Tissue Culture Techniques Lab
-
Upload
aline-rodriquez -
Category
Documents
-
view
46 -
download
0
description
Transcript of Tissue Culture Techniques Lab
![Page 1: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/1.jpg)
Islamic University _GazaIslamic University _GazaFaculty of scienceFaculty of science
Department of BiotechnologyDepartment of BiotechnologyByBy::
Mahmoud W. El-HindiMahmoud W. El-Hindi2013-20142013-2014
1By: Mahmoud El-Hindi
![Page 2: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/2.jpg)
Course Syllabus Course objectives:.
Describe the equipments used in animal tissue culture .
Understand the safety procedures need for tissue culture .
Understand techniques used in tissue culture .
2By: Mahmoud El-Hindi
![Page 3: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/3.jpg)
3By: Mahmoud El-Hindi
1 Safety Lab and Aseptic Technique.
2 Mammalian cell culture Media preparation.
3 Primary culture " Mouse or Chicken Embryo"
4 Tissue Disaggregation in Warm and Cold Trypsin
5 Chick Embryo Organ Culture
6 Routine Cell Culture Maintenance
7 Subculture Of Monolayer Cells
8 Estimation of viability by Dye exclusion
9 Suspension Culture.
10 Animal Cell line Culture “Cryopreservation and Thawing”
11 Cancer Cell Propagation "Breast Cancer"
12 MTT toxicity Assay
13 Contamination
14 Chromosome content “karyotype”:
15 Final Exam
![Page 4: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/4.jpg)
Lab 1: Aseptic TechniqueAseptic technique is a combination of
procedures designed to reduce the probability of infection.
Contamination by microorganisms remains a
major problem in tissue culture.
Bacteria, mycoplasma, yeast, and fungal spores may be introduced via the operator, the atmosphere, work surfaces, solutions, and many other sources.
4By: Mahmoud El-Hindi
![Page 5: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/5.jpg)
Cont.Aseptic technique Aseptic technique aims to exclude
contamination by establishing a strict policy of practice and ensuring that everyone using the facility adheres to it.
Contamination can be minor and confined to one or two cultures, can spread among several cultures and compromise a whole experiment, or can be widespread and wipe out your.
5By: Mahmoud El-Hindi
![Page 6: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/6.jpg)
Cont.Mycoplasmal infectionMycoplasmal infection, invisible under
regular microscopy, presents one of the major threats.
Undetected, it can spread to other cultures around the laboratory.
It is therefore essential to back up visual checks with a mycoplasma test, particularly if cell growth appears abnormal .
6By: Mahmoud El-Hindi
![Page 7: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/7.jpg)
Maintaining Sterility: Correct aseptic technique Correct aseptic technique should provide a
barrier between microorganisms in the environment outside the culture and the pure, uncontaminated culture within its flask or dish.
All materials that come into direct contact with the culture must be sterile and its non sterile surroundings.
7By: Mahmoud El-Hindi
![Page 8: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/8.jpg)
Cell culture contaminantsCell culture contaminants two types:two types:1-ChemicalsChemicals: difficult to detect caused by
endotoxins, metal ions or traces of disinfectants that are invisible.
2- Contamination by microorganisms Contamination by microorganisms remains a major problem in tissue culture.
Bacteria, mycoplasma, yeast, and fungal spores may be introduced via the operator, the atmosphere, work surfaces, solutions, and many other sources.
8By: Mahmoud El-Hindi
![Page 9: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/9.jpg)
SafetySafetyFor best results in tissue culture, we want to
work to keep microbial (bacteria, yeast and molds) contamination to a minimum.
Guidelines to follow:Guidelines to follow:Work in a culture hood set-aside for tissue
culture purposes.
Most have filtered air that blows across the surface to keep microbes from settling in the hood.
Turn off the UV/antimicrobial light and turn on the hood 30 minutes prior to entering the hood.
9By: Mahmoud El-Hindi
![Page 10: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/10.jpg)
Cont.Wash hands with soap and water before
beginning the procedure and rewash if you touch anything that is not sterile or within the hood.
Do not breathe directly into your cultures, bottles of media, etc. This also means to keep talking to a minimum.
No singing or chewing gum. 10By: Mahmoud El-Hindi
![Page 11: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/11.jpg)
Cont.Use only sterilized pipets, plates, flasks and
bottles in the hood for procedures.
Change pipettes for each manipulation.
If the tip of the pipette touches something outside of the flask or bottle, replace with a new one.
11By: Mahmoud El-Hindi
![Page 12: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/12.jpg)
12By: Mahmoud El-Hindi
![Page 13: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/13.jpg)
13By: Mahmoud El-Hindi
![Page 14: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/14.jpg)
Culture Medium Sterilization
14By: Mahmoud El-Hindi
![Page 15: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/15.jpg)
Culture flaskCulture flask
Culture PlateCulture Plate 15By: Mahmoud El-Hindi
![Page 16: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/16.jpg)
Inverted microscopy
Large stage so plates and flasks can be used.
Magnification; 5X, 10X, 20X, 40X
16By: Mahmoud El-Hindi
![Page 17: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/17.jpg)
17By: Mahmoud El-Hindi
![Page 18: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/18.jpg)
18By: Mahmoud El-Hindi
![Page 19: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/19.jpg)
19By: Mahmoud El-Hindi
![Page 20: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/20.jpg)
ASEPTIC TECHNIQUE IN VERTICAL LAMINAR ASEPTIC TECHNIQUE IN VERTICAL LAMINAR FLOWFLOWClean and swab down work area, and bring
bottles, pipettes, etc.
Carry out preparative procedures first (preparation of media and other reagents), followed by culture work.
Finally, tidy up and wipe over surface with 70% alcohol.
20By: Mahmoud El-Hindi
![Page 21: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/21.jpg)
Cont.Sterile:Sterile:_ Eagle’s 1×MEM with Hanks’ salts and
HCO3, without antibiotics . . . . . . 100 mL
_ Pipettes, graduated, and plugged.
If glass, an assortment of sizes, 1 mL, 5 mL, 10 mL, 25 mL, in a square pipette can, or, if plastic, individually wrapped and sorted by size on a rack _ Culture flasks . . 25 cm2 . . . . . . . . . 10
21By: Mahmoud El-Hindi
![Page 22: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/22.jpg)
Cont.Non sterile:Non sterile:_ Pipetting aid or bulb ._ 70% alcohol in spray bottle._ Lint-free swabs or wipes._ Absorbent paper tissues._ Pipette cylinder containing water and
disinfectant._ Scissors._ Marker pen .
22By: Mahmoud El-Hindi
![Page 23: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/23.jpg)
23By: Mahmoud El-Hindi
![Page 24: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/24.jpg)
24By: Mahmoud El-Hindi
![Page 25: Tissue Culture Techniques Lab](https://reader035.fdocuments.in/reader035/viewer/2022062221/5681320f550346895d98635c/html5/thumbnails/25.jpg)
Good Luck
25By: Mahmoud El-Hindi