CELL DISRUPTION CHEMICAL OR ENZYMATIC METHODS

Aim

To distrupt the bacterial cells for isolation of the intracellular components like protein,
nucleic acid and intracellular organelles.

Introduction
Single-cell organisms (microorganisms) consist of a semipermeable, tough, rigid,
outer cell wall surrounding the protoplasmic (cytoplasmic) membrane and cytoplasm. The
cytoplasm is made up of nucleic acids, proteins, carbohydrates, lipids, enzymes, inorganic
ions, vitamins, pigments, inclusion bodies and about 80% water. In order to isolate and
extract and of these substances from inside the cell, it is necessary to break the cell wall and
protoplasmic membrane. In some cases the cells may excrete the desired substance; but, in
most cases, the cell wall must be disrupted to release these substances.
Cell Disruption is the method or process for disrupting or lysing the cell inorder to
release the contents out of the cell. Cell disruption is done to release the cell contents. Cell
Lysis or Disruption is done for isolating Nucleic acids (DNA / Plasmids), Proteins
(Intracellular Proteins), Organelles, etc. Cell disruption categorized into physical, chemical
and enzymatic methods based on the protocol and components used for cell disruption. In
chemical method, most widely used detergents are Tween 20, tween 80, triton x, SDS and
CHAPS. In enzymatic method, lysozymes are used for bacterial cells, chitinase for yeast
cells, pectinase for lysis of plant cell walls. The advantages of chemical and enzymatic
methods are easy to perform and expeditious.
Principle
Enzymatic methods are easy and fast but cost for these methods are also high. In this
experiment we use lysozyme for cell disruption. Lysozymes, also known as muramidase or
N-acetylmuramide glycanhydrolase, are glycoside hydrolases. These are enzymes (EC
3.2.1.17) that damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages
between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and
between N-acetyl-D-glucosamine residues. This dirupts the integrity of cell wall of bacteria
and lead to release of cellular contents.Chemical agents can be used for lysing cell
membranes, chemical agents acts on the cell wall of bacteria causing it to rupture the cells
and release the product out. Detergents are used in cell lysis buffers they help to solubilize
membrane proteins and lipids there by causing the cell to lyse and release its contents outside.

In this experimen Triton X is used for cell disruption by chemical method. Triton X-100 is a
nonionic surfactant that has a hydrophilic polyethylene oxide chain (on average it has 9.5
ethylene oxide units) and an aromatic hydrocarbon lipophilic or hydrophobic group.
The polar head group of TX100 disrupts the hydrogen bonding in lipid bilayer as it becomes
inserted in the lipid bilayer and ultimately demolishing the integrity of the of the lipid
membrane, thus disrupting the cell to release the cellular contents.
Material Required

Tris HCL
NaCl
Triton X
Lysozyme
Acetone
Bacterial culture
Petri dishes
LB medium
Microfuge tubes
Pippettes and micro tips
Ice box and Ice
Water bath
PBS

Procedure
1.
2.
3.
4.
5.
6.
7.

Grow the bacterial cells overnight in LB media

Take 1 ml sample from the culture at OD600 of 1,
Centrifuge the sample at 10000 rpm for 5 minutes.
Vortex to resuspend the cells in 100uL of lysis buffer
Freeze quickly on dry ice and leave for 3 min
Thaw immediately at 42C. Vortex vigorously to mix well
Repeat the two previous steps for three more times (4 freeze-thaw-vortex cycles in

all)
8. Spin the tubes for 5 min at maximum speed in a microfuge
9. Separate the supernatant (contains soluble protein) from the pellet (contains insoluble
protein) by pipetting out the supernatant to a clean tube
10. To each supernatant, add 1 ml acetone and vortex. Freeze or leave on ice for 15 min
11. Spin 5 min at maimum speed. Remove the acetone by pipetting it out, being careful
not to disturb the pellet
12. Dissolve the protein in 500 uL of PBS
13. Estimate the protein concentration using Lowrys method (Refer protocol of protein
estimation by Lowrys method).

Preparation of Solutions
Lysis buffer
20mM Tris HCl pH 7.5,
50 mM NaCl,
0.2% triton X-100,
1 mg/ml lysozyme
PBS (for 1 litre)
Reagent

amount to add(1X)

final concentration (1X0

NaCl

8g

137 mM

KCl

0.2 g

2.7 mM

Na2HPO4

1.44 g

10 mM

KH2PO4

0.24 g

1.8 mM

Notes

Keep water bath at the given temperature ready before start of the experiment

Keep ice box and ice ready

Lysis buffer (with Triton X and Lysozyme ) lyses the cell membrane and cell wall
respectively)

PBS is normally used to dissolve protein. Other buffers like RIPA buffer can also be
used. When using PBS for dissolving protein, store the protein in 4 degree celcius

Acetone makes the protein to precipitate, by reducing the solubility in solution

Freeze thaw is done to rupture the bacterial cells completely, in this step sonication
can also be done

Practice Questions
1. What is the purpose of Acetone in this experiment?

2. Draw the chemical structure of cell wall.

3. What are the natural sources of Lyoszyme?

4. What are the detergents that can be used for cell lysis?

5. How does Triton X disrupt the cell membrane

Evaluation scheme
S. No
1
2
3
4
5

Description
Completion of all steps
Observation absorbance
reading correlated with
sonication time
Completion of calculation and
graph
Answer to the practice
questions
MCQ/Viva

Maximum
Marks

Marks
Obtained

4
2
1
1
2

Total Marks

10

Observation
Give your observation with the help of following points.
# Observe the pellet obtained at the end of the procedure
# Calculate the amount of protein using Lowrys method with standards of previous
experiment
# Plot the graph for the same

Result and Interpretation (Write in your own word)

Cell disruption by chemical/enzymatic method: Faculty use

1. Guideline for conducting the lab:
Cell disruption by chemical/enzyme method is an individual experiment done by students in
microfuge tubes. Each students have to perform the experiment and estimate the protein
concentration based on previous Lowrys standard values and they have to make graph for the
results. Lysis buffer and PBS can be prepared in common for all batches (175 students). The
culture should be made ready the previous day with appropriate bacterial sample (E.coli). The
students have to perform Lowrys estimation only for their samples, the standard values can
be obtained from previous experiments to determine the concentration.
2. Material required for conducting the practical: (15 students/individual)
Per lab session (30
S. No. Material Required
For 15 students
students)
1

LB or nutrient Broth

50ml

100 ml

Tris HCl

2 gms

4 gms

NaCl

4gms

8 gms

triton X-100

2 ml

4 ml

lysozyme

1mg

2mg

NaCl

8 gms

8 gms

KCl

4 gm

4 gms

Na2HPO4

1 gm

2 gm

KH2PO4

1 gm

2 gm

10

Microfuge stand

11

Conical flask

12

Microfuge tube 2 ml

30

60

13

1ml pipette

14

200 ul pipette

15

Acetone

15

30

S.
No.

Material
Required

Per lab
session (30
students)

For total 6
lab session

LB or nutrient
Broth

100 ml

300 ml

300gm

Nil

Tris HCl

4gms

20 ml

200 gms

nil

NaCl

8 gms

8 gms

350gms

nil

triton X-100

4 ml

4ml

600 ml

Nil

Available in
stock

Need to
purchase

lysozyme

2 mg

6 mg

nil

10 mg

NaCl

8 gms

40 gms

350 gms

Nil

KCl

4 gms

4 gms

150

30

Na2HPO4

2 gm

2 gm

12

Nil

KH2PO4

2 gm

2 gm

10

10

Microfuge stand

10

11

Conical flask

12

Microfuge tube 2
ml

60

400

10

Nil

13

1ml pipette

14

200 ul pipette

15

Acetone

3 ml

20 ml

2.5L

No need

Practice Questions
1. What is the purpose of Acetone in this experiment?
Acetone discourage the dispersion of protein molecules in the media. Thus, the solubility of
proteins can be lowered and precipitation can be induced by lowering the effective dielectric
constant of the media. This is commonly achieved by adding a water-soluble solvent such as
acetone to an aqueous solution of protein
2. Draw the chemical structure of cell wall.

3. What are the natural sources of Lyoszyme?

Lysozyme is abundant in a number of secretions, such as tears, saliva, human milk, and
mucus. It is also present in cytoplasmic granules of the macrophages and the
polymorphonuclear neutrophils (PMNs). Large amounts of lysozyme can be found in egg
white
4. What are the Chemicals and enzymes that can be used for cell lysis?
Chemicals: Tween 20, Tween 80, SDS, NP 40
Enzymes: Lysozyme, Pectinase, Hyaluronase, Chitinase

5. How does Triton X disrupt the cell membrane?

Triton X-100 is a nonionic surfactant that has a hydrophilic polyethylene oxide chain and an
aromatic hydrocarbon lipophilic or hydrophobic group. The polar head group of T-X100
disrupts the hydrogen bonding in lipid bilayer as it becomes inserted in the lipid bilayer and
ultimately demolishing the integrity of the of the lipid membrane

Feed back form

Title of the experiment :
Date :

Venue :

Faculty Incharge:
1. Whether you received the expected outcome
No

Yes

2. Able to complete the experiment in the stipulated time

Yes

No
3. Whether you learn the principle and procedure of the
experiment

Yes

No

4. Whether all the equiments are in working conditions

Yes

No

Any other suggestions: