Various Haemophilus species Identification: Introduction and using of X,V

Various Haemophilus species Identification: Introduction and Using of X,V and XV Discs

Introduction of Various Haemophilus species Identification

Before going to various Haemophilus species identification, let’s know about the scientific classification of Haemophilus.

Scientific Classification of Haemophilus

Kingdom: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Pasteurellales

Family: Pasteurellaceae

Genus: Haemophilus

Species: influenzae

other common species are

Haemophilus influenzae
Haemophilus parainfluenzae
Haemophilus aegyptius
Haemophilus ducreyi
Haemophilus haemolyticus
Haemophilus parahaemolyticus

Introduction of Haemophilus

Genus Haemophilus is a genus of Gram-negative coccobacilli bacteria and pleomorphic too. It belongs to the family Pasteurellaceae. These organisms inhabit the mucous membranes of various parts of our body like the upper respiratory tract, mouth, vagina, and intestinal tract. The genus includes commensal organisms along with some significant pathogenic species such as Haemophilus influenzae. It is a causative agent of sepsis and bacterial meningitis in young children whereas H. ducreyi, the causative agent of chancroid. Other Haemophilus are normal flora e.g. H. parainfluenzae but can also cause pneumonia and endocarditis, similarly, H. aphrophilus which causes pneumonia and endocarditis and H. aegyptius causes pink eye (purulent conjunctivitis). All species of this genus are either aerobic or facultatively anaerobic. This genus has also been found to be part of the salivary microbiome. These various Haemophilus species identification can be performed using X, V, and XV Discs.

Various Haemophilus species Identification using X, V and XV test

Haemophilus spp. have varying requirements for X, V, and XV growth factors. Consequently, the significant differences in growth factor requirements of Haemophilus spp. allows for their differentiation. Differentiation is based on the presence or absence of growth around and/or between discs impregnated with factors X, V, and XV.

Each X-Factor Disk is impregnated with hemin. Each V-Factor Disc is impregnated with NAD (nicotinamide adenine dinucleotide). Each XV-Factor Disc is impregnated with a combination of hemin and NAD.

Variety of Haemophilus species isolation on the basis of X disc, V disc, XV disc, Carbon dioxide and xylose test

#Variety of Haemophilus species identification on basis of X, V, and XV disks, blood agar, and Xylose test as shown below-

Haemophilus influenzae on chocolate agar

Haemophilus influenzae colonies on chocolate agar after overnight incubation in a carbon dioxide atmosphere as shown below the video. It must contain haemin or other iron-containing porphyrin and nicotinamide adenine dinucleotide (NAD) or its phosphate (NADP). The porphyrin requirement is referred to as growth factor X and the NAD or NADP requirement is growth factor V.

Tool and techniques for isolation of Haemophilus influenzae as shown below-

Introduction of Chocolate agar

Chocolate Agar (CHOC) is a non-selective, enriched growth medium that is the lysed blood agar. The agar is named for its color when the red blood cells (RBCs) lysis gives the medium a chocolate-brown color without having chocolate products. It is used for the isolation of fastidious bacteria, such as Haemophilus influenzae, when incubated at 35-37°C in a 5% CO₂incubator.

Principle of Chocolate Agar

The composition of chocolate agar is the same as the blood agar and the only difference is while preparing Chocolate agar, the red blood cells are lysed changing the medium color chocolate brown.

The lysis of RBC during the heating process releases intracellular coenzyme nicotinamide adenine dinucleotide (Factor V or NAD) into the agar for utilization by fastidious bacteria (the heating process also inactivates growth inhibitors). Hemin (factor X) is available from non-hemolyzed as well as hemolyzed blood cells.

The most common species that require this enriched medium for growth include Neisseria meningitidis and Haemophilus spp. H. influenzae is not able to grow on sheep blood agar.

Requirements for Chocolate Agar Preparation

Prepared blood agar
Incubator for the simplest method
But for other methods are
Blood agar base
Sheep blood
Distilled water
Measuring cylinder
Autoclave
Weighing balance
Water bath
Hot air oven ( optional)

Preparation of Chocolate Agar

It can be prepared by the following methods.

Simplest method

Take already prepared blood agar plates (5% sheep blood agar) and put those plates into a hot air oven for 2 hours at 55°C.

Take out those plates and you will get chocolate agar.

Place the plates in sterile plastic bags and store them at 4°C until use.

As a sterility test, incubate an uninoculated plate for 48 hours at 35-37°C with 5% CO₂.

Another Method

Suspend 40.5 grams in 1000 ml distilled water or deionized water.
Heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs. pressure (121°C) for 15 minutes.
Heat-lyse a volume of sheep blood that is 5% of the total volume of media being prepared very slowly to 56°C in a water bath.
Dispense 20 ml into 15×100 mm Petri dishes. Allow the media to solidify and condensation to dry.
Place the plates in sterile plastic bags and store them at 4ºC until use.
As a sterility test, incubate an uninoculated plate for 48 hours at 35-37°C with 5% CO₂ (or in a candle-jar).

Quality Control

For the quality control inoculate N. meningitidis, S. pneumoniae, and H. influenzae QC strains inoculate into prepared chocolate agar (CHOC) for 18-24 hours at 35-37°C with 5%CO₂ (or in a candle jar but it can only provide up to 3% CO₂).

Organisms growth

N. meningitidis luxuriant

S. pneumoniae luxuriant

H. influenzae luxuriant

Colony Characteristics in Chocolate Agar

Haemophilus influenzae: Non-hemolytic, opaque cream to gray colonies.
Neisseria meningitidis: Growth on chocolate agar is grayish, non-hemolytic, round, convex, smooth, moist, glistening colonies with a clearly defined edge.
Neisseria gonorrhoeae: Colonies on CHOC are pinkish-brown and translucent, exhibit smooth consistency and defined margins, and are typically 0.5-1 mm in diameter.

Uses of Chocolate Agar

It is a very useful medium to isolate fastidious organisms in Microbiology Laboratory from various clinical specimens like sputum ( H. influenzae), urethral discharge ( N. gonorrhoeae), CSF/blood (N. menigitidis) .
And thus, Chocolate agar uses to isolate and cultivate fastidious microorganisms such as Haemophilus species and Neisseria species.
It is also useful in isolating N. gonorrheae from both acute and chronic cases of gonococcal infections.
It is also useful in isolating N. meningitidis from bacterial meningitis.
Chocolate agar with bacitracin acts as a selective medium for screening H. influenzae from specimens e.g. sputum containing a mixed flora of microorganisms.
Its modified forms uses given below.

Modification of Chocolate Agar (CHOC)

Thayer-Martin agar/medium uses for the selective isolation of N. gonorrhoeae and N. meningitidis. This Media is a chocolate agar supplemented with vancomycin, colistin, and nystatin (VCN) to inhibit the normal flora, including non-pathogenic Neisseria from the clinical specimens
Chocolate Agar with bacitracin: CHOC with bacitracin is a selective medium uses to improve the primary isolation of Haemophilus influenzae from specimens containing a mixed flora of microorganisms.
Chocolate agar with GC base and growth supplement: It is a medium that supports the special growth requirements (hemin and NAD) needed for the isolation of fastidious organisms, such as H. influenzae, when incubated at 35-37°C in a 5% CO₂atmosphere.
Chocolate agar with TSA and growth supplements: It is a medium that supports the special growth requirements (hemin and NAD) needed for the isolation of fastidious organisms, such as H. influenzae, when incubated at 35-37°C in a 5%CO₂atmosphere.

E. coli on MacConkey agar, blood agar, and Chocolate agar as shown below-

Note: There is no need of using chocolate agar for cultivation of E. coli because it can grow even on an ordinary mediums like nutrient agar, but want to share one thing is that all the organisms growing on nutrient agar/ MacConkey agar/ blood agar can easily grow on chocolate agar but not vice versa.

Campylobacter on chocolate agar-

Satellitism test Positive of Haemophilus influenzae is shown below-

Haemophilus influenzae on Gram’s stained smear under the microscope showing gram-negative cocobacilli , small to the large rod, and pleomorphic forms as shown below-

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