Transcript Tissue Culture Techniques Lab

Islamic University _Gaza
Faculty of science
Department of Biotechnology
By:
Mahmoud W. El-Hindi
2013-2014
By: Mahmoud El-Hindi
1
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 .
By: Mahmoud El-Hindi
2
Course division:.
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
By: Mahmoud El-Hindi
3
Lab 1: Aseptic Technique
 Aseptic 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.
By: Mahmoud El-Hindi
4
Cont.
 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.
By: Mahmoud El-Hindi
5
Cont.
 Mycoplasmal 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 .
By: Mahmoud El-Hindi
6
Maintaining Sterility :
 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
surroundings.
be
sterile
By: Mahmoud El-Hindi
and
its
non
sterile
7
Cell culture contaminants
two types:
 1-Chemicals: difficult to detect caused by
endotoxins, metal ions or traces of disinfectants
that are invisible.
 2- 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.
By: Mahmoud El-Hindi
8
Safety
 For best results in tissue culture, we want to work to
keep microbial (bacteria, yeast and molds)
contamination to a minimum.
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.
By: Mahmoud El-Hindi
9
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.
By: Mahmoud El-Hindi
10
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.
By: Mahmoud El-Hindi
11
By: Mahmoud El-Hindi
12
By: Mahmoud El-Hindi
13
Culture Medium Sterilization
By: Mahmoud El-Hindi
14
Culture flask
Culture Plate
By: Mahmoud El-Hindi
15
Inverted microscopy
Large stage so plates and flasks
can be used.
Magnification; 5X, 10X, 20X, 40X
By: Mahmoud El-Hindi
16
By: Mahmoud El-Hindi
17
By: Mahmoud El-Hindi
18
By: Mahmoud El-Hindi
19
ASEPTIC TECHNIQUE IN VERTICAL LAMINAR
FLOW
 Clean 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.
By: Mahmoud El-Hindi
20
Cont.
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
By: Mahmoud El-Hindi
21
Cont.
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 .
By: Mahmoud El-Hindi
22
By: Mahmoud El-Hindi
23
By: Mahmoud El-Hindi
24
Good Luck
By: Mahmoud El-Hindi
25