Use of X, V, and XV Growth Factors Disks for Haemophilus species Identification: Introduction, Species Identification, Keynotes, and Haemophilus Footages

Introduction of Haemophilus

The genus Haemophilus is a pleomorphic Gram-negative coccobacilli fastidious bacteria and thus they do not grow on an ordinary medium like nutrient agar and they need chocolate agar to grow (H. influenzae) due to having X, V, and both X and V growth factors in it. They 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 similarly H. ducreyi is the causative agent of chancroid whereas H. parainfluenzae can also cause pneumonia and endocarditis.  H. aphrophilus causes pneumonia and endocarditis and H. aegyptius is the etiological agent of 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.

Haemophilus species Identification Using X, V, and XV Growth Factors Disks

Isolation of numerous Haemophilus species on the basis of growth characteristics are as follows-

Species	X Disk	V disk	CO2	Hemolysis on horse blood agar
Haemophilus influenzae	+	+	–	–
H. parainfluenzae	–	+	–	–
H. ducreyi	+	–	variable	variable
H. aegyptius	+	+	–	–
H.haemolyticus	+	+	–	+
H. parahaemolyticus	–	+	–	+
H. aphrophilus	+	–	+	–
H. paraaphrophilus	–	+	+	–

Keynotes

1. Haemophilus influenzae is xylose positive while Haemophilus aegyptius is negative.
2. The x-factor is hemin and it is required for the synthesis of respiratory enzymes and is also called a heat-stable growth-promoting substance present in RBCs.
3. V factor is nicotinamide-adenine dinucleotides (NAD)and it is applied as an electron carrier and also known as a heat-labile vitamin-like substance present in red blood cells.
4. Chocolate agar 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).
5. Nutrient agar is used for X, V and XV factors assaying for Haemophilis species identification.

Haemophilus Footagaes-

Haemophilus pleomorphic Gram-negative rods small to large and also coccobacilli in clinical sample sputum Gram-stained smear microscopy

Use of Bacitracin (10U) in chocolate agar for Sputum Culture Screening Haemophilus species

Pure isolated colony of Haemophilus influenzae on chocolate agar demonstration

Oxidase test of Haemophilus showing a positive reaction

Haemophilus influenzae in Gram staining of Culture

Haemophilus influenzae growth around XV and also between X and V growth factors disks

Satellitism Test Positive Haemophilus influenzae Demonstration

Haemophilus influenzae Antibiogram on modified MHA agar (chocolate agar)

Various Antisera (a to f) of Haemophilus influenzae Demonstration

Haemophilus parainfluenzae colony morphology on chocolate agar

Haemophilus parainfluenzae growth around V and XV disks

Haemophilus ducreyi growth around X and XV disks on MHA

Further Readings

1. Bailey and  Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
2. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
3. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
4. Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
6. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
7. Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
8. District Laboratory Practice in  Tropical Countries  –  Part-2-   Monica Cheesebrough-   2nd Edn Updated
9. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
10. Medical Microbiology-The Practice of Medical Microbiology Vol-2-12th Edn. –Robert Cruickshank