ENTEROBACTERIACEAE • (+) colorless and gas production

• Proteus mirabilis
Diagnostic Test for Gram negative bacilli:

1. Oxidase test 4. O - Nitrophenyl – beta – d - Galactopyranoside

• Principle: Cytochrome oxidase (capable of (ONPG) Test
producing cytochrome c oxidase enzyme • ONPG via beta galactosidase to Orthonitrophenol
• Used for screening test only • Beta galactosidase -
o Degrades lactose to galactose
• Use platinum wire , get colony and put in filter paper
o Do not use nichrome wire (nickel and o Separate lactose into galactose and glucose

chromium) because it will yield a false • ONPG disk is placed in test tube add small amount
positive result of saline then add colony, culture for 18-24 hrs
o Catalase test- use nichrome wire (platinum (Structure of ONPG disk is similar to lactose)
• Product: Orthonitrophenol and beta
wire will yield false positive result)
galactopyranoside
• Filter paper method uses Tetramethyl p-phenylene
• Lactose fermentation
diamine dihydrochloride (Reagent)
 Pathogenecity test for Enterobacteriaceae
• Positive result (+): Bluish purple
 (+): yellow (because of Orthonitrophenol)
• Negative result: colorless
 (-): no change in color
• Indicator: Indophenol blue
 (+) Escherichia coli – lactose fermenter
• Oxidase test differentiates enterobacteriaccae from
 (-) Salmonella typhimurium – non- lactose
diff. gram negative bacilli oxidase:
fermenter
o (+) P. aeruginosa
Take note
o (-) E. coli (All Enterobacteriaccae is
• RLF/LLF - ONPG (+)
oxidase negative except Plesiomonas)
• NLF – ONPG (-)
• RLF – additional enzyme: permease and beta
2. Nitrate reduction test
galactosidase. Permease facilitate entry of lactose in
• Screening test
bacterial cell which increase process of fermentation
• Principle:
• LLF- only have beta galactosidase
o Nitrate reductase - NO3 (Nitrate) is reduced
to NO2 (Nitrite)
5. LOA test (Decarboxylase test)
• Medium: Peptone broth (nitrate source)
• Aids in identification of enterobacteriaceae family
• Reagent: Sulfanilic acid and alpha-napthylamine
• Detect ability of bacteria to decarboxylase amino
• Result:
acids: Lysine, Ornithine, Arginine
o (+): red color (because of alpha-
• Decarboxylase: Removes carboxyl group of amino
napthylamine) acid (amine group with alkaline pH remains)
o (-): colorless (it is possible that nitrate is • Dihydrolase
reduced too much in which it turned into • Medium: Moeller’s decarboxylase medium
nitrogen or ammonia • Indicator: bromcresol purple
• If negative: add zinc dust/powder to detect • Result:
unreduced nitrate o (+) purple (alkaline)
o Turns colorless: positive o (-) yellow
o Turns red: true negative (unreduced nitrate) o Lysine-> cadaverine
o Ornithine-> putrecine
3. Nitrite reduction
o Arginine-> citrulline
• Focuses on gas production (bubble formation
o Use mineral oil for ANAEROBIC
because of N2)
• Medium: DURHAM TUBE that has peptone broth
6. TRIPLE SUGAR IRON (TSI)
(source of nitrogen) - After 24hrs observe if there is
• Glucose + Lactose + Sucrose + Iron
gas formation
• Differentiate enterobacteriaceae from other gram
• Principle: NO2-> N2 gas (nitrite to nitrogen gas)
negative bacteria in the intestinal tract
• Reagent: sulfanilic acid, alpha-napthylamine + zinc
dust =
 • Differentiate bacteria according to their ability to b. Lysine Oxidative Deamination- slant (R/A)
ferment sugars • Creates a compound that combines creating a color
• Detect ability of organism to produce gas (crack or red
gap or bubbles) • *Positive –red
• Medium: TSI agar (butt slant medium) • *negative- purple
• pH indicators: c. H2S – ferric NH4 citrate
o Phenol red o Black precipitate
o Ferrous sulfate + H2S = Black precipitate o Indicator: Bromcresol purple
• TSI reaction:
o Glucose reaction is found in the butt part 8. Indole test
medium • Principle: Indole from tryptophan due to
o Lactose and sucrose is found in slant part tryptophanase
o 10 parts of lactose, 10 parts of sucrose, 1 part • Can detect three aspects:
of glucose (10:10:1) o Hydrogen Sulfide production (Black
• Streak wire - get colony from broth and streak to precipitate)
the slant portion of butt slant then stab the butt of the o Indole (detect production of indole from
TSI medium tryptophan from tryptophanase)
• Enterobacteriaceae are all glucose fermenter o Motility- semi-solid medium; flagellated
(product is always acid, phenol red becomes yellow) organisms make solutions turbid; non-motile
• Manner of reporting (fraction-like): organism is clear medium
Sucrose∧Lactose • Butt medium – stab the agar
Glucose • Incubate in room temperature
 Yellow is letter A (Acid) • Medium: Sulfide Indole Motility medium (SIM
 Red is letter K (Alkaline) medium has tryptophan)
- There is alkaline reaction because of • Confirmatory reagent:
oxidation of peptone content; no • Kovac’s/ Erlich’s reagent =
fermentation; oxidation of medium makes p-dimethylaminobenzaldehyde
it alkaline • Result: (+) red ring formation 
• A/A = 2 - 3 sugars fermented - lactose fermenters
(yellow slant/yellow butt) 9. Rapid spot indole test (Blue)
• K/A = glucose fermenter - non lactose fermenters • Filter paper strips impregnated with p-
(red slant/yellow butt) diaminocinnamaldehyde reagent
• K/K = no sugar fermented - non fermenter • Use for anaerobic bacteria
organisms (asacarolytic organism meaning they are • Positive result: Blue color
non-fermenter)  
• Early checking result: false positive 10. Methyl red test (opposite with VP)
• Late checking result: false negative • Principle: Mixed acid fermentation
• If positive result in methyl red test then negative
KIA- Kligler’s iron Agar - (G-L-S-iron) result for VP
• Indicator: Methyl red
RDA- Russell’s double agar - (G-L)
• Medium: MRVP medium/Clark and Lubs medium
7. Lysine Iron Agar (LIA) • Result:
• Butt slanted medium o (+) red (pH below 4.4)
• Medium: Moeller’s medium o (-) yellow
• K- purple • E. coli (+)
• A- yellow • E. cloacae (-)
• R-red  
a. Lysine decarboxylation- butt 11. Voges-Proskauer
• Decarboxylase- removes carboxylic acid content • Principle: Butylene glycol of glucose fermentation
which makes the molecule alkaline product • Product is Acetoin or acetylmethylcarbinol
• *Positive- purple= K/K
• *Negative- yellow= K/A
 • Medium: MRVP medium/Clark and Lubs medium- • End product: ammonium carbonate (alkaline
uses glucose product, means bacteria has urease)
• Confirmatory reagent: • Medium: Christensens agar
• Alpha napthol • Indicator: Phenol red
• 40% KOH (barrits method) for enteric • (+) pink/red/magenta
• Alpha napthol in 40% KOH in creatine • (-) yellow
(Coblentz method)
• (+) red 17. Phenylananine Deaminase (PAD)
• (-) yellow • Phenylalanine---PPA(phenyl pyruvic acid) + 10%
• KESH (+) Klebsiella, Enterobacter, Serratia, feCl3
Hafnia • (+) green- Proteus vulgaris, Providencia
• Barrit’s Method: morganella
o (+) E. cloacae • (-) yellow- E. coli
o (-) E. coli  
• Coblentz Method: 18. KCN broth test Potassium Cyanide Broth test
o (+) Streptococcus mutans • (+) turbid
o (-) Streptococcus mitis • (-) clear
  • Klebsiella, Enterobacter, Serratia, Citrobacter
12. Citrate Utilization
• Principle: Utilize citrate as a source of carbon and 19. String test
energy • Identification test for Vibrio cholera
• Medium: Simmon’s citrate • Medium: 0.5 % sodium deoxycholate
• Indicator: Bromthymol blue • (+) string like
• End product: sodium carbonate (alkaline product)
• Positive: Blue (Alkaline pH) 20. Esculin HOH test
• Negative: Green (env. is acidic) • Hydrolyze esculin to esculitin
• (+) P. aeruginosa • (+) black
• (-) E.coli • (-) yellow
• Citrobacter- citrate (+) - royal blue • (+) Klebsiella pneumonia
• (-) Shigella flexneri
13. Acetate Utilization test
• Acetate -> alkaline pH (blue)- positive 21. MUG test ( 4-methylumbelliferyl-β-D-glucuronide)
• Acidic pH (green)- negative • All E. coli are positive for MUG test EXCEPT for
• (+) E. coli E.coli 0157H7- cause hemorrhagic colitis or bloody
• (-) Shigella flexneri diarrhea
• Medium: Simmon’s citrate agar • Detects organisms who can split beta D
glucopyranoside
14. ACETAMIDE Utilization test • 4 methylumbelliferyl-beta-D-glucuronide (UV
• ACETAMIDE-> Ammonia NH3 (Blue) Light)
• (+) P. aeruginosa • (+) blue fluorescence
• (-) S. maltophilia • (-) no fluorescence
  • If negative for MUG test, it means that clinically
15. Malonate Utilization test significant yung organism possible na E. coli
• Indicator: Bromthymol blue 0157H7
• (+) blue • P. aeruginosa
• (-) green
• Klebsiella, Enterobacter, Citrobacter, Hafnia 22. Gelatin Hydrolysis test
• (+) gel liquefies
16. Urea Hydrolysis test (UREASE) • (-) gel solidifies
• Urea - +2H2O-------- CO2 +H2O + 2NH3- • (+) Proteus vulgaris
(NH4)2CO3 • (-) Enterobacter aerogenes
 Enteric media- inhibit all Gram (+)

medium inhibitors CHO Indicator LF NLF

EMB Eosin Y Lactose Eosin Y Purple Colorless
E. coli Methylene Blue Methylene blue
Greenish metallic
sheen
MAC Crystal violet Lactose Neutral red Red/pink colorless
Bile salt
XLD Bile salt Xylose Phenol red yellow Red
Selective for SS Lactose
Sucrose
HEA Bile salt Salicin Bromthymol blue yellow Green
Lactose
Sucrose
DCA Bile salt Lactose Neutral red Red/pink Colorless
SSA Bile salt Lactose Neutral red red colorless
BSA Brilliant green Glucose Bismuth sulfite Black colonies-
Salmonella
TCBS Bile salt Sucrose Bromthymol Yellow Green
Brilliant green agar Brilliant green Phenol red