GB/T 14926.65-2026 Laboratory animal—Method for examination of Pneumocystis sp. English, Anglais, Englisch, Inglés, えいご
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ICS 13.220.10
CCS H 57
National Standard of the People's Republic of China
GB/T 14926.65-2026
Replaces GB/T 14926.65-2001
Laboratory animal - Method for examination of Pneumocystis sp.
实验动物 肺孢子菌检测方法
Issue date: 2026-01-28 Implementation date: 2027-02-01
Issued by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China
the Standardization Administration of the People's Republic of China
Contents
Foreword
Introduction
1 Scope
2 Normative References
3 Terms and Definitions
4 Principle
5 Reagents and Materials
6 Instruments and Equipment
7 Sample
8 Test Procedure
9 Test Data Processing
10 Test Report
Laboratory Animals — Detection Method for Pneumocystis spp.
1 Scope
This document describes detection methods for Pneumocystis spp. in laboratory animals.
This document applies to the detection of Pneumocystis spp. in laboratory animals such as mice, rats, rabbits, guinea pigs, and hamsters.
2 Normative References
The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
GB/T 14926.42 Laboratory animal – Bacteriological examination – Collection of specimens
GB/T 14926.43 Laboratory animal – Bacteriological examination – Staining, media and reagents
3 Terms and Definitions
No terms and definitions are required for this document.
4 Principle
Pneumocystis, which primarily infects the alveoli, can be directly observed under a microscope after fixation and staining.
5 Reagents and Materials
5.1 Glass slides.
5.2 Methanol, purity: analytical grade; CAS No.: 67-56-1.
5.3 Ethanol, purity: analytical grade; CAS No.: 64-17-5.
5.4 Giemsa stain, prepared.
5.5 Methenamine silver stain, prepared.
5.6 Chromic acid solution, concentration (mass fraction): 8%; CAS No.: 7738-94-5.
5.7 Sodium thiosulfate solution, concentration (mass fraction): 5%; CAS No.: 10102-17-7.
5.8 Gold chloride solution, concentration (mass fraction): 0.2%; CAS No.: 13453-07-1.
5.9 Light green stain, concentration (mass fraction): 2%; CAS No.: 633-03-4.
6 Instruments and Equipment
6.1 Microscope, magnification range 100x to 1000x.
6.2 Incubator or water bath, temperature control range 25°C to 100°C.
7 Sample
According to the requirements of GB/T 14926.42, euthanize the animal by anesthesia, disinfect the chest area, open the thoracic cavity, remove the lungs, and rinse with physiological saline. Cut each lobe of the lung with a scalpel, gently press and smear each lobe onto the same glass slide, and allow to air dry naturally to prepare an impression smear sample of the lung tissue.
8 Test Procedure
8.1 Giemsa Staining
According to the requirements of GB/T 14926.43, fix the air-dried lung tissue impression smear with methanol for 5 minutes, then rinse with distilled water and air dry. Stain with Giemsa stain for 15 min to 20 min, rinse with distilled water, dry, and prepare for examination.
8.2 Methenamine Silver Staining
Take the lung tissue impression smear and allow it to air dry or fix by heating. Add 8% chromic acid solution for oxidation for 20 minutes, rinse with distilled water. Prepare the methenamine silver staining working solution (prepare fresh according to Appendix A). Place the slide in a staining jar and add 1 mL of working solution. Place the staining jar in a 56°C incubator or water bath for 20 min to 25 min. Remove and rinse thoroughly with distilled water. Stain with gold chloride solution until the sample on the slide turns brown, then rinse thoroughly with distilled water. Treat with sodium thiosulfate solution for 3 minutes and rinse thoroughly with distilled water. Counterstain with light green stain for 1 minute, rinse with distilled water. Dehydrate with ethanol, dry, and prepare for examination.
9 Test Data Processing
9.1 Giemsa Staining Results
Examine the stained lung tissue impression smear under an oil immersion lens (100x). Both cysts and trophozoites of Pneumocystis can be detected. The detection of either cysts or trophozoites under the microscope is interpreted as positive.
Cysts are round or oval, with a diameter of 3 μm to 8 μm (some Pneumocystis carinii cysts can reach up to 10 μm). They contain some cytoplasm, residual mitochondria, and 2 to 8 crescent-shaped (or oval) intracystic bodies. Mature cysts contain 8 intracystic bodies, which are crescent-shaped. With Giemsa staining, the cytoplasm stains light blue, the nuclei stain dark purple, and the cyst wall remains unstained.
Trophozoites are 1.5 μm to 10 μm in size. They are mononuclear with one nucleolus, and the cytoplasm also contains some glycogen granules and lipid droplets.
Samples with suspicious results should be verified using methenamine silver staining.
9.2 Methenamine Silver Staining Results
Examine the stained lung tissue impression smear under an oil immersion lens (100x). The detection of cysts is interpreted as positive.
The cyst wall stains dark brown or black, appearing as round or oval structures. The intracystic bodies remain unstained. A typical characteristic is the presence of one black fold in the center or a darkly stained central spot.
10 Test Report
Issue a report. The report content should include at least the following:
—— Test subject;
—— Number of this document;
—— Method used;
—— Results;
—— Any observed abnormal phenomena;
—— Test date.
