UNIVERSITY OF PENNSYLVANIA

Department of Chemical and Biomolecular Engineering

CBE 580 Biotechnology and Biochemical Engineering Lab Fall 2017
Name: Ryan Fitrian Sofwan Fauzan
Title of Contents
Lab Title Page
Lab 1: Pipetting, Streaking a Plate, Serial Dilution and Plating, and Optical Density 2
Measurement of Cell Concentration

Lab 2:

Lab 3:

Lab 4:

Lab 5:
 Lab 1 9/1/2017

Name: Ryan Fitrian Sofwan Fauzan

Date: 9/1/2017
Investigators: Minsuk Song, Michael Joseph
Lab 1: Pipetting, Streaking a Plate, Serial Dilution and Plating, and Optical Density
Measurement of Cell Concentration

Objectives:
- To understand how to do serial dilution and plating of cell suspension to later count the
cell concentration as colony forming units (CFUs) per mL.
- To understand how to do indirect measurements of cell concentration by measuring the
optical density of the cell suspension.
- To understand how to properly streak a plate with bacteria cells.

Materials and Methods:

Cell counting by serial dilution and plating
Materials:
a. Tube of pUC19 BL21(D3) E. coli cell suspension grown overnight
b. 4 test tubes
c. 10 ml pipette (in the biosafety cabinet)
d. Pipet filler(in the biosafety cabinet)
e. Pipette and tips to deliver 100 l of liquid (in the biosafety cabinet)
f. 6 sterile cell spreaders (blue hockey sticks) (in the biosafety cabinet)
g. 6LB-Amp agar plates (LB-Amp contains 10 g/L tryptone, 5 g/L yeast extract, 10 g/L
NaCl, and 100 g/ml Ampicillin at pH 7.5)
h. Parafilm
i. Marker
j. Vortex mixer

Methods:
1. Make the series of sterile dilutions of the pUC19 BL21(D3) E. coli cells shown in Table
1.1. Tube 1 will be a 15 ml tube, and tubes 2-4 will be 1.5 or 2 ml tubes. Label each tube
as 1, 2, 3, or 4 as shown in the tube column in Table 1.1. Use the 10 ml pipette to add 9.9
ml of the LB Amp to tube 1. Add 100 l of cells to the LB Amp in tube 1. Invert the

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tube several times and shake it to distribute the cells uniformly in the 15 ml tube. Then
prepare tube 2using the cell suspension in tube 1; use tube 2 to make tube 3; and tube 3 to
make tube 4. Be sure to mix each tube well before removing a sample to make the next
tube. The smaller tubes can be mixed with a vortex mixer.

Table 1.1. Cell Dilutions for Serial Dilution and Plating

Tube Tube Volume of E. coli Cells Volume of LB- Dilution Agar
volume Amp Media Plate #
1 100 l cells from pUC19 BL21(D3) 9.9 ml 10-2 none
15 ml
E. coli cell culture tube
2 10 l from tube one 990 l 10-4 1
1.5 or
3 100l from tube two 900l 10-5 2
2 ml
4 100 l from tube three 900l 10-6 3

2. You will make duplicate plates for 10-4, 10-5 and 10-6 dilutions. Label six LB Amp plates
with the dilution factor, agar plate number, date, cell type, and your group number. Label
the plate along the outside edge in small letters so that you can view the surface of the
agar.
3. Shake tube 2 well before spreading the cells on the plate. Pipette 10l from tube 2onto
agar plate 1. Spread the cells over the entire agar surface with a sterile cell spreader, but
do not dig into the agar with the cell spreader. Replace the lid immediately to prevent
contamination from the air. Repeat for a second plate with tube 2.
4. Repeat step 3 by spreading 10l of cells from tube 3 on two agar plates and 10l of cells
from tube 4 on two agar plates.
5. Cut a strip of parafilm narrower than the side of the culture dish and about 1/3 the
circumference of the dish. Seal the top and bottom plates together with a parafilm strip
by stretching the parafilm around the edge of the dish. The parafilm prevents evaporation
of water from the plate during incubation and storage.
6. Wrap your stack of plates together with a long piece of lab tape and write your group
number on the tape. Leave the stack of agar plates in the incubator overnight at 37C.
Invert the plates to prevent any water droplets on the lid from falling on the agar. Liquid
droplets that fall on the agar may cause the cells to float around and clump together on
the plate.

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7. The cells will be moved to the refrigerator the next morning. The plates are inverted
when placed in the refrigerator also.

Optical density measurement of E. coli cell suspension

Materials:
a. 6 ml of pUC19 BL21(D3) E. coli cell suspension
b. Eight 1.5 ml tubes
c. LB-Amp medium
d. Tecan Infinite F200 Microplate Reader
e. 96 well plate
f. 630 nm filter
g. Vortex mixer

Methods:
1. Make the dilutions of pUC19 BL21(D3) E. coli cell suspensions in labeled 1.5 ml tubes
for samples 2 to 7 as shown in Table 1.2. Sample 1 is 100% cells and sample 8 is 100%
medium, so samples 1 and 8 can be pipetted from the stock solutions into the 96 well
plate. Be sure to swirl the flask well to suspend the cells before removing a sample.
Vortex the tube thoroughly after adding the cells to the LB Amp medium to mix the cell
suspension.
Table 1.2. Cell dilutions for OD measurements
Sample % Cell Suspension Volume of Cells Volume of LB-Amp Media
1 100 1.0 ml 0 ml
2 80 0.8 ml 0.2 ml
3 60 0.6 ml 0.4 ml
4 40 0.4 ml 0.6 ml
5 30 0.3 ml 0.7 ml
6 20 0.2 ml 0.8 ml
7 10 0.1 ml 0.9 ml
8 0 0.0 ml 1.0 ml

2. Check the 96 well plate to see which wells have not been used. Look for three
consecutive rows with empty wells. Shake each tube again before pipetting the sample
into the 96 well plate. Pipette 100 l from each tube or stock solution into the bottom of

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three microwells of a 96-well plate for triplicate samples, as illustrated by Figure 1.1.
Depress the stopper until just the first stop to avoid bubbles in the wells. Make sure that
all of the liquid is on the bottom of the well and that there are not droplets stuck on the
sides of the well. Make sure that there are no bubbles and that the well is uniformly
covered with the sample. If a sample has bubbles, add the same sample to another well
and record the sample location.

1 2 3 4 5 6 7 8

A 1 2 3 4 5 6 7 8
B 1 2 3 4 5 6 7 8
C 1 2 3 4 5 6 7 8

Figure 1.1. Locations for triplicate samples of samples 1-8 in the 96 well plate

3. Read the optical density at 630 nm for all of your wells. If the data has a lot of scatter,
check the wells for bubbles. If there are no bubbles and the data is not consistent, make a
new set of samples and repeat the OD measurement.

Streaking a plate
Materials:
a. JM109 E. coli cells
b. pUC19BL21(D3) E. coli cells
c. pGEM-luc BL21(D3) E. coli cells
d. 1 LB agar plate
e. 5 LB Amp agar plates
f. 18 sterile inoculating loops
g. Parafilm
h. Marker

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Methods:
This procedure should be done in the biosafety cabinet. Three types of cells will be streaked
(JM109 E. coli cells, pUC19 BL21(D3) E. coli cells and pGEM-lucBL21(D3) E. coli cells) on
two types of plates (LB and LB Amp plates). The ampicillin (Amp) kills cells without plasmid,
so only cells with an amplicillin resistance gene will grow on the Amp plates. JM109 E. coli
cells do not have a plasmid with antibiotic resistance, so they should grow on LB plates but not
on LB Amp plates. E. coli cells with pUC19 or pGEM-luc plasmids do have antibiotic
resistance, so these cells should grow on both types of plates. Growing JM109 cells on LB and
LB Amp plates is a control to make sure that the ampicillin is active and able to kill the JM109
cells. You can make the following sets of plates:

1. JM109 E. coli cells on one LB plate and one LB Amp plate

2. pUC19 BL21(D3) E. coli cells on two LB Amp plates
3. pGEM-luc BL21(D3) E. coli cells on two LB Amp plates

Data:
1. Cell counting by serial dilution and plating
Data for this experiment will be available in the following week.

2. Optical density measurement of E. coli cell suspension

Optical density data from of the of pUC19 BL21(D3) E. coli cell suspension with various
concentration are shown in Table 1.3.

Table 1.3. Optical density data for E. coli cells suspensions

Sample % Cell Suspension Volume of Volume of LB- Optical Density
Cells Amp Media A B C
1 100 1.0 ml 0 ml 0,4858 0,43 0,4322
2 80 0.8 ml 0.2 ml 0,2941 0,3693 0,3733
3 60 0.6 ml 0.4 ml 0,299 0,2864 0,2798
4 40 0.4 ml 0.6 ml 0,1991 0,2066 0,2064
5 30 0.3 ml 0.7 ml 0,1638 0,1605 0,1829
6 20 0.2 ml 0.8 ml 0,1342 0,1324 0,1316
7 10 0.1 ml 0.9 ml 0,0826 0,0952 0,0831
8 0 (blank) 0.0 ml 1.0 ml 0,0395 0,0589 0,0393

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3. Streaking a plate
Data for this experiment will be available in the following week.

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 Lab 1 9/1/2017

Date: 9/1/2017

Name: Ryan Fitrian Sofwan Fauzan

Lab 1: Pipetting, Streaking a Plate, Serial Dilution and Plating, and Optical Density
Measurement of Cell Concentration

Results:
1. Cell counting by serial dilution and plating
The results for this experiment will be available in the following week.
2. Optical density measurement of E. coli cell suspension
During the Lab 1 experiment, suspension of pUC19 BL21(D3) E. coli cell was diluted
into 8 dilutions. Each dilution was triplicated, and optical density of all dilutions was
measured. Each of the OD data obtained in Table 1.3 then subtracted by the blank
(Sample 8 which contains only LB-Amp medium, no cells added). The final optical
density values are shown in Table 1.4.

Table 1.4. Final optical density data (deducted by each respective blank)
Sample % Cell Suspension Optical Density
A B C
1 100 0.4463 0.3711 0.3929
2 80 0.2546 0.3104 0.334
3 60 0.2595 0.2275 0.2405
4 40 0.1596 0.1477 0.1671
5 30 0.1243 0.1016 0.1436
6 20 0.0947 0.0735 0.0923
7 10 0.0431 0.0363 0.0438
8 0 (blank) 0 0 0

The final OD data was plotted against the %cell concentration (relative to original
suspension, cell concentration data in cells/mL are yet to be obtained), as shown in Figure
1.2. Despite some scatters between each replicate, the plot shows an overall linear
correlation between %cell suspension and optical density, as the trendline gives a good
R2 value of 0.9764. The equation that relates %cell suspension to optical density is
y=249.53x-0.2413 where y is the %cell suspension and x is optical density.

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 Lab 1 9/1/2017

100
90
80

%Cell suspension
70
60
y = 249.53x + 0.2413
50 R = 0.9764
40
30
20
10
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
Optical density

Figure 1.2. Optical density vs % cell suspension curve for all data

3. Streaking a plate
The results for this experiment will be available in the following week.

Conclusions:
1. Cell counting by serial dilution and plating
Conclusion for this experiment will be available in the following week.
2. Optical density measurement of E. coli cell suspension
The OD measurement of various concentration of cell suspension shows a linear
correlation between OD and %cell concentration. Therefore, OD measurement can be
used as a means to indirectly measure concentration of a cell suspension.
3. Streaking a plate
Conclusion for this experiment will be available in the following week.