lab. no. 7. ii. enterobacteriaceae it divided into two main groups: it divided into two main groups:...
TRANSCRIPT
Lab. No. 7
Bacteria Gram’s Stain
Gram’s +ve Gram’s -ve
Cocci Bacilli Cocci Rods
II. EnterobacteriaceaeII. Enterobacteriaceae It divided into two main groups:It divided into two main groups:According to their effect on lactoseAccording to their effect on lactose Lactose Fermenters:Lactose Fermenters: (coliforms) (coliforms)Examples: Examples: E.coli, KlebsiellaE.coli, Klebsiella.. Lactose Nonfermenters:Lactose Nonfermenters:Examples: Examples: Protus, SalmonellaProtus, Salmonella. . Late Lactose Fermenters:Late Lactose Fermenters:Example: Example: ShigellaShigella..
II. EnterobacteriaceaeII. Enterobacteriaceae Gram’s Stain:Gram’s Stain:Gram’s –ve rods , having single arrangement.Gram’s –ve rods , having single arrangement.
Macroscopical Examination:Macroscopical Examination: ProteusProteus showing a swarming growth. showing a swarming growth.
KlebsiellaKlebsiella has a mucoid colonies. has a mucoid colonies.
E.ColiE.Coli has a very bad odor. has a very bad odor.
II. EnterobacteriaceaeII. Enterobacteriaceae
II. EnterobacteriaceaeII. Enterobacteriaceae Oxidase test:Oxidase test:
Principle:Tetramethyl p-phenylene diamine
(oxidase reagent)colourless
Cytochrome oxidase enzyme
Indophenol(Purple colour)
II. EnterobacteriaceaeII. Enterobacteriaceae Oxidase test:Oxidase test:
Results:+ve Test: Appearance of purple colour within 1-2 min.
purple colour
+ve test
No colour
-ve test
Pseudomonas
Enterobacteriaceae
II. EnterobacteriaceaeII. Enterobacteriaceae Oxidation Fermentation (O/F) Test:Oxidation Fermentation (O/F) Test:
Principle:
Using sensitive O/F medium ( Hugh and Leifson Medium) .
All enterobacteriaceae are O+/F+ (Fermentative)
II. EnterobacteriaceaeII. Enterobacteriaceae Nitrate Test:Nitrate Test:
Principle:
All enterobacteriaceae reduce nitrate to nitrite
II. EnterobacteriaceaeII. Enterobacteriaceae Nitrate Test:Nitrate Test:
Procedure:
Nitrate broth
test m.o
Nit.A
Nit. B
Red colourIncubate at 35oC for 24
hrs
MacConkey’s agar is a selective medium for Gram’s –ve bacteria because it contains bile salt and crystal violet to inhibit the growth of Gram’s +ve bacteria.
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
Growth on MacConkey’s agar:Growth on MacConkey’s agar:
Principle:
It also contains lactose and nutral red indicator
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
Growth on MacConkey’s agar:Growth on MacConkey’s agar:Principle:
LactoseLactose fermenter
acidNutral red
Pink colonies
Procedure:1. Inoculate MacConkey’s agar plate with the test organism by streaking.
2. Incubate the plate at 35oC for 24 hrs.
Flame & Cool
Flame & Cool
Flame & Cool
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
Growth on MacConkey’s agar:Growth on MacConkey’s agar:
Results:
Pink colonies
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
Growth on MacConkey’s agar:Growth on MacConkey’s agar:
Lactose fermenter
Pale coloniesLactose nonfermenter
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:
Principle:
This medium contains three types of sugars (lactose, sucrose and dextrose).
The conc. of lactose and sucrose is 10 times that of dextrose.
It also contains phenol red indicator.
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:
Principle:
butt slant
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:
Principle:1. No Fermentation:1. No Fermentation:
Butt: alkaline (red) Slant: alkaline (red)
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:
Principle:2. Dextrose Fermentation:2. Dextrose Fermentation:
a) Initial reaction:
Dextrose
acid
(after 10 – 12 hrs)
Butt: acidic (yellow) Slant: acidic (yellow)
Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:
Principle:2. Dextrose Fermentation:2. Dextrose Fermentation:
b) Delayed reaction:
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
(after 24 hrs)
Peptone O
2
Alkaline amines
Butt: acidic (yellow) Slant: alkaline (red)
Small amount of acid
Proteus, Salmonells & ShigellaProteus, Salmonells & Shigella
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:
Principle:3. Lactose Fermentation:3. Lactose Fermentation:
Lactose
Large amount of acid
Peptone O
2
Alkaline amines
Butt: acidic (yellow) Slant: acidic (yellow)
E.Coli & KlebsiellaE.Coli & Klebsiella
Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:
Principle:
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
This medium contains also ferrous sulfate as an indicator for H2S production.
H2S + FeSo4 FeS
Black ppt. of ferrous sulfid
Proteus & SalmonellsProteus & Salmonells
Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:
Procedure:
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
Definitive tests to differentiate betweenDefinitive tests to differentiate between Lactose fermenters & Lactose Nonfermenters
Results:
Butt:
Slant:
H2S Production:
acidic (yellow)
acidic (yellow)
-ve
E.Coli & KlebsiellaE.Coli & Klebsiella
acidic (yellow)
alkaline (red)
-ve
ShigellaShigella
acidic (yellow)
alkaline (red)
+ve
Growth on Triple Sugar Iron (TSI) agar:Growth on Triple Sugar Iron (TSI) agar:
Proteus & SalmonellsProteus & Salmonells
Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella
IMVIMViiC Test:C Test:
It is composed of a group of four tests:It is composed of a group of four tests: IIndole production test.ndole production test. MMethyl red (MR) test.ethyl red (MR) test. VVoges-Proskauer (V-P) test.oges-Proskauer (V-P) test. CCitrate Utilization test.itrate Utilization test.
Indole Production TestIndole Production Test
Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella
IMVIMViiC Test:C Test:
Principle:Tryptophane Tryptophanase
enzymeIndole
Kovac’s reagent
Purple red color in the
upper organic layer
Indole Production TestIndole Production Test
IMVIMViiC Test:C Test:
Results:
Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella
+ve -ve
MR-VP Test:MR-VP Test:
IMVIMViiC Test:C Test:
Principle:
Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella
Dextrose
Acidic pathway OROR Neutral pathway
Mixed acids pH less than
4.4MR indicator
Red colour
Acetyl methyl carbinol
α-naphtholKOH
Brick red colour
MR +ve VP +ve
Citrate Utilization TestCitrate Utilization Test
IMVIMViiC Test:C Test:
Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella
Principle:
CitrateAs a sole source of carbon
Na2CO3
alkaline
Bromothymol blueindicator
Blue colour
+ve
IndoleIndole
IMVIMViiC Test:C Test:
Results:
Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella
MRMR VPVP CitrateCitrate
IMVIMViiCC
I MR VP C
IMVIMViiC Test:C Test:
Results:
Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella
+ve +ve -ve -ve -ve -ve +ve +ve
I M Vi C+ + - -
E.Coli
I M Vi C- - + +
Klebsiella
Growth on Eosin Methylene Blue (EMB)Agar:Growth on Eosin Methylene Blue (EMB)Agar:
Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella
Principle:
This medium contains lactose, eosin and methylene blue.
It is differential medium for E.coli which produce a characteristics black colonies with metalic sheen.
Growth on Eosin Methylene Blue (EMB)Agar:Growth on Eosin Methylene Blue (EMB)Agar:
Results:
Definitive tests Definitive tests for Lactose fermentersfor Lactose fermentersE.coli & KlebsiellaE.coli & Klebsiella
black colonies with metalic sheen.E.Coli
Klebsiella Purple colonies.
Definitive tests Definitive tests for Lactose for Lactose nonfermentersnonfermenters
Proteus & SalmonellaProteus & Salmonella Urease Production Test:Urease Production Test:
Principle:
UreaUrease enzyme
NH3 ( alkaline)
Phenol red indicator
Pink colour
Definitive tests Definitive tests for Lactose for Lactose nonfermentersnonfermenters
Proteus & SalmonellaProteus & Salmonella Urease Production Test:Urease Production Test:
Results:
+ve -ve
Proteus Salmonella
Definitive tests Definitive tests for Lactose for Lactose nonfermentersnonfermenters
Proteus & SalmonellaProteus & Salmonella Gelatin Liquefaction Test:Gelatin Liquefaction Test:
Principle:
Gelatin Gelatinase enzyme
Peptides
gel liquid
Definitive tests Definitive tests for Lactose for Lactose nonfermentersnonfermenters
Proteus & SalmonellaProteus & Salmonella Gelatin Liquefaction Test:Gelatin Liquefaction Test:
Results:Positive test: Gelatin medium remain liquid even after refrigeration.
Gelatinase +ve Proteus
SalmonellaGelatinase -ve
It is selective medium for Salmonella It is selective medium for Salmonella and Shigella.and Shigella.
It contains sodium thiosulfate and It contains sodium thiosulfate and ferric citrate as an indicator for Hferric citrate as an indicator for H22S.S.
Definitive tests for Definitive tests for Salmonella & ShigellaSalmonella & Shigella
Growth on Salmonella-Shigella (S-S) Agar:Growth on Salmonella-Shigella (S-S) Agar:
Principle:
Definitive tests for Definitive tests for Salmonella & ShigellaSalmonella & Shigella
Growth on Salmonella-Shigella (S-S) Agar:Growth on Salmonella-Shigella (S-S) Agar:
Results:
Salmonella Pale colonies with black centers
Shigella Pink colonies
Flame & Cool
Flame & Cool
Flame & Cool
Practicle WorkPracticle Work Oxidase test.Oxidase test. O/F test.O/F test. Nitrate broth.Nitrate broth. Indole broth.Indole broth. MR-VP broth.MR-VP broth. Citrate slant.Citrate slant. Urea Slant.Urea Slant. Gelatin test. Gelatin test. Growth on MacConkey’s agar.Growth on MacConkey’s agar. Growth on EMB agar.Growth on EMB agar. Growth on S-S agar.Growth on S-S agar.
ResultsResults Nitrate Test:Nitrate Test:
Nit.A
Nit. B
Red colour
No red colour
Add zinc dust
ResultsResults Indole Production TestIndole Production Test
Indole
Kovac’s reagent
Purple red color in the
upper organic layer
I
ResultsResults MR-VP Test:MR-VP Test:
MR-VPbroth
MR
VP
MR indicator
Red colour
MR +ve
α-naphtholKOH
Brick red colour
VP +ve
Oxidase TestOxidase Test
+ve-ve
Pseudomonas Enterobacteriaceae
O/F test: O+/F-
Nitrate test: +ve further reduction to N2
Growth on cetrimide agar: Pale colonies with green pigmentation
MacConkey’s agar& TSI
Lactose fermenter
Pink colonies on MacConkey& acidic butt and slant onTSI
Pale colonies on MacConkey& acidic butt alkaline slant onTSI
Lactose non-fermenter
IMViC test& EMB
IMViC++ - -& black colonies with metalic shines on EMB E.coli
IMViC- - ++
Klebsiella
No H2S production(no blacking in TSI)
H2S production(blacking in TSI)
ShigellaUrease production
& gelatin liquefaction
+ve
Proteus
-ve
S-S agar
Pale colonies with black centers
Salmonella
Pseudomonas
E.coli Klebsiella
shigella
Proteus Salmonella
ResultsResults
Gram’s Stain Gram’s –ve rods, single arrangement
O/F testO+/F- O+/F+ O+/F+ O+/F+ O+/F+O+/F+
Nitrate test
No red colour after addition of zinc dust+ve further reduction to N2
+ve reduction from nitrate into nitrite
Oxidase test +ve -ve -ve -ve -ve-ve
Cetrimide agar
Pale colonies with green pigmentation
- - -- -
E.coli Klebsiella shigella Proteus Salmonella
ResultsResults
MacConkey’s agar
Pink colonies Pink mucoid colonies
Pale colonies Pale coloniesPale colonies
TSI agarButt:Slant:H2S:
Acidic (yellow)Acidic (yellow)-ve
Acidic (yellow)Acidic (yellow)-ve
Acidic (yellow)Alkaline (red)-ve
Acidic (yellow)Alkaline (red)+ve
Acidic (yellow)Alkaline (red)+ve
IMViC test IMViC++ - -
IMViC- - ++
IMViC- + - -
IMViC- - - +
IMViC- + - +
EMB agar Black colonies with metalic sheen
Pink colonies Pale coloniesPale colonies Pale colonies
ResultsResultsE.coli Klebsiella shigella Proteus Salmonell
a
Urease production test
Gelatin liquefaction test
+ve (pink)
-ve-ve +ve -ve
+ve (liquid)
-ve-ve -ve -ve
S – S agar Pink coloniesPink coloniesPale coloniesPale colonies Pale colonies with black centers
With my Best Wishes,,,Manal Abu El-Khair