Scribd
Upload
168 views

Biochemical Tests Lab7

Uploaded by

skystears07
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
168 views

Biochemical Tests Lab7

Uploaded by

skystears07
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
You are on page 1/ 19

Biochemical Tests

Objectives
Tests to know
 Indole
 Methyl Red/Voges Proskauer
 Citrate
 H2S production in SIM
 Urea hydrolysis
 Sugar fermentation
 Lactose fermentation
 Oxidase test
 Catalase test
Indole Production Test
 The amino acid tryptophan can be broken down by enzyme
tryptophanase to form indole.
 Tryptophanase differentiates indole-positive enterics, such as
Escherchia coli and Proteus vulgaris from indole-negative
enterics, such as Serratia marcescens.
 Media and Reagents: SIM with tryptophan and Kovac’s reagent.
 Method: Inoculate medium and incubate at 37°C for 24-48 hours.
After incubation, add five drops of Kovac’s reagent to the surface.
Do not stir or shake the tube.
 Expected Results:
 Positive test : Kovac’s reagent combines with indole and turns the surface
red.
 Negative test: No red color development
Results for indole test
Methyl Red/Voges Proskauer
(MR/VP)
 How to Perform Tests: Inoculate 2 glucose broths
with inoculating loop. After 48 hours of incubation, add a
few drops of MR to one tube, and VP reagents to the other
tube.
 Properties they test for: Both tests are used to help
differentiate species of the family Enterobacteriaceae.
 MR—tests for acid end products from glucose fermentation.
 VP—tests for acetoin production from glucose fermentation.
 Media and Reagents Used:
 Glucose Broth
 Methyl Red indicator for acid
 Voges Proskauer reagents—A: Alpha-Naphthol, B: Potassium
Hydroxide (KOH).
MR/VP continued
 Reading Results:
 MR— a + result is red (indicating pH below 6) and a – result is yellow
(indicating no acid production)
 VP—A + result is red pinkish after VP reagents are added (indicating
the presence of acetoin) and a – result is no color change.

Methyl Red: left – and right + VP: left + and right –


Citrate Utilization
 Citrate is an organic molecule that can be utilized by bacteria
that produce the enzyme citrase. Citrase is produced by some
bacteria such as E. aerogenes but not by others like E. Coli

 Media and Reagent: Simmon’s Citrate Agar. It has citrase as


the only carbon source and PH indicator bromothymol blue
 Method: Inoculate the slant and incubate at 37°C for 24-48
hours.
 Expected results:
 Positive test: Growth and color changes to blue
 Negative test: No growth and color remains green
Results for Citrate Test

Negative
Positive
H2S Production
 Bacteria use enzyme cysteine desulfurase to hydrolyze the
amino acid cysteine, forming hydrogen sulfide as end-product.

 Media and Reagent: SIM with cysteine and ferrous sulfate


(detects H2S)
 Method: Inoculate the media and incubate at 37°C for 24-48
hours.
 Expected Results:
 Positive Test: H2S production = Black
 Negative Test: No H2S production = No blackening of medium
Results of H2S production

Negative Positive
Urea Utilization
 Some bacteria produce urease, an enzyme capable of
breaking down urea and produce alkaline end
products. This distinguishes Proteus from other
bacteria
 Media and Reagent: Urea Broth with phenol red
 Method: Inoculate the media with a loop and
incubate at 37°C for 24 hours.
 Expected Results:
 Positive test: production of alkaline end products = pinkish
red color
 Negative test: No color change
Results for Urea Test

Positive
Negative
Glucose Fermentation & Gas
Production
 How to Perform Test: Inoculate broth with inoculating loop.

 Property it tests for: This tests for the bacteria’s ability to


ferment glucose and produce gas and/or an acid end-product..

 Media and Reagents Used: Glucose broth contains phenol


red as indicator to indicate an acid end product. A Durham tube is
added to indicate gas production.
 Results
 A positive result for acid is yellow after indicator is added
(indicating glucose fermentation)
 A positive result for gas is a bubble in the Durham tube.
 A completely negative result has no color change or reddish
color and no bubble.
Sugar Fermentation Tests

Tube 1: Negative acid /Negative gas


Tube 2A: Must incubate longer (ambiguous result)
Tube 2B: Positive acid /Negative gas
Tube 3A: Positive acid/ Positive gas
Lactose Fermentation
 MacConkey Agar differentiates lactose-fermenting bacteria,
such as E. Coli from non-lactose fermenting bacteria.
 Media and Reagent: MacConkey Agar and neutral red dye
 Method: Streak MAC plate and incubate at 37°C for 2 days.
 Expected results:
 Positive test: Lactose fermentation = Growth and color change to pink
 Negative test: No lactose fermentation = May or may not grow and no
color change
Results of Lactose Fermentation
- Oxidase test:
Some bacteria produce
Oxidase enzyme
(Pseudomonas )
Detection by adding few
drops of colorless oxidase
reagent
Colonies turn deep
purple in color (positive)
Catalase Test
The Catalase test determines if the organism produces the
enzyme “Catalase”, which breaks down hydrogen peroxide
(H2O2) to water and oxygen (O2).

Catalase
2 H2O2 2 H2O + O2 (gas)

and may be seen in aerobic and facultatively anaerobic


organisms like Staphylococcus bacteria.

Addition of H2O2 lead to production of gas bubbles


(O2 production)
Examples of Biochemical Tests

API 50 Test

You might also like