Bakteriell fysiologi och patogenes, 7.5 hp

Transcription

Bakteriell fysiologi
och patogenes, 7.5 hp
(Bacterial physiology and
pathogenesis, 7.5 ECTS)
5MO077, HT 2015
5MO077 Manuel Contents
Course plan
Schedule
Dry Lab
Wet Lab
Safety/Security Instructions
Plagiarism
Laboratory Examination I
Laboratory Examination II
Laboratory Examination III
Unofficial translation
Sid 1 (3)
Sun Nyunt Wai
2015-08-24
Bacterial Physiology and Pathogenesis , 7.5 Credits
Credits: 7.5 hp
Course code: 5MO077
Responsible Department : Molecular Biology ( Biology )
Date of establishment: 2012-09-21
Approved by: Technical Science Faculty
Valid from: 2012-10-08
Level: Undergraduate
Main Field of Study: Molecular biology (basic level with previous academic training). Should have at
least 60 credits in previous courses at the first two years of combined studies (see eligibility
requirements admission requirements).
Grading scale:
Pass with distinction (VG), Pass (G), Fail (U)
Contents
Part 1, theory module, 4.5 hp
The course deals with bacteria diverse physiology and pathogenesis , which provides important
information on the fundamental biological processes that are key to life.
The lectures cover four core areas.
"Bacterial diversity." The lectures focus on bacterial diversity and taxonomic classification , the
molecular basis of life in extreme environments. Basic concepts of bacterial bioenergetics will also be
discussed .
"Bacterial regulatory networks." The ability of bacteria to preserve biological functions when exposed
to harmful environmental factors is a hallmark of their ability to adapt and recover. The lectures deal
with the mechanisms underlying this adaptation , how bacteria regulate gene expression, transcriptional ,
post - transcriptional , translational and post-translationally .
"Physiological processes in bacteria." Lectures address global regulatory mechanisms and the use of "
omics -based" techniques to study this regulation. Mechanistic aspects of important biological processes
will be discussed including molecular transport across membranes, directional movement ,
communication, differentiation and development in both the "single cell" and " multicellular bacterial
communities ."
"Bacterial pathogenesis and host response to infection." This section covers : i) What changes /
mutations in the genome of harmless non-pathogenic bacteria can lead to pathogens that can infect new
Sid 2 (3)
hosts , such as humans. ii ) the host's response to this - the strategies that non- specific and specific
immune system uses to fight bacterial infections. iii) how bacteria recognize and respond to the host
cell's response by regulating the expression of virulence genes to the precise time and place during the
infection . iv ) The alarming increase in multidrug-resistant clinically relevant bacteria in the population
and the implications for antibacterial treatment , and the use of vaccines as a strategy to prevent
infection.
Part 2 , laboratory module, 3 hp
Laboratory part gives practical application of theory content . Students will independently plan
experiments in which both genetic methods and classical phenotypic characterization is used to
exemplify the classification of bacteria
Expected learning outcomes
After completing the course students should be able to:
- Demonstrate knowledge of bacterial diversity, its importance to life on Earth and to use appropriate
methods to study this
- Give examples of the principles behind transcriptional , post-transcriptional , translational and posttranslational bacterial gene regulatory mechanisms and their linkage to changes in the environment
- Summarize the bioenergetics of bacteria , including electron transport and how proton motif force is
generated and its impact on the uptake and transport across the bacterial inner membrane
- Discern the various fundamental physiological processes that are essential for the bacterial life cycle ,
such as stress sensing, motion, communication , differentiation, and protein secretion
- Distinguish the normal flora or bacterial colonization of humans can affect health and disease
- Explain how non -pathogenic bacteria can develop into disease-causing bacteria and how bacterial
pathogens and their virulence factors can be studied in research
- Use the central concepts in infection biology to distinguish bacterial infection strategies , the types of
virulence factors that are involved in the different processes and how these factors are regulated in time
and space during the course of infection
- Demonstrate knowledge of and be able to discuss current issues related to the treatment and
prevention of bacterial infectious diseases
- Demonstrate practical skills in cultivation and analysis of bacteria
Sid 3 (3)
Eligibility Requirements
Completed courses of at least 45 credits in biology , including classical genetics , cell biology ,
molecular biology and physiology. Completed courses in chemistry at least 30 credits , including at least
7.5 credits biochemistry. Demonstrated skill in experimental work involving at least 5 credits. English B
from upper secondary school. Or the equivalent.
Form of instruction
Instruction is in English. The course includes lectures, laboratory exercises and presentations.
Laboratory exercises and presentations are mandatory. The labs will begin with theoretical
introductions and then the students themselves plan the flow of the laboratory work. This planning is
reviewed by the course assistants and must be approved before the lab may begin. The labs are
documented according to the instructions in a laboratory diary ("labnotes"). The results obtained from
laboratory work is reported with an oral presentations and with short written answers to a series of
relevant questions.
In case of absence from compulsory education, the examiner will decide on an appropriate extra
assignment or if the student should complete the module at a later date.
Examination
The examination is a written exam on the theory module and through written and oral examination of
the laboratory modulet with certain aspects assessed continuously (i.e.: maintaining a laboratory
notebook). To pass the course it is required that all mandatory elements are approved . Grades given on
theory module are Fail ( U), Pass (G) and Pass with Distinction (VG). The lab work is given either
Pass (G) or fail (F ) . For the final grade VG requires that this rating is obtained in theory torque and
G on the laboratory portion.
Students who fail , have the right to undergo re- examination to receive a passing grade . The approved
must not undergo re- examination for a higher grade.
A student who has failed two tests for a course or part of a course entitled, upon written request to the
director of studies / head of the Department of Molecular Biology , have another examiner appointed,
unless there are specific reasons against it
Other Regulations
Literature
The literature is not available through the web. Please contact the faculty .
UMEÅ UNIVERSITY
Department of Molecular Biology
Sun Nyunt Wai
SCHEDULE
5MO077 Bacterial Physiology and Pathogenesis, HT15
2015-09-30
BACTERIAL PHYSIOLOGY &
PATHOGENESIS, 7.5 ECTS
COURSE TIME:
30th September, 2015 – 30th October, 2015
LOCATION:
Lectures: Minor Groove, Building 6L, Försörjningsvägen
Practicals: Red & Green Laboratories, Building 6L, Försörjningsvägen
LITERATURE: For example:
1. Online resource: Todar’s Online textbook of Bacteriology at
http://www.textbookofbacteriology.net/index.html
2. Online resource: Microbiology and Immunology Online at
http://pathmicro.med.sc.edu/book/bact-sta.html
3. Other material provided by the Department.
EXAMINATION:
Friday, October 30th, 9.00-13.00, Öp
RE-EXAMINATION: Monday, December 14th, 16.00-20.00, ÖP
COURSE LEADER: Sun Nyunt Wai
Phone: 785 6704; Email: [email protected]
ADMINISTRATOR: Eva-Christine LUNDSTRÖM
Phone: 785 2869; Email: [email protected])
LABORATORY ASSISTANTS:
Lisa WIREBRAND (LWi)
Email: [email protected]
[[email protected] (report submissions only)]
Nikola ZLATKOV (NZl)
Email: [email protected]
[[email protected] (report submissions only)]
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SCHEDULE
2015-09-30
LABORATORY SESSIONS: (Strictly Mandatory)
“Genetic and physiological characterization of enriched bacterial isolates” (LWi, NZl)
LABORATORY EXAMINATION: (Strictly Mandatory)
 White-board presentation of the laboratory project divided into introduction, goals, methods,
results and discussion; a group-based exercise! (Lab examination I)
 Laboratory “Quiz” (short answer and multiple choice questions); a group-based exercise!
(Lab examination II)
Answers are to be submitted electronically to the Laboratory assistants’ (LWi or NZl)
“Urkund” email addresses.
 In addition, your own personal laboratory notebook MUST be utilized on every single
laboratory session. It is also your responsibility to have this CERTIFIED (signed and dated)
by a lab assistant (LWi, NZl) at the conclusion of EVERY laboratory session.
Note: You must obtain a grade ‘G’ to gain credit for this course module.
LECTURERS
Victoria SHINGLER (VSh)
Jörgen JOHANSSON (JJo)
Matthew FRANCIS (MFr)
Bernt Eric Uhlin (BEU)
Sun Nyunt Wai (SNW)
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
LECTURER THEMES
Principles and Applications of Bacterial Diversity
 Bacterial Diversity (Lecture 1; SNW)
 Extreme Environments (Lecture 2; SNW)
 Bioenergetics (Lecture 3; MFr)
General Principles of Bacterial Regulatory Networks
 Transcriptional Regulation (Lecture 4; VSh)
 Post-transcriptional Regulation (Lecture 5; JJo)
 Translational Regulation (Lecture 6; JJo)
 Post-translational Regulation (Lecture 7; SNW)
Physiological Processes
 Bacterial Stress Responses – Global Regulation (Lecture 8; VSh)
 Solute Transport (Lecture 9; SNW)
 Prokaryotic Differentiation and Development (Lecture 10; BEU)
 Bacterial Motion (Lecture 11; VSh)
 Protein Secretion Systems (Lecture 12; SNW)
Bacterial Virulence Strategies
 Bacterial Pathogenesis I– the infection process and virulence mechanisms (Lecture 13; BEU)
 Bacterial Pathogenesis II and III– Studies of bacterial pathogens (Lecture 14 ; SNW)
 Bacterial Responses to the Host Cell – virulence gene regulation (Lecture 15; BEU)
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SCHEDULE
2015-09-30
TUTORIAL
One tutorial session has been planned.
This time is allocated for YOU to address YOUR questions to the course leader and/or other
participating lecturers. Make full use of this available time by ensuring your advanced preparation
(i.e. go through the lecture material before the tutorial).
Remember that:
a) all group members must attend all laboratory orientation/introductory sessions and the full
duration of every experimental session
b) any absence, even for a short period, must be first reported to, and/or agreed upon by the
Course assistant in charge (i.e. you CAN NOT come and go when you please)
c) both group members are expected to participate equally in all laboratory examinations and to
maintain their own individual laboratory notebook that is to be certified by a lab assistant at
the conclusion of each session
Please use any “free” time wisely!
Important Information:
i.
Lab equipment is very expensive – use with extreme care and concentration. If you are unsure
as to how to properly use a particular piece of lab equipment, before use ask one of the lab
assistants.
ii. In the labs, you will be potentially working with pathogenic bacteria deserving of your respect.
Follow all advice given to you about safety precautions.
iii. Treat your lab assistants with respect; it is not an easy job and they do have more laboratory
experience than you.
iv. To be allowed to sit the exam, all laboratory examinations must be completed and/or
submitted.
During a fire alarm, evacuate promptly to the clearing, a safe distance
from the outside entrance to building 6L.
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UMEÅ UNIVERSITY
Sun Nyunt Wai
SCHEDULE
2015-09-30
Week 40
(working week 1)
Wednesday (30-09-2015)
09.00 – 09.45
Course orientation & Roll-call (SNW)
Minor Groove
10.00 – 12.00
Lecture 1:
Minor Groove
“Bacterial Diversity” (SNW)
Lunch
13.00 – 13.45
Laboratory Safety (SNW)
Minor Groove
13.45 – 14.30
Introduction to the laboratory course
(SNW; LWi; NZl)
Minor Groove
Thursday (01-10-2015)
09.00 – 12.00
Laboration: “Day 1 – dry lab”
Minor Groove
sequence data analysis (bacterial identity/clustal W analysis)
(LWi, NZl)
Lunch
13.00 – 14.00
Lecture 2: “Extreme Environments” (SNW)
Minor Groove
Literature study of sequenced bacteria for experimental plan discussions.
Friday (02-10-2015)
09.00 – 11.00
Lecture 3: “Bioenergetics” (MFr)
Minor Groove
Lecture 4: “Transcriptional Regulation” (VSh)
Minor Groove
Lunch
12.00 – 14.00
Literature study of sequenced bacteria for experimental plan discussions.
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UMEÅ UNIVERSITY
Sun Nyunt Wai
SCHEDULE
2015-09-30
Week 41
(working week 2)
Monday (05-10-2015)
9.00 – 11.00
Lunch
12.00 – 13.00
13.00 – 15.00
Lecture 5:
“Post-transcriptional Regulation” (JJo)
Laboration: “Day 2 – dry lab” (LWi; NZl)
Bacterial strain selection
Minor Groove
Red & Green Laboratory
Wet-lab experimental plan discussions/work flow
(LWi; NZl)
Minor Groove
Tuesday (06-10-2015)
9.00 – 11.00
Lunch
12.00 – 14.00
Lecture 6:
“Translational Regulation” (JJo)
Minor Groove
Lecture 7:
“Post-Translational Regulation” (SNW)
Minor Groove
14.30 – 15.30
Laboration:
“Day 3” (LWi; NZl)
08.30 – 10.00
Laboratory
Laboration:
“Day 4 – morning” (LWi; NZl)
Red & Green
10.30 – 12.30
Lunch
13.30 – 17.00
Lecture 8:
“Bacterial Stress – Global Regulation” (VSh)
Laboration:
“Day 4 – afternoon” (LWi; NZl)
Minor Groove
Thursday (08-10-2015)
08.00 – 09.00
Laboratory
Laboration:
“Day 5 – morning” (LWi; NZl)
Red & Green
09.30 – 12.00
Lunch
13.00 – 17.30
Lecture 9:
“Solute Transport” (SNW)
Minor Groove
Laboration:
“Day 5 – afternoon” (LWi; NZl)
Friday (09-10-2015)
9.00 – 12.30
Laboration:
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UMEÅ UNIVERSITY
Sun Nyunt Wai
SCHEDULE
2015-09-30
Week 42
(working week 3)
Monday (12-10-2015)
09.30 – 12.00
Lecture 10: “Prokaryotic Differentiation and
Development” (BEU)
Minor Groove
Lecture 11:
“Bacterial Motion” (VSh)
Minor Groove
Lecture 12:
“Protein secretion systems” (SNW)
Minor Groove
Laboration:
Lunch
13.00 – 15.30
Tuesday (13-10-2015)
09.00 – 11.00
Lunch
12.00 – 17.00
9:00-11:00
Lecture 13:
“Bacterial Pathogenesis I" (BEU)
Laboration:
Minor Groove
Lunch
12.00 – 17.30
Thursday (15-10-2015)
9:00-12:00
Lecture 14:
“Bacterial Pathogenesis II and III” (SNW)
Minor Groove
Lecture 15:
“Bacterial Responses to the Host Cell” (BEU)
(virulence gene regulation)
Minor Groove
Lunch
13:00-15:00
Friday (16-10-2015)
9.00 – 12.00
Laboration: “Day 9” (LWi; NZl)
(Laboratory Roundup!)
To attend the Symposium in connection with Umeå University EC Jubilee Award
Preparation for Lab examinations I and II
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UMEÅ UNIVERSITY
Sun Nyunt Wai
SCHEDULE
2015-09-30
Week 43
(working week 4)
Monday (19-10-2015)
Session 1
08.00 -10.00
10.00-10.15
10.15-12.15
(Break)
Lab examination I “Group presentations - Oral”
(Examinators: LWi and SNW)
Bring your laboratory notebooks
Mandatory
Individual group examination – one at a time (Not open to any audience)
Note: Schedule will follow
Minor Groove
Tuesday (20-10-2015)
Session II
8.00-10.00
10.00-10.15 (Break)
10.15-12.15
Lab examination I “Group presentations - Oral”
(Examinators: NZl and SNW)
Bring your laboratory notebooks
Mandatory
Individual group examination – one at a time (Not open to any audience)
Note: Schedule will follow
16.00
Lab examination II – deadline for “Written Quiz Responses”
(Examinators: LWi; NZl)
Submit electronically to: [email protected]
[email protected]
Thursday (22-10-2015)
Private study
Friday (23-10-2015)
Private study
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Minor Groove
UMEÅ UNIVERSITY
Sun Nyunt Wai
SCHEDULE
2015-09-30
Week 44
(working week 5)
Monday (26-10-2015)
09.00 – 12.00
Q&A session:
“Tutorial” (SNW)
Tuesday (27-10-2015)
Private study
Private study
Thursday (29-10-2015)
Private study
Friday (30-10-2015)
09.00 – 13.00
“Theory Examination”
Location: ÖP
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Minor Groove
UMEÅ UNIVERSITY
Sun Nyunt Wai
SCHEDULE
2015-09-30
Week 51
(working week 6)
Monday (14-12-2015)
16.00 – 20.00
“Re-Examination”
Location: ÖP
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UMEÅ UNIVERSITY
Sun Nyunt Wai
SCHEDULE
2015-09-30
Summary of course composition
Week 40
Week 41
Week 42
Week 43
Week 44
Week 51
Lectures
Tutorial
Laboratory sessions (in working groups of
two)
mandatory
Laboratory
‘Round-up’
Laboratory
Examination I
mandatory
Laboratory
Examination II
mandatory
Theory exam
(4h)
Literature study of sequenced bacteria; preparation for experimental plan discussions
10
Theory Reexam (4h)
Dry Lab
UMEÅ UNIVERSITY
Sun Nyunt Wai
LABORATION PROTOCOL
5MO077 Bacterial Physiology and Pathogenesis, HT15, 7.5 ECTS
Isolation and Identification of Bacteria from Environmental
Samples (Dry Lab)
Introduction:
We live on a microbial planet (1). Consider that forty billion bacteria live in a gram of ordinary
soil and one million live in a millilitre of fresh water. In fact, there are estimated to be ~5×1030
bacteria on earth, forming much of the world's biomass. This represents thousands of different
bacterial species, although almost none of these are known to science. Primarily, this is because
of an inability to culture many of these in the laboratory. This is unfortunate since all bacteria
influence the function of the global ecosystem in some way – predominately through
biogeochemical cycling of carbon, hydrogen, nitrogen, oxygen, phosphorous and sulphur.
Without them we could simply not live!
At the core of bacterial diversity is the dynamic variability of genes within the genome. Gene
variability evolves new traits that enable bacteria to inhabit environmental niches that we as
humans would consider very extreme. In addition, bacteria often associate within mixed
communities, such as a biofilm. As different bacteria within these communities possess
different complementary attributes, this extends niche adaptation beyond what would be
possible for a single homogenous population of bacteria.
Bacterial variability can be seen in many ways. The most obvious of these is differences in
individual cell and colony morphology. Cells can vary in size and shape. Colonies can vary in
shape (overall form, shape of edge, elevation), surface texture, optical characteristics (opaque,
translucent, glistening, dull), consistency (slimy, dry, wet, sticky etc) and pigmentation.
However, numerous other features can define variability including cellular constituents and
structures, metabolic traits, motility, mechanism of cell division and developmental forms as
well as properties that enable adaptation to environmental extremes. Many of these attributes
can be distinguished by microscopy techniques, cultivation techniques, biochemical analyses
and molecular/genomic analyses. While all of these can have their place in a standard
microbiology laboratory, without any doubt the most discriminatory of all available techniques
is direct nucleic acid sequencing (2).
Being a pioneer of molecular phylogenetic studies, the late Professor Carl Woese examined the
sequence structure of genes for 16S rRNA and 18S rRNA for ribosomal subunits of prokaryotes
and eukaryotes, respectively (3). As a direct result from his work, a universal phylogenetic tree
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LABORATION PROTOCOL
(the so-called ‘Tree of Life’) emerged that contained three domains of life – bacteria, archaea
and eurkarya. Archaea defined a new domain of life that was formerly grouped together with
bacteria in a domain called prokaryote. Today, multiple typing methods based upon
compositional analysis of the small ribosomal 16S rRNA gene represent the gold-standards for
the classification and identification of bacterial species (4).
References:
1.
2.
3.
4.
Oren, A. (2004) Philos Trans R Soc Lond B Biol Sci 359, 623-638
Li, W., Raoult, D., and Fournier, P. E. (2009) FEMS Microbiol Rev 33, 892-916
Carl Woese bibliography (http://www.igb.illinois.edu/about/archaea)
Rajendhran, J., and Gunasekaran, P. (2011) Microbiol Res 166, 99-110
The following has been done for you.
Bacterial enrichment and isolation. Water and soil samples were collected. Aliquots were
mixed into nutritionally rich liquid media (eg. BHI broth), and minority bacteria underwent a
growth enrichment phase for 2-3 hours at 30°C. Ten-fold serial dilutions (10-1, 10-2, 10-3, 10-4) of
the cultures were plated onto solid BHI plates and then incubated for 2 days at 30°C.
PCR amplification and sequence analysis. Only well isolated and morphologically distinct
colonies were selected for further analysis. A portion of the colony was re-streaked onto a
master plate (to be provided to you for use in the wet-lab). The remaining portion of the colony
was suspended in a small aliquot of water, lysed for 5 mins at 95°C. This served as the DNA
template for the PCR amplification of the 16S rRNA gene. Amplified DNA product was purified
from an agarose gel, and then subjected to DNA sequencing by Capillary Electrophoresis using
the BigDye sequencing reaction. The result of this DNA sequence analysis is provided for your
analysis in this dry lab.
In case you are interested, see the link for information on DNA sequencing by Capillary
Electrophoresis:
http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/dnasequencing-fragment-analysis.html
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LABORATION PROTOCOL
Now it is your turn. What must you do?
Step 1 (day 1):
Each group will be given two undisclosed DNA sequences.
1) The first task is to utilise public domain software (eg. BlastN) to define what organism
your DNA sequences belong to. (This will be demonstrated to you by the course
assistants.) What is the name of the sequenced gene? What is the genetic context of
this gene on the bacterial chromosome? What part of the gene has been sequenced?
2) The second task is to utilise other public domain software (eg. ClustalW) and perform an
alignment of ALL DNA sequences provided to EVERY group. The idea with this exercise is
to gain some insight into the phylogenetic relationship between the different bacterial
isolates recovered from soil and/or water. (Again, this will be demonstrated to you by
the course assistants.) What is the percent nucleotide identity between your own two
DNA sequences? Which bacteria are the most closely related? Which bacteria are more
distantly related (perhaps even unrelated)?
3) The third task is to perform a literature based search to gain some basic understanding
of the two bacteria species identified by DNA sequencing. This should include salient
features of the bacteria such as colony morphology, pigmentation, Gram stain, motile,
environmental niche, pathogen/non pathogen, lifecycle etc. Armed with this
information, the goal is to achieve three things:
 Compare your information to the individual streaked plates (see Step 2, day 2
and Table 2), to endeavour to select your two isolates for further wet-lab
analysis
 Get the feel for hypothesis-driven research. In other words, you must form a
hypothesis as to whether you predict strain 1 and strain 2 have the following
properties as listed in Table 1. Discuss your hypothesis in your notebook.
 Develop an experimental plan/flow chart that can test your hypotheses. Write
it down in your notebook. That is to say that on Step 2 (day 2), you will present
to the course assistants four potential experimental methods, one for each
hypothesis, which is capable of testing whether your hypothesis is correct or
incorrect. (Note: your methodology must come from a scientific research
publication i.e.: derived from PubMed searches, and not something that you
have pulled directly from the internet.)
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LABORATION PROTOCOL
UMEÅ UNIVERSITY
Sun Nyunt Wai
Table 1: Example of hypothesis driven research
Hypothesis
Yes
Experimental investigation
No
Hypothesis correct
Hypothesis incorrect
Quorum sensing and AHL
production
Flagella based swimming
motility
Biofilm formation
Eukaryotic cell attachment
Step 2 (day 2):
Today, you have two tasks to fulfil in preparation for your hypothesis-driven wet-lab based
experimental research.
1) The first task is to inspect all the agar plates (labelled plate A through to plate P) that
contain single cell streaks of isolated bacteria from soil or water samples. Write down in
Table 2 and your notebook any distinguishing features based upon colony
morphology/appearance. Two of these plates belong to you, and will be used by you in
the wet-labs. Which ones are they? Attempt to match information from your DNA
sequence analysis and subsequent literature review (from Step 1), with the information
recorded in Table 2 and your notebook. Those two that match are (hopefully) your
strains. Check your decision with the course assistants. They can help you select the
correct ones.
2) The second task is to discuss your experimental proposal/flow chart for the wet-lab
component of this course with assistants
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Sun Nyunt Wai
LABORATION PROTOCOL
Table 2: Sequenced bacteria enriched from soil and water samples
Bacterial isolate
Some characteristic
Plate A
•
•
•
Gram negative
Likes the intestinal tract of cattle
Non-lactose fermenter
•
•
•
Gram negative
Coliform
Lactose fermenter
•
•
•
Gram negative
Inhabitants of aquatic ecosystems
Broad temperature range
•
•
•
Gram negative
A-layer production
Flourishes in poor quality water
•
•
•
Gram negative
Facultatively aerobic
Arthritis sequelae
•
•
•
Gram positive
Possesses an array of environmentally adapted survival
mechanisms
Rod shaped
•
•
•
Gram positive
Catalase positive
Spore forming
•
•
•
Gram negative
Strict aerobe
Highly nutritionally versatile
•
•
•
Gram negative
Strict aerobe
Soil remediation
•
•
•
Gram negative
Found on human skin and tissues
Often encapsulated
•
•
•
Gram negative
Can cause water to freeze
Gene-for-gene hypothesis
•
•
•
Gram negative
Infamous relative
Non-lactose fermenter
•
•
•
Gram negative
Environmental
High incidence of antibiotic resistance
•
•
•
Gram positive
Widely found in the environment
non-spore forming
•
•
•
Gram positive
Crystals protein inclusions production
Spore forming
37°C
Plate B
37°C
Plate C
26°C
Plate D
26°C
Plate E
26°C
Plate F
37°C
Plate G
37°C
Plate H
37°C
Plate I
26°C
Plate J
37°C
Plate K
26°C
Plate L
26°C
Plate M
37°C
Plate N
37°C
Plate O
26°C
Identity based on 16S rRNA sequence
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Wet Lab
UMEÅ UNIVERSITY
Sun Nyunt Wai
LABORATION PROTOCOL
Bacterial motility assay
Introduction:
Bacteria can move in many diverse ways (1). It is most common for many bacteria to exhibit
active motility, where they produce organelles that provide the force to propel their motion (2).
Most often motility organelles are flagella, long, rotating rigid structures which protrude from
the cell surface; their propeller-like rotation propels bacterial motion that is termed swimming
motility. However, other modes of flagella-dependent movement are also possible. Some
bacteria use flagella to move over the tops of solid surfaces in a form of movement called
swarming (3), while spiral bacteria have specialized internal flagella that cause the entire cell to
rotate in a ‘corkscrew’ like-fashion (4). Other examples are independent of flagella. Gliding
bacteria move over solid surfaces on a layer of secrete slime (5); this form of motility is
commonly referred to as social motility because colonies of bacteria move together in unison,
and this relies on intimate bacterial communication mechanisms. Some other bacteria also
move independent of flagella by producing type IV pili that extend and retract, and this leads to
a motion termed twitching motility (6).
Why do bacteria move? Motility is an energetically expensive process, so motion must provide
tangible benefits to bacteria. The most obvious of these is the ability to follow concentration
gradients to encounter solute or nutrients essential for growth (chemotaxis), or to avoid
noxious environments that are deleterious for growth. This might even include escape from
predators. In addition, many bacteria preferentially attach to surfaces to form a biofilm. These
surfaces might be particulate matter that is also a source of nutrient to enhance growth. In
order to attach, bacteria must use some form of motility to seek out these favourable surface
hot-spots for growth. Having multiple ways to move, e.g. in liquid or over a solid surface,
enables bacteria to maintain motility even under diverse environment circumstances.
In this laboratory, you will have the opportunity to investigate whether your environmental
samples are motile by way of flagella-mediated swimming. While other forms of motility exist,
this is by far the most likely form for bacteria to exhibit and is easily testable. However, as you
perform this assay, consider what factors are influencing this technique and whether better
assays exist.
For controls, you will be given parental Yersinia (as a positive control) and laboratory
engineered Yersinia that is unable to assembly flagella (as a negative control) control strains.
References:
1.
Jarrell, K. F., and McBride, M. J. (2008) Nat Rev Microbiol 6, 466-476
1
UMEÅ UNIVERSITY
Sun Nyunt Wai
2.
3.
4.
5.
6.
LABORATION PROTOCOL
Erhardt, M., Namba, K., and Hughes, K. T. (2010) Cold Spring Harb Perspect Biol 2, a000299
Partridge, J. D., and Harshey, R. M. (2013) J Bacteriol 195, 909-918
Charon, N. W., Cockburn, A., Li, C., Liu, J., Miller, K. A., Miller, M. R., Motaleb, M. A., and
Wolgemuth, C. W. (2012) Annu Rev Microbiol 66, 349-370
Nan, B., and Zusman, D. R. (2011) Annu Rev Genet 45, 21-39
Burrows, L. L. (2012) Annu Rev Microbiol 66, 493-520
2
UMEÅ UNIVERSITY
Sun Nyunt Wai
LABORATION PROTOCOL
Quorum-sensing response assay
Introduction:
Quorum sensing in bacteria is the ability to communicate to each other via small chemical
molecules to coordinate activities as a population instead of as single cells (1,2). This means
that regulation of gene expression occurs in response to the density of a population; only once
a certain density (number) has been reached – known as a quorum – does a reprogramming of
gene expression occur. This is the way that bacteria regulate multicellular behaviour. The types
of multicellular behavioural patterns that regulated by quorum sensing include: light emission,
motility, cell division, virulence, biofilm formation, antibiotic production and swarming.
The pattern of gene expression is determined by the concentration of signal molecules
produced by bacteria. The concentration of signal molecules accumulates at high cell density
because their production is maximised at high cell populations. There are a number of different
types of quorum sensing signalling molecules e.g.: N-acyl-homoserine lactones (AHLs),
hydroxyquinolones (HQs), furanosyl borate, cyclic dipeptides (cDPs), and oligopeptides. Some of
these molecules are made only by Gram-negative bacteria (e.g.: AHLs, HQs and cDPs). Bacteria
sense when concentrations of signalling molecules are high to alter gene expression.
However, specificity comes from the molecule chemistry and the production of cognate sensing
pathways that recognize these specific molecules. There are at least four basic types of sensing
pathway (1,2). By far the most common are the LuxI/R signalling systems, which are general
Gram negative bacteria. Freely diffusible signal synthesized by LuxI-like proteins is bound
directly by LuxR-like transcriptional regulators. Bound LuxR-like proteins become activated to
modulate target gene transcription (3). A second pathway are hybrid signalling systems that
transmit chemical signals via a phosophorelay transduction system based upon the three
components histidine kinase, histidine phosphotransferase and the, response regulator. So far
these appear to be unique to Vibrio spp (4). The third pathway involves the peptide signalling
systems that also involve signal transmission by phosphorylation (5). These are unique to Gram
positive bacteria. The fourth system is again a phosphorelay dependent system, responding to
autoinducer 3 (AI-3) signals. Besides being used in bacterial interspecies signaling, AI-3 has an
intrinsic role in interkingdom communication because of the similarity to the eukaryotic
hormones epinephrine/norepinephrine (6).
Given the influence that quorum sensing has virulence factor expression, antibiotic production
and biofilm production, there is a great interest to study these pathways. Knowledge of these
quorum sensing pathways could identify novel aspects of bacterial physiology important for
virulence or environmental survival as well as for biotechnological development (7). In turn,
UMEÅ UNIVERSITY
Sun Nyunt Wai
LABORATION PROTOCOL
mechanisms of signal interference could be established as a novel application to control
bacterial virulence or bio-fouling through biofilm formation (8,9).
Given the phenomenal impact that quorum sensing systems have on bacterial physiology, your
samples will be tested for the production of quorum sensing molecule production using a
biosensor. When performing this assay, realise that there are severe limitations to the amount
of information that can be obtained.
As a positive control, you will utilise the clinical bacteria Yersinia YPIII, and as a negative control
you will use the laboratory bacteria E. coli K-12.
References:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Ng, W. L., and Bassler, B. L. (2009) Annu Rev Genet 43, 197-222
Reading, N. C., and Sperandio, V. (2006) FEMS Microbiol Lett 254, 1-11
Nasser, W., and Reverchon, S. (2007) Anal Bioanal Chem 387, 381-390
Milton, D. L. (2006) Int J Med Microbiol 296, 61-71
Lyon, G. J., and Novick, R. P. (2004) Peptides 25, 1389-1403
Hughes, D. T., and Sperandio, V. (2008) Nat Rev Microbiol 6, 111-120
Choudhary, S., and Schmidt-Dannert, C. (2010) Appl Microbiol Biotechnol 86, 1267-1279
Uroz, S., Dessaux, Y., and Oger, P. (2009) Chembiochem 10, 205-216
Njoroge, J., and Sperandio, V. (2009) EMBO Mol Med 1, 201-210
UMEÅ UNIVERSITY
Sun Nyunt Wai
LABORATION INFORMATION
Biofilm formation
Introduction:
Axenic (pure) cultures of planktonic cells grown in the laboratory have given microbiologist a
wealth of information about individual organisms. In reality however, microbes naturally exist as
mixed biofilms. Broadly put, biofilms are often a mixed multicellular community of microbes
associated with any surface (sessile) and are typically encased in an extracellular polymeric
substance (EPS) matrix (1,2).
EPS is the ‘lifeblood’ of the biofilm structure (3). It is responsible for biofilm cohesion and
architecture (biofilm structure). It functions as the “cirulatory system” in terms of channeling
water, the provision of nutrients and providing a reservoir of DNA to facilitate the exchange of
genetic material. Finally, it generates a protective barrier against antimicrobials. This could
either be a physical barrier or a chemical barrier (neutralization of toxic substances and sorption
of heavy metals).
Biofilm formation is complex, but it is generally consider to take shape in 5 major steps: 1) initial
attachment to the substratum, which can be reversible, 2) irreversible attachment, 3) cell
proliferation and microcolony formation, 4) macrocolony formation i.e.: generation of a mature
fully developed biofilm encased in EPS, and 5) Biofilm disintegration (bacterial release). All these
individual biofilm formation stages require certain subsets of proteins e.g. motility and adhesion
factors are required for initial stages of formation, while EPS biosynthesis and secretion is only
required later during biofilm formation (during maturation). Hence, quite an array of regulatory
pathways is required to maintain a dynamic biofilm. None more important than quorum sensing
or cell-to-cell communication (2,4), which we also examine in this laboratory course.
Biofilm formation must be urgently understood because it has a dramatic effect on our natural
environment, industry and human health. Since they are a natural part of the ecosystems,
effects can be positive or negative (5). However, their profound effect on clinical and veterinary
and agricultural infectious disease has commanded the most attention (6), in order to search for
and develop anti-biofilm treatments.
In this laboratory, you will have the opportunity to investigate whether your environmental
samples can form biofilm. As you perform your assay, consider how different biofilm formation
might be in the natural environment.
Page 1 of 2
UMEÅ UNIVERSITY
Sun Nyunt Wai
LABORATION INFORMATION
References:
1.
2.
3.
4.
5.
6.
McDougald, D., Rice, S. A., Barraud, N., Steinberg, P. D., and Kjelleberg, S. (2011) Nat Rev
Microbiol 10, 39-50
Elias, S., and Banin, E. (2012) FEMS Microbiol Rev 36, 990-1004
Flemming, H. C., and Wingender, J. (2010) Nat Rev Microbiol 8, 623-633
Jayaraman, A., and Wood, T. K. (2008) Annu Rev Biomed Eng 10, 145-167
http://www.biofilm.montana.edu/node/2409
Hall-Stoodley, L., and Stoodley, P. (2009) Cell Microbiol 11, 1034-1043
Page 2 of 2
UMEÅ UNIVERSITY
Sun Nyunt Wai
LABORATION INTRODUCTION
HeLa cell association assay
Introduction:
Attachment and colonization of host eukaryotic cells is a fundamental property of bacterial
pathogenesis. Without this feature, bacterial pathogens would be unable to resists clearance by
the washing (flushing) action of host body fluids. Therefore, attachment and colonization allows
a build-up of bacteria at the attachment foci. With greater numbers of bacteria are they better
able to resist the host immune system. The types of adherence factors (ligands) can be many
and varied e.g. hair-like structures termed pili (fimbriae), non-piliated structures, soluble
(secreted) factors and biofilms. While biofilms are a non-specific form of attachment (bacteria
attach on any surface so long as conditions are favourable), the other three represent specific
ligands that must engage with a cognate receptor on the host cell surface. If that receptor is
only produced on certain cell types, or in certain organs, or in certain animals, it establishes a
specific bacterial infection niche; they can only colonize on eukaryotic cell expressing the
correct receptor. This is one reason why some host animals are resistant to infection by a
certain bacteria, but other hosts are susceptible to the same bacterial infection.
Yersinia pseudotuberculosis is an enteropathogen commonly transmitted to humans through
contaminated food or liquid. This pathogen causes a gastrointestinal disorder that is usually
self-limiting and rarely results in a systemic disease. Important for Yersinia virulence is host cell
contact. Yersinia produces several surface-localized adhesins (1-3). The most notable Yersinia
adhesins are invasin, Ail, YadA and the pH 6 antigen. For example, invasin is an important
enteropathogenic Yersinia adhesin that engages β-1 integrins on the host cell surface. Ail
promotes the binding of Yersinia to host cells, and also contributes to serum resistance and the
inhibition of host inflammatory responsiveness. The pH 6 antigen plays an important role in Y.
pseudotuberculosis binding to host cells by interacting with β1-linked galactosyl residues in
glycosphingolipids and phosphatidylcholine on the surface of host cells.
These attachment factors assemble on the bacterial surface. Like all bacterial proteins, surface
proteins are made on the inside of the cell. To reach the surface they must be transported
across four layers of the cell envelope (the inner membrane, the periplasm, the peptidoglycan
layer and the outer membrane). In the absence of certain surface proteins the bacterium is not
able to colonise the host, thus both the surface protein and its trafficking pathway are obvious
targets for antimicrobial development. Within the periplasm are piloting, folding and
degradation factors necessary for correct protein trafficking and protein surface assembly. For
example, at least five periplasmic chaperones (SurA, PpiA, PpiD, FkpA and FklB) are thought to
be involved in the trafficking of surface proteins. One of these chaperones (i.e. SurA) is essential
for the virulence of Yersinia in a mouse infection model (4). In fact, the Yersinia surface
adhesins – invasin and Ail – depend upon SurA for periplasmic trafficking and outer membrane
1
UMEÅ UNIVERSITY
Sun Nyunt Wai
LABORATION INTRODUCTION
assembly; a situation that leaves surA minus bacteria unable to associate with eukaryotic cells
(5).
References:
1.
2.
3.
4.
5.
Kolodziejek, A. M., C. J. Hovde, and S. A. Minnich. 2012. Yersinia pestis Ail: multiple roles
of a single protein. Front Cell Infect Microbiol 2:103.
Mikula, K. M., R. Kolodziejczyk, and A. Goldman. 2012. Yersinia infection toolscharacterization of structure and function of adhesins. Front Cell Infect Microbiol 2:169.
Leo, J. C., and M. Skurnik. 2011. Adhesins of human pathogens from the genus Yersinia.
Adv Exp Med Biol 715:1-15.
Obi, I. R., R. Nordfelth, and M. S. Francis. 2011. Varying dependency of periplasmic
peptidylprolyl cis-trans isomerases in promoting Yersinia pseudotuberculosis stress
tolerance and pathogenicity. Biochem J 439:321-32.
Obi, I. R., and M. S. Francis. 2013. Demarcating SurA activities required for outer
membrane targeting of Yersinia pseudotuberculosis adhesins. Infect Immun 81:2296-308.
2
Safety/Security Instructions
Order and safety directions concerning the course laboratories at Molecular Biology
1)
Lab coats should always be worn in the laboratory
2)
Smoking, eating, use of snuff, application of cosmetics, licking of labels etc are not allowed in the
laboratory.
3)
Gloves should always be worn and trashed after.
4)
Bags should be left outside of the laboratory. Binders and writing pads are placed in the cabinet
under the bench. The floor must be cleared at all time.
5)
Hands should be washed and rinsed with disinfectant before leaving the laboratory.
6)
To decrease the risk for contamination and infection, the lab bench should be washed with 70%
ethanol before and after work.
7)
Immediately label all infected material. Agar plates are labeled on the bottom to prevent mixing
plates up when viewing the plates.
8)
All material that has been used should be treated as infective. Treat laboratory bacterial strains as
pathogens. Never leave bacterial cultures open.
9)
If infectious material is spilt, or if glassware containing such material is broken, the area of
contamination should be thoroughly disinfected with 70% ethanol. Immediately report the
accident to the teaching assistant in charge.
10) All infected material should be thrown away to the yellow autoclave bins which will be replaced
by the teaching assistants when it is full.
11) Empty plastic pipet tip boxes will be trashed to the black bags which is provided by teaching
assistants for recycling.
12) Remove labeling from all glassware before placing it into the washing basin.
13) Never get close to the bunsen burner if you have rinsed your hands with ethanol.
14) Tail up your hair if it is long.
15) Before leaving the laboratory, check that the gas, water baths and other equipment are turned off.
16) Carefully document all laboratory work and calculations done on to your notebook which will be
checked and signed by teaching assistants every day.
How to use the pipette pump
Be careful not to break the pipette when placing it into the pipette pump. A number of accidents have
happened, resulting in hand or wrist wounds. Please follow the recommendations below.
1)
Hold the pipette close to top. This is also important to keep it sterile.
2)
Gently twist the pipette into the pump. Don`t use too much force. If too much force is required,
loosen the pressure on the rubber gasket holding the pipette.
Plagiarism
Laboratory Examination I
UMEÅ UNIVERSITY
Sun Nyunt Wai
LABORATORY GROUP ORAL PRESENTATION – EXAMINATION I
Formalities
•
Oral presentations are performed together in laboratory group (usually a group of two)
•
Total allotted examination time per group is 20 min.
•
Each student presents consecutively for an absolute maximum of 7.5 mins (i.e. you will be
stopped immediately at this point) – you will be given a ‘stop’ warning after 5 min
•
What will follow is then a 5 min combined discussion with the examination panel (to
include course leader and at least one course assistant)
•
The only presentation aid allowed is a whiteboard and whiteboard markers i.e. this is NOT
to be a PowerPoint presentation. (Of course, you are allowed to bring any notes to help you
in your presentation)
•
Prior to the start of your presentation, each student must submit their completed laboratory
notebook (i.e. examination III) for a final appraisal
•
It is a closed room session i.e. no other classmates are present
Your presentation content
• As a group, you are to explain all your activities in the laboratory course. (How you divide the
content is up to you – see below)
• This must involve a description of the goals, methodology (briefly), results and analysis,
conclusions and your reflections.
• The point of this exercise is to provide us with an insight into your understanding and
comprehension of why and what it was that you did. For example, you must demonstrate an
ability to troubleshoot in the event your experiments failed. Moreover, you must provide
insight into the limitation(s) of your experimental methodology and propose how this could
be improved.
• You should also discuss a little about the bacteria you worked with. Critical would be your
interpretation of the data and how this is seen to fit with the known lifestyle of your organism
(as judged from your own literature searches)
• You have complete freedom with how you wish to portion responsibilities among the two of
you, but ensure that both share responsibility equally and that the two presentations are of
similar duration (i.e. each has a 7.5 min limit)
• Remember, this is a whiteboard presentation. Therefore, part of the exercise requires that
you figure out a way to clearly and effectively describe your data using only the whiteboard
and marker pens as tools.
1
UMEÅ UNIVERSITY
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LABORATION QUIZ EXERCISE
Deadline for electronic submission ([email protected] or
st
[email protected]) is kl.16:00 on Wednesday, 21 October, 2015
Note: This exercise is to be performed in pairs with your laboratory partner. For each set of
questions, state the extent of contribution by each partner.
If your partner does not contribute sufficiently, do not submit as a co-authored document. In
this case, seek advice from the course assistants or course leader in good time before the
submission deadline.
Some questions may only require short answer responses. In others, you are expected to
formulate more detailed responses of at least 5 to 10 sentences.
If your answers are insufficient and/or incorrect, the document will be graded as failed (‘U’).
During this semester you will have one guaranteed opportunity for re-examination (i.e. a
corrected resubmission). If resubmission is late – occurring well after the date of course
completion, the course assistants have the right to delay grading until the next instalment of this
course (i.e. the following year).
A pass grade of ‘G’ is only awarded once answers to all questions are considered adequate.
Under no circumstances will any form of plagiarism be tolerated.
Clear definitions of plagiarism are given in your course manual.
1
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Bacterial diversity
1) How can different species be distinguished by growth on agar plates?
2) What advantages does a solid medium have over a liquid medium when culturing bacteria?
3) Why are culture enrichment steps usually necessary prior to diluting out onto agar plates
bacteria derived from some environmental or clinical source?
4) Describe how would you use PCR to amplify the 16S rRNA gene?
5) Why use 16S rRNA sequencing to define a bacterial species? Explain!
6) Instead of using nucleic acid sequencing, describe briefly two other techniques that could be
used to identify a bacterial species?
Bacterial motility
1) Why are flagella important for bacteria?
2) What do flagella arrangements look like? Sketch the four major arrangements.
3) Is flagella-mediated swimming motility the only form of bacterial locomotion? Explain!
4) What is the limitation of the motility assay used in this course?
5) Describe other methods that could be used to demonstrate bacterial motility?
Biofilm formation
1) Describe in detail the structure of a mature biofilm
2) Why do bacteria form these biofilms?
3) In what ways may biofilm formation benefit a bacterial pathogen to mount a successful
infection?
4) How well does the biofilm formation assay used in this laboratory course reflect the true
nature of biofilm formation? Explain!
5) What other method(s) could be used instead?
Quorum sensing
1) What is the principle of Quorum sensing?
2) Read the article: (doi: 10.1099/00221287-143-12-3703). Briefly describe the properties of
Chromobacterium violaceum and how it works as a quorum sensing biosensor?
3) Is it useful for sensing all types of quorum sensing molecules? Explain!
2
UMEÅ UNIVERSITY
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Bacterial colonization
1) Why is colonization an important bacterial pathogenesis strategy?
2) Describe the different factors that bacteria might produce that enable them to colonise
surfaces?
3) How do bacteria utilise these colonization factors to establish a niche tropism during an
infection?
4) What are HeLa cells? How might the use of HeLa cells in this assay impact on the
experimental readout?
5) Discuss the advantages and disadvantages encountered by a bacterium that can induce their
own uptake into eukaryotic cells.
General
1) What is the purpose of controls in experimentation? Have your controls used here fulfilled
their role? Why? Why not?
2) Choose any one of the phenotypic experiments. In a few lines, indicate how it could be
better optimised to provide a more robust readout.
3
Laboratory Examination III
UMEÅ UNIVERSITY
Sun Nyunt Wai
LABORATION NOTEBOOK – EXAMINATION III
What is a laboratory notebook?
A lab notebook is a legal document. It is a permanent, chronological record of experiments that
you performed. It records your thoughts and approaches taken during the course of a project. It
documents the timing and outcome of experiments.
Why is a laboratory notebook important?
As it is a legal document, it authenticates your experimental approach and your collected data.
• First and foremost, it allows you to keep track of what and how you did an experiment, giving
you every chance to reproduce your data again and again. This is critical to move forward in
your research and to produce data of a high international standard appropriate for
publication.
• Second, a well-maintained notebook will avoid any academic integrity issues in the event of
you being accused of falsification and/or fabrication of data. There are many examples
nowadays of international scientists succumbing to the temptations of fraudulent behavior in
order to meet deadlines or simply to get ahead quicker. The accuracy of your notebook is the
key to avoid any such issues of mistrust.
• Finally, many experiments may contain data of commercial interest. To claim ownership of
this intellectual property prior to commercialization, you will probably need to file a patent
claiming novel discovery. The only way for your interests to be fully protected is to present a
notebook that represents your observations, data and interpretations seamlessly, so there can
be no doubt that the discovery is YOUR intellectual property.
Should your laboratory notebook have a particular format?
The answer is both yes and no! Let me explain! Depending on your place of employment/study,
the exact format demanded of a laboratory notebook can vary tremendously among course
leaders (or their course assistants), researchers, principal investigators, academic departments,
universities or private companies. Hence, as you progress through your professional career, you
will most probably need to adapt and change your notebook style depending upon the specific
standards of the individual employers.
Why should you keep a notebook for this series of courses (i.e. the
term 5 block of courses)?
You need to start practicing good habits. It is absolutely critical that:
……….as a scientist, an important skill you must develop is the self-discipline to
maintain an accurate, clear record of your daily experiments in your own
laboratory notebook.
1
UMEÅ UNIVERSITY
Sun Nyunt Wai
LABORATION NOTEBOOK – EXAMINATION III
If you are not convinced, it is a good idea to remember that the laboratory notebook you
generate as an undergraduate is a tangible product of your studies. When looking for a thesis
project placement (or even a job), it could be a good idea to show your potential supervisor (or
potential employer) your lab notebook as a way to demonstrate your ability to keep accurate,
clear records of experiments.
This is what we expect for our course!
Normally, you should endeavor to document records with sufficient detail and completeness so
that an outsider, with background in your field of study, could understand what was done
without your aid.
In your case however, we ask that you do the following each and every day of the laboratory part
of this course.
This is mandatory and takes the form of “Examination III” for this component of the
course.
Notebook checklist (i.e. what you must do!):
(Remember to write neatly and clearly with good detail – after all this is an examination!!!)
1) Date the top of your page
2) State the goal(s) of the day’s exercise. What are you doing? Why are you doing it?
3) State the material you are working with (e.g. bacterial strains). Why?
4) State the types of methodologies you will be using. Why?
5) Make clear notes of any changes to the protocol (e.g. as discussed by course assistants during
pre-lab or at any other time)?
6) Record ALL your results (e.g. raw data such as absorbance readings and other measurements
as well as any observable traits) from the experiment. You must state the time in which you
began your experiments and also again when you make your observations. All raw
data/images need to be entered into your notebook.
7) Reflect on the outcome of the experiment (e.g. discussion, interpretation and conclusions)
8) Given what you discovered from this experiment, make some comment concerning what you
plan to do next?
9) At the end of the day – it is critical for both you and the course assistant to sign you notebook
(with signature and time)
If this is not done correctly, you will be given the grade ‘U’ for this examination.
2

Bakteriell fysiologi och patogenes, 7.5 hp

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