enterococcus speci
red minute, round
es
Crystal Violet 0.001gm/1ltr.
Staphylococcus sp
Agar 13.5gm/1ltr. pale pink opaque
ecies
ph should be 7.1±0.2 Pseudomonas fluorescent
green-brown
aeruginosa growth
Procedure
1.Suspend 49.53 grams of dehydrated
medium in 1000 ml of distilled water.
2.Heat to boiling to dissolve the
medium completely.
3.Sterilize by autoclaving at 15 lbs
pressure (121°C) for 15 minutes.
4.Cool to 45°C -50°C.
5.Mix well before pouring into sterile
Petri plates.
MSA media (Mannitol-Salt agar)
Composition
Peptone 10gm/1ltr.
Nacl 75gm/1ltr.
D-mannitol 10gm/1ltr.
Yeast extract 1gm/1ltr.
Phenol red 0.025gm/1ltr.
Organisms Results
Agar 15gm/1ltr. Yellow colonies surrounded
Staphylococcus aureus
ph should be 7.2±0.2 by the yellow zone
Staphylococcus epidermidis Pink or Red colonies
Micrococci Red colonies
Escherichia coli No growth
Procedure
1.Suspend 111 grams of Mannitol Salt Agar in 1000 ml of distilled
water.
2.Boil to dissolve the medium completely.
3.Sterilize by autoclaving at 15 lbs. pressure (121°C) for 15 minutes.
4.If desired, sterile Egg Yolk Emulsion (E7899) can be added to a final
concentration of 5% v/v after autoclaving.
5.Pour cooled Mannitol Salt Agar into sterile petri dishes and allow to
cool to room temperature.
EMB (Eosin-methylene blue)
Composition
Peptic digest of animal tissue 10gm/1ltr.
Dipotassium phosphate 2gm/1ltr.
Lactose 5gm/1ltr. Organisms Growth
Sucrose 5gm/1ltr. Blue-black bull’s eye; may
Escherichia coli
have a green metallic sheen
Eosin – Y 0.400gm/1ltr.
Methylene blue 0.065gm/1ltr. Pseudomonas aeruginosa Colorless
Agar 13.5gm/1ltr. Good growth; pink, without
Enterobacter aerogenes
sheen
ph should be 7.2±0.2
Klebsiella pneumoniae Pink, mucoid colonies
Luxuriant growth; colorless
Proteus mirabilis
colonies
Luxuriant growth; colorless
Salmonella Typhimurium
colonies
Proedure
1. Suspend 35.96 grams in 1000 ml distilled water.
2. Mix until the suspension is uniform. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. AVOID OVERHEATING.
4. Cool to 45-50°C and shake the medium in order to oxidize the methylene blue (i.e. to restore its blue
color) and to suspend the flocculent precipitate.
5. Pour into sterile Petri plates.
6. Allow plates to warm to room temperature.
7. The agar surface should be dry before inoculating.
8. Inoculate and streak the specimen as soon as possible after collection.
9. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface and
streak for isolation with a sterile loop.
10.Incubate plates aerobically at 35-37°C for 18-24 hours and protect from light.
11.Examine plates for colonial morphology. If negative after 24 hours, reincubate an additional 24 hours.
Catalase Test
slide method
use a loop or sterile wooden stick to
transfer a small amount of colony growth to
the surface of a clean, dry slide.
 Place a drop of 3% H2O2 in the glass
slide.
 Observe the evolution of oxygen
bubbles.
Urease test
Composition
Peptone 1 gm/L
Dextrose 1gm/L
NaCl 5gm/L Positive Reaction: Development of an
K2HPO4 2gm/L intense magenta to bright pink color in
15 min to 24 h.
Phenol red few drops Examples: Proteus spp, Cryptococcus spp,
Agar 15gm/L Corynebacterium spp, Helicobacter
Urea 20gm/L pylori, Yersinia spp, Brucella spp, etc.
Negative Reaction: No color change.
ph should be 6.8±0.2 Examples: Escherichia, Shigella,
Salmonella, etc.
procedure
 Take 20 ml dil. Water in a clean and dry culture bottle.
 Then weight all composition for 20 ml (peptone 0.02 gm, dextrose 0.02 gm, NaCl 0.1 gm K 2HPO4 0.04 gm,
phenol red 4 drops, agar 0.30 gm, urea 0.40 gm) and add in the bottle one by one.
 After that, maintain the pH and transfer 10-10 ml media in two clean and dry tests then add 0.15 gm
agar/test tube and cover the test tube tightly with 4 layer paper.
 After that, autoclave the media for 45 minutes. Then add 0.20 gm urea/ test tube under the laminar air flow
and vertex it for a few seconds then prepare a slant in the test tube for streaking.
 After solidifying the agar well, a cool streak isolation bacteria (E. coli) in test tube slant media.
 Incubate it 48 hours. After incubation, observe the result.
Citrate test
Composition
Sodium chloride 5gm/L
Sodium citrate 2gm/L
Ammonium dihydrogen phosphate 1gm/L
Dipotassium phosphate 1gm/L
Magnesium sulphate 0.2gm/L Positive Reaction: Growth with color change
Bromothymol blue 0.08gm/L from green to intense blue along the slant.
Examples: Salmonella, Edwardsiella,
Agar 15gm/L
Citrobacter, Klebsiella, Enterobacter,
ph should be 6.9±0.2 Serratia, Providencia, etc.
Negative Reaction: No growth and No color
change; Slant remains green.
Examples: Escherichia, Shigella,
Morganella, Yersinia etc.
procedure
 Take 20 ml dil. Water in a clean and dry culture bottle.
 Weight all composition (sodium chloride 0.10 gm, sodium citrate 0.04 gm, ammonium dihydrogen
phosphate 0.02 gm, dipotassium phosphate 0.02, magnesium sulphate 0.004 gm, bromothymol few drop)
and add it in the dil. Water.
 After that, maintain the pH and transfer 10-10 ml media in two clean and dry test tubes then add 0.15 gm
agar/ test tube and cover the test tube tightly from 4 layer paper.
 After solidifying the media, cool streak the isolation bacteria (E. Coli) in the test tube slant media.
 Incubate it for 24 hours and observe the result.
 Nitrate Reduction Test
Composition
Peptone 5gm/ltr.
Meat extract 3gm/ltr.
Potassium nitrate 1gm/ltr.
ph should be 7.0± 0.2

Preparation of 0.8% sulfanilic acid (Regent A)

Sunfanilic acid 0.8 grams
Distilled water 70 mL
Glacial acetic acid 30 mL
Preparation of 0.5% -naphthol (Reagent B)
-naphthol (N, N-dimethyl- α-napthylamine) 0.5 grams
Distilled water 70 mL
Glacial acetic acid 30 mL
Procedure
• Determination of nitrate reduction to nitrite is a two step process. First, the reduction
of nitrate to nitrite is determined by the addition of Nitrate Reagents A and B, then if
necessary, the reduction of nitrate beyond nitrite is determined by the addition of
Nitrate Reagent C (zinc dust).
1.Inoculate the nitrate broths with bacterial suspension.
2.Incubate the tubes at the optimal temperature 30°C or 37°C for 24 hours.
3.After incubation look for N2 gas first before adding reagents.
4.Add 6-8 drops of nitrite reagent A and add the 6-8 drops of nitrite reagent B.
5.Observe for the reaction (color development) within a minute or less.
6.If no color develops add zinc powder.
7.Observe for at least 3 minutes for a red color to develop after addition of zinc.
Lipase Test Method
Composition
Peptone 5gm/ltr.
Sodium chloride 5gm/ltr.
Yeast extract 3gm/ltr
Agar 15gm/ltr.
Egg yolk 40ml/ltr.
ph should be 7.0±0.3

Positive Control: Staphylococcus aureus ATCC

12600
Negative Control: Clostridium difficile ATCC
9689/ Clostridium perfringens ATCC 12924
Procedure
 take 25 ml dil. Water in a clean and dry culture bottle.
 All composition (peptone0.125 gm, yeast 0.075 gm, sodium chloride 0.125 gm) are added to the dill. Water. Then maintain
the pH and add the 0.375 gm agar.
 After that autoclave the media for 45 min. during the autoclave, prepare the petri plate to give the UV for pouring the media.
 After autoclave and before pouring the media add 1 ml egg yolk in the shake and pour it under the laminar air flow 10 min.
UV of media.
 After solidifying the agar and drying media, take a sterile loop full of isolated bacteria and streak it as a straight line on the
plate.
 Incubate anaerobically in a gas pack jar immediately after streaking and transfer into the incubator maintained at 35-37°C
for 24 to 48 hours for anaerobes and for aerobes incubate the plate at 35-37°C for 24 to 48 hours.
 Positive test – A positive lipase test is noted by the appearance of an iridescent sheen.
 Immediately around colonies that can be seen when the plate is held at an angle to a light source.
 Negative test – A negative lipase test is indicated by the absence of an iridescent sheen.
Proteus test
Composition
Peptone 5gm/ltr.
Dextrose 1gm/ltr.
Yeast extract 3gm/ltr.
Agar 15gm/ltr.
Milk powder 20gm/ltr.
ph should be 6.8± 0.2

Positive should be create zone around bacteria

Procedure
 take 25 ml of dill water in a clean and dry culture bottle.
 Weigh all composition (peptone 0.125 gm, dextrose 0.025 gm, yeast exract 0.075 gm, milk powder 0.50 gm)
and add I dilute water.
 After that, maintain the pH and add 0.375 gm agar and autoclave the media for 45 min.
 During the autoclave, prepare the petri plate for pouring the media, so give the UV of the petri plate for 10
min.
 After autoclave, pour the media in the ready plate then leave the media in UV for 15 min. after that dry the
media for 15 min.
 After that, strip the isolated bacteria with the help of a sterile wire loop (E. Coli) on agar media to a straight
line.
 Incubate it for 24 hours and observe the result.
Hydrogen sulphate (H2S) Test
Composition
Yeast extract 3gm/ltr.
Peptone 30gm/ltr.
Ammonium ferrous sulphate 0.2gm/ltr.
Sodium thiosulphate 0.025gm/ltr.
Agar 3gm/ltr.
ph should be 7.3± 0.2

H2S Test Positive Bacteria

Proteus spp., Citrobacter spp., Salmonella spp., Staphylococcus saprophyticus,
Campylobacter spp., etc.
H2S Test Negative Bacteria
Klebsiella pneumoniae, Shigella spp., Staphylococcus aureus, E. coli, Pseudomonas aeruginosa,
Neisseria gonorrhoeae, Vibrio cholerae, Yersinia pestis, etc.
Procedure
 An iron compound and a sulphur compound are included in the test medium to test for the production of
hydrogen sulphide gas.
 Hydrogen sulphate is production if the sulphur compound is reduced by the bacterial strain. This test thus
determines whether the microbe reduces sulphur- containing compound to sulphide during the process of
metabolism.
 H2S is produced by certain bacteria through reduction of sulphur containing amino acids like cystine,
methionine or through the reduction of inorganic sulphur compound such as thiosulphates, sulphate or
sulphites during protein degradation or when anaerobic respiration shuttles the electrons to sulphur instead of
to oxygen.
 In either case H2S is produced (hydrogen sulphide gas) which reacts with the iron compound to from the back
precipitate of ferric sulphide.
 The lack color acts as an indicator for the presence of hydrogen sulphide. The detection of hydrogen sulphide
(H2S) gas produced by an organism is used mainly to assist in the identification of that particular organism.
Starch hydrolysis
Composition
Yeast extract 3gm/ltr.
Starch 5gm/ltr.
Agar 15gm/ltr.
ph should be 6.8±0.2

•Starch hydrolyzing (amylase producing) bacteria: S. bovis, Bacillus subtilis,

B. cereus, B. megaterium, Clostridium perfringens, etc.
•Starch non-hydrolyzing (amylase non-producing)
bacteria: Corynebacterium diphtheria, Clostridium difficile, C. botulinum,
bile-esculin positive viridans Streptococci except S. bovis, E. coli, S. aureus,
Pseudomonas aeruginosa, P. putida, etc.
Procedure
1. Take a clean and washed culture bottle and pour 25 ml distilled water in the bottle.
2. Weigh these reagets : 0.07 gm of beef extract, 0.25 gm of starch and 0.37 gm of agar added in the culture bottle.
3. Maintain the pH at 6.8 ± 0.2, diluted HCL or NaOH to maintain pH.
4. Pour the media into test tubes and cover it with 4 layers paper and a rubber band.
5. Autoclave the media at 121°C for 15 minutes at 15 PSI pressure.
6. After autoclave, let the media cool down for some time.
7. During the autoclave, prepare the petri plate for pouring the media, so give the UV of the petri plate for 10 minutes.
8. After the autoclave, pour the media in the ready plate. Then leave the media in UV for 15 minutes, after drying the media for 15
minutes.
9. After that strip, the isolated bacteria with the help of a sterile wire loop on agar media to a straight line.
10. Incubate it for 24 hours and observe the result.

11. Following incubation, add a few drops of iodine solution directly over the colonies and observe for the formation of a clear halo
around the colonies.
 Methyl red (MR)
Composition
Yeast extract 7gm/ltr.
Dextrose 5gm/ltr.
Dipotassium phosphate 5gm/ltr.
ph should be 6.9± 0.2

MR positive: Escherichia coli (ATCC25922)

MR negative: Enterobacter aerogenes (ATCC13048)
Procedure
1.Inoculate MRVP broth with a pure culture of the organism.
2.Incubate at 35°-37°C for a minimum of 48 hours in ambient air.
3.Add 5 or 6 drops of methyl red reagent per 5 mL of broth.
4.Observe for the color change in the broth medium.
 Voges-Proskauer (VP)
Composition
Yeast extract 7gm/ltr.
Dextrose 5gm/ltr.
Dipotassium phosphate 5gm/ltr.
ph should be 6.9± 0.2 VP Positive Bacteria VP Negative Bacteria

5% Alpha-naphthol Solution (Barritt’s Reagent A) Klebsiella spp.,

and 40% KOH or NaOH solution (Barritt’s Enterobacter spp.,
Escherichia spp.,
Reagent B) are required. Viridans Streptococci (except S. mitis,
Proteus vulgaris,
Preparation of Barritt’s Reagent A and S. vestibularis)
Citrobacter freundii,
Dissolve 5 grams of α-naphthol reagent in 100 mL Proteus mirabilis,
of 95% ethanol. The reagent can be stored for up Morganella morganii,
Hafnia spp.,
to 3 weeks in a dark place at 4 to 8°C. Shigella spp.,
Serratia spp.,
Preparation of Barritt’s Reagent B Yersinia spp.,
Staphylococcus aureus,
Dissolve 40 grams of KOH pellet in 100 mL of V. parahaemolyticus
sterile distilled water. The reagent can be stored
Listeria spp.,
for up to 3 weeks at 4 to 8°C. V. cholerae
Procedure
• Using a sterile inoculating loop, pick up well-isolated colonies of sample bacteria
from 18 to 24 hours old culture and inoculate the broth.
• Incubate the tubes aerobically for 18 to 24 hours at 35±2°C.
• Following incubation, transfer 2 mL of broth to a clean (sterile if possible) test tube.
• Add 6 drops of Reagent A (5% α-naphthol solution) and mix properly by shaking.
• Add 2 drops of Reagent B (40% KOH solution) and mix properly by shaking.
• Observe for the formation of red-pink color at the surface of the medium within 30
minutes. Continuously shake the tube vigorously during the 30-minute waiting
period.
• If no color is developed (negative reaction) re-incubate the remaining broth for
additional 24 hours and test again.
 Indole test
Composition
Tryptophan 10gm/ltr.
Sodium chloride 5gm/ltr.

Indole Kovacs Reagent: •Positive: A positive reaction is denoted by the appearance of a

p-Dimethylaminobenzaldehyde
blue to blue-green color change on the bacterial smear within 2-
3 minutes.
•Negative: Negative reactions remain colorless or light pink.
50.0 gm Note: Positive reaction is Red-violet in the case of Providencia
alcalifaciens.

Hydrochloric Acid, 37% 250.0 ml

Amyl Alcohol 750.0 ml

 Procedure
 Take sterilized test tubes containing 4 ml of tryptophan broth.
 inoculate the tube aseptically by taking the growth from 18-24 hrs. ’culture.
 Add 0.5 ml of Kovac’s reagent to the broth culture.
 Observe the presence or absence of a ring.

Positive: Formation of a pink to red color (“cherry-red ring”) in the reagent layer on top of the medium within seconds of adding
the reagent.
Examples: Aeromonas hydrophila, Aeromonas punctata, Bacillus alvei,Edwardsiella sp., Escherichia
coli, Flavobacterium sp., Haemophilus influenzae, Klebsiella oxytoca, Proteus sp. (not P. mirabilis and P. penneri), Plesiomonas
shigelloides,Pasteurella multocida, Pasteurella pneumotropica, Enterococcus faecalis, and Vibrio sp.
Negative: No color change even after the addition of appropriate reagent.
Examples: Actinobacillus spp., Aeromonas salmonicida, Alcaligenes sp.,
most Bacillus sp., Bordetella sp., Enterobacter sp., Lactobacillus spp., most Haemophilus sp.,
most Klebsiella sp., Neisseria sp., Pasteurella haemolytica, Pasteurella ureae, Proteus mirabilis, P.
penneri, Pseudomonas sp.,Salmonella sp., Serratia sp., Yersinia sp.
Carbohydrate utilization test
1. Dextrose test Procedure
 Take 10 ml dil. Water in a bicker and weigh all the ingredients
Composition (peptones 0.05 gm, NaCl 0.05 gm, yeast 0.03 gm, dextrose 0.10
Peptone 5gm/ltr. gm, phenol red few drop) add in dil. Water.
Sodium chloride 5gm/ltr.  After that, maintain the pH then transfer 5-5 ml media I two
clean and dry tests and cover the test tube with 4 layer of paper.
Yeast extract 3gm/ltr.
 After that place the media in the autoclave for 45 min.
Dextrose 10gm/ltr.
 After the autoclave, place the media in cool water to cool it
Phenol red few drops down. after that take culture broth and sterile pipette ad tips from
ph should be 7.0± 0.2 UV then inoculate 100 µl culture broth of salmonella and E. Coli
Media color should be pink, red or dark orange in dextrose broth separately.
 Place the inoculated tube into the incubator at 35-37°C for 24 to
48 hours.
 Positive result – change the media color into yellow from orange.
 Negative result – media color is not changed.
 2. Sucrose test
Procedure
 Take 10 ml dil. water in a bicker and weight the
Composition compositions add in dil. water.
Peptone 5gm/ltr.  After that, maintain the pH then transfer 5-5 ml media in
Sodium chloride 5gm/ltr. two clean and dry tests and cover the test tube with 4
Yeast extract 3gm/ltr. layers of paper.
sucrose 10gm/ltr.  After that place the media in the autoclave for 45 min.
Phenol red few drops
 After the autoclave, place the media in cool water to cool
down. after that take culture broth and sterile pipette and
ph should be 7.0± 0.2
tips from UV then inoculate 100 µl culture broth of
Media color should be pink, red or dark orange
Salmonella and E. coli in sucrose broth separately.
 Place the inoculated tube into the 35-37 °C incubator.
 Positive result – media colour changes from orange.
 Negative result – media colour is not changed.
3. Lactose test
Procedure
Composition  Take 10 ml dil. water in a bicker and weight all the ingredients add
I dil. water.
Peptone 5gm/ltr.  After that, maintain the pH then transfer 5-5 ml media in two clean
Sodium chloride 5gm/ltr. and dry tests and cover the test tube with 4 layers of paper.
Yeast extract 3gm/ltr.  After that place the media I the autoclave for 45 min.
lactose 10gm/ltr.  After that autoclave, place the media I cool water to cool water to
Phenol red few drops cool down after that take culture broth and sterile pipette and tips
ph should be 7.0± 0.2 from UV then inoculate 100 µl culture broth of salmonella and E.
Media color should be pink, red or dark orange coli in lactose broth separately.
 Place the inoculated tube into the 35-37° C incubator.
 Positive result – change the media colour into yellow from orange.
 Negative result – media colour is not change.
4. Maltose test Procedure
 Take 10 ml dil. water in bicker and weight all the ingredients
add in dil. water.
Composition  After that, maintain the pH then transfer 5-5 ml media in two
clean and dry tests and cover the test tube with 4 layers of
Peptone 5gm/ltr.
paper.
Sodium chloride 5gm/ltr.
 After that place the media in the autoclave for 45 min.
Yeast extract 3gm/ltr.  After the autoclave, place the media in cool water to cool
maltose 10gm/ltr. down. After that take culture broth and sterile pipette and tips
Phenol red few drops from UV then inoculate 100 µl culture broth of salmonella
ph should be 7.0± 0.2 and E. coli in maltose broth separately.
Media color should be pink, red or dark orange  Place the inoculated tube into the 35-37°C incubator for 24-48
hours.
 Positive result – change the media colour into yellow from
orange
 Negative result – media colour is not change.
5. D- mannitol
Procedure
 Take 10 ml dil. water in a bicker and weight all the
Composition 
ingredients add in dil. water.
After that, maintain the pH then transfer 5-5 ml media in
Peptone 5gm/ltr.
two clean and dry tests and cover the tests tube with 4
Sodium chloride 5gm/ltr. layers of paper.
Yeast extract 3gm/ltr.  After that place the media in the autoclave for 45 min.
D- mannitol 10gm/ltr.  After the autoclave, place the media in cool water to cool
Phenol red few drops down after that take culture broth and sterile pipette and
ph should be 7.0± 0.2 tips from UV then inoculate 100 µl culture broth of
Media color should be pink, red or dark orange salmonella and E. coli in mannitol broth separately.
 Place the inoculated tube into the 35-37°C incubator.
 Positive result – change media colour into yellow from
orange.
 Negative result – media colour is not changed.
6. Sorbitol test Procedure
 Take 10 ml dil. water in bicker and weight all the ingredients
add in dil. water.
Composition  After that, maintain the pH then transfer 5-5 ml media in two
clean and dry tests and cover the test tube with 4 layers of
Peptone 5gm/ltr.
paper.
Sodium chloride 5gm/ltr.
 After that place the media in the autoclave for 45 min.
Yeast extract 3gm/ltr.
 After the autoclave, place the media in cool water to cool
Sorbitol 10gm/ltr. down. After that take culture broth and sterile pipette and tips
Phenol red few drops from UV then inoculate 100 µl culture broth of salmonella and
ph should be 7.0± 0.2 E. coli in maltose broth separately.
Media color should be pink, red or dark orange  Place the inoculated tube into the 35-37°C incubator for 24-48
hours.
 Positive result – change the media colour into yellow from
orange
 Negative result – media colour is not change.
7. Glycerol test Procedure
 after the maintenance the pH then transfer 5-5 ml of media in 2
clean and dry test tubes and covers the test tube from 2 layers
Composition paper.
Peptone 5gm/ltr.  After that place the media to autoclave.
Sodium chloride 5gm/ltr.  After autoclave, place the media I cool water to cool down.
Yeast extract 3gm/ltr. After that take culture growth ad sterlite pipette ad tips from
glycerol 10ml/ltr. UV then inoculate 100 ml broth culture salmonella and E. coli
Phenol red few drops in glycerol broth separately.
ph should be 7.0± 0.2
 Place the test tube in the incubator at 35°C for 24-48 hours.
Media color should be pink, red or dark orange  Positive result – change the media colour into yellow from
orange
 Negative result – media colour is not change.