Tissue Culture Techniques Lab

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

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 .



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

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.


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.


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 .


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.


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.


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.


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

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.




Culture Medium Sterilization


Culture flaskCulture flask

Culture PlateCulture Plate 15By: Mahmoud El-Hindi

Inverted microscopy

Large stage so plates and flasks can be used.

Magnification; 5X, 10X, 20X, 40X





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.


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


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 .




Good Luck


Documents

Tissue Culture Techniques Lab