PAGES: 583-586 DOI: Full paper
A Comparative Study of Preservation and Storage of Haemophilus influenzae

Olga C Aulet de Saab, Marta C de Castillo +, Aida P de Ruiz Holgado, Olga M de Nader

Instituto de Microbiología "Dr. Luis C. Verna", Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Ayacucho 491, (4000) San Miguel de Tucumán, Argentina


The aim of this study was to compare the efficacy of conservation by freezing the strains of Haemophilus influenzae at -20ºC and -70ºC.

Skim milk supplemented with glucose, yeast extract and glycerol allowed highest viability of H. influenzae both at -20ºC and -70ºC from the media analyzed. Trypticase soy broth and brain heart infusion broth supplemented with glycerol, allowed excellent recovery. Use of cotton swaps as supporting material, with or without addition of cryoprotective agents, did not modify H. influenzae viability after six months of storage. Concentration of the initial inoculum positively affected viability when stored at -20ºC. Initial concentration did not influence survival after storage at -70ºC. Thawing at room temperature should not exceed 3 h as to get highest survival percentage.

Haemophilus influenzae is an etiological agent of a wide variety of infectious diseases in human beings, from non-complicated ones such as conjunctivitis and otitis media, to invasive, potentially life-threatening ones like bacterial meningitis and acute respiratory tract infections. This pathogen holds a serious agent responsible for numerous infections in paediatric patients in the Tucumán province, Argentina, mainly in infants less than two years of age.

It is very important to maintain strains isolated from this region viable, in order to observe physiopathogenic aspects, virulence factors and anti-microbial resistance. As these microorganisms are demanding in their nutrition requirements, they need well-supplemented, complex culture media for isolation. Due to the difficulties presented by H. influenzae with respect to its preservation and storage through time, an analysis of factors enhancing recovery of the microorganism after freezing has been proposed. Freezing is a very common way of preservation and storage of microorganisms (Calcott 1986).

The aim of this study was to carry out a comparative study of the efficiency of preservation of H. influenzae at -20ºC and -70ºC, evaluating the influence of (1) different suspension media, with or without addition of protective agents; (2) supporting material; (3) initial inoculum concentration; and (4) thawing time at room temperature of the stored strains. Statistical analysis of the results is given.




This study was carried out with five H. influenzae type b strains, random by selected from isolates from clinical samples from patients treated for different infectious diseases at Niño Jesús Children's Hospital, Tucumán, Argentina.

Methodology - Samples were subjected to a direct bacteriological assay and then seeded on chocolate agar plates. Plates were incubated at 35-37ºC for 24 h under a humid and CO2 (5-10%) atmosphere. Serological classification of the strains studied was carried out with monovalent anti-
sera, using agglutination techniques (Bacto-Haemophilus influenzae Antiserum Type a, b, c, d, e and f, Difco (Campos 1999).

Suspension media and cryoprotective agents

Suspension media assayed were brain heart infusion broth (BHI) and trypticase soy broth (TSB), either with or without addition of one or more of the following cryoprotective agents: 4% milk, 6% lactose, 10% sucrose, or 25% glycerol. A third medium assayed consisted of 10% skim milk, supplemented with 1% glucose, 0.5% yeast extract and 10% glycerol (MGY).

Methodology - Selected strains were transferred onto BHI chocolate agar, supplemented with 1% yeast extract (BYB), and incubated at 35-37ºC for 24 h. Microorganisms were then harvested, suspended at a concentration of 1015 cells/ml in 0.3 ml of each suspension medium, supplemented or not with cryoprotective agents, and stored at -20ºC and -70ºC. Viable cell counts were carried out after 0 (initial inoculum), 10, 30 and 60 days and 4, 6 and 12 months of storage to determine viability.

Supporting material

Cotton swaps were used as supporting material, either untreated or soaked in BHI + 25% glycerol and MGY.

Methodology - A H. influenzae suspension of 1015 CFU/ml was obtained in BHI; 0.1 ml was seeded on BYB agar plates, spread with a Drigalsky spatula, and incubated at 35-37ºC for 24 h. With a single cotton swap 25% of the plated cultures was harvested; the swaps were then placed in sterile tubes and stored at -20ºC and -70ºC. Viability was studied after 24 h, and 1, 2, 3 and 6 months of storage, placing the swaps in 1 ml of BHI. Suspensions were homogenized by vortexing, and viable cell counts were carried out immediately afterwards.

Influence of initial inoculum concentration

H. influenzae viability at different initial inocula was studied in BHI + 25% glycerol and MGY.

Methodology - Microorganisms were suspended at final concentrations of 102, 104, 108 and 1015 CFU/ml in either of the media assayed; 0.3 ml of each concentration was stored at -20ºC and -70ºC. Viable cell counts were carried out after 0 h (initial concentration), 24 h, and 1, 3 and 6 months of storage.

Influence of thawing at room temperature

Methodology - Suspensions of H. influenzae (1015 CFU/ml) in BHI + 25% glycerol were prepared and then 0.3 ml aliquots were stored in cryo-vials at -20ºC and -70ºC. After 30 days the samples were left at room temperature for 15, 30 and 60 min, and 3, 6, 12, 24, 48 and 72 h, avoiding agitation and formation of foam. Viable cell counts were carried out after each thawing time.

Viable cell counts - Viable cell counts were carried out in duplicate on BYB according to the technique by Clark et al. (1987).

Statistics - Factor analysis with three variables (Sigma Stat) was used to analyze the influence of suspension medium, protective agents, supporting material, initial inoculum concentration, and thawing at room temperature. Percentage of viability was studied at different temperatures and after different storage times.



Influence of suspension media and cryoprotective agents on survival of H. influenzae

Storage at -20ºC - Results are shown in Tables I and II. BHI and TSB without addition of cryoprotective agents as suspension media showed a substantial loss in viability; only 23.3 and 10% respectively was recovered after 60 days. The same media, but with addition of 25% glycerol, yielded a recovery of viable cells of 98 and 100% respectively after 60 days. After 12 months recovery dropped to 66% for BHI-glycerol and 68.6% for TSB-glycerol. Addition of sugars yielded a satisfactory recovery after four months of storage (between 60 and 73.3%), decreasing to 42% or less after 12 months. Supplement of 4% milk demonstrated a similar behaviour. Addition of glycerol significantly increased survival of the microorganism through time. MGY as suspension medium yielded excellent viability after 12 months (86.3%).

Storage at -70ºC - BHI and TSB supplemented with sugars and/or glycerol allowed a 100% recovery of H. influenzaeafter 12 months of storage. After storage of BHI and TSB without supplements, viability of the microorganism was only 20% after one year (results not shown).

Factor analysis between suspension media with or without addition of cryoprotective agents, storage time and storage temperature revealed significant differences (p<0.001) in the percentage of survival of H. influenzae (results not shown).

Influence of supporting material

Statistical analysis showed no significant difference in viability of the strains stored at -20oC and -70oC with or without supporting material and supplemented or not with cryoprotective agents; and it was possible to recuperate more than 90% of the viable strains in all the cases after six months of storage (results not shown).

Influence of initial inoculum concentration

At -20oC - At an initial concentration of 1015 CFU/ml in BHI + 25% glycerol or MGY, 90% was recovered after six months; more than 75% was recovered after three months at an initial concentration of 108 CFU/ml in both media, supplemented with glycerol. Starting with an inoculum of 102 CFU/ml in BHI + 25% glycerol or MGY, recovery percentages of 15 and 40 were obtained respectively after 6 months (Figs 12).

At -70oC - Initial concentration and medium did not influence viability, because after six months a recovery of 100% was obtained for either medium and all the initial inocula assayed (results not shown).

Significant differences (p<0.001) in viability were observed for the various initial inocula when stored at -20oC. Storage at -70oC did not show significant differences in survival for initial concentrations.

Influence of thawing at room temperature

Strains stored at -20oC showed a recovery of more than 90% after thawing at room temperature for 3 h. Viability decreased as thawing time increased, dropping to 59% after 12 h and disappearing completely after 24 h. At -70oC viability patterns were similar (Fig. 3).

Statistical analysis of the influence of thawing time at room temperature showed significant differences (p<0.001) for the percentage of H. influenzae survival both at -20oC and -70oC.



Use of cryoprotective agents in preservation media was first implemented in 1941 when Woodcock et al. used sugars in order to protect erythrocytes from freezing injury. Since that time numerous researchers have used different additives to enhance survival of frozen bacteria. Ling (1965), Merymann and Hornblower (1972), Pribor (1974) and Nei (1981) consider that freezing injury is less with addition of cryoprotective agents such as glycerol, sugar or milk, that inhibit intracellular iceforming during rapid freezing. Addition of carbohydrates decreases inactivation of dried Loctobacillus plantarum cells due to dehydration (Lievense et al. 1992, Leoni Linders et al. 1997). In our study addition of glycerol allowed hight recovery of the frozen microorganisms, with coincides with results obtained by Castillo et al. (1985).

Major (1953), Major et al. (1955) and Castillo et al. (1985) maintain that an increase in the initial cell concentration enhances the percentage of viable microorganisms after freezing, which agrees with our results.

During thawing alterations in cell wall permeability occur, and therefore careful precautions should be adopted. Agitation, foam formation and sudden temperature changes should be avoided, in order to prevent bacterial injury (Wagman 1960). Our results indicate that microorganisms should not be left for more than 3 h at room temperature in order to achieve highest recovery.

From the results obtained in the present study we recommend (a) starting with a concentrated inoculum; (b) suspending viable cells in MGY, BHI or TSB, the two latter ones supplemented with 25% glycerol; and (c) thawing at room temperature for not more than 3 h in order to obtain a satisfactory survival and assure preservation of H. influenzaestrains through time at -20oC and -70oC.



To Mr Eric Fengler for language advice and drawing of the graphics.



Calcott PH 1986. Cryopreservation of microorganisms. CRC Crit Rev Biotechnol 4: 279-298.

Campos JM 1999. Haemophilus. In RP Murray, J Baron, MA Faller, FC Tenover, RH Yolken (eds), Manual of Clinical Microbiology, 7 ed., ASM Press, Washington, p. 604-613.

Castillo MC de, Nader OM de, Gutiérrez RC de, Ruiz Holgado AP de 1985. Estudio comparativo de métodos de conservación aplicados a Neisseria gonor-rhoeaeBact Clin Argentina 3/4: 54-60.

Clark WS, Brazis AR, Fowler JL, Johns CK, Nelson FE 1978. Standard plate count methods. In EM Marth,Standard Methods for the Examination of Dairy Products, 4th ed., American Public Health Association, Washington, p. 77-94.

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Ling GN 1965. Physiology and anatomy of the cell membrane: the physical state of water in the living cell. Fed Proc 24 (Suppl. 15): 103-112.

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Meryman HT, Hornblower M 1972. Changes in red blood cells following rapid freezing with extracellular cryoprotective agents. Cryobiology 9: 262-267.

Nei T 1981. Mechanism of freezing injury to erythrocytes. Effect of initial cell concentration on the post thaw hemolysis. Cryobiology 18: 229-237.

Pribor DB 1974. PVP contrasted with dextran and the multifactor theory of cryoprotection. Cryobiology 11: 60-72.

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Woodcock AH, Thistle MW, Cook WH, Gibbons NE 1941. The ability of sheep's erythrocytes to survive freezing.Canad J Res 19 D: 206-212.


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