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Year : 2003  |  Volume : 21  |  Issue : 3  |  Page : 221-

Chocolate glycerol broth for maintenance of Haemophilus influenzae

NS Srikanth, R Macaden 
 Department of Microbiology, St. John's Medical College, Bangalore - 560 034, Karnataka, India

Correspondence Address:
N S Srikanth
Department of Microbiology, St. JohnSQs Medical College, Bangalore - 560 034, Karnataka

How to cite this article:
Srikanth N S, Macaden R. Chocolate glycerol broth for maintenance of Haemophilus influenzae.Indian J Med Microbiol 2003;21:221-221

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Srikanth N S, Macaden R. Chocolate glycerol broth for maintenance of Haemophilus influenzae. Indian J Med Microbiol [serial online] 2003 [cited 2020 Oct 28 ];21:221-221
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Dear Editor,

Haemophilus influenzae has been incriminated as the leading causative agent of several diseases in humans, particularly pneumonia and meningitis.[1] Preservation of these strains is essential for further studies. Maintenance of H. influenzae on chocolate agar slope at 35-37C is a recommended method.[2] They can also be maintained on Dorset egg media.[3]

In this study, several preservation methods were tested to devise a means for preserving H. influenzae in small laboratories. Five strains of H. influenzae identified by standard methods[4] were obtained from clinical specimens (CSF-1, blood-1, sputum-3). All five strains of H. influenzae were plated on two sets of chocolate agar, blood agar streaked with Staphylococcus aureus (satellitism), Dorset egg media slants, chocolate glycerol broth, blood glycerol broth and brain heart infusion glycerol broth. They were incubated at 37C in candle extinction jars. After 24 hours incubation, 1 set was incubated at room temperature while the other set remained at 37C in candle extinction jars.

Chocolate and blood glycerol broth were prepared by adding 67.2 mL of glycerol to 100 mL of nutrient broth. After autoclaving, 5 mL of heated sheep blood or normal sheep blood were added respectively using sterile precautions. They were then dispensed in 2-3 mL amounts in previously sterilized screw capped plastic vials.

Daily subcultures were made from the above media onto chocolate agar and incubated at 37C in a candle jar to determine viability. The primary media were incubated till the daily subcultures showed no growth. Standard methods were used to identify the organism after each subculture.

The H. influenzae strains were found to be on chocolate agar - 3 days, satellitism plate - 5 days, Dorset egg slants - 7 days, chocolate glycerol broth - 5 weeks, blood glycerol broth - 2 weeks and brain heart influsion glycerol broth - 1 week, at 37C. H. influenzae remained viable in brain heart infusion glycerol broth alone for 2 days at room temperature. In earlier studies H. influenzae has been found to survive on Dorset egg medium for 21 days, whereas in this study they were viable for one week.

Thus, chocolate glycerol broth and brain heart infusion broth afford economical methods to preserve H. influenzae with easily available ingredients. The advantage of brain heart infusion glycerol broth lies in the absence of requirement for blood and maintenance of viability of H. influenzae for two days at room temperature, allowing transport to a bigger laboratory with adequate facilities. In chocolate glycerol broth, which has been tested for the first time, the H. influenzae remained viable for 5 weeks allowing preservation at 37C.


1Shann F. Haemophilus influenzae pneumonia. Type b or nontype b. Lancet 1999, 353:1488-1490.
2Cheesebrough M. Culturing bacterial pathogens Chapter 7.4. In : District Laboratory Practice in Tropical Countries. Part 2 (Cambridge University Press, Cambridge) 2000:45-62.
3Wasas AD. Use of Dorset egg medium for maintenance and transport of Neisseria meningitidis and Haemophilus influenzae type b. J Clin Microbiol 1999;37(6):2045-2046.
4Betty AF, Daniel FS, Alice SW. (Eds) Gram negative bacilli and coccobacilli (MacConkey - Negative, Oxidase variable) Section 4, In : Bailey and Scott's Diagnostic Microbiology, 10th ed. (The CV Mosby Company, St. Louis), 1998:555.