Media & Biochemical TestsMedia & Biochemical Tests

Laboratory Objectives

Extracellular EnzymesExtracellular Enzymes

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

Intracellular enzymesIntracellular enzymes

Catalse Oxidase IMViC Urease Carbohydrate Fermentation Nitrate reduction

Blood agar 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 hemolysisTypes of hemolysis1. Beta hemolysis refers to colorless zone surrounding the colony, where a

complete lysis of the red blood cells.

2. Alpha hemolysis appears as a zone of partial hemolysis surrounding the colony, often accompanied by a greenish discoloration of the agar.

3. Gamma reaction refers to no hemolysis or discoloration of the agar surrounding the colony.

-hemolysis

-hemolysis

-hemolysis

MSA is selective differential medium for staphylococciIt contains: NaCl (7.5%), Mannitol, & Phenol RedThe cause of selectivity due to presence of high salt concentrationThe 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 fermentedYellow colonies:

S. aureus

Mannitol nonfermenterRed colonies:

S. epidermidis& S. saprophyticus

MACCONKEY AGARMACCONKEY AGAR It is selective differential medium for used in identification of

lactose fermenting, Gram-negative bacteria The cause of selectivity due to presence of crystal violet

and bile salt thatprevent growth of G+ bacteriaThe cause of differential because contains lactose (sugar) and Neutral red (pH indicators turns red in acidic pH

Classification of BacteriaClassification of Bacteria

Bacteria

Gram-Positive Gram-negative

Gram-Positive BacteriaGram-Positive Bacteria

I- Gram Positive bacteria

A- Gram positive cocci B- Gram positive rods

Spore-formingNon spore-formingCorynebacterium

AerobicBacillus anthracis

AnaerobicClostridium

Gram-Positive CocciGram-Positive Cocci

A- Gram-positive cocci

I- staphylococci II- streptococci

Species of SatphylococciSpecies 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 StaphylococcusGram stain of Staphylococcus

The catalase test is distinguished streptococci from staphylococci flood culture with drops of 3% H2O2 Catalase-positive cultures bubble at once

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

Catalase test

PositiveMicrocococcaceae

Staphylococci

NegativeStreptococcaceae

Streptococci

H2O2 H2O + O2 (gas, ↑)

Staphylococci

Catalase

Catalase test

Coagulase TestCoagulase TestPrinciple: This test used to differentiate between S. aureus& other

Staphylococcus species

Coagulase test

Coagulase PositiveStaphylococus aureus

Coagulase-NegativeS. epidermidis & S. saprophyticus

Fibrinogen (Plasma)

Coagulase Fibrin (Clot)

Coagulase TestCoagulase Test The tube coagulase test (Free): Procedure:

Mix 0.1 ml of culture + 0.5 ml of plasmaIncubate at 37C for 4 hObserving the tube for clot formationAny degree of clotting constitutes a positive test

AdvantageMore accurate

DisadvantageTime consumed

S. aureus S. epidermidis

Coagulase TestCoagulase Test Two Methods:

The slide MethodTube Method

The slide coagulase test Used to detect bound coagulase or clumping factorAdd one drop heavy bacterial suspension and one drop of plasma on clean slideMixing well and observing for clumping within 10 seconds

AdvantageRapid diagnosis

DisadvantageLess accurate

Oxidase testOxidase 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 KnowTests To Know Case Study Tests

IndoleMethyl Red/Voges ProskauerCitrateH2S production in SIMUrea hydrolysisMotilityLactose fermentationSucrose fermentationGlucose fermentation & gas production

Indole TestIndole 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. Kovac’s reagent—yellow in color.

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

IndoleIndole

Methyl Red/Voges Proskauer (MR/VP)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 BrothMethyl Red indicator for acidVoges Proskauer reagents—A: 5% Alpha-Naphthol, B: Potassium Hydroxide.

MR/VP continuedMR/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 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 –

CitrateCitrate 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: Simmon’s 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

CitrateCitrate

Left positive and right negative.

Urea HydrolysisUrea Hydrolysis How to Perform Test: Inoculate Urea broth with

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

bacteria’s 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)

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

Sulfur Reduction Purpose: This test is used to

identify those bacteria capable of reducing sulfur.

MotilePositive for H2S production

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

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.

Kovac’s Reagent

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

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

Motility Purpose: This medium can

be stab-inoculated with an inoculating needle to indicate motility.

SIM MediumControl

S. aureus Non-motile

. SIM Tube Results . 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) Symbol Interpretation

Red/yellow K/A Glucose fermentation only, Peptone catabolized

Yellow/yellow A/A Glucose and lactose and/or sucrose fermentation

Red/red K/K No fermentation, Peptone catabolized

Red/no color change K/NC No fermentation, Peptone used aerobically

Yellow/yellow with bubbles A/A,G Glucose and lactose and/or sucrose fermentation, Gas produced

Red/yellow with bubbles K/A,G Glucose fermented only, Gas produced

Red/yellow with bubbles and black precipitate K/A,G, H2S Glucose fermentation only, Gas produced,

H2S produced

Red/yellow with black precipitate K/A, H2S Glucose fermentation only, H2S produced

Yellow/yellow with black precipitate A/A, H2S Glucose and lactose and/or sucrose fermentation, H2S produced

No change/no change NC/NC No fermentation

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

Tube deamination glucose lactose H2S production

amino acids fermentation fermentation (black color) 1 + – – – 2 + + – – 3 + + – + 4 + + + – 5 + + + +

Triple Sugar Iron Agar (TSI) . 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.