Biotechnology In The Classroomx

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22 Φεβ 2013 (πριν από 4 χρόνια και 10 μήνες)

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Chris Mitchell

Benton High School

Advanced Biology

Goals



Introduce you to some of the new microbiology labs
that I use in my classroom.


Take a look at the technology available to demonstrate
tough concepts to students.


Review the labs and the concepts which they help to
reinforce.


Share examples of what works and what doesn’t work.

Background


Labs are used in my 12
th

grade Advanced Biology
course.


Students have a double lab period, twice a week on
back to back days.


The other days consist of 42 minute class periods, used
to cover the theory.


Much of the equipment in the lab was purchased as we
began to offer Advanced Placement Biology, with
required labs as part of the curriculum.


Many manufacturers offer similar lab kits as well as
replacement materials.

Lab # 1 An Introduction to
Microbiology Techniques


Learning Objectives
:


Students will use aseptic technique to culture
microorganisms.


Students will use the pour plate technique to prepare
agar dishes.


Students will distinguish between colonies of bacteria
and fungi.


Students will heat fix and stain bacteria for
identification purposes.

Rationale


This is a good introductory lab which can be used to teach
sterile technique, omnipresence of microorganisms, and
simple culturing techniques.


Students get hands
-
on practice using the instruments
needed for collecting and transferring bacteria.


You can introduce bacterial staining techniques i.e.. Gram
Staining, which can be used for identification purposes.


Groups can prepare slides to identify the general shape of
bacteria which they have collected.


Students enjoy being able to choose sites for collecting and
comparing which group(s) obtained the largest growth.


Procedure


Part 1: Media Preparation
:


Can be done with all types of agar (I usually just use
what I have left over from last year)


Be sure to emphasize sterile technique (washing hands,
wiping down tables, care for Petri dishes)


Agar can be melted in hot water bath (heat ahead of
time), or a microwave works well.


Allow plates to cool before attempting to transfer any
microbes.



Procedure Continued


Part II: Collection and Culture:


Allow students to choose a sampling sight of interest around
the school (remember to inform staff of what is going on!)


Provide students with sterile swabs to collect samples.


Use streak plate technique to transfer microbes from swab to
plates. (No poking holes in agar)


Incubate at 30⁰C for 4
-
5 days or at room temp for 5 days,
having students check plates daily for growth.


Tip
: Plates may have to be incubated upside down to prevent
moisture from dropping on agar and inhibiting growth.

Procedure Continued


Part III: Observing Colonies and Isolating Pure
Cultures


Compare bacterial growth (circular colonies) to any fungal
growth (white or yellow fuzz).


Students can now prepare slides for examination using
various staining techniques. ( we Gram stain to ID Gm+ or
Gm
-
).


Have students practice flame sterilizing inoculating loops for
bacterial transfer. (Stress sterile technique here!)


Have students classify bacteria collected by shape (cocci,
bacilli, or spirilla and by Gm+ or Gm
-
)


Colonies can be isolated and transferred to additional agar
plates for growth and further study.

Bacterial Culturing

Clean Up / Analysis/ Extension


Clean up:

Autoclave (if available) or place in autoclave bag
and soak with bleach. I would
strongly

recommend using
disposable plates. Also, sterile disposable inoculating loops
are available.


Analysis:
Students can report on the type of bacteria which
was cultured and the area of the school which the sample was
taken from. Also, the teacher can confirm their findings by
examining their stained slides.


Extension:

Now is also a great time to introduce the concept
of antibiotics. Antibiotic soaked discs (or varying
antibiotics?) can be placed on areas of growth and then
incubated to see the possible effect.


Sanitizers (germ
-
x, hand sanitizers, and other “antibacterial”
products) can also be tested for effectiveness.




Lab #2: Colony Transformation


Learning Objectives:


Students will genetically modify a strain of
E. coli
bacteria by inserting a plasmid.


Students will gain an understanding of the principles
used in bacterial transformation.


Students will use a selectable marker found in bacterial
DNA to alter the phenotype of bacteria.


Students will transfer antibiotic resistance to a strain of
bacteria to use for selection purposes.


Rationale


This lab satisfies AP Lab #6 requirements for AP Biology.


Several different plasmids are available for genetic
modification (pBlu, pGreen, pAmp, pVIB,…) from several
manufacturers.


I recommend using the pBlu or Pgreen, since both have
easily observable phenotypes.


The plasmid not only alters the overall appearance of the
bacteria, but also makes the bacteria antibiotic resistant.


This activity allows students to gain an understanding of
the process used to insert plasmids into “competent”
bacteria and also the effectiveness of such techniques.

Practice Before the Lab


I have students practice the theory behind the lab
before doing the actual activity.


Students access the free Prentice Hall


Lab Bench
website at
http://www.phschool.com/science/biology_place/labb
ench/lab6/design1.html


Students work through online tutorial and complete
quiz at the end to show me they “know what they are
doing”. (

I highly recommend doing this before
attempting to do the lab.)

Transformation Lab Procedure


Part 1: Plate and Colony Preparation:


Students need to prepare their agar and pour their
plates. Label plates before pouring.



They need to prepare 3 different types of plates:


Plate 1 = Simple Luria Broth Agar plates (LB)


Plate 2 = Luria Broth Agar + Ampicillin (LB + Amp)


Plate 3 = Luria Broth Agar +Ampicillin + X
-
Gal


What is X
-
gal?


a substance when metabolized by the
transformed bacteria which makes them turn blue!


Once plates are poured, they will need to be cooled
down before use.



Part 1 Procedure Continued


Colony Transfer:


The
E. coli
bacteria supplied in the stock culture need to
be transferred onto individual Luria Broth agar plates
and grown.


Plates need to be incubated at 37⁰C for a 22 hour period.
Timing is critical, since bacteria need to be in the
most rapid phase of growth for the transformation
to work!


Tip:

Plates need to be incubated upside down to prevent
moisture from falling onto the agar.

Day 2 Procedure


Day 2: Colony Selection and Transformation


After 22 hours of incubation, bacteria should be in the log
phase (most rapid growth)


Locate areas of the plate that show individual (satellite)
colonies


avoid areas where lawns have formed.


Using a sterile inoculating loop, transfer a loop full of bacteria
to the test tube provided and follow the transformation
procedure.


Also


as a
Control
, some of the non
-
transformed (
-

plasmid)
bacteria should be applied to each of the three types of plates.
(If I am running low on materials, we have just used one set of
plates as a “class control”)


It is extremely important to follow the times listed in order to
“heat


shock” the bacteria to get them to take the plasmid. If
temps are too hot or too cold, or if not enough time is
allowed, transformation will not occur!



Testing for Transformation


Once bacteria have been transformed, they will need
to be applied to the three types of agar: LB, LB+Amp,
and the LB+Amp+X
-
Gal.


To do so, glass beads are supplied with most kits to
spread bacteria evenly over the surface of the agar.


Plates need to be incubated at 37⁰C overnight or at
room temp. if no incubator is available. Have students
check the results on each of the plates for a three day
period. ( I have students do a daily count for each
plate)


I also have students predict the outcomes on each of
the plates before viewing their results.

Expected Results

Control Group (
-

Plasmid)

Experimental Group (+ Plasmid)


LB agar
= Lawn of Growth


LB + Amp
= No growth


bacteria were not
transformed and were thus
killed by ampicillin


LB+Amp+X
-
Gal

= No Growth

bacteria were not
transformed and were killed
by ampicillin and thus
couldn’t metabolize X
-
gal


LB agar
= Lawn of Growth


LB+Amp

= many clear
-
white
circular colonies formed,
indicating resistance to
ampicillin was achieved.


LB+Amp+X
-
Gal

= several
blue colonies formed,
indicating ampicillin
resistance and blue color due
to metabolizing X
-
gal!

Questions? Comments?


Has anyone done either of these experiments?


How did they work out for your students?


What changes would you make?


What other labs are offered for senior biology
students?


Does anyone else make use of the Prentice Hall Lab
-
Bench website? Any other lab simulation web
-
sites?


Contact email:
cmitchell@bentonsd.k12.pa.us