Helicobacter Pylori Causative Factor For Peptic Ulcer Disease Biology Essay


Helicobacter pylori (Hp) is a well-recognized gastroduodenal pathogen and a class I carcinogen which successfully colonizes the harsh acidic environment of the stomach. Hp is the causative factor for peptic ulcer disease (PUD) and an independent risk factor for gastric adenocarcinoma development. Therefore, accurate detection of infection is crucial for devising eradication regimens and preventing the more severe GI complications.

Detection of Hp in the gastric mucosa can be performed via 1) direct detection of the bacterium; bacterial culture, histology and polymerase chain reaction (PCR)or 2) indirect detection of its enzymatic products particularly urease as well as serum Hp-specific immune response, which can be detected by, rapid urease test (RUT) and serology respectively.

The accuracy of these diagnostic tests are reported as follows: 98.1% (bacterial culture),98.1% (histology), 94.3% (PCR), 96.2% (RUT), serology (84.9%) [3] .

The sensitivity and specificity of RUT, culture, histology, UBT and serology are 100% and 100%, 77.3% and 100%, 81.8% and 94.6%, 90.9% and 91.9%, 77.3% and 97.3% respectively [4] .

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Bacterial culture is preferable for: 1)Diagnostics: to determine its antibiotic sensitivity and devising proper eradication regimens; 2)Research: to investigate microbial host-cell interaction, pathophysiology, growth requirements and metabolism; and 3) Production: vaccines and therapeutics [5] , [6] .

Despite these advantages, Hpculture from gastric biopsy specimens is becoming less popular among clinical laboratories for its time/care demanding and fastidious nature. Some of these disadvantages include requirements for 1) special transport conditions, (2) rapid sample processing, (3) costly and complicated culture media with specific incubation conditions, 4) lengthy time before confirmation and devising treatment regimens for patients.

Hp is a fastidious organism which requires a microaerophilic environment and complex nutrient media for its growth. Several selective and differential media have been evaluated and recommended [7] , [8] , [9] , [10] , [11] , [12] . These media usually (but not exclusively) are based on Columbia, Brucella or brain heart infusion agar base containing either blood or blood products [13] or an additive, such as starch, charcoal, cyclodextrin, or bovine serum albumin (BSA). These media, depending on the conditions, can be supplemented with antimicrobial agents to prevent the overgrowth of contaminants, present in the gastrointestinal tract and/or the endoscopy/transportation environment.

Rapid urease test (RUT) is the most reliable indirect means of detecting viable Hp organisms in the gastric lumen. This test is highly preferred by the endoscopists due to its ease of use, rapidity, and cost-effectiveness. Hp produces abundant amounts of cell surface-associated urease as a catalytically active heterodimer. It is estimated that urease comprises more than 5% of bacterial protein [14] . In the gastric environment urease catalyzes urea into carbon dioxide and ammonia. The produced hydroxyl ions then react with carbon dioxide, producing bicarbonate, which neutralizes gastric acid, providing means for successful colonization of Hp in the otherwise acidic gastric lumen.is dependent on the acid-neutralizing activity of its urease. Such that urease-negative Hp mutants fail to cause persistent infection in Hp animal models (ref).

In this section, we present the methodology for Hp bacterial culture and rapid urease test on gastric biopsy specimens (RUT).

Serologyis the most widely used method of detecting Hp infection and is most applicable for non-invasive population screening approaches as well as monitoring success. of bacterial eradication regimens. After successful recovery of Hp strains from agar plates, first serological results were published [2007; 17, 18]. The applicable biopsy based invasive methods for detecting active infection (the gold standard tests), prevalence rate of Hp infection in different geographic regions and the studied population are the main considerable factors in developing serology diagnosis tests.

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Systemic tests such as urea breath tests (UBT) and stool antigen assays are preferred for detecting acute infection as well as monitoring treatment success. Nevertheless, serology is the method of choice because of its cost efficiency and ease of performance applicable in low-tech laboratories making it a powerful tool in epidemiological screening programs and vaccination studies [2009; 37]. Disadvantages of serology include false negative results (in case of recent eradication or anti-acid treatment) [2007; 20] or false positive results (due to the prolonged persistence of Hp specific antibodies despite its successful eradication)which is a major consideration in its applications [2007; 21].

Most commercial serology assays detect Hp-specific IgG antibodies whereas detecting specific Hp IgA molecules will just provide a small increased sensitivity of the test [2007, 19].

Different antigens

Previous studies have recommended severalHp antigens/immunogens for incorporation into serology assays. These antigens are presented as components of intact cells, sonicated cell extracts as well as purified antigens in native or recombinant format [2007; 19]. The applied antigens are secretory or surface proteins such CagA, Urease subunits, heat shock proteins, catalase and other immuno-reactive antigens.

Why a mixture of native antigens

It has been suggested that serological tests including a mixture of different Hp antigens have higher immunologic criteria due to simultaneous assess of antibodies toward different conserved and non conserved proteins [Michel, 2009; Selgrad, 2009].Extensive antigenic heterogeneity amongst Hp strains isolated from different geographic locations has created demand for development of home made assays incorporating antigens from local clinical strains.

Different methods:

Amongst the various Hp-specific serology assays, enzyme immunoassays (EIA) with varying immunologic criteria.aremost frequent with sensitivity and specificity range from 60 to 100% [2007, 19]. According to a meta-analysis carried out by Leal et al, the immunological criteria of different sero-diagnosis tests were evaluated [2009; 27]. The obtained results showed that western blot tests have high immunologic criteria in comparison with other included assays. In contrast,commercial IgG based enzyme linked immunosorbent assays (ELISA) showed low sensitivity but high specificity with varied performance due to the heterogeneity of included antigens originated from different Hp strains from various geographic locations.This analysisPrevious studies have shown that home-made ELISA tests using native whole cell extracts has the highest diagnostic accuracy amongst other serologic assays [2009; 27]. It also has been documented that tests based on whole blood samples or detecting Hp antibodies in urine or saliva do not have the same criteria in comparison with home made tests and they can not replace current sero-diagnosis approaches [2007, 20].

2. Materials

2.1. Rapid Urease Test (RUT) media

Dissolve the following ingredients in 800ml dH2Oand bring up the volume to a total of 1 Litre: 0.1g yeast extract, 20.0g urea, 0.091g monopotassium phosphate (KH2PO4), 0.095g disodium phosphate (Na2HPO4), 0.01g phenol red. Adjust the pH to 6.9 (See Note [15] ).

Sterilize the solution by passing it through 0.45-μm filters (Solution D).

Aliquot solution D in sterile microtubes (600μl/tube).

Store stock solution in dark bottles at 4°C (See Note [16] , [17] )for a maximum of 48 hours. However, it is best used on the day of preparation (See Note [18] ).

2.2. Transfer media

Dissolve the following ingredients in 150 ml dH2O and bring up a total volume of 200ml : 2g casamino acid, 2g peptone, 0.4g yeast extract, 1g NaCl, 0.32g agar.

Autoclave at 120°C, 15 psi (1.05 kg/cm2) for 20 min, cool and store at 4°C (Solution A).

Dissolve 0.04g L-cystein in 20ml dH2O and adjust pH to 7.0. Add 0.2g glucose to the solution and bring up the volume to a total of 40 ml. Sterilize the solution by passing it through 0.45nm filter (Millipore) and store at 4°C (Solution B).

Combine solutions A and B and add 28ml autoclaved glycerol (87%) to the combination (Solution C).

Aliquot solution C in sterile microtubes (600μl/tube).

This media can be stored at 4°C for up to 6 months (See Note [19] ).

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2.3. HPSPA Culture Media (H. pylori Special Peptone Agar) (See Note [20] ).

1. Rehydrate specified amounts of Brucella agar (as directed by the manufacturer) with 900 ml dH2O (for a final volume of 1 Litre).

2. Add 3.0g yeast extract and 5.0g beef extract as growth supplements.

3. Autoclave at 120°C, 15 psi (1.05 kg/cm2) for 20 min. Cool down to 56°C in a water bath (See Note [21] ).

4. Dissolve 0.5g ferrous sulfate in 2-4 ml dH2O and sterilize by passing through 0.2-μm filters. Add to the cooled culture media.

5. Dissolve 0.5g sodium pyruvate in 2-4 ml dH2O and sterilize by passing through 0.2-μm filters. Add to the cooled culture media. (See Note [22] )

6. Prepare the following stock solutions in dH2O and sterilize by passing through 0.2-μm filters: 5mg/ml fungizone, 5mg/ml vancomycin, 10mg/ml trimetoprim. Add to the culture media; 400μl of fungizone, 1.2ml vancomycin and 500 μltrimetorpim from these stock solutions to create the following final concentrations: 6.0 mg/L vancomycin, 20.0mg/L trimetoprim, and 2.0 mg/L fungizone.

6. Finally add 70 ml (7%) defibrinated sheep blood (5-10% is desirable). Mix gently and dispense 15-20 ml per plate (15100 mm) under sterile conditions. Following solidification in room temperature, stock and store in plastic bags at 4°C for up to one week (See Note [23] , [24] ).

2.4. Helicobacter pylori Identification

A. Gram staining

Gram staining materials may be purchased commercially or prepared in the laboratory.

Crystal Violet Staining Solution

Dissolve 2.0g crystal violet in the 20ml alcohol (95%).

Dissolve 0.8g ammonium oxalate in 80ml dH2O.

Mixing the above two solutions. The mixture is stable for 2-3 yrs at room temperature.

Iodide Staining Solution

Dissolve 2.0g potassium iodide in 2-3 ml dH2O (the crystals will dissolve and the solution temperature will fall).

Dissolve 1.0g iodine crystals in the concentrated potassium iodide solution. Dilute the mixture to 300 ml with dH2O.

Destaining Solution (Acetone-Alcohol)

Mix 100 ml acetone (reagent grade) with 100 ml ethanol (95%). Combine in a brown-glass bottle, label with 1-year expiration date, and store at room temp.

Counterstaining Solution

- Dissolve 0.25g safranin in 10 ml ethanol (95%). Dilute the mixture up to 100 ml with dH2O.

B. Catalase test (See Note [25] )

- Prepare 3 - 6 % percent hydrogen peroxide solution (supplied in various concentrations by commercial manufacturers) (See Note [26] , [27] ).

C. Oxidase test (See Note [28] )

There are different disks that are commercially available and may contain N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-Dimethyl-p-phenylenediamine (DMPD) (BBL, Difco, REMEL), which are also a redox indicator. The reagent is a dark blue to maroon color when oxidized, and colorless when reduced.

3. Methods

The entire handling of this section should be conducted under a horizontal laminar-flow cabinet with a high-efficiency particulate air (HEPA) filter which provides contamination-free handling of sterile media and accessories.

Flame sterilization can be additionally used to further decontaminate the immediate working environment, in order to (1) eliminate externally introduced contaminants from the exposed sides of media bottles, culture flasks, or test tubes, (2) sterilize small instruments such as forceps and wire inoculating loops.

3.1. Transfer of gastric specimens

Gastric specimens are obtained by medical experts during gastroscopy, preferably according to Sydney protocols (Correa, 2003, See Note [29] ).

RUT analysis (See Note [30] )

Place the biopsy obtained from the IncisuraAngularis (See Note [31] ) in microtubes containing RUT media by sterile forceps (See Note [32] ).

The color change of RUT media from pale yellow to deep pink is considered as a positive test result (See Note [33] , [34] , [35] ).

B.Transfer of gastric specimens (See Note [36] 

Place gastric biopsies in microtubes containing transfer media (Solution C) by sterile forceps and transfer them to the bacterial culture lab within 4 hours (See Note [37] ).

Mince and homogenize biopsies with tissue grinder (Wheaton, Millville, NJ) in the transfer media (Solution C) prior to culture (See Note [38] ). Thoroughly pipette the homogenized biopsy.

Dispense and spread 300 μl of homogenized biopsies on HPSPA culture plates (See Note [39] ).

3.2. Culture Media

1. Keep inoculated culture media in CO2 Incubators at 37°C. (See Note [40] )

2. Turn over culture media after one day when homogenized biopsies absorbed to the media (See Note [41] ).

3. Check media for detecting Hp colonies after 5-7 days of incubation (See Note [42] , [43] ).

3.3.Helicobacter pylori Identification

Culture H. pylori suspected colonies on without antibiotic media and keep inoculated culture media in CO2 Incubators at 37°C, for further analysis.

A. Gram staining

Smear preparation (see Note [44] ): Place a drop of sterile saline or water on the slide, transfer a sample of Hp colony using a sterile applicator, wire needle, or loop. Gently mix to emulsify. (See Note [45] ). Avoid mixing vigorously and creating aerosols.

Air dry smears on a flat surface. Fix the slides by passing them two or three times through a flame (See Note [46] ). To avoid distortions, do not overheat. Allow slide to cool before staining.

Immerse the fixed slides in the crystal violet solution; allow the stain to remain for 30 s.

Discard the solution, and rinse slide gently under running water (See Note [47] ).

Rinse off excess water with iodine solution, and then flood the slide with fresh iodine solution. Allow the iodine to remain for 30 s.

Rinse gently with flowing tap water.

Decolorize by letting the reagent flow over the smear while the slide is held at an angle. Stop when the runoff becomes clear. Remove excess decolorizer with gentle flow of tap water (See Note [48] 

Flood the slide with safranin and allow counterstain to remain for 30 s.

Remove excess counterstain with a gentle flow of tap water.

Drain slide, and air dry in an upright position. Clean off the bottom of the slide by wiping the alcohol or aceto ne on a tissue or paper towel.

Examine the smear microscopically.

Microscopy of gram-stained smears reveals curved gram-negative rods resembling Helicobacter.

B. Catalase test

Discrete bacterial colonies on solid medium (See Note [49] ).

Place approximately 0.2 ml of hydrogen peroxide solution in a test tube

Carefully pick a colony to be tested with a wire/loop or disposable alternative

Rub the colony on the inside wall of the bottle above the surface of the hydrogen peroxide solution.

Cap the tube or bottle and tilt it to allow the hydrogen peroxide solution to cover the colony

Look for vigorous bubbling occurring within 10 seconds.

Helicobacter pylori is catalase positive bacteria.

C. Oxidase test

Moisten disk (See Note [50] ) with sterile dH2O and place them in a perti dish and aadd a loopful of H. pylori suspected colony that is grown on medium.

H. pylori will change a color to pink, through maroon and into black, within 10-30 seconds.

D. Wet mounts (See Note [51] )

The wet mount is test for observation of motility (movement) and analyzing the structure of microorganisms.

Put a drop of dH2 O in the middle of a slide.

Allow the loop to cool transfer a small portion of a single colony to the drop with a loop in sterile condition and dissolve it.

Put a clean cover over the drop.

Find a bubble in the liquid suspension by 4x objective and regulate the focus on the edge of the bubble.

Then switch to the 10x and 40 x objectives carefully.

Suspended H. pylori in the water possess a spiral-shaped body and moves in Brownian motion which is look like a random shimmying-shaking and cork-screw spiraling motility.

2. Materials

2.1 Culture of multiple clinical strains of Hp (local antigenic cocktail)

This section should be performed according to methodology provided in the Hp culture section.

Antigen preparation

Phosphate Buffered Saline (PBS, pH 7.2)

Add the following ingredients into a graduated 1 liter cylinder containing 800ml dH2O: 8g NaCl, 0.2g KCl, 1.1g Na2HPO4, 0.2g KH2PO4. Adjust the pH to 7.2 using HCl. Bring up the volume to 1 liter with dH2O. Autoclave the solution at 121°C for 20 minutes at 15psi (1.05kg/cm2) and store at 4°C (solution A).

Protease inhibitor solution: Dissolve one tablet (Protease inhibitor cocktail, Roche, Germany, see Note 1) in 10ml of solution A (Solution B).


The following buffers should be prepared to the volume required by the number of selected wells/strips.

Coating buffer (Carbonate-Bicarbonate Buffer):

Dissolve 1.59g Na2CO3, 2.93g NaHCO3, 0.01% (w/v) sodium azide (NaN3, see Note 2)in 800 ml dH2O. Adjust the pH to 9.6 with NaOH or HCl . Bring up the volume to 1 liter with dH2O and store at 4°C. The coating buffer can be stored at 4°C for up to two weeks.

Blocking buffer:

Dissolve 1% (w/v) bovine serum albumin (BSA, Sigma, USA) in solution A (see Note 3). Freshly prepared buffer is highly recommended.

Diluent buffer (pH 7.2):

Mix 0.1% (v/v) tween 20 in desired amount of blocking buffer (see Notes 3 and 4). Freshly prepared buffer is highly recommended.


Standard A: Hp negative serum

Standard B (cut off control): 10U/ml anti Hp specific IgG (IBL, Germany)

Standard C (weakly positive control): 25U/ml anti Hp specific IgG (IBL, Germany)

Standard D (strongly positive control): 150U/ml anti Hp specific IgG (IBL, Germany).

Washing buffer:

Mix 0.1% (v/v) tween 20 in solution A. Washing buffer can be stored at 4°C for up to two weeks.

3. Methods

3.1 Hp culture for collecting multiple strains and antigen preparation (according to the mentioned protocols in culture section, identity confirmation of grown bacteria by gram staining, urease, catalase and motility test)

3.2 Antigen preparation

The following steps should be performed on ice:

Harvest Hp colonies from the surface of 5-10 (3-5 day old) culture plates with 1ml of solution A /plate into corresponding number of microtubes (see Note 5).

Centrifuge the bacterial suspension at 2300 rcf at 4°C for 10 minutes. Remove the supernatants and resuspend the pellets in equivalent volume of solution A (1ml) and repeat the centrifugation procedure.

Combine and measure the wet weight of the pellet and resuspend it in sufficient volume of solution B to make the final concentration of 0.5g/ml.

Sonicate the bacterial suspension at 20,000Hz on ice 5 times for 45sec with 45sec intervals (Sonicator, Hielscher GmbH, Germany).

Centrifuge the bacterial sonicate at 7,200 rcf for 1hour at 4°C. Carefully transfer the supernatant to a new tube and discard the pellet (see Note 6).

Sterilize the obtained supernatant by sequentially passing it through 0.8, 0.45 and 0.22µm (Millipore) filters. This step will also remove bacterial cell debris (Solution C).

Measure and label the protein concentration of solution C at 280nm by spectrophotometer (a protein concentration of 30-50mg/ml is expected). Aliquot and store at -20°C until further use.


All buffers and reagents should be brought to room temperature (21-25°C) prior to use (see Note 7).

Antigen coating:

Select the number of needed wells/strips according to Figure-1.

Place the assigned number of strips into an ELISA microwell frame (Maxisorb ELISA plate, Nunc, Denmark).

Dilute solution C to a final concentration of 5μg/ml with the coating buffer. Add 100μl to each microwell. Cover the frame with a plastic seal and incubate it at 4°C over night.

Wash the coated wells three times with solution A (300μl/well, see Note 8). Invert the plate and gently tap it on a clean dry paper towel after each washing step.


Add 100μl blocking buffer to each previously coated well (see Note 9), cover the frame with a plastic seal and incubate for 1hour at room temperature.

Remove the blocking solution and tap the inverted plate on a clean, dry paper towel.

Serum and standard addition:

Dilute test sera at a 1:100 rate using the diluent buffer (e.g. 2µl + 198µl).

Add 100μl of standards A-D and diluted test sera to the assigned blocked wells (Figure-1). Cover the frame and incubate it at room temperature for 1hour.

Remove serum samples/standards and wash the wells five times with the washing buffer (300μl/well). After the final wash, tap the plate on clean, dry paper towel to remove all liquids from the wells.

Conjugate antibody addition

Dilute polyclonal rabbit anti human IgG conjugated with Horse Radish Peroxidase (HRP, Dako, Denmark) at a 1:10,000 rate in the diluent buffer.

Add 100μl diluted conjugated antibody to each well, cover the frame and incubate at room temperature for 1hour.

Remove the conjugated antibody and wash the wells five times with the washing buffer (300μl/well). After the final wash, tap the plate on clean, dry paper towel to remove all liquids from the wells.

Substrate Addition:

Add 100μl of ready to use Tetramethylbenzidine (TMB, Sigma) solution to each well

Cover the frame and incubate at room temperature for 10minutes. Avoid direct exposure to light during incubation (see Note 10).

Stopping the reaction:

Add 100μl stop solution (2M H2SO4) to each well in the same order as substrate addition (see Notes 11 and 12).


Read the strips at 450nm by a microtiter plate reader capable of reading absorbance at 450nm (BIOHIT, Finland)

Dispose the plate after reading.

Interpretation of the results:

The cut off value is determined by the optical density (OD) measured for Standard B. Samples in the range of 20% above or below the cut off value are considered as borderline (see Note 13). Optical densities higher and lower than the borderline zone are interpreted as positive and negative readings respectively.

For quantitative determination of antibody titers, calculate the estimated IgG concentration of each serum sample according to standard readings. Graph the OD values of standards on the X axis (linear) and their IgG international units on the Y axis (logarithmic) on a semi-logarithmic graph paper. Based on the obtained OD value of each serum sample, its concentration of anti HpIgG can be deduced from the standard curve (Figure-2).


The sensitivity of test depends on the pH, urea concentration, type of indicator and the temperature of incubation [52] .

Do not heat or reheat urea base into solution; urea decomposes upon heating [53] .

Urea is known to undergo auto hydrolysis; therefore, it is advisable to store urea-based media at 4-8°C. Color change may take slightly longer when media is refrigerated 2.

Urea media exposed to light may develop peroxide, which could interfere with the urease reaction2.

Prior to use, make sure solution C is clear with no turbidity. You can also check for contamination by incubation of an aliquot overnight at 37°C.

H. pylori can grow on different media containing blood supplements. Most studies have used Brucella or Columbia as the agar base. An amount of 7 to 10% blood cause better growth of H. pylori as compared with 5% blood. Horse blood better supports H. pylori growth as compared to sheep blood [54] .

56°C is the optimum temperature for addition of blood supplements and also to prevent solidification of the media.

Do not heat or autoclave sodium Pyruvate or ferrous sulfate.

For contamination control, place a sample plate at 37°C and check for growth. Lack of growth authorizes the use of remaining plates for H. pylori culture [55] .

The mentioned antibiotics will select for the growth of H. pylori from gastric specimens but also limits the rate of growth. For the following H. pylori passages, the addition of antibiotics is optional [56] .

The catalase test is used to detect the presence of catalase enzymes produced by H. pylori which decomposes hydrogen peroxide and releases oxygen and water (2 H2O2 → 2 H2O + O2) [57] 

Hydrogen peroxide is unstable and should be stored at 4°C, avoiding light.

Hydrogen peroxide can cause irritation and is harmful if swallowed.

This test principle is to determine the presence of the oxidase enzymes.

Fifteen percent decrease in culture rate is observed when biopsies are transported or stored overnight [58] . Lengthy transportation period decreases culture yield, particularly if the patient has received antibiotic treatment or the numbers of colonizing H. pylori in the gastric biopsies are minimal, in which case may result in no bacterial growth or false negative results. This situation can be remedied by prolonged incubation period for up to 12 days [59] .

Rapid urease test is based on the principle that abundant urease enzyme produced by H. pylori hydrolyses urea into ammonia and carbon dioxide. The produced ammonia raises the medium pH which is detected by phenol red indicator [60] .

The sharp angular depression in the lesser curvature which separates the fundus from the antrum.

Large biopsies or multiple biopsies which are placed in a single test may enhance the speed of the reaction. The sites with the highest yield are the gastric angle and the greater curve of the corpus [61] .

An increase in pH due to the production of ammonia results in a color change from yellow (pH 6.8) to bright pink (pH 8.2) [62] .

For preventing false-negative results, high bacterial density is required for rapid urease tests especially in the case of recent patient use of bismuth-containing compounds, antibiotics, or proton pump inhibitors may cause [63] .

It is recommended that RUT tubes be monitored for the desired color change for up to 24 hours. Bacterial culture is however recommended despite RUT negative results [64] .

H. pylori is recognized as a sensitive organism to drying, and many laboratories have reported that rapid transportation of biopsy specimens to microbiologic laboratories is crucial to guarantee the isolation of the organism [65] .

The majority of gastric sampling is obtained from the antral region. However, for optimal results obtaining several biopsies from various gastric locations is preferred [66] .

It is recommended that the biopsies grinding before culture may increase the number of H. pylori colonies and improve H. pylori isolation [67] 

Spreading tissue homogenate on the entire plate surface increases the rate of H. pylori recovery and prevents the overgrowth of potential contaminants [68] .

In general, primary cultures of H. pylori are sensitive to oxygen in comparison with most Campylobacter species. H. pylori is usually grown a standard microaerobic condition, in CO2 incubators or anaerobic chambers with a microaerobic condition or in jars with gas-generation kits. 2 to 5% O2, 5 to 10% (optimal closer to 10%) CO2 and 0 to 10% H2 are used in studies with standardized atmospheres for Hp culture [69] .

Be careful about contaminations! Check media every days and if you saw suspicious colonies, sub culture Hp colonies.

The question of how long cultures should be incubated has been evaluated by several investigators, and regardless of the type of media or the selected atmosphere, plates should be kept for 10 days before they can be considered negative for primary isolation of H. pylori. The length of incubation depends on whether selective or nonselective media is used [70] .

H. pylori colonies are yellowish 0.5 to 2 mm colonies on 7% lysed horse blood agar and with 0.5 to 1 mm pale grayish colonies on sheep blood agar.

Proper smear preparation should produce a monolayer of organisms sufficiently dense for easy visualization but thin enough to reveal characteristic morphological characteristics. Use clean, new glass slides.

Avoid mixing vigorously and creating aerosols.

To avoid distortions, do not overheat

Excessive rinsing may cause the detachment of crystal violet from gram-positive cells and produce false positive results. The flow of water may be applied to the underside of the angled slide to ensure a gentle flow across the smeared side.

A properly decolorized smear appears with an almost olive-green hue and without observable evidence of crystal/gentian violet

The catalase test should not be performed on colonies taken from media containing whole red blood cells which may cause false positive.

For control all disks must be tested with Known positive and negative cultures.

Fresh cultures must be used for demonstrating maximum motility. Never use stain since most stains kill the organisms. Dark field capabilities of microscope are ideal illumination for representing motility. You can put a small dark paper disc in the center of condenser to make approximate dark field optics.



Protease inhibitor cocktail is used for the inhibition of several metalloproteases in cellular extracts of bacteria, mammalian, yeast and plant cells including serine and cysteine metalloproteases. One tablet is sufficient for the inhibition of the proteolytic activity in 10ml protein extraction solution.

Sodium azide (NaN3) is used for increasing the shelf life of material preventing bacterial contamination. It is a severely irritating material. Solutions containing NaN3 must be clearly labeled. Disposal of solutions containing NaN3 should be followed by large volumes of water to avoid interaction with the plumbing system. Handling should be performed with great care using safety gloves and goggles. Upon exposure, thoroughly wash the area with water. NaN3 can also inhibit the activity of conjugated antibodies, therefore use clean tips for addition of this antibody.

Avoid harsh agitation of blocking buffer to prevent BSA proteolysis.

Tween 20 is a non ionic detergent which binds to water insoluble components rendering them hydrophilic.

Bacterial colonies can be previously collected during several bacterial passages, in order to yield sufficient bacterial proteins. To avoid bacterial lysis the collected strains should be kept at -70°C.

It is important to avoid collecting pellet material into the supernatant as it will block the sterilizing filters.

Use latex gloves throughout the procedure. Avoid cross contamination of reagents as it may produce false results.

Insufficient washing will result in assay variations affecting the validity of test. Completely remove the wash buffer completely after the last wash (remove bubbles).

Avoid bubbles upon addition of buffers as they may yield false results.

Presence of Hp-specific antibodies will turn the colorless substrate solution to blue.

Addition of the stop solution will transform the color to yellow. The stopped reaction can be read up to 1hour.

Handle sulfuric acid under chemical fume hood as this acid is severely irritating to the skin, eyes and the respiratory tract. It may create severe damages including fires upon contact with other material. Upon exposure, thoroughly wash the area with water.

Samples with borderline values are reported as equivocal or borderline after having repeated the test.

Michel A, Waterboer T, Kist M, Pawlita M. 2009. Helicobacter pylori multiplex serology. Helicobacter 14: 525-535.

Selgrad M, Kandulski A, Malfertheiner P. 2009. Helicobacter pylori: diagnosis and treatment. Cur OpinGastroenterol 25: 549-556.

Michel A, Waterboer T, Kist M, Pawlita M. 2009. Helicobacter pylori multiplex serology. Helicobacter 14: 525-535.

Selgrad M, Kandulski A, Malfertheiner P. 2009. Helicobacter pylori: diagnosis and treatment. Cur OpinGastroenterol 25: 549-556.