Biotechnology for Higher - Culturing Microbes - Education Scotland

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Feb 12, 2013 (4 years and 4 months ago)

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Biotechnology

Protocols


Culturing Microbes:

General Points


27









CULTURING MICROBES:

GENERAL POINTS
Biotechnology

Protocols


Culturing Microbes:

General Points


28

CULTURE TECHNIQUES


Source: HSDU Biology and Biotechnology Microbiological Techniques
(Intermediate 1
-
Advance Higher)
Folder


GENERAL TIPS




All work should be completed where possible within a nine inch radius of
the bunsen burner (on a
blue

flame). This is because the updraft of hot air
carries
micro
-
organisms

away from the experiment (i.e. reduces
contamination problems) as well as carrying them away from you (i.e.
reduces
h
ealth &
s
afety risks).




Label all tube
s, bottles, plates etc.
before

commencing inoculation.




Loosen all screw
-
caps beforehand so that it requires only one half
-
turn to
remove
cap
.




Make a habit of flaming the loop as soon as you take it up and immediately
before you put it down, even if you d
on’t use it.




Similarly, you should flame the necks of all bottles when you open and
when you close them.


Biotechnology

Protocols


Culturing Microbes:

General Points


29

PREPARATION OF SELF AND WORKSPACE


Source: HSDU Biology and Biotechnology Microbiological Techniques
(Intermediate 1
-
Advance Higher)
Folder


Materia
ls


Benchkote and non
-
absorbent tape if required

Disinfectant and paper towel

Discard jar with disinfectant

Bunsen burner

Wire loop/inoculating instrument

Cultures and media


Instructions


The following applies to right
-
handed operators. Left
-
handed peopl
e should
reverse the arrangement on the bench.


1

Tie back long hair.


2

Wash and dry hands thoroughly.


3

Cover any cuts/grazes with waterproof plaster.


4

Put on lab coat.


5

Collect materials.


6

Attach benchkote to the bench if necessary using sticky tape.


7

Swab t
he surface of your work space with 1% hypochlorite or 1% bleach
using the paper towel. Discard paper towel.


8

Keep workspace clear of non
-
essential items.


9

Place the wire loop/inoculating implement to the right of the bench so that
you can reach it with ea
se.


10

Place the Bunsen burner centrally so that you can reach it with ease but
not so close that you are likely to burn yourself.


11

Place cultures and media to the left but still within easy reach. Place
discard jar containing clear phenolic disinfectant su
ch as Stericol to the
right within easy reach.


The bench must be set up in this way
(see diagram on page 30)
each time

a microbiology practical is carried out.


Biotechnology

Protocols


Culturing Microbes:

General Points


30

Source: HSDU Biology and Biotechnology Microbiological Techniques
(Intermediate 1
-
Advance Hi
gher)
Folder









Basic layout of work space:





Biotechnology

Protocols


Culturing Microbes:

General Points


31

FLAMING A LOOP


Source: HSDU Biology and Biotechnology Microbiological Techniques
(Intermediate 1
-
Advance Higher)
Folder


Loops are used to transfer microbes from one growth medium/vessel to
another.
Metal (nichrome) loops have the advantage over plastic loops in that
they can be re
-
used, but they must be sterilised before every application.
Sterilisation is achieved using heat, as detailed here.



Biotechnology

Protocols


Culturing Microbes:

General Points


32

FLAMING WITH ALCOHOL


Source: HSDU Biology and Biot
echnology Microbiological Techniques
(Intermediate 1
-
Advance Higher)
Folder



Materials


Implement


spreader, forceps or mounted needle

20 ml alcohol in closed bottle

Shallow glass dish such as Petri dish with glass or fireproof cover


Instructions


1

Pour
alcohol into shallow glass dish.


2

Dip implement in alcohol and shake off excess.


3

Place lid over alcohol.


4

Place implement in flame to allow alcohol to catch fire.


5

Remove implement from flame and allow alcohol to burn off.

Do not

put the implement down
on bench.


6

Carry out the
procedure

using the implement.


7

Repeat steps 2


5. Place implement on heat resistant mat.


Biotechnology

Protocols


Culturing Microbes:

General Points


33

FLAMING A NECK


Source: HSDU Biology and Biotechnology Microbiological Techniques
(Intermediate 1
-
Advance Higher)
Folder


The necks of a
ll bottles, flasks and test tubes should be flamed before and
after opening to reduce the chance of contaminants entering the vessel. This
is done by the process shown in the fo
llow
ing diagram:



Biotechnology

Protocols


Culturing Microbes:

General Points


34

TREATMENT OF SPILLAGES


Source: HSDU Biology and Biotechn
ology Microbiological Techniques
(Intermediate 1
-
Advance Higher)
Folder



Spillage Kit Contents




Disposable plastic gloves




Undiluted clear phenolic disinfectant (e.g. Stericol, Hycolin, Clearsil) or
Virkon in a bottle marked with a dilution line




Disposab
le cloths or paper towels for soaking with disinfectant and
applying to spill




A small plastic dustpan and paper towels




Autoclavable plastic tongs




An autoclavable waste disposal bag


To deal with a small scale spill


1

Wear a lab coat, disposable plastic g
loves and use eye protection.


2

Cover spillage with paper towels.


3

Dilute the disinfectant to the recommended concentration

(see
manufacturers guidelines for specific disinfectants and also the Code of
Practice detailed on p8 of these notes)
.


4

Pour a ring o
f freshly made disinfectant around the spill and over the paper
towels covering the spill.


5

Leave for at least ten minutes.


6

Using paper towels, sweep the debris on to the plastic dustpan.


7

Using tongs if necessary, place the debris into an autoclavable di
sposal
bag (broken glass should be placed in a separate solid autoclavable
container or 'double
-
bagged').


8

Autoclave the debris.


9

Place in a bin bag and dispose of with domestic waste.


10

Autoclave the dustpan and tongs. If not autoclave safe, immerse in cl
ear
phenolic disinfectant for at least 24 hours.


Biotechnology

Protocols


Culturing Microbes:

General Points


35

STERILISATION USING A PRESSURE COOKER

/

AUTOCLAVE

Source: HSDU Biology and Biotechnology Microbiological Techniques
(Intermediate 1
-
Advance Higher)
Folder


Materials

Protective gloves

Pressure cooker/autoc
lave

Water

Materials to be sterilised


Method


1

Wear a lab coat and use eye protection. Wear protective gloves when
handling hot equipment.


2

Add water to the recommended depth (3 cm
)

to the base of the pressure
cooker.


3

Place the trivet/stand in the base i
f required.


4

Loosen caps slightly (tighten then loosen a quarter turn) on bottles and
place the materials to be sterilised in cooker/autoclave. Note that bottles
should contain a maximum of 500 cm
3

of medium and there should be
space for expansion above t
he medium in the bottle.


5

Place Browne’s tube/spore strip as close as possible to centre of materials
to be sterilised.


6

Secure the lid of the pressure cooker/autoclave according to the
manufacturer’s instructions.


7

Heat on an electric or gas ring till ste
am issues evenly or switch on if the
heater is integral.


8

Place on the valve (pressure cooker and some autoclaves).


9

When cooker begins to ‘hiss’ evenly, turn down heat.


10

Continue heating gently for 15 minutes. Note that steam should continue
to issue gen
tly from the valve


if it does not, pressure and temperature
are likely to have fallen.


11

Turn off heat and allow to cool.
Do not attempt to speed up cooling


this can distort the pressure cooker and can cause media to boil
over.


12

Take care when opening
lid that steam does not issue towards the
operator.


13

Allow sterilised materials to cool before removing from the vessel.

Biotechnology

Protocols


Culturing Microbes:

General Points


36

AGAR PLATES


Source:
NQ Curriculum Support

Intermediate 2
Biotechnolo
gy


(
Unit 2 Student Materials
)



Micro
-
organisms are grown in
or on a
culture medium

containing all the
required nutrients for growth. Culture media can be liquid or solid. Liquid or
broth media although convenient have some disadvantages. Growths usually
do not exhibit characteristic appearances in them and, exce
pt when they are
designed for a specific biochemical test, they are of limited use in identifying
species. Also, contaminating organisms cannot be seen readily in liquid
media and liquid media are more difficult to handle in ways which avoid
formation of
aerosols.


On solid media, micro
-
organisms grow to form discrete colonies. Each colony
is a clone of cells originating from a single organism and represents the
growth of a single species. Micro
-
organisms can be identified by the
appearance exhibited by
the different colonies.








Bacterial colonies on a nutrient agar plate











Biotechnology

Protocols


Culturing Microbes:

General Points


37

Agar


Liquid media can be made solid by the addition of
agar
. Agar has no
nutritional value nor does it inhibit growth, it is simply a solidifying agent.


The
melting a
nd solidifying points of agar are not the same
. At the
concentrations normally used, most agars melt at about 95

C when heated
but solidify only when cooled to about 42

C. This low solidifying point allows
heat
-
sensitive nutrients to be mixed with molten

agar at temperatures as low
as 45

C before pouring. The high melting point ensures that the medium
remains solid at all laboratory incubation temperatures.





MAKING NUTRIENT AGAR

(always check individual suppliers instructions
for quantities to use)




W
eigh out 2 g agar powder (
use masks
)




Dissolve in 100 ml distilled water
whilst stirring




Heat until boiling (
stir continuously
)




Remove heat




Leave to cool

i.e. until you can pick up the beaker
NOT

until the agar sets




Pour into
labelled

‘medical flats’
i.e. flat
-
sided glass bottles




Put the tops on


NOT

fully tightened




AUTOCLAVE

the bottles of agar at 121
0
C for 15 minutes

i.e. high
TEMPERATURE

and
PRESSURE

.




Allow to

cool to 55
0
C before pouring.






Pouring Agar Plates


Agar plates are prepared by p
ouring liquid agar at 55

C into sterile Petri
dishes and allowing it to solidify.


Before pouring, unopened
plates are labelled on the underside

using an
indelible pen or wax pencil with initials, date and type of agar. This prevents
Biotechnology

Protocols


Culturing Microbes:

General Points


38

confusion should lids

inadvertently be swapped. The plates are then placed
the right way up
prior to pouring of

molten agar.

If sterile agar in universal bottles or other containers has cooled and solidified,
then it must be melted by heating
to 100

C then cooled in a water b
ath to
55

C. Using as
eptic technique, a bottle neck is flamed and
the agar poured

gently into a Petri dish on a flat surface, raising the lid of the dish only far
enough for the mouth of the bottle to enter.
About 15mls of molten agar is
poured into each

Petri dish.
The lid is then replaced and the plate left
undisturbed until the agar has cooled and set.


It is essential that the
surface of the medium should be dry

in order to
maintain single colonies. Condensation produced from the cooling of the agar

can make the agar surface wet but is normally reduced by pouring the agar
when it is
at 55

C. The agar will still be molten so must set before the plates
can be inverted, however

if
condensation is
s
till an issue then
the plates can

be dried open and up
side down in an undisturbed area.


Any plates showing lumps, bubbles or growth should be discarded.
Acceptable plates should be stored upside down until required.


Plates are always incubated and stored in the inverted position to prevent
condensation dro
pping on the agar surface.


Biotechnology

Protocols


Culturing Microbes:

General Points


39

POURING PLATES


Source:
NQ Curriculum Support

Intermediate 2
Biotechnolo
gy


(
Unit 2 Student Materials
)


Materials


Universals or bottles containing larger volumes
*

of sterile molten nutrient agar
(at 55

C)

Sterile plastic Pe
tri dishes

Bunsen burner



Instructions


This method is written for right
-
handed people. If you are left handed, please
reverse handling instructions.


1

Wear a lab coat and use eye protection.

2

Label the empty sterile Petri dishes on the base with name, da
te and type
of agar. (N.B. if the lid comes off, the plate is no longer sterile and you
must discard it).

3

Light the Bunsen burner.

4

Collect one bottle of sterile molten agar from the water bath. Check it is
not too hot
nor

that it has not started to soli
dify.

5

Place a Petri dish right way up on the bench.

6

Check that the top of the bottle of agar is loose.

7

Hold the bottle of agar in the left hand.

8

Unscrew and remove the cap of the bottle with the little finger of the right
hand.

9

Flame the neck of the bottle
.

10

With the right hand lift the lid of the Petri dish a little and gently pour in the
molten agar.




11

Replace the lid of the Petri dish.

12

Flame neck and then r
eplace the cap of the bottle and put it down.

13

Swirl the plate very gently to distribute the agar
evenly. (N.B. The base of
the plate must be covered, agar must not touch the lid of the plate and the
surface must be smooth with no bubbles).






Biotechnology

Protocols


Culturing Microbes:

General Points


40

14

Repeat for the other bottles and plates.

15

Leave the agar to solidify.

16

Once cool, turn the plates upside down.



*

Note: if larger volumes of agar are to be poured, lay out the appropriate number of


sterile Petri dishes and flame the neck of the bottle before each plate is poured
.