Maintenance of Helicobacter pylori Cultures in Agar Stabs

Abstract

Background

Helicobacter pylori requires frequent passage at 37 °C with reduced oxygen tension to maintain viability, and recovery from frozen stocks can be unpredictable and slow. Agar stab cultures were assessed as a possible means of maintaining viability without the need to passage every 4–7 days.

Materials and Methods

Agar stabs prepared from either Brucella or Brain Heart Infusion media were inoculated deeply with H. pylori strains or H. felis and grown under varying conditions for up to 13 weeks. Subcultures were prepared from these stabs at various intervals to test for viability.

Results

Established cultures in agar stabs failed to survive at room temperature but did survive at 37 °C with 10% CO₂ for up to 56 days. H. felis remained viable for up to 28 days. No difference was observed between the two media formulations.

Conclusion

H. pylori grown in agar stabs remains viable for prolonged periods of time without the need to subculture and may represent an improved method for storing H. pylori for infrequent use.

Helicobacter pylori (H. pylori) can be maintained on both solid [1-4] and liquid media [5-7] but maintaining viable bacteria for prolonged periods can be expensive or impractical. Growth of plate cultures of H. pylori under aerobic conditions has been reported [8], and we routinely grow cultures in standard tissue culture incubators set at either 5% or 10% CO₂ (unpublished results). Most laboratories however must rely on the use of closed container systems complete with gas generating envelope technology to create the appropriate microaerobic environment. These envelope systems can constitute significant costs when performing frequent or multiple cultures. Additionally, one of the manufacturers of these systems has recently discontinued their product, resulting in limited options in the market place.

Continuous culture of H. pylori requires frequent subculture of viable bacteria to fresh plates. Many laboratory and pathogenic bacterial strains can be kept viable on sealed plates and stored at 4 °C to arrest growth. H. pylori survival at 4 °C, however, varies significantly with the media being used and it typically dies rapidly within 2 days [3,9-12]. Optimal methods for storing plate cultures at 4 °C have achieved viability for up to 9 days [13]. Typically, however, H. pylori must be passaged every 3–5 days to prevent the culture from dying. Although liquid cultures of H. pylori can be maintained in standard tissue culture incubators under static conditions, long-term maintenance requires frequent passage and ultimately consumes media supplemented with fetal bovine serum. While many of these issues can be circumvented simply by recovering H. pylori from frozen stocks when needed, viability varies depending upon the freezing media being employed [5,14,15]. Additionally, recovery times can take up to 7 days, result in light growth, and occasionally fail outright (unpublished observations).

The purpose of this study was to assess a mechanism for maintaining viable H. pylori cultures in the laboratory without having to perform frequent passaging. We now report that H. pylori strains grown in agar stabs can be stored for at least 8 weeks in agar stab cultures kept at 37 °C.

Methods

Bacterial Strains

Helicobacter felis was obtained from a domestic cat by endoscopy as previously reported [16]. H. pylori strains included clinical isolates p82 and p89 from our own H. pylori culture archive, one of our own mouse-adapted clinical isolates, HpM5 [17], the widely used mouse-adapted H. pylori Sydney Strain (HpSS1) [18], and two common laboratory strains, H. pylori 26695 (Manassas, VA), and H. pylori 60190, (a generous gift from Dr. Richard Peek, Vanderbilt University, Nashville, TN, USA).

Plate Cultures

All bacteria were initially grown on Columbia blood agar (Difco, Detroit, MI, USA) containing 7% defribrinated horse blood (HemoStat laboratories, Dixon, CA, USA), amphotericin B (2.5 μg/mL) and selective antibiotics trimethoprim (20 μg/mL), and vancomycin (6 μg/mL). Plates for H. pylori also contained cefsulodin (16 μg/mL), whereas plates for H. felis contained Polymyxin B (0.125 μg/mL). Antibiotics were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cultures were grown in a designated CO₂ incubator with humidity tray at 37 °C and 10% CO₂ for 72–96 hours.

Agar Stabs

Agar stabs were prepared from Brucella broth containing 0.6% agar. Media was dispensed at four ml per tube into 5-mL round-bottom 12 × 75 mm polypropylene tubes with snap on caps (BD Biosciences, San Jose, CA, USA). Agar stabs were inoculated by transfer of a heavy and visible bacterial inoculum from plate cultures. A 1-μL disposable loop was used to make several 5-cm stabs with a single inoculum. Stab cultures were grown at either room temperature or 37 °C. Each strain was examined in duplicate for each condition being assessed.

Results

Long-term Culture of H. felis and H. pylori in Agar Stabs

Two clinical isolates of H. pylori, p82 and p89, as well as H. felis, were grown on Columbia blood agar with their respective selective antibiotics. Plates exhibiting good growth at 3 days were selected for inoculation. Visible amounts of bacteria were removed from each plate using 1-μL disposable bacterial inoculation loops and used to passage bacteria to sets of agar stabs of either Brucella agar or Brain Heart Infusion agar. Each tube was stabbed approximately 5 cm deep three to four times with a single inoculum of bacteria. The tubes were capped loosely and placed in a tissue culture incubator complete with humidity pan and set at 37 °C with 10% CO₂ for 7 days. Sets of paired tubes were split on day seven with a duplicate set remaining in the incubator and another set removed and placed on a laboratory bench at room temperature for the duration of the experiment. Cloudy growth was evident within the top 0.5 cm of the agar and to a lesser extent along the stabbing track throughout the depth of the agar. Samples were removed from each tube periodically for passage onto Columbia blood agar plates by inserting a disposable inoculation loop into the top 0.5 cm where cloudy growth was most evident and transferring to plates. No viable bacteria could be recovered from any of the samples stored at room temperature (Table 1). However, all three bacteria were recovered at days 14 and 28 from samples stored in the incubator. On day 49, strains p82 and p89 were found to be viable but H. felis failed to grow. No bacteria could be recovered from any sample at day 91. Results were the same for bacteria grown in Brucella agar or Brain Heart Infusion agar stabs.

Table 1.

Viability of H. pylori and H. felis in agar stabs under differing temperatures

	Room temperature/ atmosphere days				37 °C, 10% CO2 days
	14	28	49	91	14	28	49	91
B-Agar
H. felis	−	−	−	−	+	+	−	−
p82	−	−	−	−	+	+	+	−
p89	−	−	−	−	+	+	+	−
BHI-Agar
H. felis	−	−	−	−	+	+	−	−
p82	−	−	−	−	+	+	+	−
p89	−	−	−	−	+	+	+	−

Long-term Viability of H. pylori in Sealed Vials

Storage of stab cultures with loose caps for multiple weeks resulted in evaporation of water from the agar and a noticeable change in agar volume in the previous experiment. A reduction in water content increases the concentration of agar and other media components and may impact its suitability for sustained growth of Helicobacter organisms. Therefore, the experiment was repeated to compare bacterial stabs stored with loose caps to those stored under similar conditions with tightly sealed caps. H. felis, along with common laboratory strains of H. pylori including HpSS1, 26695, and 60190, was passaged to Brucella agar stabs and placed in the same incubator. Sets of companion stabs were stored with the manufacturer’s snap on cap firmly in place to provide a water tight seal. No difference in viability was observed when the method of tube capping was compared (Table 2). All four strains were viable at day seven. The three H. pylori isolates remained viable at subsequent time points of days 28 and 56. Small numbers of colonies were recovered from only one strain, 60,190 at 100 days.

Table 2.

Viability of H. pylori and H. felis in sealed agar stabs

	Airtight caps days			Gas exchange days
	7	28	56	7	28	56
H. felis	+	−	−	+	−	−
HpSS1	+	+	+	+	+	+
26695	+	+	+	+	+	+
60190	+	+	+	+	+	+

Discussion

Many studies have been performed to evaluate the utility of various media for the isolation and recovery of H. pylori from gastric biopsies and for maintenance in the laboratory [1-7]. Most laboratories that routinely work with H. pylori isolates have adopted rich medias designed for the growth of fastidious organisms supplemented with sheep or horse blood when using agar-based media, or supplemented with horse serum or fetal bovine serum when growing in liquid cultures. Long-term storage of H. pylori isolates has been less successful. H. pylori cultures do not survive longer than 12 hours at room temperature or for more than 48 hour at 4 °C [3,9-12]. The preparation of frozen stocks held at −70 °C is widely used and represents the only practical means of storing viable H. pylori but successful recovery of isolates from the frozen state can vary among isolates. Additionally, recovery from frozen stocks is often slow, requiring up to 7 days to generate cultures suitable for secondary passage. There is also always the potential for freezer failure or loss of liquid nitrogen in deep freeze systems that can result in the complete loss of important or unique strains. A reliable stab culture storage system could serve as a back-up for such critical strains.

Conditions for achieving prolonged viability in culture have been investigated in the context of developing transport media for H. pylori but these cultures generally remained viable for only several days [12,13]. A previous study in which transport media was tested for long distance shipping achieved some success in maintaining viability for several weeks [19]. Bacterial isolates grown on either plates or slants in Ireland were shipped to Ireland, France, and China where cultures were tested for viability through passage to fresh plates. Isolates received on slants within 6 days were recovered at rates of 90–100% when excluding contaminated samples, whereas isolates received on plates were recovered at rates of 30–100%. Notably, slants received at day six and then subsequently stored at 4 °C remained viable at 50% for four more days and 10% for nine more days for a total of 10 and 15 days, respectively. These results suggested that under conditions of sufficient nutrients, moisture, and environment, H. pylori might remain viable for prolonged periods without the need for frequent subculture.

This study demonstrates that H. pylori grown in deeply inoculated agar stabs remains viable for at least 8 weeks when kept at 37 °C. The identical results achieved with both brucella agar and brain heart infusion agar indicate that other nutrient rich medias commonly used for the growth of H. pylori would also be successful. Although prolonged viability at 4 °C might be possible, those conditions were not assessed in this study. The requirements for growth are minimal when compared to other forms of culture, including only the agar stabs and a 37 °C CO₂ incubator. Liquid cultures can also be grown in tissue culture incubators but require expensive animal sera and frequent passage. Although some laboratories have been able to grow H. pylori on agar plates in incubators at 5% or 10% CO₂, this does not seem to work uniformly well for many laboratories. The only recourse has been to grow H. pylori under microaerophilic conditions using expensive jars and gas generating envelope systems. These systems were not necessary to start and maintain cultures in agar stabs.

This new method has several benefits in addition to limiting the use of expensive gas generating systems and reducing the amount of media supplements required for frequent passage. It may serve to improve the survival rates of cultures that are shipped between laboratories, and it would also reduce the need to start new cultures from frozen stocks. Finally, it would provide a more predictable means of starting cultures to coordinate with timing of specific experimental needs. The use of agar stabs, therefore, will provide the microbiologist with an additional tool with which to maintain, share, and experiment with H. pylori.