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Water Analysis: - Detect The Presence of Coliform Bacteria

This document describes various tests used to identify bacteria in the Enterobacteriaceae family. It discusses tests such as indole, methyl red, Voges-Proskauer, citrate, triple sugar iron, urease, nitrate reduction, cytochrome oxidase, phenylalanine deaminase, and decarboxylase that are used to differentiate bacteria based on their biochemical characteristics and reactions. The tests yield results like gas production, color changes, or precipitate formation that can identify specific bacteria.

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Zhon Cabitac
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66 views35 pages

Water Analysis: - Detect The Presence of Coliform Bacteria

This document describes various tests used to identify bacteria in the Enterobacteriaceae family. It discusses tests such as indole, methyl red, Voges-Proskauer, citrate, triple sugar iron, urease, nitrate reduction, cytochrome oxidase, phenylalanine deaminase, and decarboxylase that are used to differentiate bacteria based on their biochemical characteristics and reactions. The tests yield results like gas production, color changes, or precipitate formation that can identify specific bacteria.

Uploaded by

Zhon Cabitac
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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Water analysis

• Detect the presence of coliform bacteria


have been used as the principal indicator
of suitability of a particular water source
for domestic, dietetic or other uses.
• Multiple tube fermentation technique
used the Most Probable Number (MPN)
Index
• Three steps: A. Presumptive test
B. Confirmatory test
C. Completed test
• Presumptive test:
a.Lactose broth (LB) w/ Durham
fermentation tube
• Completed test:
a.Brilliant green lactose broth (BGLB) w/
Durham ferment. tube
b. EMB agar or Endo agar
• Confirmatory test –
a. Lactose broth w/ Durham ferment. tube
b. Gram staining reagents
• Presumptive test (LB tube)  +gas

• Completed BGLB EMB or Endo


Test (+) gas (+) growth

• Confirmatory LB tube Gm stain


Test (+) gas gm (-) bacilli
non-spore former
Tests for Identification
Enterobacteriaceae
IMViC
• Indole
• Methyl red
• Voges-Proskauer
• Citrate
• 3 groups are separated :
++-- Escherichia coli
--++ Aerobacter-Klebsiella group
-+-+ Citrobacter group
• Indole test - separates E. coli from K-E-S-H (Klebsiella,
Enterobacter, serratia, Hafnia )grp.
SIM medium in tube (other media tryptophan borth,
nitrate-indole )
Kovac’s or Ehrlich’s reagent & chloroform
Test organism

SIM medium + organism  35 C x18-24 hrs. + chloroform + 15


drops Ehrlich’s reagent

tryptophanase
Tryptophan ---------> indole + pyruvic acid + NH3
red color ring
positive negative
(+) motility (+) H2S
Methyl red
• Quantitative test for production of lactic,
acetic & formic acid from glucose thru the
Mixed acid fermentation pathway
• Positive reaction with red color if pH < 4.4
• Intermediate color (orange) – negative rxn

MR-VP medium + organism incubate at 35 C


x 48-72 hrs + 5 drops methyl red
Voges -Proskauer
• Materials: MR-VP medium 40% KOH
alpha-napthol Test organism

• Principle: Degradative fermentation of


Glucose  pyruvic acid acetoin (chief end
product) by Butylene glycol pathway +
6 qtts alpha-napthol + 2 qtts. 40% KOH +O 2
 Diacetyl (red color complex)
Citrate Test
• Citrate – utilized as a source of Carbon
with the production of alkaline by-
products (NH4OH) or alkalinization
• Materials: test organism
Simmon citrate (bromthymol blue as
indicator)
(+) result if green color medium  blue
due to a pH 7.6 and above or presence of
growth
Triple Sugar Iron Test (TSI)
• Utilization of glucose (1) & lactose (10) /sucrose
(10) acid + gas + H2S
• TSI medium (slant/butt)– 3 sugars, phenol red as
indicator, ferrous thiosulfate

• Interpretation: acid – yellow; alkaline – red


gas – bubble/break; H2S-black precipitate

K/K – no fermentation K/A – glucose only


A/A – glucose, sucrose &/or lactose fermentation
Urease test
• Materials: test org. Stuart broth/Christen’s agar
• Principle
Urease  acts on urea
O
||
NH2 - C - NH2 + 2HOH  CO2 + H2O + 2NH3  (NH4)2 CO3

• The ammonia produced during the reaction reacts


in solution to form ammonium carbonate resulting to
alkalinization of the medium (fuschia red)
(+) urease – Proteus & Klebsiella
Nitrate Reduction test
• Used to differentiate Enterobacteriaceae,
Haemophilus, Neisseria, Branhamella And
Mycobacterium
• Principle:
NO3 -- + 2e -- + 2 H  NO2 + H2O
nitrate nitrite

Nitrite + a.-naphthylamine + sulfanilic acid  bloody


red color
• Materials: Test org. Nitrate broth
Zinc dust a.-naphthylamine Sulfanilic acid
Cytochrome Oxidase test
• Used to differentiate the gm(-); All Enterobacteriaceae
(-); All Pseudomonas (+)
• Materials : Kovac’s reagent test organism
filter paper Applicator stick

• Principle: Cytochromes (iron containing hemoproteins )


function as link in transferring H to O to form H2O
Test org. (oxidase) + reduced state Kovac’s reagant
+ atmospheric O2  purple color
(+) Oxidase – purple color on line of
streak of organism
Phenylalanine deaminase test
• Deamination of phenylalanine by Proteus,
Morganella and Providencia
• Principle:
Phenylalanine  Phenylpyruvic acid
deaminase

Phenylpyruvic acid + 10% FeCl3  dark


green
Decarboxylase test
• Enzymes reacting on the carboxyl
(COOH) of A.A.  alkaline amines + CO2
• Lysine, ornithine, arginine – most commonly used

• Lysine  Cadverine
• Ornithine  Putrescine
• Arginine  Citrulline Ornithine  Putrescine
(+)purple (-) yellow

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