MICROBIAL REACTION IN CULTERED MEDIA

A. Mannitol Salt Agar (MSA) C. Eosin methylene blue agar (EMB)

-contains NaCl -pH indicator: Eosin y & methylene blue
-pH dye: Phenol Red -isolate, identify, and differentiation of lactose and non-lactose
fermenting gram-negative bacteria
Results:
Mannitol fermenter: yellow (acidic) Results:
Non-mannitol fermenter: red Lactose fermenter: green metallic sheen
Non-lactose fermenter: no metallic sheen
Organism:
-Staphylococcus cohnii Organism:
-Staphylococcus aureus -Escheria coli -Pseudomonas putida
-Klebsiella oxytoca

B. Macckonkey Agar (MAC) D. Catalase Test (NA)

-nitrogen source: peptone -ability to produce catalase and breakdown H2O2 to water and
-carbon source: lactose oxygen
-Bile salts = reduce surface tension -Nutrient Agar medium

Results: Organism:
Lactose fermenter: red colonies -Enterobacter aerogenes
Non-lactose fermenter: colorless or translucent

Organism:
-Escheria coli -Pseudomonas putida
-Klebsiella oxytoca

E. Starch Hydrolysis (Starch agar)

-enzyme: amylase
-w/ gram iodine solution to react w/ starch = blue black

Result:
Positive: colorless zone surrounding colonies

Organism:
-Staphylococcus cohnii -Bacillus subtilis
MICROBIAL REACTION IN CULTERED MEDIA

I. Reactions in TSIA/KIA
F. Carbohydrate/Sugar Fermentation Medium K:red/alkaline slant/butt
-Durham Tube method A:yellow/acid
-acid production: red to yellow
-gas production: bubbles K/A (glucose only) – non-lactose fermenter
-pH indicator: phenol red or bromcresol purple A/A (glucose, lactose, sucrose) – acid slant and butt
K/K (no glucose,lactose,sucrose) – degrade peptone from
Organism: presend and absence of Oxygen
-Escherichia coli K/NC (no glucose, lactose, sucrose) – only utilize peptone
aerobically
G. Methylene Red Voges-Proskauer test (NB + 0.5%
Glucose) Black ppt = acid condition exists
-test for production acid or acetoin Cracking/splitting = gas produced
-MR pH indicator: methyl red
-VP reagent: A alpha-naphthol
40% KOH Organism:
-with butylene-glycol or butaneidol fermentation as basis -Escherichia coli -Pseudomonas putida
-Klebsiella oxytoca
Result:
Acid production: red
Negative MR: yellow

AMC production: pink to red

Negative AMC: no change in color

Organism:
-Escherichia coli -Pseudomonas putida
-Klebsiella oxytoca

J. Gelatin Hydrolysis Test

-test for proteases
-enzyme: gelatinase
-degrade to amino acids and peptides

Result:
gelatinase produced: gelatin remains fluid
H. Citrate Utilization (SCA)
-more citrate = more ammonia Organism:
-source of nitrogen: ammonium dihydrogen phosphate -Bacillus subtilis
-ph indicator: bromthymol blue

Results:
Citrate as source of Carbon: blue
-turns yellow when acidic

Organism:
-Escherichia coli
-Enterobacter aerogenes
MICROBIAL REACTION IN CULTERED MEDIA
K. Indole Production (Tryptone Broth) M. Tributyrin Agar (lipase test)
-breakdown of tryptophan and Kovac’s reagent (p- -reactant: glycerol tributyrate
dimethylamniobenzaldehyde) -lipolytic degrades glycerol tributyrate
-enzyme: lipase
Result:
Indole present: red ring at surface Result:
Lipase and breakdown of tributyrin: clear zones around
Organism: colonies
-Escherichia coli -Pseudomonas putida
-Klebsiella oxytoca -Enterobacter aerogenes

L. Lysine Decarboxylation and deamination (LIA)
-pH indicator: bromcresol purple
-decarboxylation only occurs at acidic medium
-acidified by glucose fermentation
-Lysine give rise to ammonia, reacts with ferric ammonium
citrate = red color (slant)
-Iron sulfide formation = blackening of medium
Results:
purple:alkaline, red:ammonia and ferric ammonium
citrate, yellow: acid
N. Litmus Milk
-dehydrated skim milk + litmus
Results:
1.Acid pH – pink (fermentation of lactose)
2. Alkaline pH – purple/blue (no fermentation of lactose)
3. Reduction – white (litmus as electron acceptor)
4. Acid curd – precipitation of casein by acid from
lactose
5. Rennet Curd – soft curd followed by peptonization
6. Peptonization – dissolution of clot (digestion of
curd/milk by proteolytic enzymes)
7. Gas production – bubbles

1. Negative – purple/yellow
2. Lysine Deamination – red/yellow
3. Lysine Decarboxylation – purple/purple
4. H2S – blackening

Organisms:
-Escherichia coli -Pseudomonas putida
-Klebsiella oxytoca -Seratia marcesens

Organism:
-Escherichia coli -Pseudomonas putida
-Klebsiella oxytoca -Seratia marcesens
MICROBIAL REACTION IN CULTERED MEDIA
O. Nitrate Reduction Test (Nitrate Broth) Q. Urease Test (Stuart’s Urea Broth)
-reagent A: sulfanillic acid -Nitrogen metabolism
-reagent B: naphthylamine
-test for presence of unreduced nitrate: zinc powder Results:
Urease production: red
Result:
Negative: red color Organisms:
Positive: no red color appears -Escherichia coli -Pseudomonas putida
=reduced to N2 or NH3 -Klebsiella oxytoca -Seratia marcesens

Organism:
-Escherichia coli -Pseudomonas putida
-Klebsiella oxytoca -Seratia marcesens

P. Oxidative-Fermentative Test
-carbohydrate 1% and peptone 0.02%
-oxidative use of carbohydrates by gram-negative bacilli
-pH indicator: bromthymol blue

Result:
Positive: yellow color (acidic environment)
Negative: green or blue-green

Organism:
-Pseudomonas putida