Media & Biochemical Tests

Laboratory Objectives

Extracellular Enzymes
Starch hydrolysis (Amylase)
Lipid hydrolysis (Lipase) Casein hydrolysis (protease) Gelatin hydrolysis (Gelatinase) Blood hydrolysis (Streptolysin)

Intracellular enzymes
Catalse
Oxidase IMViC Urease Carbohydrate Fermentation

Nitrate reduction

Blood agar
It

is Enriched- Differential media contains general nutrients and 5-10 % sheep blood. It is useful for cultivating fastidious organisms and for determining the hemolytic patterns

Types of hemolysis
1.

Beta hemolysis refers to colorless zone surrounding the colony, where a complete lysis of the red blood cells. Alpha hemolysis appears as a zone of partial hemolysis surrounding the colony, often accompanied by a greenish discoloration of the agar. Gamma reaction refers to no hemolysis or discoloration of the agar surrounding the colony.

2.

3.

-hemolysis

-hemolysis
-hemolysis

MSA is selective differential medium for staphylococci It contains: NaCl (7.5%), Mannitol, & Phenol Red The cause of selectivity due to presence of high salt concentration The cause of differential because contains mannitol (sugar) and phenol red (pH indicators turns yellow in acidic pH and turns red in alkaline pH).

Mannitol fermentation on MSA

Mannitol fermented Yellow colonies: S. aureus

Mannitol nonfermenter Red colonies: S. epidermidis& S. saprophyticus

MACCONKEY AGAR

lactose fermenting, Gram-negative bacteria The cause of selectivity due to presence of crystal violet and bile salt thatprevent growth of G+ bacteria
The cause of differential because contains lactose (sugar) and Neutral red (pH indicators turns red in acidic pH

It is selective differential medium for used in identification of

Classification of Bacteria
Bacteria

Gram-Positive

Gram-negative

Gram-Positive Bacteria
I- Gram Positive bacteria

A- Gram positive cocci

B- Gram positive rods

Spore-forming

Non spore-forming Corynebacterium

Anaerobic Clostridium

Aerobic Bacillus anthracis

Gram-Positive Cocci
A- Gram-positive cocci

I- staphylococci

II- streptococci

Species of Satphylococci

Three species of staphyloccoci have medical importance:

S. aureus: Pathogenic & commensally found in nose (nares) S. epidermidis: non pathogenic & common commensals in nares & skin S. saprophyticus: Cause UTI in female & occasionally commensally found skin

Gram stain of Staphylococcus

Catalase test

The catalase test is distinguished streptococci from staphylococci flood culture with drops of 3% H2O2 Catalase-positive cultures bubble at once
H2O2 Catalase Staphylococci H2O + O2 (gas, )

The test should not be done on blood agar because blood itself will produce bubbles
Catalase test

Positive
Microcococcaceae Staphylococci

Negative
Streptococcaceae Streptococci

Coagulase Test Principle:

This test used to differentiate between S. aureus& other Staphylococcus species

Coagulase Fibrin (Clot)

Fibrinogen (Plasma)

Coagulase test
Coagulase Positive Staphylococus aureus Coagulase-Negative S. epidermidis & S. saprophyticus

Coagulase Test

The tube coagulase test (Free):

Procedure:
Mix 0.1 ml of culture + 0.5 ml of plasma Incubate at 37C for 4 h Observing the tube for clot formation Any degree of clotting constitutes a positive test

Advantage
More accurate

Disadvantage
Time consumed

S. aureus

S. epidermidis

Coagulase Test

Two Methods: The slide Method Tube Method The slide coagulase test
Used to detect bound coagulase or clumping factor Add one drop heavy bacterial suspension and one drop of plasma on clean slide Mixing well and observing for clumping within 10 seconds

Advantage
Rapid diagnosis

Disadvantage
Less accurate

Oxidase test

Basically, this is a test to see if an organism is an aerobe. It is a check for the presence of the electron transport chain that is the final phase of aerobic respiration. Normally, oxygen is the final electron acceptor for this system. In the oxidase test, an artificial final electron acceptor (N,N,N,N-tetramethyl phenylenediamine dihydrochloride) TPD is used in the place of oxygen. This acceptor is a chemical that changes color to a dark blue/purple when it takes the electron from the last element (cytochrome oxidase) in the electron transport chain.

Tests To Know

Case Study Tests

Indole Methyl Red/Voges Proskauer Citrate H2S production in SIM Urea hydrolysis Motility Lactose fermentation Sucrose fermentation Glucose fermentation & gas production

Indole Test

How to Perform Test: Inoculate Tryptone broth with inoculating loop. Property it tests for: This test is performed to help differentiate species
of the family Enterobacteriaceae. It tests for the bacteria species ability to produce indole. Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan, which makes by-products, of which, indole is one.

Media and Reagents Used: Tryptone broth contains tryptophan.

Kovacs reagentyellow in color.

Reading Results: Kovacs reagent reacts with indole and creates a red
color at the top part of the test tube.

Indole

Methyl Red/Voges Proskauer (MR/VP)

How to Perform Tests: Inoculate 2 glucose broths with

Properties they test for: Both tests are used to help
differentiate species of the family Enterobacteriaceae.
MRtests for acid end products from glucose fermentation. VPtests for acetoin production from glucose fermentation.

inoculating loop. After 48 hours of incubation, add a few drops of MR to one tube, and VP reagents to the other tube.

Media and Reagents Used:

Glucose Broth Methyl Red indicator for acid Voges Proskauer reagentsA: 5% Alpha-Naphthol, B: Potassium Hydroxide.

MR/VP continued

Reading Results:
MR a + result is red (indicating pH below 6) and a result is yellow (indicating no acid production) VPA + result is red 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

How to Perform Test: Inoculate slant with inoculating loop. Property it tests for: This test is used to help differentiate species of the
family Enterobacteriaceae. It is selective for bacteria that has the ability to consume citrate as its sole source of carbon and ammonium as sole nitrogen source.

Media and Reagents Used: Simmons Citrate Agar contains sodium

citrate (carbon source), ammonium ion (nitrogen source), & pH indicator bromthymol blue.

Reading Results:
A + result is blue (meaning the bacteria metabolised citrate and produced an acid end product) and a result remains green

Citrate

Left positive and right negative.

Urea Hydrolysis

How to Perform Test: Inoculate Urea broth with

inoculating loop.

Property it tests for: This test is done to determine a

bacterias ability to hydrolyze urea to make ammonia using the enzyme urease.

Media and Reagents Used: Urea broth contains a

urea, and phenol red indicator.

Reading Results: Urea broth is a yellow-orange color.

The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made.

SIM Medium
(Sulfur Reduction Test, Indole Production, Motility)

Sulfur Reduction

Purpose: This test is used to identify those bacteria capable of reducing sulfur.

Motile Positive for H2S production

SIM Medium
(Sulfur Reduction Test, Indole Production, Motility)

Indole Production

Purpose: The indole test is used to identify bacteria capable of producing indole using the enzyme tryptophanase.

Kovacs Reagent

SIM Medium
(Sulfur Reduction Test, Indole Production, Motility)
SIM Medium Control

Motility

Purpose: This medium can be stab-inoculated with an inoculating needle to indicate motility.

S. aureus Non-motile

. SIM Tube Results

A. Indole positive and hydrogen sulfide positive B. Hydrogen sulfide positive C. Indole positive and motility positive (note fuzzy growth away from stab line) D. Negative Control

Kligler Iron agar is a combination medium. While fermentation of various sugars can be tested to characterize different bacteria, the ability to ferment glucose and lactose are of special importance since these can be used to classify bacteria to various groups. Gram negative bacteria can be classified as lactose fermenters and non-lactose fermenters. To facilitate the testing of both sugars a combination medium is used. Kligler Iron agar also tests for the production of H2S by the bacteria. KIA contains the 2 sugars: glucose and lactose in a ratio of 1:10. The medium is used in the form of slants.

The abundance of lactose in the medium compared to glucose allows for differentiation of fermentation of one sugar against that of both sugars. Since lactose is a disaccharide (glucose and galactose) if lactose is fermented then glucose must be fermented too. The reaction is made visible by the accumulation of various amounts of acids altering the pH of the medium to varying extents (either the slant alone glucose alone fermented; or both slant and the butt of the agar changing color due to increased amounts of acids produced by both sugars being fermented).

The production of gas in these reactions is made visible by cracks or displacement of the agar in the medium. Production of H2S is made visible by the addition of ferrous salts that precipitate the H2S and form a black precipitate.

Results (slant/butt) Red/yellow Yellow/yellow

Red/red

Symbol K/A A/A

K/K

Interpretation Glucose fermentation only, Peptone catabolized Glucose and lactose and/or sucrose fermentation
No fermentation, Peptone catabolized

Red/no color change

Yellow/yellow with bubbles Red/yellow with bubbles Red/yellow with bubbles and black precipitate Red/yellow with black precipitate Yellow/yellow with black precipitate No change/no change

K/NC
A/A,G K/A,G K/A,G, H2S K/A, H2S A/A, H2S NC/NC

No fermentation, Peptone used aerobically

Glucose and lactose and/or sucrose fermentation, Gas produced Glucose fermented only, Gas produced Glucose fermentation only, Gas produced, H2S produced Glucose fermentation only, H2S produced Glucose and lactose and/or sucrose fermentation, H2S produced No fermentation

Fermentation reactions have to be read within 18-24 hours before sugar reversion can occur.

Tube deamination amino acids 1 + 2 + 3 + 4 + 5 +

glucose lactose H2S production fermentation fermentation (black color) + + + + + + + +

Triple Sugar Iron Agar (TSI) .

Purpose: To differentiate bacteria based on their ability to ferment glucose, lactose and/or sucrose, and to reduce sulfur to hydrogen sulfide.