SOP-MI05/V01
Title
Liquid and solid culture of bacteria
Code
SOP-MI05/V01
Pages
10
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SOP-MI05/V01 - 28th, February 2011 - Page 1 of 10
SOP-MI05/V01
Table of Contents
I.
Objective ............................................................................................................................. 3
II. Definitions........................................................................................................................... 3
III. Abbreviations ...................................................................................................................... 4
IV. Procedures ........................................................................................................................... 4
A.
Materials, furniture, reagents ...................................................................................... 4
1)
Chemicals .................................................................................................................... 4
2)
Material ........................................................................................................................ 4
3)
Instruments .................................................................................................................. 4
B.
Description of analysis ................................................................................................ 5
1)
Bench organisation ...................................................................................................... 5
2)
« Receptor » tubes or plates preparation ..................................................................... 5
3)
Sterilization techniques................................................................................................ 5
4)
Culture tubes and plates preparation ........................................................................... 5
5)
Picking bacteria ........................................................................................................... 6
6)
Inoculation of liquid media.......................................................................................... 6
7)
Isolation on solid media ............................................................................................... 7
8)
Incubation .................................................................................................................... 7
C.
Safety ........................................................................................................................... 8
D.
Quality control management ....................................................................................... 8
E.
Waste and decontamination ........................................................................................ 9
F.
Cleaning .................................................................................................................... 10
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SOP-MI05/V01
I. Objective
Microbiological culture is a method of multiplying microbial organisms by letting them to
reproduce in predetermined culture media under controlled laboratory conditions.
In theory, from a single cell, a population composed of millions of identical cells and identical
to the mother cell can be obtained.
In a liquid media, bacteria diffuse throughout the liquid: the growth results in a turbulence of
the media a few hours to a few days after incubation at optimized conditions.
On a solid media, bacteria are not able to diffuse. The mother cell divides and the cells formed
stay close to each other, forming a mass called colony.
This procedure describes how to cultivate bacteria in aseptic conditions, in accordance with
the GLP, from different kind of samples, on a solid and/or liquid media.
Also read the procedure “SOP-MI01/V01: General information in a Microbiology laboratory”
before starting to work in the microbiology laboratory.
II. Definitions

Aseptic conditions/Aseptically: Environment where no micro organism is present. This
can be obtained using a Bunsen burner (the aseptic zone is the spheric area around the
flame with a diameter of approximately 15 cm), or a laminar flow hood (sterile air is
continously produced and present in the hood and the difference of pressures between the
inside and outside of the hood prevents the air outside to come inside and contaminate the
environment under the hood).

Contaminated by microorganisms: Every plate, tube, pipette, or other instruments
(glassware, pestles, ependorff tube...) which has been in contact with microorganisms and
cannot be sterilized by the flame of a Bunsen burner is considered as contaminated.

Contaminated by toxic chemicals: Every tube, flask, pipette or other instruments
(weighing boats, glassware…) which has been in contact with toxic chemicals is
considered as contaminated.

Good Laboratory Practices (GLP): The Principles of Good Laboratory Practices (GLP)
have been developed to promote the quality and validity of results and of the analysis
conducted in a laboratory. It is a managerial concept covering the organization and the
conditions under which laboratory studies are planned, performed, monitored, recorded
and reported. Its principles also include the protection of man and the environment.

Mother tube/plate/product: Tube/plate/product which the bacteria are picked from. The
result of the growth of this inoculation is considered as the daughter which can become
the mother for the next inoculation...
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SOP-MI05/V01
III. Abbreviations
- oC: Celsius degree
- g: gram
- GO: Gelose Ordinaire
- GLP: Good Laboratory Practices
- g/l: gram per litre
- h: hour
- ml: millilitre
- min: minute
- MSDS: Material Safety Data Sheet
- rpm: rotation per minute
- x of n: x is the number of the item, n the total number of the items
IV. Procedures
A. Materials, furniture, reagents
1)
Chemicals
- Mother plate/tube/product
- Liquid media (tubes, flasks…)
- Physiological water in tubes (NaCl, 9 g/l)
- Sodium hypochlorite solution (Jik)
- Solid media (in plates or tubes)
2)
Material
- Bunsen burner
- Incubators and rotative incubator
- Laminar hood
- Vortex
3)
Instruments
- Forceps
- Loop
- Mother plates, tubes or products
- Plastic box for incubation
- Racks for test tubes
- Sterile Pasteur pipettes
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SOP-MI05/V01
B. Description of analysis
1)
Bench organisation
- Put the rack with the tubes or the plates from which the bacteria will be picked on the side
near the burner and the pipettes or the loop on the opposite side.
- Let the rest of the bench to be clean. Other plates and tubes which are not being used can be
placed upside down on the side of the bench (or on a trolley next to you), as far as possible
from the place where you’re working. Anything not useful has to be out of the bench.
2)
« Receptor » tubes or plates preparation
- All the plates and tubes have to be labelled before starting any manipulation. Note the date
of the manipulation, the kind of sample you’re using (name of the strain, origin, number of the
sample…) and any other information that might be useful. If many samples won’t be
incubated at the same temperature, also write the incubating temperature. On the plates, write
upside down to be able to read what is written once the plate is incubated.
3)
Sterilization techniques
- To pick the bacteria, loop or pipettes can be used.
- To sterilize the loop, pass it horizontally through the blue flame (all the metallic part), from
the handle to the end (all the part which may be in contact with the tube). Then put it
vertically on the flame. The platinum part has to become red hot. Let it cool down for a while
before using it.
- If Pasteur pipettes are being used to pick from a solid culture, sterilize it through the flame.
From liquid, the inside part of the pipette must be already sterile (cottoned pipettes). Break the
end of the pipette (red mark) then sterilize the outside by passing it through the flame.
- Pass the pipette through the fire but not for a too long time for the glass to melt. Then be
careful not to touch the part which will be in contact with the tube with the hands.
4)
Culture tubes and plates preparation
- Open the cap a little bit if they are closed tightly or remove the foil from the tubes.
- If a solid culture is used as the mother, select an isolated colony (as big as possible) and note
it with a marker pen at the back of the plate.
- Try to organize your rack and the order of your plates depending on the rest of the
manipulation.
- Shake the suspension to make it homogeneous. Shake the tube with a vortex or by rotations
keeping it vertically. Ensure that no liquid is touching the cap or the cotton.
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5)

Picking bacteria
From liquid culture
- Once the loop/pipette is sterilized, let it cool down for a while not to kill the bacteria in the
samples. Keep the loop/pipette in one hand, and with the same hand, open the cap with your
little finger. Keep the cap in this hand until the bacteria are picked. Never put the cap on the
bench. Once the tube is open, pass it through the flame to sterilize the mouth. Be careful not
to incline the tube to much: the suspension mustn’t be in contact with the flame.
- Put the loop/pipette inside the liquid media and remove it without touching the sides of the
tubes. Hold the pipette/loop horizontally not to drop the liquid on the bench or in the top of
the pipette (where the cotton wool is).
- Pass the mouth of the tube through the flame before closing it. The cap should still be held
with your little finger. Be careful not to put the pipette/loop through the flame or the bacteria
will die. Put the tube on the rack and inoculate the media as described below.

From a solid media
- Either a pipette (don’t break the end) or a loop can be used. Once the tool is sterilized, let it
cool down for a while so as not to kill the bacteria in the samples.
- Open the cap of the mother plate/tube and hold it in one hand. With the loop/pipette, pick
the top of the selected isolated colony without touching the cap of the plate or the sides of the
tube.
- Close the plate/tube and proceed to inoculation as explained below.

From other products (soil, sand, commercial product, nodules…)
- Depending on the nature of the samples, the quantity to be picked is different.
- For solids, a suspension has to be done before inoculation. In most cases, 10 g of products
are diluted in 90 ml of physiological water to get a representative quantity of the sample.
- Liquids can be inoculated as explained previously for liquid culture but can also be diluted
to make the sample more representative of the initial product. 10 ml are mixed in 90 ml of
physiological water.
- Shake well with a vortex or preferably put under agitation in a rotative incubator to get a
better suspension of the bacteria contained in the initial product. Then proceed as from a
liquid culture.
- For nodule, crash the nodule in a small volume of distilled water with a sterile pestle and
proceed as if it was a liquid suspension.
6)
Inoculation of liquid media
- Open the tube containing the sterile media as explained before (using your little finger), pass
it through the flame and while holding the cap, put the loop/pipette into the media without
touching the sides of the tube. Shake the media with the loop/pipette, remove it, pass the tube
through the flame and close the tube. To check on the purity of the liquid media (by
inoculation of a solid media) inoculate directly on the solid media. Once done, sterilize the
loop as described above or put the pipette into a hypochlorite solution.
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SOP-MI05/V01
7)

Isolation on solid media
Solid media on plates
- Once inoculum is picked from the mother plate, open the daughter plate and hold the cap in
one hand (the second hand holds the loop).
- There are many ways to streak: one can use any as long as isolated colonies are obtained
after incubation.
- One example is explained below in details and sketches of the others are also given.
1
2
3
- Start to streak on one half on the plate: the lines have to be very close to each other (as much
as possible). Then turn your plate by 90° and streak again one half. Finish in the last quarter,
without touching the space you have already streaked. Close the plate, turn it upside down and
sterilize the loop as described above or put the pipette into a hypochlorite solution.
4
1
2

3
3
Solid media in tubes
- Open the tubes as explained before and streak the slope of the gel. Try to make the lines as
close as possible and avoid touching the side of the tubes.
8)
Incubation
- Put the plates into a box and then place the box in an incubator. Put the tubes in an incubator
which is also an agitator if expected bacteria are oxygen dependant (aerobic). Agitation
optimizes the oxygen gradient.
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2
SOP-MI05/V01
- The temperature of incubation is set at the optimal growth temperature or at a selective
temperature to allow the growth of the targeted bacteria and/or to avoid the growth of the
other microorganisms.
- Speed of the agitation depends on the volume and the needs of the bacteria. Usually, a speed
of 200 rpm is used.
C. Safety





Autoclave: The autoclave can pose a great danger if not used correctly because of the high
pressure and temperature. Before using, visually check the general aspect (no corrosion,
no leak), the quantity of water and the settings. Strictly follow the instructions to start it.
During the heating phase, the pressure increases. Be sure that the door is correctly closed
so that there is no leakage. Don’t try to force the autoclave to open. The autoclave has a
safety device and will refuse to open if the temperature is still high or if the pressure is not
back to the atmospheric pressure. The maintenance has to be done regularly and the
results recorded in a specific file (Maintenance file, available in the office).
Biological hazards: Manipulating microorganisms poses a risk not only to the one who is
working, but also to other people in the lab and potentially to the environment in case of
dissemination. The rules of safety have to be well understood and respected in order to
avoid any contamination of the staff and/or environment (Read the “Hygiene and Security
rules in a laboratory” document for more details). In case of accidental contamination
(broken test tubes, suspension spilled on the bench, direct contact with microorganisms,
wounds with contaminated material...), clean and disinfect properly before the activities
can be restarted. Dispose contaminated waste as indicated in the section E. For more
details, consult also the “Hygiene and Safety rules in a laboratory” document.
Bunsen burner: The risk of fire can be minimised by following a few simple rules. If the
hood is being used, turn on the fire only when it is needed, don’t cross your arms, don’t
pass your arm on the burner... Also see the safety rules about fire in the “Hygiene and
Safety rules in a laboratory” document.
Chemicals: Before using a new chemical, the information about the toxicity, conditions of
use, risks and safety phrases... have to be understood and observed. Use the equipments
for protection if needed (gloves, masks, hood...). The MSDS (Material Safety Data Sheet)
of the different chemicals are also available to get more details about the products. MSDS
files are available in the Preparation Room and in the office.
Special caution for:
o Sodium hypochlorite: Contact with acids liberates toxic gas. Causes burns.
Laminar flow hood: If the hood is not working properly, it can lead to a fire risk. The
maintenance has to be done regularly and the results recorded in a specific file
(Maintenance file, available in the office).
D. Quality control management



The protocol, date of preparation, name and origin of the samples, name of the plates and
any other relevant information are recorded in the lab book.
The reagents are labelled with the name of the contents, date of preparation, name or
initials of the person who prepared them and any other relevant information.
Before inoculation, the plates of media are visually checked. The plates which are
contaminated or wet are discarded.
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

A data base can be done to follow the plates with time.
The plates are observed every day, at least once a day.

Equipment maintenance: All the equipments are regularly checked in regard to the
specific specifications. The results are recorded and in case of repairs, the details about the
intervention are recorded. Details about maintenance services and repairs are compiled in
the Maintenance file, available in the office.
Hood maintenance: In addition to the general maintenance, simple tests can be run to
assess the good functioning of the hood: Petri plates of GO (or other media depending on
the targeted microorganisms) are left open for some time under the hood while on and
then incubated. If the plates are highly contaminated, then the hood is not working
properly. Bacteriological control can also be performed, as explained in the SOPGA02/V01 “Cleaning plan”.
The general Good Laboratory Practices: They must be respected by everyone in the lab.
The “Hygiene and Safety rules in a laboratory” document describes the general rules to be
observed in the laboratories.
In case of accident: Every accident must be reported to the lab manager and the staff if
needed. Lab manager must put necessary measures in place to avoid the accident to occur
again. For details on what to do in case of accident, read the “Hygiene and Safety rules in
a laboratory” document.
Use of the equipments: For specific equipment, a form has to be filled when used. This
may include the date and time of use (start and end), the name of the user, the notification
of any deviations or problems and any relevant information about the equipment. These
forms are also used to estimate the needs and the frequency of the maintenance. Books are
available near the specific equipments and should not be taken away.




E. Waste and decontamination




Non contaminated waste is eliminated in the normal bin.
Non contaminated glass waste (Pasteur pipette, slides, broken glassware...) are put in a
separate container labelled with the mention: “Broken glass”.
Anything contaminated by microorganisms should be decontaminated before appropriate
elimination/cleaning. Waste are put in a special autoclave bag and autoclaved for 20 min
at 121oC. The autoclave bag can then be disposed off as non contaminated waste. Re used
material (glassware, small tools as sieves, pestles, ...) are autoclaved and then cleaned as
non contaminated items. Not reused glass instruments (pipettes, slides, cover glasses,
broken glassware...) are put in a beaker containing Sodium hypochlorite solution
(commercial Jik) for decontamination before being eliminated as non contaminated glass
waste.
Solid (including broken glass) and liquid waste contaminated by toxic chemicals are
placed in separate containers (labelled with the mention: “Toxic waste, Danger”). A
private company (ECC) comes regularly to collect waste for elimination. Contaminated
glassware is properly rinsed with tap water and the water is collected in a specific
container for toxic liquid waste. The glassware can then be cleaned as non contaminated
items. Consult the MSDS of the product for more information since non compatible
products should not be put in the same container.
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SOP-MI05/V01
F. Cleaning
A complete Cleaning Plan is available for details. Consult SOP-GA02/V01 “Cleaning plan”.





The rooms are cleaned on a daily basis.
Before starting and after manipulation, the hood and/or the bench is/are cleaned with a
disinfectant and/or with 70% Ethanol.
In case of contamination of the bench, floor, user ... it has to be cleaned and disinfected
before the work can be continued (cf. “Hygiene and Safety rules in a laboratory”
document).
Non contaminated or decontaminated items are cleaned with soap, rinsed with water and
eventually rinsed with distilled water.
Equipments: cf the SOP-GA02/V01 “Cleaning plan” for details.
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