Chick Culture, Propagation, Fixation and Staining

TEACHER COPYDr. Nowicki 2013

Lab Overview

• Background– 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

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

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

Animal Tissue Culture Intro

• Current applications and studies:– Inheritance– Embryonic development– Drug action mechanisms on cells– Cellular immunity– Cellular disease processes

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

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

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

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

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

Materials

• Curved dissecting forceps• 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

Pre-lab

• Incubate chick egg of interest for 7 days– Normal incubation is 21 days @ 102-103°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)

Establishment of Primary Cell Culture

1. Obtain egg incubated for 7 days and wipe with alcohol pad to sterilize shell

2. Keep egg w/ large end facing up into sterile, cushioned petri dish

3. Use sterile forceps to crack and remove shell and membrane around air sac inside of shell

4. Remove embryo by either: a) pouring egg contents into sterile petri dish and

removing from membrane with 2 sterile forceps

b) by using sterile forceps and dissecting scissors to cut embryo out of egg and vascularization

Establishment of Primary Cell Culture

5. Place embryo into sterile culture tube and cap tube

6. Add 1mL versene solution to explant using a sterile disposable pipet

7. Using sterile glass rod, gently grind explant and versene and replace cap on tube then set aside for 20-30 minutes

8. Using sterile disposable pipet, draw up and expel tissue several times to further homogenize it

9. Using sterile disposable pipet, add one tube of medium 199 to culture tube

Establishment of Primary Cell Culture

10.Shake tube to mix suspension well then let stand for 5 minutes

11.Using sterile disposable pipet, transfer 3mL of cell suspension to sterile culture flask and cap flask. DO NOT transfer large particulates.

12.Label flask and incubate with largest flat surface facing downward at 35°C

13.Once incubated, invert flask and view cells in compound microscope at 100X

14.If cultures turn yellow or become cloudy they are contaminated and should be discarded

Propagation of Chick Cells onto Coverslips

1. After culture flask was incubated for 6-7 days, pour off medium and replace cap

2. Using sterile disposable pipet, add 1mL versene, swirl, then pour off versene and replace cap

3. Using sterile disposable pipet, add 1mL versene and replace cap

4. Place flask on flat surface so solution completely covers layer of cells and leave @ room temp for 15 minutes

a) Cells will loosen and float off surface; may need extended incubation or shaking to dislodge cells

Propagation of Chick Cells onto Coverslips

5. Using sterile disposable pipet, draw up and expel suspension several times to break up larger clumps

6. Using sterile disposable pipet, transfer all suspension into sterile vial of medium 199

a) Pool cells from 5 flasks into 1 medium vial

7. Swirl vial gently to mix transferred cells with fresh medium

8. Using sterile disposable pipet, add 2mL cell suspension to sterile vial containing coverslip and cap tightly

Propagation of Chick Cells onto Coverslips

9. Label vial and place in incubator upright so coverslip remains flat on bottom of vial at 35°C for 4-5 days

a) Cells should settle and attach to coverslip in a few hours

Fixation of Chick Cell Monolayer

1. Heat Hank’s balanced salt solution to 35°C and place in staining jar

2. Remove coverslip from culture vessel with sterile forceps and transfer to staining jar, keeping side w/ cells upright to see better

3. Pour off Hank’s solution and add fresh, warm Hank’s solution for 2nd washing

a) Repeat for 3 rinses

4. Pour enough methanol in staining jar to just submerge coverslip and let fix for 7-10 minutes, agitating staining jar occasionally

Staining of Chick Cell Monolayer5. Pour off methanol and rinse coverslip w/ water to

remove excess methanol

6. Add hematoxylin stain to completely submerge coverslip and allow cells to sit for 10 minutes

7. Pour off stain and rinse coverslip w/ water

8. Add Hank’s solution and set aside for 2 minutes, then pour off and rinse coverslip w/ water

9. Add enough eosin stain to completely submerge coverslip and allow cells to sit for 5 minutes

10.Pour off stain and rinse coverslip w/ 99% isopropyl 3 times, 1 minute per rinse

Staining of Chick Cell Monolayer

11.Add Histoclear and allow to sit for 2 minutes, agitating occasionally

12.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

13.Remove coverslip from Histoclear and place it cell-side down onto center of microscope slide

14.Allow slide to dry for 1 day before viewing

15.Examine at 100X