It is not difficult to obtain H. pylori colonies, even in blood agar plates. If you have gastric biopsies recently obtained to culture, you can simply pass them with a sterile inoculator loop, over the agar plate. Then you must incubate the plate as soon as possible at 35 to 37ºC inside a box or bag with an microaerophilic atmosphera. There are several pre-prepared commercial sistems or even you can use a candel. After inoculation of the plate, you can prepare a smear to stain wiht GRAM coloration and easily check if you can see the typical shaped rods. So you will confirm previously that you have a positive sample that should grow. After 2 to 5 days of incubation, check the growth of small, brilliant colonies that you can use for the antibiotic tests.

Thank you Isabel.Do you have any experience recovering it from gastric biopsies stored at -70C.

Yes. You can keep the biopsies at -70 degrees. It is very important to freeze the biopsy as soon as possible, in fresh. This will preserve H.pylori alive. Doing in this way you will recover the bacteria many times later, just when you need to do different analysis or studies. Simply defrost to inoculate on to the plate and then keep it frozen again.

Thank you once again for your suggestions.

Guinaz-Bashir, I did a lot of work on Isolation and antimicrobial susceptibilities on H. pylori. I will direct you to the following publications. if further assistance is required, pls feel free to get in touch:

1. Antibiotic Resistance of H. pylor from patients in Ile-Ife, south-west, Nigeria: Aboderin et al., 2007-African Health Sciences 7(5), 143-147

2. Antimicrobial Susceptibility of H. pylori isolates of dyspeptic Nigerian patients. Abdulrasheed Abdu et al. (2005). Tropical Gastroenterology 26(2), 85-88.

3. Helicobacter pylori in the dental plaque and gastric mucosa of dyspeptic Nigerian patients. Ogunbodede et al., (2002): Tropical Gastroenterology 23(3) 127-33

hope these will answer your questions.

Dear Gulnaz,

We cultivated H.pylori in the microbiology dept. at The Aga Khan University Hospital, Nairobi ,Kenya during a doctoral thesis project for Dr Kimanga Nyerere of Jomo Kenyatta University of Agricultural technology in Naiorobi Kenya. The culture is not extremely difficult but not very easy either. Gastric biopsy speciemns have to be inoculated as soon as possible after collection on freshly made supplemented media and incubated in microaerophilic jars.

Kimang’a, A., Revathi, G; Kariuki, S, Shahin, S; Smita, D. (2010). Helicobacter pylori: Prevalence and antibiotic susceptibility among Kenyans.S. Afri. Med. J. 100 (1): 53 – 57.

Thank you Abdu Abdulrasheed and Gunturu Revathi for your comments and suggestions

Thank you for your reply Samaneh .

We are using E-Test to measure antibiotic susceptibility of H. pylori strains in vitro.

One should recognize that while breakpoints have been defined to indicate susceptibility, http://www.eucast.org/clinical_breakpoints/ the method is poorly standardized (choice of media, details of inoculum, and incubation conditions). Whether nonsusceptible strains - using the definition above - are indeed clinically resistant remains to be defined.

A significant reason for the failure to eradicate H. pylori despite in vitro susceptibility may be due to the fact that in vivo conditions (gastric pH, possible low concentrations of antibiotic at the site of the organisms residence) may lead to suboptimal action of antimicrobial drugs.

Thank you Andre. I am currently storing gastric biopsies in BHI broth with glycerol at -70C.Once my consumables for culture are ready, I will start culturing them in batches. I am also planning to do susceptibility by E-test. Thank you for your reference.

We have good experiences too in culturing H.pylori on frozen gastric biopsies at -70°C.

however for unclear reason we tend to loose H.pylori viability after subculturing.

Any experience on what could be the main reasons ?

that is a difficult question - many possible reasons

When subculturing H. pylori we try to limit exposure to ambient ais as much as possible - working rapidly and in small batches. Placing a cotton pad wetted with sterile water in the jar to prevent desiccation of media during incubation. Use an evacuation-replacement system to create the desired incubation atmosphere. Above all: we subculture frequently - prolonged incubation tends to give bigger, but less viable colonies.

Merci André !

Thank you Andre. May need your help once I start my project.

In the attached paper you will find the experience of our group in comparing Helicobacter pylori antibiotic resistances by culture (fenotypic resistance) and genetic mutations in bacterial DNA inducing resistances (genotypic resistances). As you will argue this comparison allows sharing the possibilities and the limits of both investigations. As you can see the main limits of culture is that it represents a complex procedure even in expert hands and the lack of detection of heteroresistance (i.e. simultaneous presence of both susceptible and resistant strains).

Thank You Lerardi for sharing your experience.

I had the little beasties. I also have problems with antibiotics. I took a natural supplement: MASTIC GUM.l. I don't have the reference here, but an article in J Am Med Atss. Compared the gum to several antibiotics. The gum was better. I have been cleari of the beasties for two years

JJS

Thank you John for your reply.

Hi

15th Iranian and International Congress of Microbiology

Tehran university of Medical Sciences (TUMS) during of August 2014 , Iran

Thank you for your comments Lyudmila.

Thanks Amin. We are doing this study for research to generate baseline data for antimicrobial susceptibility in H. pylori in our state and not for diagnostic purpose.

Thank you Seyyed Mehdi for your information.

Dear All

I have started culturing gastric biopsies stored in BHI broth with glycerol for 4-5 months on BHIA with 5 % sheep blood and skirrow's medium with 5% sheep blood. Today was the 5th day of incubation in microaerophilic atmosphere using campygen pouches. However,  could see lot of contaminants growing on BHIA and no colonies or barely visible colonies on skirrow's. Am growing a bit anxious. I am planning to do blind subcultures today. Can anybody give his/ her suggestions.

Thanks Amin

I am using skirrows medium with supplement containing polymyxin B, trimethoprim and vancomycin. On skirrow's medium there is no contamination but no growth either. Contamination is seen on BHIA with blood. 

Dear All

After blind subculturing from Skirrow's onto BHIA,I have grown 3 isolates that are oxidase, rapid urease and catalase positive. On Gram staining they appear cocco bacillary. I have again subcultured them and am planning to  do their sensitivity tomorrow. Can I use these cocco bacillary forms for doing sensitivity

We were doing H. pylori culture from gastric biopsies and susceptibility testing in microbiology lab at the Aga Khan University Hospital Nairobi for a doctoral thesis work of Dr Kimanga Nyerere. It is a very tedious, time and labor intensive process. The resource demand for this work is not sustainable out side a research setting. Usually funding should be sourced as a research grant.

Thanks Revathi

I have received research grant from my institute for this project. Truly it is quite tedious and a costly affair. 

With rapid urease test, catalase and oxidase positive tests can you be 100% sure that the isolate is H.pylori.

I subcultured all the three isolates on Mac Conkey Agar and incubated them aerobically.

All of them showed aerobic growth. Hence, they were not H.pylori but pseudomonads.

 What to do next. ................... 

Dear all

I use Columbia chocolate agar plus VCNT antibiotics and Vitox supplement with good recovery of colonies and scarse contamination. In the case of antibiotic susceptibility testing, we use Columbia blood agar, a young (48 h) and heavy inoculum (McFarland 3.0).