Abstract
Haemophilus species are Gram-negative coccobacilli that require factor X and factor V for growth. Beyond this, it is a finicky bacterium to culture, and any modification of culture procedures greatly reduces isolation rates. Poor quality of laboratories in developing countries results in its poor isolation rates. This study was done with the objective of finding out the optimal cultural environment and media so that it could be maintained for a longer period in economical settings like ours which was done using H. influenzae ATCC 49,766. In this study, several culture media were tested as a means to preserve H. influenzae ATCC like TSB + glycerol + sheep blood, BHI broth, BHI broth + glycerol, BHI broth+ glycerol + sheep blood, Chocolate agar slant and satellitism plate. Three sets of respective media were inoculated with 18 - 24 hours growth of H. influenzae. They were incubated at 37 oC 48 hours in a candle extinction jar. The media were checked for growth by subculturing them on chocolate agar plates and identified by biochemical reactions. Each set was maintained at 2 oC - 8 oC, -20 oC and at room temperature and checked for the viability 24 hourly by subculturing them on chocolate agar. Results showed best growth of H. influenza on chocolate agar slants for 15 - 20 days, followed by BHI + glycerol + sheep blood broth and satellitism plate for 4 - 6 days followed by BHI broth for 2 - 4 days. There was no growth in TSB + glycerol + sheep blood broth and BHI + glycerol broth media. Present study showed similar results as done by NS Srikanth et al. 2003 with growth on chocolate agar & satellitism plate for 3 - 5 days but no growth in TSB + Glycerol + Sheep blood broth media. Chocolate agar slant is by far the most long term preserving media for H. influenzae. However, growth on BHI broth with various modifications is also showed a good preservation for 3 - 5 days, so with further experiments we can hope to maintain the organism in these media also.
Highlights
The genus Haemophilus includes a number of species that cause a wide variety of infections but share a common morphology and a requirement for bloodderived factors during growth that has given the genus its name
H. influenzae was best preserved in the media that were kept at room temperature compared to lower temperatures that is at 2 ̊C - 8 ̊C and at −20 ̊C
H. influenzae remains viable at room temperature for 15 - 20 days in chocolate agar slants, 4 - 6 days in satellitism plate and in BHI + Glycerol & sheep blood
Summary
The genus Haemophilus includes a number of species that cause a wide variety of infections but share a common morphology and a requirement for bloodderived factors during growth that has given the genus its name. Haemophilus species have generally been thought not to make toxins or other extracellular products that account for their ability to produce infection These organisms require hemin (factor X) and/or nicotinamide adenine dinucleotide (NAD+) (factor V) for growth. Whereas NAD+ is released into the medium by red blood cells and is available to the bacteria in blood agar, hemin is bound to red blood cells and is not released into the medium unless the cells are broken up, as in chocolate agar Haemophilus influenzae requires both factors X and V; it grows on chocolate agar but not on blood agar. It may appear on a blood agar plate as tiny satellite colonies around the colonies of other bacteria that have lysed red blood cells [2]. Lack of proper facilities in laboratories of developing countries has resulted in poor isolation rates of H. influenzae
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