In order to further improve the quality of the "Microbiome Experiment Manual", a new public review link has been added to this project. After the article passes the peer review, the full text is shared by means of the official account push, and anyone can submit the modification comments online. There is a slight problem with the format of the official account, it is recommended that the computer click at the end of the article to read the original article to download the PDF review. The Online Documentation (https://kdocs.cn/l/cL8RRqHIL) Public Review page registers suggestions for changes to the name, unit, and line number index. The deadline for solicitation of amendments is 72 hours after the tweet is posted, and the article will be further revised with constructive revisions and will be published online by DOI, and will be listed as a reviewer or acknowledgment according to the degree of contribution. Thank you to your peers for their valuable comments.
Isolation culture and morphological analysis of ruminant rumen protozoa
Separation and Morphological Analysis of Protozoa from Rumen Contents
GAO Jian, ZHEN Yongkang, WANG Mengzhi*, WANG Hongrong
College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu
*Corresponding Author Email: [email protected]
Abstract: Ruminants rumen contain a large number of protozoa, which play an important role in maintaining the stability of the rumen microecological environment and promoting feed decomposition and fermentation. Rumen protozoa mainly include endophyllum, diptera, isophyllum, cephalopods and anterior hairy genus, and the number and morphology of their nuclei are important markers of classification. In this paper, protozoa cells are sorted by microscope, then cultured rumen protozoa, and finally morphologically analyzed under the microscope in an anaerobic workstation.
Key words: Rumen protozoa, microbial isolation, morphological analysis
Materials and reagents
1.16 × 150-mm tubes (with rubber stopper)
2. Capillary straw
3. Large diameter straw
4. Slides
5. Gauze
6. CO2 gas, purity of 99.999%
7. Dipotassium hydrogen phosphate, K2HPO4
8. Potassium dihydrogen phosphate, KH2PO4
9. Ammonium sulfate, (NH4) 2SO4
10. Sodium chloride, NaCl
11. Magnesium sulfate, MgSO4
12. Calcium chloride dihydrate, CaCl2 ·2H2 O
13. Cysteine hydrochloride
14. Sodium carbonate, Na₂CO₃
15. Blade Azure
16. Sodium acetate, CH3COONa
17. Sodium bicarbonate, NaHCO3
18. Flour
19. Duck grass powder
20. Anaerobic dilution solution (see Solution Recipes)
21. Culture medium M (see Solution Recipes)
22. Culture solution SP (see Solution Recipes)
23. Substrate suspension (see Solution Recipes)
Instrumentation
1.40 mesh sieve
2. Dissecting microscope
3. Constant temperature water bath pot
4. Anaerobic workstation
5. Centrifuge
Experimental steps
1. After morning feeding, the rumen juice is collected from the rumen of the ruminant equipped with a permanent fistula by a negative pressure device, filtered by double-layer gauze, and put into a CO2-filled and 39 °C pre-warmed thermos bottle and brought back to the laboratory.
2. The filtered rumen juice is put into the anaerobic workstation, and the anaerobic dilution is used to dilute the anaerobic dilution in turn for ten-fold gradient dilution (10-2, 10-3 or other multiples, subject to 6 to 8 protozoa per counter), and the diluted rumen juice is placed in the anaerobic workstation at 39 °C for preservation.
3. Pipette 0.1 mL of diluted rumen juice into a slide, examine it microscopically in an anaerobic workstation (100 ×), and isolate the protozoa.
4. Aspirate individual protozoan cells into capillaries under the microscope and check whether protozoal cells are viable before transferring to the culture tube, transfer the protozoan cells to 16 × 150 mm culture tubes, placing 1 to 3 cells per culture tube.
5. Add 10 mL of culture medium to the culture tube (choose different culture solutions according to different protozoal growth states, such as the anterior hairy protozoa using culture medium M culture growth is better, the internal hair protozoa is suitable for using culture medium SP culture growth is better) and 1 mL substrate suspension, anaerobic closed culture tube, the culture tube is placed at a 10 ° angle in the anaerobic workstation at 39 °C culture.
6. Add 0.1 mL of substrate suspension to the culture tube in the anaerobic workstation every day, observe the growth state of the protozoa under a dissecting microscope, and grow to 6 to 8 protozoa per counter.
7. Transfer the cultured protozoa to the new culture tube twice a week in the anaerobic workstation, aspirate 5 mL of culture medium using a large-bore pipette, add it to the culture tube that has been filled with 5 mL fresh culture solution and 0.1 mL substrate suspension, and after inoculation to the new culture tube, observe daily, based on 6 to 8 protozoa per counting cell.
8. Using a dissecting microscope, the cultured protozoa are identified according to the size and morphology of the protozoa under the condition of magnification of 400 ×, and the specific morphology can refer to the common protozoal morphology and pictures in Dehority (1993) and precautions.
Precautions
Morphological characteristics of common protozoa in the rumen:
1. Entodinium has an average body size of about 2-168 μm × 13-104 μm, most of the body cilia disappear, and only a small part of the cilia have a composite cilia band on the front surface of the body, which is retractable. The worm body begins with a relatively straight, 6-sided orientation partition of the worm body, the mouth is located in the anterior segment, and the rod-like nucleus is to the right. A contracted vesicle, no bone plate, some species with caudal spines. Ciliates of this genus account for a large proportion, about 50%.
Fig. 1 Morphological diagram of entodinium protozoa (400 ×)
2. Diplodinium is approximately square (77-86 μm × 53-61 μm), oriented into 6 zones, the large nucleus is thick and thin, the shape is complex, located on the left side of the body, the staining limit is not obvious, and the small nucleus is generally located on the large nucleus. There are two bundles of compound ciliary bands in front of the body, namely the mouth and the left anterior part of the body, with a distinctly raised roof between them. Most have two shrinking vesicles, no bone plates, and no tail spines.
Fig. 2 Morphology of diplodinium protozoa (400 ×)
3. Isotricha insects have simple morphological structure and are larger (108-207 μm × 73-128 μm), but have thinner walls (10 nm). It is shaped like a flat egg and covered with isolong cilia on the surface of the body. There is a mouth, a slightly curved rod-shaped large nucleus, a small nucleus, and 1-4 unequal shrinking vesicles.
Fig. 3 Morphology diagram of Isotricha protozoa (400 ×)
4. Ophryosolex is larger (140-160 μm × 80-110 μm), tapered or irregular, and harder. It has 2 ciliary bands, and the dorsal ciliary band forms a band-like shape in the center of the body. There are 9 constrictive vesicles, arranged in 2 rows, without gill caps, with 3 bone plates, 2 on the body, one on the right side of the body, and a number of tail spines behind the body.
Fig. 4 Morphology diagram of protozoa of The genus Ophryosolex (400 ×)
5. Epidinium is large and long (80-150 μm × 40-70 μm) and tapered at the posterior end. It has two ciliary bands, both in the anterior part of the body, the proximal ciliary band is near the apex, the left ciliary band on the back is 1/5 of the top, and the 2 shrinking vesicles are longitudinally arranged in the left anterior and posterior parts of the rod-like large nucleus, and the small nucleus is located at the left anterior groove of the large nucleus. No gill caps, three bone plates, with or without tail spines.
Fig. 5 Morphology of Epidinium protozoa (400 ×)
6. Osteracodinium is oval (100-130 μm × 50-60 μm) and is characterized by a large bone plate occupying most of the upper body surface of the insect. The front has two ciliary bands. The large nucleus is long, with a depression in the middle of the left side, in which the small nucleus is located. More than 6 shrinking vesicles are longitudinal with the left side of the large nucleus.
Fig. 6 Morphology of osteracodinium (400 ×)
Solution formulation
1. Anaerobic dilutions (Bryant and Burkey, 1953)
The 100 mL anaerobic diluent contains 45 mL mineral solution No.1 (0.3% K2HPO4), 45 mL mineral solution No.2 (0.3% KH2PO4, 0.6% (NH4)2SO4, 0.6% NaCl, 0.06% MgSO4 and 0.08% CaCl2·2H2O, add 1.67 mL 3% cysteine hydrochloride, 5 mL 0.3% sodium carbonate solution, add distilled water to 100 mL. In addition, the anaerobic dilution contains 0.0001% edge azure. The dilution is sterilized in an autoclave (121 °C, 0.1 MPa sterilized for 20 min), when the sterilizer is opened, the sterile rubber stopper is used immediately to block the dilution, and when the temperature of the dilution is cooled to 45 ~ 50 °C, fill the solution with CO2 to saturation.
2. Medium M (Dehority, 1998)
Each 100 mL of culture medium M contains 50 mL of mixed mineral solutions M (6.0 g/L NaCl, 0.2 g/L MgSO4, 0.26 g/L CaCl2·2H2O and 2.0 g/L KH2PO4), 5 mL of 1.5% sodium acetate solution, 8.33 mL of 6% sodium bicarbonate solution, 26 mL distilled water, 10 mL rumen fluid (rumen solution is an supernatant filtered by 2 layers of gauze, 1,000 × g centrifugation for 10 min), 0.67 mL contains 3% cysteine hydrochloride solution (anaerobically stored in a sterile centrifuge tube under nitrogen conditions until removed before use). Before the addition of cysteine, the culture solution should be mixed for 15 to 20 min, and if necessary, the 1 mol/L NaOH or 1 mol/L HCl adjustment solution pH to 6.6 to 6.8 should be used, and then the 3% cysteine hydrochloride solution containing cysteine was added under anaerobic conditions. Culture medium M was sterilized using an autoclave (121 °C, 0.1 MPa sterilized for 20 min).
3. Culture medium SP (Dehority, 1998)
Each 100 mL of culture solution SP contains 25 mL of mixed mineral solution SP-1 (20 g/L K2HPO4), 25 mL of mixed mineral solution SP-2 (16 g/L KH2PO4, 4.0 g/L NaCl, 0.212 g/L CaCl2·2H2O and 0.154 g/L MgSO4), 10 mL rumen solution (rumen solution is an supernatant filtered by 2 layers of gauze, 1,000 × g centrifugation for 10 min), 10 mL 6% sodium bicarbonate solution , 29.33 mL distilled water, 0.67 mL containing 3% cysteine hydrochloride solution (anaerobically stored in a sterile centrifuge tube under nitrogen conditions until removed before use). Before the addition of cysteine, the culture solution should be mixed for 15 to 20 minutes, and if necessary, naOH or HCl can be used to adjust the solution pH to 6.6 to 6.8, and then add a solution containing 3% cysteine hydrochloride under anaerobic conditions. The culture solution SP was sterilized using an autoclave (121 °C, 0.1 MPa sterilized for 20 min).
4. Substrate suspension
Each 100 mL of distilled water is added 1.5 g of wheat flour, 1.0 g of duck grass powder to make a substrate suspension, wheat flour and duck grass powder are crushed through 40 to 60 mesh standard sieve, and the substrate suspension is CO215 to 20 min to ensure anaerobic. The substrate suspension was sterilized using an autoclave (121 °C, 0.1 MPa sterilized for 20 min). The substrate suspension is frozen and thawed before use.
Thanks
1. Thank you to the National Natural Science Foundation of China (30771567) for its support of this experiment.
2. Wang Mengzhi,Cheng Xin,Xie Wenwen,Zhang Baisong,Liu Xiang,Wang Hongrong. (2010). In vitro study on the effects of different lipids on bacterial microcirculation of rumen protozoa. China Agriculture Science, 43(18): 3831-3837.
3. Wang Mengzhi. Study on the relationship between goat rumen protozoa and bacterial phagocytosis and the mechanism of microbial AA change[D]. Yangzhou University, 2008.
4. Thanks to Wang Mengzhi of the College of Animal Science and Technology of Yangzhou University for her research work, she has provided great help for this experiment.
bibliography
1.Dehority, B. A. (1998). Generation times of Epidinium caudatum and Entodinium caudatum, determined in vitro by transferring at various time intervals. J Anim Sci 76(4): 1189-1196.
2.Bryant, M. P., Burkey, L. A. (1953). Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. J Dairy Sci 36: 205-217.
3.Dehority, B. A. (1993). Laboratory manual for classification and morphology of rumen cilate protozoa. CRC Press, Boca Raton, FL.
10,000+: Microbiota Analysis Baby & Dog Syphilis Rhapsody Mention DNA Hair Nature Cell Special Issue Intestinal Conduction Brain
Tutorial Series: Getting Started with the Microbiome Biostar Microbiome
Professional Skills: Academic Charts High Score Essays Student Letter Treasure Book Indispensable Person