1. Chick Culture, Propagation, Fixation and Staining
Found in Ward’s Teacher’s Guide packet in “Chick” section in “Animal Tissue Culture” half of binder
Chick Culture, Propagation, Fixation and Staining
TEACHER COPY
Dr. Nowicki 2013

2. Lab Overview Background Materials Procedures
Animal Tissue Culture Introduction
General Techniques
Sterile Technique
Materials
Procedures
Pre-lab
Establishment of Primary Cell Culture
Propagation of Chick Cells onto Coverslips
Fixation and Staining of Chick Cell Monolayer

3. Animal Tissue Culture Intro
Cell and organ cultures maintained in vitro
Easier to study
Kept alive for different period of time
Taken from variety of species
Humans, monkeys, mice, dogs, cats, frogs, insects, fish, and others
Taken from variety of organs
Heart, kidney, lungs, liver, blood, skin, and others

4. Animal Tissue Culture Intro
Cancer studies compare normal and cancerous tissues
Used to test potential cancer treatment drugs
More cost-effective and faster than using animals
Tissue cultures used for other viral diseases
Vaccines made from viruses for diseases such as polio and measles
Diseases discovered using isolating agents

5. Animal Tissue Culture Intro
Current applications and studies:
Inheritance
Embryonic development
Drug action mechanisms on cells
Cellular immunity
Cellular disease processes

6. Primary Culture
Refers to the stage of the culture after the cells are isolated from the tissue and divide under the appropriate conditions
Until they occupy the entire surface
Reach Confluence
Then need to be subcultured

7. General Techniques Tissues kept at 35°C in balanced salt solution
1% blood serum helps protect cells
Tissues cut into small explants then placed in vessels w/ nutrient medium
Sometimes attached to walls of vessel w/ blood plasma to clot
Cells migrate from explant into medium to divide and produce “halo” of growth around original tissue

8. General Techniques
Can also treat culture w/ chemicals to dissociate cells into a suspension
Then place cells in nutrient medium and some suspension in culture vessel
Culture vessel incubated so cells settle out, attach to vessel walls, and grow
Monolayer= sheet of cells covering vessel wall
Cell lines grow as monolayers
Scrape vessel with rubber spatula or remove medium then remove cells from medium w/ a chemical
Suspend cells and dilute w/ medium to obtain desired cells

9. General Techniques
Nutrient medium for cultures can be solution of sterile chemicals or natural materials
pH must be controlled
CO2 partly control acidity- keep airtight vessels
Animal cells are often chick embryos after 21 days of incubation but can be obtained at any time
Embryonic tissues have greater growth potential
Shell protects cells from possible contamination

10. Sterile Technique Wipe down equipment and lab areas with 70% ethanol
Minimize airflow- keep door closed
Turn on sterile hoods about 20 minutes before experiment and clean with 70% ETOH
Wash hands and arms, but don’t scrub too hard- can promote flaking
Spray outside of gloves and packages to enter the sterile hoods with 70% ETOH
Keep equipment such as forceps and scalpels in 95% ETOH then dip in sterile water- makes equipment sterile for use

11. Materials Curved dissecting forceps 100mL Hank’s solution
1 sterile culture tube
1 sterile petri dish
25 sterile pipets
10 sterile culture flasks
1 sterile glass rod
2 sterile Versene tubes, 10mL each
2 sterile medium 199 tubes, 20mL each
5 sterile alcohol pads
1 staining jar
100mL Hank’s solution
30mL methanol
30mL Hematoxylin stain
30mL Eosin stain
100mL 99% isopropyl
4 oz. Histoclear
15mL Piccolyte II mounting medium
*egg incubated 7 days
*70% isopropyl
*incubator
*microscope
*safety gear
*= not provided in kit!
Also, can substitute ethanol for isopropyl alcohol

12. Pre-lab Incubate chick egg of interest for 7 days
Normal incubation is °C at 57% humidity
Label eggs with date incubation began
Lay eggs larger side facing up
Rotate egg 2 or 3 times a day until used
Prepare any chemicals or media for class as needed
Take out given materials from kit(s)

13. Establishment of Primary Cell Culture
Obtain egg incubated for 7 days and wipe with alcohol pad to sterilize shell
Keep egg w/ large end facing up into sterile, cushioned petri dish
Use sterile forceps to crack and remove shell and membrane around air sac inside of shell
Remove embryo by either:
pouring egg contents into sterile petri dish and removing from membrane with 2 sterile forceps
by using sterile forceps and dissecting scissors to cut embryo out of egg and vascularization
*for this experiment, either dip tools from 95% ethanol into sterile water or flame sterilize them, i.e. sterile forceps can be flame sterilized

14. Establishment of Primary Cell Culture
Place embryo into sterile culture tube and cap tube
Add 1mL versene solution to explant using a sterile disposable pipet
Using sterile glass rod, gently grind explant and versene and replace cap on tube then set aside for minutes
Using sterile disposable pipet, draw up and expel tissue several times to further homogenize it
Using sterile disposable pipet, add one tube of medium 199 to culture tube

15. Establishment of Primary Cell Culture
Shake tube to mix suspension well then let stand for 5 minutes
Using sterile disposable pipet, transfer 3mL of cell suspension to sterile culture flask and cap flask. DO NOT transfer large particulates.
Label flask and incubate with largest flat surface facing downward at 35°C
Once incubated, invert flask and view cells in compound microscope at 100X
If cultures turn yellow or become cloudy they are contaminated and should be discarded

16. Propagation of Chick Cells onto Coverslips
After culture flask was incubated for 6-7 days, pour off medium and replace cap
Using sterile disposable pipet, add 1mL versene, swirl, then pour off versene and replace cap
Using sterile disposable pipet, add 1mL versene and replace cap
Place flask on flat surface so solution completely covers layer of cells and room temp for 15 minutes
Cells will loosen and float off surface; may need extended incubation or shaking to dislodge cells

17. Propagation of Chick Cells onto Coverslips
Using sterile disposable pipet, draw up and expel suspension several times to break up larger clumps
Using sterile disposable pipet, transfer all suspension into sterile vial of medium 199
Pool cells from 5 flasks into 1 medium vial
Swirl vial gently to mix transferred cells with fresh medium
Using sterile disposable pipet, add 2mL cell suspension to sterile vial containing coverslip and cap tightly

18. Propagation of Chick Cells onto Coverslips
Label vial and place in incubator upright so coverslip remains flat on bottom of vial at 35°C for 4-5 days
Cells should settle and attach to coverslip in a few hours

19. Fixation of Chick Cell Monolayer
Heat Hank’s balanced salt solution to 35°C and place in staining jar
Remove coverslip from culture vessel with sterile forceps and transfer to staining jar, keeping side w/ cells upright to see better
Pour off Hank’s solution and add fresh, warm Hank’s solution for 2nd washing
Repeat for 3 rinses
Pour enough methanol in staining jar to just submerge coverslip and let fix for 7-10 minutes, agitating staining jar occasionally

20. Staining of Chick Cell Monolayer
Pour off methanol and rinse coverslip w/ water to remove excess methanol
Add hematoxylin stain to completely submerge coverslip and allow cells to sit for 10 minutes
Pour off stain and rinse coverslip w/ water
Add Hank’s solution and set aside for 2 minutes, then pour off and rinse coverslip w/ water
Add enough eosin stain to completely submerge coverslip and allow cells to sit for 5 minutes
Pour off stain and rinse coverslip w/ 99% isopropyl 3 times, 1 minute per rinse

21. Staining of Chick Cell Monolayer
Add Histoclear and allow to sit for 2 minutes, agitating occasionally
Pour off Histoclear and repeat step 11. Just before 2 minutes, add 1 drop of Piccolyte II mounting medium to center of clean microscope slide
Remove coverslip from Histoclear and place it cell-side down onto center of microscope slide
Allow slide to dry for 1 day before viewing
Examine at 100X