Helicobacter pylori May-Grünwald-Giemsa (MGG) Staining Protocol
Helicobacter pylori May-Grünwald-Giemsa (MGG) Staining Protocol
1 Overview
1.1 Purpose and Scope
This protocol is used to establish a routine procedure for Helicobacter pylori May-Grünwald-Giemsa (MGG) staining. It is suitable for the morphological visualization of Helicobacter pylori in paraffin-embedded gastric mucosal tissue sections and can be used for auxiliary observation and preliminary interpretation of Helicobacter pylori in gastric mucosal biopsy samples.
1.2 Method Principle
(1) The MGG method belongs to the Romanowsky-type staining system. Through sequential staining with May-Grünwald working solution and Giemsa working solution, relatively stable combined color differences are produced between tissue components and bacterial structures.
(2) Helicobacter pylori usually appears blue in this staining system, which facilitates localization and observation in the gastric mucus layer, gastric pits, and epithelial surface regions.
(3) Cell nuclei, gastric mucosal epithelium, collagen fibers, and red blood cells show different color tones after staining, helping distinguish background tissue from target bacteria.
1.3 Staining Quality Requirements
(1) Helicobacter pylori should be clearly stained, with recognizable outlines.
(2) The gastric mucosal background tissue should show natural color differentiation, and cell nuclei should be completely displayed.
(3) The section background should be relatively clean, without obvious dye precipitates or extensive destaining.
2 Materials and Reagents
2.1 Sample Types
(1) Paraffin-embedded gastric mucosal tissue sections.
(2) Paraffin sections of gastroscopic biopsy tissue.
2.2 Main Reagents
(1) 10% formalin fixative.
(2) May-Grünwald working solution.
(3) Giemsa working solution.
(4) Distilled water.
(5) Absolute ethanol.
(6) Xylene or dewaxing clearing solution.
(7) Neutral balsam.
2.3 Consumables and Equipment
(1) Glass slides and coverslips.
(2) Staining jars or drop-staining platform.
(3) Paraffin microtome.
(4) Forceps and absorbent paper.
(5) Microscope.
3 Staining Solution and Sample Preparation
3.1 Fixation Requirements
(1) Tissue should be fixed in 10% formalin.
(2) After fixation, dehydration and embedding should be completed according to the routine workflow.
(3) Samples from the same batch should preferably be fixed under consistent conditions to reduce staining variation.
3.2 Sectioning Requirements
(1) Paraffin sections should be 4 μm thick.
(2) Sections should adhere firmly to avoid detachment during drop staining.
(3) Section thickness should be kept as consistent as possible to improve result comparability.
3.3 Staining Solution Requirements
(1) May-Grünwald working solution must be prepared immediately before use and should not be stored for a long time.
(2) Giemsa working solution must be prepared immediately before use and should not be stored.
(3) Reagents should be used as soon as possible after opening to avoid affecting subsequent staining performance.
4 Experimental Procedure
4.1 Fixation, Embedding, and Dewaxing
(1) After tissue is fixed in 10% formalin, perform routine dehydration and embedding.
(2) Prepare paraffin sections at a thickness of 4 μm.
(3) Dewax paraffin sections routinely with xylene or dewaxing clearing solution and hydrate to water.
(4) Wash once with distilled water.
4.2 May-Grünwald Staining
(1) Gently blot excess water around the section without allowing the tissue surface to dry completely.
(2) Add an appropriate amount of May-Grünwald working solution dropwise onto the section, ensuring that the tissue area is fully covered.
(3) Stain for 10 min.
(4) After staining, pour off the staining solution. Prolonged washing is not required.
4.3 Giemsa Staining
(1) Add Giemsa working solution dropwise onto the same section, covering the entire tissue area.
(2) Stain for 20 min.
(3) After staining, pour off the staining solution.
4.4 Dehydration, Clearing, and Mounting
(1) Quickly rinse off residual liquid with absolute ethanol.
(2) After ethanol treatment, slightly dry by gentle heating or brief air drying.
(3) Transfer to xylene or dewaxing clearing solution for clearing.
(4) Add neutral balsam and cover with a coverslip to complete mounting.
5 Staining Results
5.1 Target Bacteria
(1) Helicobacter pylori appears blue.
5.2 Background Tissue
(1) Collagen fibers appear red.
(2) Red blood cells appear green.
(3) Gastric mucosal epithelium appears light blue.
(4) Cell nuclei appear dark blue.
6 Key Points for Result Interpretation
6.1 Bacterial Localization
(1) Helicobacter pylori is mostly located in the gastric mucus layer, on the surface of gastric pits, or near the epithelium.
(2) During observation, special attention should be paid to the gastric surface mucus layer and the areas near glandular pit openings.
6.2 Bacterial Morphology
(1) When staining is satisfactory, the bacteria may show fine curved, short rod-like, or slightly spiral outlines.
(2) When the bacterial load is low, locate the relevant area under low magnification first, then examine the mucus layer edge carefully under high magnification.
6.3 Background Interpretation
(1) Gastric mucosal epithelium and nuclear staining should be clear enough to distinguish bacteria from deposits.
(2) If the background is too dark or shows obvious mixed colors, bacterial recognition may be affected, and staining uniformity should be considered during interpretation.
7 Common Problems and Cause Analysis
7.1 Bacteria Are Unclear or Weakly Stained
(1) Giemsa working solution staining time is insufficient.
(2) The staining solution was not freshly or adequately prepared before use, or it was left standing for too long.
(3) The bacterial load in the tissue is low, requiring observation over a larger area.
7.2 Overall Section Destaining
(1) Absolute ethanol washing time is too long.
(2) Treatment before clearing is too strong, causing loss of color from target bacteria.
(3) In the MGG method, the final absolute ethanol step should be a rapid rinse and should not be prolonged.
7.3 Excessive Background Staining
(1) May-Grünwald or Giemsa staining time is too long.
(2) Water around the section was not handled properly, resulting in uneven local dye concentration.
(3) The staining solution is aged or unstable after preparation.
7.4 Uneven Staining or Local Precipitation
(1) The section was incompletely dewaxed.
(2) Residual water on the section surface is uneven.
(3) The staining solution does not fully cover the tissue, or the section surface is contaminated.
8 Application Scope
8.1 Observation of Gastric Mucosal Biopsy Samples
(1) Used for auxiliary visualization of Helicobacter pylori in gastric mucosal tissue.
(2) Suitable as an auxiliary pathological observation method for gastritis, gastric mucosal injury, and related biopsy specimens.
8.2 Routine Histological Screening
(1) Suitable for preliminary screening of suspected Helicobacter pylori infection in paraffin sections.
(2) Can be used as a supplementary method for further targeted observation after HE staining.
9 Safety and Operating Standards
9.1 Personal Protection
(1) Wear a lab coat and disposable gloves during the experiment.
(2) Handle staining solutions, ethanol, and xylene in a well-ventilated environment.
9.2 Operating Precautions
(1) May-Grünwald working solution and Giemsa working solution must be prepared immediately before use and should not be stored.
(2) The final absolute ethanol step should quickly remove residual liquid; otherwise, destaining may easily occur.
(3) In some cases, Helicobacter pylori can also be displayed using Giemsa working solution alone, but the MGG method usually provides better combined color differentiation between background tissue and bacteria.
9.3 Waste Disposal
(1) Staining waste, ethanol waste, and xylene waste should be collected separately.
(2) All contaminated consumables should be disposed of according to laboratory chemical and pathological sample handling regulations.
10 Related Reagent and Material Selection
Table 1 Reagent Selection for Helicobacter pylori May-Grünwald-Giemsa (MGG) Staining
Cat. No. | Product Name | Grade and Purity/Specification | Corresponding Step | Application |
Helicobacter pylori Stain Solution (MGG Method) | BioReagent,Suitable for microbiology, Biological Stain, for microscopy | Core staining step | Directly corresponds to this protocol and can be used for May-Grünwald and Giemsa system staining | |
Neutral Formalin Buffered Solution | 10% | Fixation | Corresponds to the step “tissue fixed in 10% formalin” | |
Water | Ultra pure | Distilled water wash / solution preparation | Suitable for staining systems requiring higher background cleanliness | |
Xylene | Premium-Grade Reagents, ≥99%, xylene isomer and ethyl benzene | Dewaxing / clearing | Corresponds to paraffin section dewaxing and post-staining clearing steps | |
Xylene | ACS, ≥98.5%, isomers plus ethylbenzene | Dewaxing / clearing | Suitable for standardized dewaxing and clearing treatment | |
Xylene | Anhydrous Grade, ≥98%, mixture of isomers | Dewaxing / clearing | Can be used in routine xylene clearing systems | |
Environment Friendly Wax Impregnation Dewaxing Transparent Solution | BioReagent,for microscopy, Suitable for Immunohistochemistry(IHC) | Dewaxing / clearing | Can replace xylene for dewaxing and clearing steps | |
Neutral gum | FMP | Mounting | Corresponds to the neutral balsam mounting step |
For more related articles, please see below:
[1] Principles and methods of smear staining, microbiological staining, and fundamental dye systems