Media Biochemical Tests

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 510 % 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.

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.

β-hemolysis α-hemolysis γ-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 

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+ bacteria The cause of differential because contains lactose (sugar) and Neutral red (pH indicators turns red in acidic pH

Classification of Bacteria Bacteria

Gram-Positive

Gram-negative

Gram-Positive Bacteria I- Gram Positive bacteria

A- Gram positive cocci

B- Gram positive rods

Non spore-forming Corynebacterium

Spore-forming

Aerobic Bacillus anthracis

Anaerobic Clostridium

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

, ↑(H2O + O2 (gas

Catalase Staphylococci



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

Positive

Negative

Microcococcaceae Staphylococci

Streptococcaceae Streptococci

Coagulase Test Principle: 

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

Fibrinogen (Plasma)

Coagulase

Fibrin (Clot)

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

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. 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.

Indole

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

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 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: 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

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 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)

 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.

 Kovac’s

Reagent

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

 Motility 

Purpose: This medium can be stabinoculated 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)

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

glucose lactose fermentation fermentation – – + – + – + + + +

H2S production (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.