CHEMICAL ENGINEERING LABORATORY 1

CHE-290

DEPARTMENT OF CHEMICAL ENGINEERING

Fall 2016 Instructor: Jennifer Moll
Edition: Sept 2016

TABLE OF CONTENTS (with Ctrl + click quicklinks)

INTRODUCTION
........................................................................................................1
Basic Laboratory Safety Rules ........................................................................................2
Pipetting...........................................................................................................................3
How to Use Autopipettors ...............................................................................................3
GENERAL INSTRUCTIONS ..........................................................................................4
IN-LABORATORY EVALUATIONS .............................................................................5
Lab Quiz ..........................................................................................................................5
Professional Conduct/Safety Assessments ......................................................................5
LABORATORY REPORTS .............................................................................................7
A. Preliminary Reports ....................................................................................................7
B. Full Memo Lab Reports ..............................................................................................8
Evaluating Errors ...........................................................................................................10
Propagation of Error Equations .....................................................................................11
Error in Linear Regression Analysis .............................................................................12
Assessing Quality of Fitted Data ...................................................................................13
Tables and Figures.........................................................................................................13
C. Data Analysis Reports ..............................................................................................17
LENGTH of MEMO REPORTS for ChE290 ...............................................................18
ACADEMIC OFFENCES UW Policy #71 ....................................................................19
EXPERIMENT 1: INTRO TO THE BIOLAB TECHNIQUES ................................20
Part A: Aseptic Technique ...........................................................................................21
Ai. Transfer Of Microorganisms From Flask To Culture Tube ................................21
Aii. Transfer Of Microorganisms From Flask To Culture Plates ..............................22
Part B: Microscopic Examination of Prepared Slides ..................................................24
Bi: Parts of the Light Microscope ..............................................................................24
Bii Operation of the Microscope .................................................................................26
Part C: Differentiating Bacteria ....................................................................................28
Part D: Morphology of Bacterial and Fungal Colonies ................................................29

ChE290 Chem Eng Lab 1

General Info

INTRODUCTION
Progress in the field of science or technology depends not only on a clear grasp of relevant
theoretical principles, but also on the quality of experimental investigations carried out in that
field. Solutions to many complex problems of interest to chemical engineers can be approached
by a carefully designed and properly executed experimental program.
Laboratory courses allow the student to study numerous experimental arrangements, equipment
details, and measuring devices which cannot be covered adequately in a lecture course. An
appreciation is also gained of the difficulties involved in obtaining accurate data under properly
controlled conditions.
Basically, the three main course objectives of this laboratory course are:

To assist in the understanding of basic principles of chemical engineering through actual

observations of the behaviour of physicochemical systems.

To help develop skills in experimentation, data analysis and interpretation of results.

To practice in the art of writing effective engineering reports.

The laboratory may also be viewed as an opportunity to gain experience working in a laboratory
setting and in the safe handling of hazardous materials. The experiments have been designed to
minimize hazards however, some materials pose potential health risks if not handled properly.
For your safety, as well as, that of others in the lab you should ALWAYS work in accordance
with the safe laboratory procedures listed below. It is essential that you carefully follow the
instructions of the teaching assistant and the experimental procedure outlined in the lab manual.
BEFORE BEGINNING THE EXPERIMENT, review the procedure and familiarize yourself
with the potential hazards of the materials with which you will be working. Information on all the
hazardous materials used in the ChE 290 laboratory can be found on the Safety Data Sheets
(SDS) binder available in the laboratory or can be found online through UW Safety Office
http://www.safetyoffice.uwaterloo.ca/hse/chemicals/whmis/msds.html

The following safety practices are useful not only in the ChE 290 laboratories, but may also
serve to minimize the risks to yourself and your colleagues in any laboratory, industrial, or other
situation where chemicals are utilized or handled.

ChE290 Chem Eng Lab 1

General Info

Basic Laboratory Safety Rules

All students are required to have completed the WHMIS 2015 course. Use common sense.
If you have any doubts about a procedure, ask the TA for assistance.

Safety goggles must be worn when labs are in operation. Contact lenses only permitted with
goggles that seal properly around eyes.

Lab coats, closed-toe shoes and long pants must be worn when working in the labs.

Food and beverages are strictly prohibited from the lab.

Tie long hair in a ponytail. Confine loose clothing, ties or jewellery. Be extra cautious
when working near open flames.

Familiarize yourself with the hazards associated with the materials and equipment to be
used. Consult Safety Data Sheets (SDS) prior to experiment. Wear lab gloves.

No chemicals to be released down the sink. All hazardous wastes must be disposed of in
appropriate waste containers. Read label before disposing into any receptacle. *Note*
solvent and aqueous wastes are collected separately. All materials that contact microbe
must be disposed in orange biowaste bags or containers.

Keep chemical bottles closed tightly when not in use.

Immediately inform your Teaching Assistant of any injuries or spills. For cuts, burns and if
a chemical comes in contacts with your skin, eyes or mouth, flush immediately with water at
the sink or safety station. You must file an accident report with the department. In the
event of a serious injury, inform TA that you must go to Health Services. TA will assign
someone to accompany you.

Dispose of any broken glass in the bucket labeled Broken Glass. Place bucket in
fumehood if glass is contaminated with liquid. Do not pick up shards with bare hands.

Clean up your work area and wash hands thoroughly before leaving the lab. No gloves in
hall.

Know the location of the following:

Fire extinguisher

Chemical Spill Kit

Safety shower

Telephone

First aid kit

Emergency Exit Route

Fire alarm

IMMEDIATELY REPORT ALL ACCIDENTS AND INJURIES

TO THE DEMONSTRATOR OR TEACHING ASSISTANT
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ChE290 Chem Eng Lab 1

General Info

Pipetting
Pipetting is required in most of the ChE 290 experiments and your results will depend upon
transferring an exact quantity of uncontaminated fluid. The following are a few notes to help
ensure an effective pipetting technique.
To avoid contamination:

Never pipette directly from the stock bottle. Transfer a small amount of stock fluid to a
clean, dry beaker and pipette from there. Unless one must retain the full volume of the
sample, rinse the pipette once with the solution to be used. Discard excess.

Ensure that no fluid enters the rubber pipette bulb. Keep the pipette tip immersed as a
sudden influx of air will cause fluid to enter the bulb. Do not lay the pipette horizontally
with the bulb still attached.

Rinse the pipette well with deionized water between transfers of different solutions.

To ensure accuracy:

Use volumetric rather than graduated pipettes wherever possible. Volumetric pipettes are
more accurate for the same volume. When using graduated pipettes, transfer desired volume
by emptying between graduations (i.e. to transfer 4 mL from a 5 mL pipette, drain from the 5
mL mark to 1 mL mark and discard remainder).

Do not blow out volumetric pipettes. Most are calibrated to be emptied by gravity with a
drop remaining in the tip. Touch the tip to the glass wall of the receiving vessel to remove
any drops from the exterior of the pipette.

How to Use Autopipettors

1. Ensure that pipette tip is attached securely
2. Set the desired volume on dial by twisting plunger head. Do not twist beyond the maximum
specified on the autopipettor body!
*NOTE* Step 3 is important to ensure proper hydrostatic pressure for pipettor accuracy:
3. Insert pipette tip just a few mm below the liquid surface and hold the pipettor at a 90 angle
to surface of liquid when aspirating.
4. Press the plunger down just until you feel resistance then slowly release making sure to keep
tip within liquid.
5. Transfer to desired receptacle and empty pipette by pressing plunger all the way down a few
times.

ChE290 Chem Eng Lab 1

General Info

GENERAL INSTRUCTIONS
Most of the experiments require several students working together in order to manipulate the
equipment, take readings, and record the data. Accordingly, students are assigned to groups of
two to four members.
Before commencing an experiment, all members of a group should have a thorough
understanding of:

Theoretical principles involved in the experiment.

Type and amount of data to be recorded.

Expected results.

Whenever the foregoing requirements are met, the experiment can become a true learning
experience which will immeasurably assist in understanding the underlying principles;
otherwise, the exercise becomes merely one of manipulation of equipment and the reading of
various instruments.

Every member of a lab group is expected to actively participate in the lab. The
pre-assigning of duties to each member of the group is strongly advised in order
for the experiment to be performed effectively and quickly.

ATTENDANCE:
Marks are reduced for latecomers to the lab. If you have a conflict with a co-op interview, you
must inform the TA in advance in order to make alternate arrangements to fulfill your lab
requirements.
In the event of a lab missed due to an illness, contact the TA or lab instructor asap. You must
provide the chemical engineering undergraduate secretary with a Verification of Illness form
completed by a doctor. Arrangements will be made for you to make up for the missed lab.
Verification of Illness form can be downloaded from:
https://uwaterloo.ca/health-services/student-medical-clinic/services/verification-illness

ChE290 Chem Eng Lab 1

General Info

IN-LABORATORY EVALUATIONS
Lab Quiz
Quizzes are held over the first 10 minutes of the lab session in experiments #2 & #3. Please note
that latecomers will not be given extra time. Maximum of 5 short answer questions to test your
knowledge of the theory, safety and procedures of the experiment. To prepare, read over the
experiment theory and procedure in the lab manual and attend the lab preview to look over the
equipment in the lab.
Professional Conduct/Safety Assessments
Although you are going to work as a group, some marks are allocated for individual professional
conduct. You are expected to come to the lab session on-time and well-prepared for the
experiment. Marks may also be deducted for lack of participation or focus during the
experiment, not complying with safety protocol, damaging/mishandling lab equipment or leaving
station untidy, inefficient use of lab time, and failing to assess observed data.
Before starting a new project or working with unfamiliar equipment, it is extremely important for
your own personal safety, as well as that of others working nearby, that a review of the
procedure is made including an assessment of the potential hazards involved. Below is a list of
potential sources of hazards to consider in our labs:

SAFETY ASSESSMENT CHECKLIST

When evaluating the safety hazards involved in an experiment, consider the follow hazard categories:
Chemical Hazards (e.g. exposure, toxicity, incompatibilities, waste handling, by-products formed)
Electrical shock
Burn/scald potential or risk of fire
Radiation exposure
Pinch points or Rotating parts
Cuts or Stabs potential
Pressurized lines (explosions), vacuum systems (implosions)
Potential Projectiles
Suffocation hazard or low O2 level potential
Trips/falls
Overhead hazards (falling objects, low-clearance zones)

ChE290 Chem Eng Lab 1

General Info

Also consider the precautions that must be taken to ensure your personal safety and that of the people
working nearby.
o
o
o
o
o
o

Proper handling of chemicals

Proper operation of equipment
Personal Protection Equipment (PPE) required
Protective devices needed on equipment (i.e. Guarding on rotating parts)
Preventative behavior (i.e. securing loose clothing and hair, not wearing contact lenses when
working with chemicals, checking integrity of PPE, etc.)
Emergency response (avoiding injuries or contamination from hazardous incidents: what to do if
there is a spill of chemical, basic first aid, etc.)

During the lab session, the TA will ask you to review the safety hazards involved with the
experiment. They might ask each group member a few questions to review the purpose of the
experiment, highlight potential hazards in the procedure and/or to discuss steps that will be taken
to circumvent these hazards.
For example, comment on the class of chemicals to be used in the experiment.

General

precautions would be to wear safety glasses and gloves when working with these chemicals to
reduce risk of personal exposure and in the event of a small spill, clean-up will be done with
paper towel but for larger spills the TA will be informed. It is good lab practice to consult the
SDS before handling any chemicals in the lab, particularly concentrated sources, to check for
any special precautionary warnings regarding the use and disposal of such chemicals.
The TA may also ask you to explain the function of key parts of the experimental set-up (e.g. the
spectrophotometer in lab 2, the potentiostat in lab 4, etc.) or the purpose of specific steps in the
procedure. The safety assessment is part of your professional conduct mark.

ChE290 Chem Eng Lab 1

Lab Reports

LABORATORY REPORTS
A scientific report is not merely a list of facts or observations; it must provide an interpretation
of the findings and show their significance. A good engineering report is organized logically and
the wording chosen carefully so that the reader knows exactly what you mean and where to find
the evidence that supports your conclusions.
The requirements for memo reports are outlined below:
Three types of laboratory reports are required for each lab in this course: Preliminary Report,
Lab Memo Reports and Data Analysis Report. The requirements for each type of report are
outlined below:
A. Preliminary Reports
The purpose of a Preliminary Report is to prepare for the experiment. Each group prepares one
Preliminary Report for experiments that involve in-lab submission of a Data Analysis or Results
Report. The marks for these reports count toward the course mark of each group member. This
report should be submitted to the online dropbox by noon of the day before your lab session. It
will be reviewed by the TA to check that the group is not pursuing some erroneous task and
graded. Your TA will address any major concerns at the start of the lab session.
The prelab report should contain the following:
1)

Introduction and Statement of the objective.

2)

Equations to be used in calculation of results including details of assumptions made.

3)

General summary of the experimental design in your own words (i.e. number of runs and
range over which the experiment, equipment used, diagram of experimental set-up, etc.).

4)

Possible hazards to be encountered in the experiment and safety precautions to be taken.

(refer to safety assessment checklist, highlighted hazards in lab manual procedures and
SDS sheets for chemicals to be used).

5)

Blank datasheet with appropriate headings (bring a copy to lab).

6)

Answers to Prelab Report Questions from lab manual.

Be sure to leave some time before submission is due to read over the entire report from
cover to cover to check grammar, spelling, consistency of thought and proper
referencing to data, tables, figures, equations and literature sources. This review is
especially important when the report is a compilation of efforts by several group
members.

ChE290 Chem Eng Lab 1

Lab Reports

B. Full Memo Lab Reports

A Memo Report is a complete record of your experiment. It describes:

Why the experiment was performed.

What data were obtained, and

Your interpretation of the data

The Memo Report should include the following:

1)

2)

Title Page

Name of the University and Department.

Course name and number.

Experiment number and title

Group number and names all the members of the group contributing to report.

Date experiment was performed.

To whom and the date that the report was submitted.

Abstract
Provide a brief overview of the purpose of the experiment, the procedure and equipment
used, key observations, findings, and recommendations drawn from the experiment.

3)

Table of Contents
List numbered divisions of the report (e.g. 1.0 Introduction) with page numbers opposite.
Lengthy divisions should be subdivided into sub-numbered headings (e.g.1.1 and 1.1.1) as
needed.

4)

Introduction
The purpose of the Introduction is to guide the reader to consider the importance of the topic
being presented. It should include:

General background information relating to the experiment.

Brief statement of the objective.

Significance of the data obtained to Chemical Engineering (i.e. current and future
applications).
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ChE290 Chem Eng Lab 1

5)

Lab Reports

Theoretical Principles
This section should include:

Brief discussion of the theoretical foundation of the experiment.

Include all necessary equations to be used in the calculations. Label each with a unique
id. Derivations of equations go in the Appendix. Remember to comment on any
assumptions associated with the derivation of the equation.

Conclude with a brief statement indicating how the objectives will be met using the
theoretical principles involved.

The Introduction and Theoretical Principles must be written in your own words. Do not
copy from the Lab Manual. Research your topic. Use library resources.

6)

Experimental
In this section:
a) Summarize, in your own words, the laboratory procedure performed. DO NOT JUST
COPY PROCEDURE FROM LAB MANUAL.
b) Include a labeled sketch or schematic of the experimental set-up.
c) State all modifications made to the procedure.

7)

Observations and Results

In this section, you should present your qualitative and quantitative observations. Guide the
reader through the manipulation of the data in fitting it to the theoretical model.
Qualitative Observations
a)

Describe the experimental observations and, if required, refer to the appendix which
contains your original measurements.

b)

Report any irregularities in the experimental procedure that might explain outlying
data points.
Original datasheet should go in an appendix

ChE290 Chem Eng Lab 1

Lab Reports

Quantitative Results
c)

Present results which were calculated based on the data recorded during the
experiment. Remember to add reference to which equation from theoretical
principles section was used. Place sample calculations in the appendix but refer to
them here.

d)

Use tables or figures to clearly demonstrate trends in data (Refer to Tables and
Figures section below).

e)

List the estimated precision of your measurements, as well as, any other
observations which may help to explain the results. Refer to the Evaluating Error
section. Compare to expected values based on referenced literature sources.

f)

Use significant digits. Remember to include units.

Always maintain significant figures when reporting measured or calculated values. The
number of digits used to express a value is extremely important as it indicates the precision
of the value. The last significant digit is considered to vary by 10 % unless the error is
specified. It is incorrect and misleading to report values to higher precision than possible
from the equipment used in the experiment. Refer to Chapter 11 section 6.2 in Introduction
to Professional Engineering in Canada by Andrews et. al. (2003) for more information on
determining significant figures through algebraic operations.

Evaluating Errors
Unless one is counting individual objects, there is error inherent in all measurements. Two types
of errors occur: Systemic (or determinate) and Random errors.
Systemic errors are constant or proportional biases in a measurement occurring due to the
investigators habits or poor calibration or drift of the analytical equipment. For example, the
ruler was misread by +0.5 mm each time, or the pressure gauge was zeroed when the system pressure
was actually 10 psi however, the upper range was accurate. Large differences between

experimentally-derived results and commonly-accepted or cited values of a parameter are likely

due to the occurrence of systemic errors. Fortunately, systemic errors may be identified and
correction factors used to compensate for them if they can be quantified. Systemic errors can
also be minimized by regular recalibration of analytical equipment and by standardizing
experimental procedures.
Random errors, on the other hand, are inevitable and vary from reading to reading. It is an
estimate of these random errors which must be included when reporting any measurement or
derived experimental value. Unlike systemic errors, random errors cannot be quantified exactly
but they can be approximated. For example, you measure the length of your pen to be 16.0 cm using a
ruler with gradations to the nearest 0.5 cm. Those markings are thick and imprecise so you might assume
that you can read precisely to, say, the nearest 0.1 cm. This is the external error of this measurement.
You would report the length as 16.0 0.1 cm. Depending on the size of the scale of measurement,

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ChE290 Chem Eng Lab 1

Lab Reports

the human eye can differentiate up to 1/10 of the interval between markings. For digital
displays, the precision is to the manufacturers specifications, if given or half of one decimal
place below the last stable digit. Use judgment in assessing the precision of the measurement.
Random errors tend to offset each other so, for any given parameter the average of several
repeated measurements will be more precise than any single measurement. You can estimate
this higher precision by calculating the average value ( x ) and the standard deviation (s) of n
repeats. Your measurement would be reported as x SE where the standard error SE can be
approximated by:

SE

s
n

Propagation of Error Equations

If the precision of individual measurements are known, the overall precision of any value
derived from these measured values can be approximated using the equations shown below:
Addition and Subtraction

y k k1a k 2 b k 3c ...
y

Multiplication and Division

k1a 2 k 2 b2 k3c 2 ...

kab
cd
2

y
a
b
c
d

y
a
b
c
d

Exponential

y bn
y n b

y
b

In General

y f ( x)
y x

dy
dx

Where: k, n are constants, a, b, c, d, x and y are measured variables, x is the standard

deviation of x which can be approximated by the standard error of a repeated measurement or
the estimated precision or external error of a single measurement.

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ChE290 Chem Eng Lab 1

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Error in Linear Regression Analysis

Linear regression uses the Method of Least Squares to determine the line which best fits N
experimental data points. The relationship is plotted as: y = mx + b. Generally in the 290
experiments, the values of interest from a regression line are the intercept, b or the slope of line,
m. Unless the data fit perfectly to a straight (i.e. R2=1), then some error is inherent in these
regressed values and this error must be reported.
Generally in linear regression, the known value is plotted on the x-axis while the measured value
which has some error associated with it is plotted on the y-axis. Linear regression attempts to
determine the error in the y-value associated with a given x-value.

To simplify the calculations, some key quantities need first be defined:

S xx

Sr

xi2

xi 2

S yy m 2 S xx
N 2

S yy

yi2

yi 2
N

where (xi,yi) are the individual data points.

Use the following equation to find the standard deviation of the slope, sm:

sm sr / S xx
The standard deviation of the y-intercept, sb, is given by:

Sb

Sr

xi 2
N
xi2

The standard deviation, sc, of any point taken from the regressed line ( =
+ )
based on an average of M replicate measurements, is found using:
S
Sc r
m

1
1 y y 2
c
M N
m 2 S xx

The line over y indicates the mean (average) of all values of yi (where i = 1.N) used to
determine the regression line.

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Assessing Quality of Fitted Data

When regressing data, it is assumed that the random deviations from the model are
independent, normally distributed, and have a constant variance. For any modeling of fitted
data, you should assess the quality of the fit of the model using the coefficient of
determination and residual plots. Outliers should be identified and justification provided if
they are removed from the evaluation of the data.
Tables and Figures
Any tables and figures must be explicitly mentioned (cited) in the text and should be
inserted in the report as closely after they have been cited as possible. All figures and
tables must have titles and they must give units for all data presented. Identify tables using
Roman numerals (i.e. Table I).
Tables must contain enough information to stand alone. The reader should not have to refer
to the report to understand it. An example of a table is shown below.
The experimental conditions and the measurement results are shown in Table .
TABLE I: pH of the reaction product in the titration of 1.0 mmol/L Copper perchlorate using
0.04 wt % Ethylenediamine [en] solution (298 K).
[en] in Cu solution

pH

mmol/L

0.00
1.46
2.93
4.41

2.80
2.89
3.05
3.05

Figures should be prepared by computer. Label all figures with Arabic numerals (i.e. Figure
1, Figure 2, etc.) and a relevant title. As with tables, be sure to include all information that is
necessary for the reader to interpret or identify the image (i.e. reaction temperature, pressure,
etc.) in the title or in a caption below the graph or image.
For graphs:
Label each axis with a scale and title. Use appropriate scales (i.e. 2s, 5s, or 10s).
Remember to include units
If several sets of data being plotted, use a legend to identify the different symbols
Depict observed data as discrete points
Plot trendlines or fitted models using lines
Initial and date all figures

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Microbial Growth Curves

ChE290 Chem Eng Lab 1(grown at 37C in Tryptose Phosphate broth

Lab Reports

with 2.5 g/L dextrose added)

3.000

Bacillus subtilis

2.500

Optical Density (ABS)

Streptococcus faecalis
2.000

1.500

1.000

0.500

0.000
0

100

200

300

400

500

600

Incubation Time (min)

Figure 1: Microbial Growth Curves for Bacillus subtilis and Streptococcus faecalis cultures
grown at 37C in Tryptose phosphate broth with 2.5 g/L dextrose added.

8)

Discussion of Results and Error Analysis - MOST IMPORTANT PART!

Many students have trouble knowing what to discuss. A good guideline is to think of the
experiment not merely as a school exercise but as an experiment being performed as
engineering research. Indicate which observations or calculated results would be useful to
other engineers who might read your report. Guide the reader through your analysis of the
observed results. Explain any discrepancies with the theory and what may have occurred to
cause the discrepancy?
Discuss the quality of your experimental data. Are there any data points which may be
unreliable? State the reasons for the unreliability. Consider the purity of the reagents
used.

All the equations should be presented in the theoretical principles section and
referred here in the discussion section only by equation number.
Note that you must support your ideas and this will usually require finding and
reading reference material.

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ChE290 Chem Eng Lab 1

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Discuss your calculated results. It may be necessary to review briefly any key
assumption made in the derivation of the equation used in the calculations and how they
might affect the result obtained. Mention any assumptions made in the calcualtions, such
as, values assigned as constants or data points that were excluded from the calculations.
Relate your results to the theory. Do your results agree with values presented in
literature? Include references. Check overall error relative to error propagation results.
Are your values reasonable? If not, try to explain the course of the discrepancy.
Discuss the error analysis. Include a discussion of the individual measurement errors that
contributed to the overall error in the calculated results. Which measurement error had
the largest effect on the results? Could the results be improved by taking replicate
measurements, by using different equipment, or by taking measurements under different
conditions? Is the precision in your results comparable to experiments in the literature?
Comment on how well objectives of experiment were met.
Discuss the significance of your results, and any new information that has been learned.
9)

Conclusions
This section should include a summary statement of the relation between the results and the
objectives of the experiment.
What are the principal results of the experiment? They can be listed in point form stating
the most important ones first.
Summarize the significance of the results obtained as they relate to the objective stated
and any applications in engineering.

10) Recommendations
Recommendations to improve the experiment should be included here.
Most important recommendation should be made first.
Base recommendations on lab experiences, observations and error analysis. Consider
how the experiment could be improved. Make sure your recommendations are feasible
from a practical point of view.
Approximate the % improvement in the quality of the results that could be obtained.
11) Nomenclature
Ensure consistency and uniqueness of symbols used to define variables used in calculations.
Define all symbols used in the report in alphabetical order.

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12) References
For journals, give complete information, including names of all the authors, title of the
article, name of the journal, volume, page number(s) and the year of publication. For
example:
Reilly, P.M., B.M.E. van der Hoff, and M. Ziogas, Statistical study of the application
of the Huggins equation to measure intrinsic viscocity, J. Appl. Polym. Sci., 24, 20872100 (1979)

1.

For references to books give author(s), Title, edition, publisher, place and (year of
publication), and the page number(s).
2.

Laidler, K.J. and Meisner, J.H., "Physical Chemistry", Benjamin/Cummings Publ. Co.
Inc., Menlo Park, California (1982), p. 789.

For websites give the authors name, Title of section used, URL, (date accessed)
3.

Shuzon Ohe, Distillation Computation, http://www.s-ohe.com/McCabe-Thiele.html

(accessed Jan. 19, 2005).

The abbreviation et al. should not be used in the reference list. List the names of all authors.
Do not repeat references. If the next reference refers to the same article or book write:
4.

Ibid., p.238.

If referring to a reference already listed, refer to its reference number and give page number:
5.

Ref. 2, p. 237.

13) Appendices
The purpose of putting extraneous material, such as, experimental data into the appendix is to
prevent disruption of the coherence of the report by long derivations or large columns of
numbers. Materials that should be in the appendix include:

Long tables of original observations.

Lengthy derivations of equations

Sample calculations

Answers to Memo Report Questions.

Material essential for understanding the report must remain in the body of the report.

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C. Data Analysis Reports

A Data Analysis Report is a partial lab report that focuses on presenting the experimental results.
It includes the following sections:

Title Page

Experimental procedure highlighting modifications

Results and Discussion inc error analysis

Conclusions and Recommendations

Although an abbreviated lab report, it should follow the proper lab report format with
nomenclature, proper referencing (internal to figures/tables/equations and to literature), sample
calculations and other appendix material

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LENGTH of MEMO REPORTS for ChE290

To reduce the time spent writing and to help you produce organized and concise reports, the
following guidelines regarding the length of Memo Reports for the course are recommended (not
required):

Title or cover page

Table of contents

1 page

Abstract

< 150 words

Introduction

1/2 page

Theoretical principles

1-2 pages

Experimental

1 page

Observation of results

1-2 pages

Discussion of results and Error Analysis

2-3 pages

Conclusions and Recommendations

1/2 page each

References

page

Appendices

2+ pages

Your reports should be as straightforward and concise as possible.

Note that Figures and Tables are not included in the guidelines above.

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ACADEMIC OFFENCES
EXCERPTS FROM THE UNIVERSITY OF WATERLOO POLICY #71
"STUDENT ACADEMIC DISCIPLINE POLICY"
Original text available at: http://www.adm.uwaterloo.ca/infosec/Policies/policy71.htm
"Communication, inquiry and the free exchange of ideas are fundamental to a university
education, and require an environment of tolerance and respect. Academic freedom provides for
the freedom to study, learn, publish and debate, independent of current opinion, subject to
commonly accepted scholarly standards. Academic freedom is protected and carries with it the
duty to use that freedom in a responsible and ethical way. A student's academic freedom does not
extend to disruption of other students, faculty or staff members, or their work/study/residence
environments.
Academic integrity is a commitment to five basic values: honesty, trust, fairness, respect and
responsibility. It applies to all academic endeavours teaching, learning and scholarship, and
applies to a range of academic activities, from conduct in research to the writing of co-op work
term reports.
Students are expected to know what constitutes academic integrity, to avoid committing
offences, and to take responsibility for their actions."

Some of the academic offences outlined by the University include:

Infringing unreasonably on the work of other members of the University community

(disrupting classes or examinations; harassing, intimidating or threatening others).

Cheating on examinations, assignments, or any other work used to judge student

performance. Cheating includes copying from another student's work or allowing
another student to copy from one's own work, submitting another person's work as
one's own, fabrication of data, consultation with an unauthorized person during an
examination or test, and use of unauthorized aids.

Plagiarism, which is the act of presenting the ideas, words or other intellectual property
of another as one's own. The use of other people's work must be properly
acknowledged and referenced in all written material.

Submitting an essay, report, or assignment when a major portion has been previously
submitted or is being submitted for another course without the express permission of all
instructors involved.

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ChE290 Chem Eng Lab 1

Intro to Biolab

EXPERIMENT 1: INTRO TO THE BIOLAB TECHNIQUES

Special skills are needed when working with living cells in a biological lab. Microorganisms
pose a serious risk of infection if they enter to our bloodstream, digestive tract or mucous
membranes. All biological cultures should be treated as potentially pathogenic and so, be
handled with the proper respect and care. Special procedures, collectively called the Aseptic
Technique, have been developed to minimize the risks of exposure of the handler, as well as, to
prevent contamination of the work environment and maintain the purity of the culture being
transferred from medium to medium. For your safety and that of your fellow lab mates, it is
imperative that the proper procedures be followed. The Aseptic Technique should become
second nature to you when working in the biolab.
Another important skill when handling microbes is to be familiar with the use of the microscope.
Microorganisms come in a variety of shapes and sizes with specific features and responses to
environmental factors that can be used to identify and isolate specific cell types. Microscopy
enables the determination of the morphology and ultrastructure of cells. Ultrastructure refers to
structures found internally and externally of the cells. The type of microscopy used is dictated
largely by the size of the object one wishes to resolve. The resolving power of a light
microscope is limited to the wavelength of light. Generally, light microscopy only allows for
viewing of cell membranes/walls, nuclei, and mobility structures such as flagella. Biological
staining or phase contrast can improve their visibility. However, higher resolution microscopy is
needed to view most cell organelles and is achieved by moving to shorter wavelengths in the
electromagnetic spectrum (i.e. electron microscopy TEM or SEM).
Other characteristics that are used in classifying microbial cells are those associated with growth
behaviour (i.e. colonial morphology, pigmentation, etc.); physiological properties (optimum pH
and temperature for growth, antibiotic resistance, etc.); nutritional requirements (C and N source,
metabolic pathway, etc.) and biochemical cell structures (cell wall constituents, pigmentation,
RNA, etc). In this lab, you will be introduced to techniques that take advantage of such
properties to effectively isolate or identify specific cell cultures, such as, differential staining
(e.g. Gram stain) and selective growth media (e.g. NA-EMB, LB broth with ampicillin, etc.).
Before leaving this lab, you should have demonstrated for the TA competence in using the
Aseptic Technique, be able to quickly locate and focus on cells at 1000 magnification using a
microscope, have identified the biological characteristics which can be exploited to classify
and/or isolate living cells and have discovered the ways in which various microorganisms are
applied in industrial-scale biotechnology. No report is required but you will be tested on the
contents of this lab in future quizzes and/or the lab final.
TASKS AT A GLANCE
Perform aseptic technique.
Observe prepared slides under the microscope.
Gram stain a bacterial culture and observe under the microscope.
Observe colony morphology under stereomicroscope.

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ChE290 Chem Eng Lab 1

Intro to Biolab

Part A: Aseptic Technique

In order to study the properties and behaviours of a particular microbe, one must isolate and
concentrate a single species in a particular medium and be able to maintain the culture purity
during transfers to new media. Microorganisms cling to surfaces and are easily transferred from
one surface to another. Any objects that make contact with a cell culture will become
contaminated. Practicing the Aseptic Technique essentially means to take all prudent steps
necessary to prevent this contamination. In addition to protecting your culture from
contamination by unwanted microorganisms from the surrounding environment, use of the
Aseptic Technique will protect you from contamination by your culture.
All capped glassware and fresh, uninoculated media used in the preparation has been sterilized in
an autoclave (i.e. exposed to steam at 121C for at least 20 minutes) to ensure that it is free from
any viable cells, viruses or spores. By following the steps outlined below, you will learn the
techniques required to aseptically isolate and transfer a culture from one medium to another.

Ai.

Transfer Of Microorganisms From Flask To Culture Tube

Materials
70% ethanol

test tubes of 9 mL of sterile culture medium

Bunsen burner

culture broth

inoculating loop
Procedure
1. Secure hair, roll up sleeves and remove any rings, watches, etc. Clear a work area of the
bench and sterilize the surface with 70% ethanol. Allow to air dry.
2. Thoroughly wash hands and wrists with soap and towel dry. Rinse hands with 70% ethanol
and air dry.
Why use 70% ethanol? Why not higher or lower concentrations of ethanol? And why air dry?

3. Obtain a shaker flask of culture broth containing the seed culture.

4. Light the Bunsen burner.
5. If you are right-handed, hold the shake flask of culture broth in the left hand and the wire
inoculating loop in the right hand. If you are left-handed you may wish to reverse the use of
the hands in each of the steps.
6. Sterilize the inoculating loop by flaming the entire wire portion until red hot.
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ChE290 Chem Eng Lab 1

Intro to Biolab

7. Swirl the culture broth gently to ensure a homogenous suspension. Holding the flask at about
a 45o angle, remove the foam plug with the last two fingers of the right hand. Sterilize the
8. Dip the inoculating loop into the flask. Allow the loop to quench (reach the temperature of
the culture). Shake the loop to remove any heat-killed microorganisms. Carefully remove
the loop from the flask. Quickly flame the mouth of the flask, replace the foam plug and
place the flask on the bench.
9. As quickly as possible, pick up the capped sterile test tube of culture medium with the left
hand, remove the cap with the last two fingers of the right hand and quickly flame the mouth
of the test tube. Insert the inoculating loop. Be careful not to touch the inoculating loop to
the lip or sides of the test tube. Shake the loop within the medium to dislodge the
microorganisms.
10. Remove the inoculating loop and again flame the mouth of the test tube. Replace the test
tube cap and return tube to tray.
11. Flame the inoculating loop until red hot.
12. Return the flask to the incubator. Re-sterilize the bench space with ethanol. Wash hands
with soap thoroughly.

Aii.

Transfer Of Microorganisms From Flask To Culture Plates

(Spread Plate Technique)

Materials

70% ethanol

10 mL sterile pipette

Bunsen burner

culture broth

inoculating loop

culture plates

Procedure
1. Secure hair, roll up sleeves and remove any rings, watches, etc. Clear a work area of the
bench and sterilize the surface with 70% ethanol. Allow to air dry.
2. Thoroughly wash hands and wrists with soap and towel dry. Rinse hands with 70% ethanol
and air dry.
3. Obtain a shaker flask of culture broth containing the seed culture.
4. Light the Bunsen burner.
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Intro to Biolab

5. Obtain an individually sealed pipette. If you are right-handed, hold the flask in the left hand
and the sterile pipette in the right hand. If you are left handed you may wish to reverse the
use of the hands in each of the steps.
6. Open the pre-sterilized pipette package only at the blunt, cotton plugged end.
Do not touch the pipette with your hands
7. Insert the pipette securely into the bulb. Remove the pipette from the packaging. Do not
allow to contact benchtop or any other surface.
8. Holding the flask at about a 45o angle, remove the foam plug with the last two fingers of the
right hand. Flame the mouth of the flask briefly.
9. At the same time, holding the pipette in the right hand, quickly flame tip of the pipette.
Insert the pipette into the flask and allow the pipette to quench. Swirl the flask gently to
obtain a homogenous mixture. Withdraw 0.2 mL of culture broth.
10. Quickly flame the mouth of the flask, replace the foam plug and place the flask on the bench.
11. As quickly as possible, tip the lid of the culture plate AWAY from you and deposit the
culture from the pipette onto the centre of the plate. Replace the lid of the culture plate.
Discard the pipette into the biohazard bag/labeled bucket.
12. Holding the glass spreader in the RIGHT hand, lightly spray with 70% ethanol into a beaker.
KEEP THE FLAME AWAY FROM THE ALCOHOL. Pass the spreader very carefully over
the flame just once to ignite the excess alcohol and sterilize the spreader. DO NOT
OVERHEAT THE GLASS SPREADER.
13. Tip the lid of the culture plate AWAY from you but keep it over the plate as you spread the
inoculum evenly over the entire surface of the solid medium by rotating the plate with the
fingertips of your LEFT hand as you rest the spreader lightly on the surface of the solid
medium.
DO NOT PENETRATE THE SURFACE OF THE AGAR!
14. Replace the lid of the culture plate. Re-sterilize the glass spreader.
15. Return the flask to the incubator. Wait 5 minutes, and then invert the plates and incubate.
16. Re-sterilize the bench space with ethanol. Wash hands with soap thoroughly.

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Part B: Microscopic Examination of Prepared Slides

Materials

Prepared slides (Escherichia coli, Bacillus subtilis, Streptococcus lactis, Spirillum sp.,
Saccharomyces sp., Mold types)
Microscope
Ethanol spray bottle
Immersion oil
Lens Paper

Procedure
Bi: Parts of the Light Microscope
Using Figure 1-1, identify and become familiar with the following parts of the microscope:
Brightness Control Dial/Power Source Switch - this switch controls the power and brightness of
the light source. It should be adjusted until adequate brightness is obtained.
When photomicrography is not being performed, brightness is largely subjective.
Revolving nosepiece - rotating part to which the objectives are attached. When rotating from
one objective to another, make certain by looking from the side, that the objective
will clear the slide on the stage otherwise this may result in damage to the slide or
the objective. Also ensure that the objective clicks into position.
Objectives -

lenses that magnify the image by the power shown on the side of the objective
lens, e.g. lower power objective (10) gives the smallest image magnification,
high power objective (40) gives a larger image magnification, and the oil
immersion objective (100) gives the largest image magnification.

Eyepiece -

a lens that magnifies the real image an additional 10X. The overall magnification
of the image seen by the eye is, therefore, the product of the magnifying power of
the objective and the eyepiece lenses.

Stage -

supports the microscope slide, which is held on the stage by a mechanical slide
holder. The slide is moved on the stage by the back and forth travel knob and the
lateral travel knob.

Condenser - a lens located directly below the microscope stage that concentrates the light
before it passes through the specimen. During magnification the intensity of the
high flux decreases. The condenser compensates for this by focusing the light on
the specimen. The condenser lens is focused by means of the condenser focus
knob.

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ChE290 Chem Eng Lab 1

Intro to Biolab

Condenser aperture diaphragm ring - an iris diaphragm on the bottom of the condenser that
regulates the amount of light entering the condenser lens.
Coarse focus knob - a rotating knob that moves the stage up or down, and in this manner the
specimen is brought into initial focus. This knob is used ONLY with low power
objectives (10). When used with high power objectives, there is the danger of
driving the objective into the microscope slide.
Fine focus knob - a rotating knob that brings the specimen into fine (sharp) focus. It is used to
achieve fine focus with low power objectives (10) and for ALL focusing with
high power objectives (40, 100).

Diopter Ring

Eyepiece
Headpiece
Revolving Nosepiece

Condenser Focus Knob

Objectives

Coarse Focus Knob

Stage

Fine Focus Knob

Brightness Control Dial

Stage Motion
Travel Knobs
Aperture
Diaphragm Ring

Filter Receptacle
Field Lens Unit

Figure 1-1: Compound Microscope

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ChE290 Chem Eng Lab 1

Intro to Biolab

Bii Operation of the Microscope

Please note that ONLY USE LENS PAPER to clean the ocular and objective lenses of the
microscope. Kimwipes and paper towel can scratch the glass.
1. Turn the power source switch ON and adjust to adequate brightness.
2. With the stage at its lowest position of travel, rotate the 10 objective into position and place
the slide with cover glass on the stage. Move the specimen into the light path by adjusting
the stage position with the travel knobs on the right-hand-side.
3. Taking care to ensure that the objective does not contact the stage, bring the stage to its
highest point.
4. Then, looking through the eyepiece, gradually lower the stage using the coarse focus knob
until shadows of cells are evident. Once the specimen is in coarse focus, clarify the image
using the outer (fine) focus knob.
5. Adjust the interpupillary distance by moving the eyepieces until the right and left images
become one circular view field.
6. Adjust for difference in eyesight between your two eyes by looking into the right hand
eyepiece with the right eye and focusing the image with the fine focus knob. Then, look into
the left hand eyepiece with the left eye, and focus the image for this eye by turning the
DIOPTER RING located on the eyepiece section.
7. Adjust the aperture diaphragm by removing the right eyepiece and closing it until the border
can be seen to be projected on the image plane. Then, adjust the condenser focus knob until
the border is in focus. Next, gradually shrink the diaphragm until the field of view and
eyepiece are about the same diameter. Replace eyepiece.
8. Once the image is focused at 100 magnification (10ocular 10objective = 100), do not
touch the course focus knob. The microscopes are parfocal which means that they are
designed such that the 40 and 100 objectives can be rotated into position and the image
focused with only FINE FOCUS (outer knob) adjustments. When rotating objectives, take
precautions to protect the lens from hitting the slide.
9. The refractive index of the 100 objective is such that observations must be performed with
immersion oil between the cover glass and the front lens of the objective. The black band
marking around the barrel end of the 100 objective indicates that it is of the oil immersion
type. Place one or two drops of immersion oil on the area of interest on the cover glass, then
carefully rotate the 100 objective into place, viewing from the side while doing it. Focusing
will only require small changes in the FINE focus knob.
After finishing the oil immersion observation, be sure to clean the 100X objective lens and
prepared slide with ethanol and LENS PAPER!

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10. Use the microscope to view each of the prepared slides listed below. Describe the cell
morphology - is it rod, coccus, or spiral shaped? Consider which features differentiate this
culture from the others?

Prepared slides are fragile, please handle them with care. Clean them with 70% ethanol and
lens paper after use. Use only the FINE focus knob for all examinations at magnifications
above the low power objective (10) to reduce the risk of running the objective lens through
the slide.

Escherichia coli are enterobacteria that typically ferment lactose and produce indole from
tryptophan. It will not grow on media containing citrate as the sole carbon source. E.coli
is one of the most widely studied microorganisms. The wealth of knowledge on its
physiology makes it a traditional workhorse of biotechnology.
Bacillus subtilis is a soil bacterium, used commercially to make several biological catalysts and
antibiotics. It too is well characterized, and depending on the application, presents some
advantages over E. coli industrially.
Streptococcus lactis are one of the important milk bacteria that break down the milk sugar
lactose to lactic acid. Although very common, this is not the only microorganism that can
cause milk to curdle.
Spirillum spp. is a genera of bacteria that possess a characteristic helical or spiral morphology.
Saccharomyces sp. is a fungus, commonly referred to as yeast. It has traditionally been used in
fermentation for the production of alcohol. Yeasts have several advantages compared to
bacteria in industrial applications and it is commonly used when bacterial cells are not
suitable. (See the description of reproduction of yeast on pg 23 of Shuler & Kargi., pg 17
of Bailey and Ollis).
Mold types - This slide contains at least three different types of fungi. Note the different
vegetative structures, which are collectively referred to as the mycelium. It is composed
of long, thin filaments, which are called hyphae. Note also the variety of spore bearing
structures.

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Part C: Differentiating Bacteria

One of the most important and widely used differential staining techniques is the Gram stain. It
is a four-part procedure, which uses organic dyes to make a bacterial cell stand out against its
background for easy determination of its shape. In addition, the Gram stain allows
discrimination of cell wall characteristics based on the retention of a dye complex. The cell wall
is porous to most small molecules but is essential to maintaining the structure of the cell.
Organisms that retain the crystal violet-iodine complex are called Gram-positive (Gm +) and
appear bluish-purple under the light microscope. In these cells, the majority of the cell wall
(~90%) is comprised of a peptidoglycan or murein layer and may contain teichoic acids that help
to bind substances to the cell surface. Organisms that lose the complex and take-up the colour of
the counter-stain are called Gram-negative (Gm -) and will stain reddish-pink. Gram-negative
cells have a more complex cell wall structure which is comprised of only a thin peptidoglycan
layer (~10 % of the cell wall) surrounded by an outer cell membrane called a cell envelope. This
envelope is composed of phospholipids and lipopolysaccharides that are readily removed by the
decolourizer agent. Some organisms partially retain the dye complex will appear as Gm + while
others as Gm -. These are referred to as Gram-variable.
Materials

light microscope
Bunsen burner
clean glass slide
coverslip
inoculating loop
stopwatch

bacteria culture
Gram stain kit (crystal violet, iodine,
decolourizer, safranin solutions)
70% ethanol
distilled water
immersion oil

Procedure
1. Using a wax marker, draw a circle approx. the size of a dime on a clean glass slide.
2. Obtain a loop of bacteria from the plate/culture flask with a sterilized inoculating loop and
spread within the marked circle. If necessary, use a drop of water to thin out. Remember to
note the name of the culture being tested.
3. Heat-fix the smear by slowly passing the slide face-up through the flame three times. Avoid
excessive heating of the slide. Be careful to not burn yourself. Allow the slide to cool.
4. Place slide on mesh. Flood the smear with crystal violet and let rest for 60 seconds.
5. Wash the slide with distilled water to remove excess dye. Flood the smear with the iodine
solution and allow to stand for 1 to 2 minutes.
6. Wash the slide with distilled water. Add decolourizer drop-wise until no colour is emitted
from the smear. Allow to stand only briefly for 30 seconds then wash slide with distilled
water.
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7. Flood the slide with safranin; and let rest for 60 seconds.
8. Wash slide with distilled water to remove excess dye. Gently blot the slide dry with bibulous
paper, and allow it to air dry before viewing. Do not rub the smear.
9. Make microscopic observations working up to 100 X magnification with oil immersion.

Part D: Morphology of Bacterial and Fungal Colonies

During fermentation, samples are routinely taken and their microscopic and cultural
characteristics determined to detect possible contamination of the fermentation broth. When
microorganisms in a liquid suspension are dispersed on the surface of a semi-solid culture
medium in a Petri dish and then incubated, individual colonies will develop. These colonies
consist of millions of cells that have originated from the division of a single, viable cell. The
characteristics of a colony are specific to the species and therefore, are useful in identifying and
classifying bacteria and fungi.
Several procedures are available to isolate a single microorganism from a mixed population.
Physical techniques can be applied when plating a culture on a solid medium. Streaking of a
plate involves dragging a loop of cells from a concentrated region across the fresh media surface
while Spread plates are prepared by diluting a concentrated culture before applying to the agar
surface. Both techniques can sufficiently disperse the original culture to isolate individual
colonies on the plate. Chemical techniques of isolation involve tailoring the growth media to the
culture of interest. Selective media which contain a specific carbon or nitrogen source or
chemical additive (e.g. antibiotics, EMB, etc.) can be used to enrich specific microbial species
over others. A combination of physical and chemical techniques can be applied in a series of
platings to gradually isolate specific species.
Materials

Culture plates
Stereomicroscope

RulerProcedure

The cultures on display represent a variety of microorganisms that are used in industrial
biotechnology applications. Examine the demonstration material. For each culture plate, use the
colony properties tablet provided to describe the following characteristics:
i.
Relative colony size
ii.

Pigmentation or colour

iii.

Elevation of the colony

iv.

Appearance of the edge or margin

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