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PRACTICAL
BIOTECHNOLOGY
ADDITIONALINFORMATION
Byrom, J. and Byrom,D. (1991) Biopol
Natures plasticNCBE NewsletterSummer 1991. pp. 911.
A broader perspectiveis provided in:Whatever happenedto
bioplastics?byHannah PearceScientific Europeanpp.1417.(Supplement
toScientific American,December 1990)
Practical worksuitable forundergraduates issuggested in:
Huisman, L. A. (1982)Isolation andidentification of thereserve
material ofBacillus megaterium,in Sourcebook ofexperiments for
theteaching ofmicrobiology,Primrose andWardlaw (Eds), pp.233241,
AcademicPress.
POLY--HYDROXYBUTYRATE (PHB) is an energy reservepolyester
naturally accumulated by a wide variety of microorganisms.This
plastic can comprise up to 70% of the dry weight of some
species.
The build-up of PHB during growth on carbohydrates is promoted
by
phosphorus or nitrogen limitation.
Unfortunately it is not possible to produce large quantities PHB
in the
school laboratory. However, the accumulation of PHB inside
bacterial
cells can be observed if a sufficiently high-powered microscope
(with
an oil immersion lens) is available.
Materials
Culture ofAlcaligenes eutrophus
(available from Philip Harris Limited)
Half strength nutrient broth
Sudan Black B stain (0.3% in 70% ethanol)
Safranine stain (0.5% in water)
Xylene (dimethylbenzene see safety note below)
Universal bottle and incubation facilities (25C)or a fermenter
e.g.NCBE Bioreactorin which to
culture microbes
Microscope with oil immersion objective
(at least x100)
Forceps, inoculating loop, Bunsen burner etc.
Prac tical details
Growing the bacteriaAfter complete utilisation of the nitrogen
in the nutrient
broth, the bacteria can no longer grow and energy derived
from the sugar in the medium is used for the production of
the reserve material, PHB. Sufficient bacteria for staining
may be conveniently cultured in McCartney bottles or in an
NCBE Bioreactor.
1. Aseptically transfer a loopful ofAlcaligenes
eutrophus from a slope to a Universal bottle
containing 20 cm3 of half-strength nutrient broth.Alternatively,
prepare an inoculum and fermenter
for larger scale cultivation.
2. Incubate the bacteria at 25C for 48 hours.
Staining for PHB
1. Flame the loop and allow it to cool. Remove thecap from the
culture bottle, flame the neck,
remove a loopful of broth, flame the neck again
and replace the cap.
2. Spread the culture on a clean, grease-free slide,
using the loop. The smear should cover an area
about 10 mm x 30 mm. Flame the loop.
Allow the smear to dry in the air.
3. Fix the smear by holding the slide with forceps
and passing it horizontally through a small
Bunsen flame 23 times. Do not overheat the
slide. Fixing kills the bacteria by coagulating
the cytoplasm. It also sticks them to the slide.
4. Place a few drops of Sudan Black solution on
the fixed preparation.
5. After 510 minutes the ethanol in the stain should
have evaporated. Any excess liquid can be
carefully drawn off using the edge of a piece of
filter paper.
6. Immerse the slide in xylene until it is completely
decolorized (this takes about 10 seconds).
Allow the slide to dry.
7. Flood the slide with the counterstain, Safranine
solution.
8. After 10 seconds, gently rinse the slide with
running water and allow it to dry again.
9. When the slide is completely dry add a drop of
immersion oil directly to the slide (no cover
slip is needed). Examine with an oil immersion
lens. The PHB can be seen as very dark granules
inside pink cells.
Safety
Standard microbiological safety procedures, including
aseptic
techniques, mustbe observed by teachers, technicians and
students when carrying out this work.
Teachers in England and Wales are referred to:Microbiology.
An HMI guide for schools and further education(1990)
HMSO [Second Edition] , as well as any safety guidelines
produced by their LEA and / or school governing body.
Xylene (dimethylbenzene) is highly flammable and produces
a toxic vapour. It should be used with care, in a fume
cupboard. Plastic Petri dishes will dissolve in xylene!
Further activities
1. Bacillus megaterium (available from Philip
Harris Limited) also produces PHB in
nutrient-limiting conditions. It has much larger
cells thanAlcaligenes, and is therefore easier to
examine under a microscope but this still
requires an oil immersion lens.
2. Investigate the effect of adding sterile glucose
solution to the bacterial growth medium once
the bacteria have exhausted the nitrogen
supply.
Plastic
from bacteria
