Joint Clinical Research Centre
Laboratory Policy and Procedure Manual
Preparation of Culture Media
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Author: JCRC
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Heidi Hanes
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Effective (or Post) Date:
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Review date
Pro67-A-11
13-Oct-09
JCRC
Peggy
8-Feb-12
SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s
specific processes and/or specific protocol requirements. Users are directed to countercheck facts when considering
their use in other applications. If you have any questions contact SMILE.
Joint Clinical Research Centre
Index code
Name of policy/procedure
Media 402
Prepared by
Preparation of Culture Media
Date to be adopted
Juliet Kitabalwa
Signature Laboratory Manager or Section
Head
01 Oct 2009
Reason for procedure (check one)
New procedure
X
Checkmark one
Annual review
Signature Director
Supercedes retired
procedure index code
______________
SIGNATURE
DATE
REVIEWED BY
Annual review
Annual review
Annual review
Annual review
REVISED BY
VERSION No.
SIGNATURE
REVISED DATE
SUMMARY OF CHANGES TO THIS SOP
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Media 402
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ACKNOWLEDGEMENT OF READING AND UNDERSTANDING SOP:
MEDIA 402
NAME
SIGNATURE
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DATE
Media 402
Version 1.0
Joint Clinical Research Centre
1.0
Principle:
Using Cultural techniques in microbiology helps demonstrate the presence of organisms which may
be causing disease, and when indicated, to test the susceptibility of pathogens to antimicrobial agents.
If pathogens are to be isolated successfully, standard operating procedures are needed which detail
for each culture medium used in the laboratory, its purpose, from where it can be obtained, its
preparation, and how it is sterilized, dispensed, labeled, stored and performance tested.
2.0
Scope:
This procedure is intended for all technologists involved in preparing culture media in the
Microbiology laboratory at JCRC.
3.0
Responsibility:
It is the responsibility of every technologist working in the Microbiology laboratory at JCRC to
follow this procedure.
4.0
Procedures for various media
4.1
Blood Agar
Source: Liofilchem S.R.L., Italy
Purpose: Blood agar is blood agar base II enriched with 5-10 % sheep blood. Blood agar is used to
isolate organisms with exacting growth requirements, including streptococci. It is appropriate for
sputum, throat swabs, urine, urogenital swabs, pus aspirates, eye and ear swabs, wound swabs,
aspirates (for aerobes and anaerobes), and some fungi. It is appropriate for culturing almost all
specimens for bacteriology.
Procedure for preparation:
i)
Suspend 40.0g in 1 litre of distilled water.
ii)
Heat until completely dissolved.
iii)
Autoclave at 1210C for 15 minutes.
iv)
Cool to 45-500C.
v)
Aseptically add 50 ml of defibrinated sheep blood.
vi)
Pour 15-20 ml of the ready media on to petri dishes.
vii)
Leave standing for thirty minutes to solidify.
viii) Perform sterility testing as described in Section 7.
ix)
Label the bottom of each plate with date of preparation and batch number.
x)
Store the culture media plates upside down at 2-80C sealed in plastic bags to reduce chances
of contamination.
xi)
Test Samples for performance, using stable, typical control cultures.
 pH of the medium 7.2-7.6 at room temperature depending on the agar base used.
 Shelf life: up to sixteen weeks provided there is no change in the appearance of the medium to
suggest contamination, haemolysis, or deteroriation.
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4.2
Chocolate (Heated Blood) Agar
Source: Depends on source of blood agar
Purpose: Chocolate agar supplies the factors required for the proper growth of Haemophilus
influenzae, the X and V factors. It is also used to culture nutritionally demanding pathogens such
Neisseria meningitidis and Streptococcus pneumoniae. When Blood agar is heated, the red cells are
lysed and the medium becomes brown in colour; it is referred to as chocolate agar. It is appropriate
for isolating pathogenic bacteria in sputum, throat swabs, eye swabs, ear swabs, urogenital swabs,
cerebrospinal fluid.
Procedure for preparation:
i)
Prepare as described for Blood agar except after adding blood, heat the medium in a
700C water bath until it becomes brown in colour. This takes about 10-15 minutes during
which time the medium should be mixed gently several times.
ii)
Allow the medium to cool to about 450C, remix and dispense in sterile petri dishes as
described for blood agar.
iii)
Leave standing for thirty minutes to solidify.
iv)
Perform sterility testing as described in Section 7.
v)
Label the bottom of each plate with date of preparation and batch number.
vi)
Store the culture media plates upside down at 2-80C sealed in plastic bags to reduce chances
of contamination.
vii)
Test Samples for performance, using stable, typical control cultures.
 pH of the medium 7.2-7.6 at room temperature depending on the agar base used.
 Shelf life: up to sixteen weeks provided there is no change in the appearance of the medium to
suggest contamination or deteroriation.
Important: Care must be taken not to over heat or prolong the heating of the medium because this
will cause it to become granular and unfit for use.
4.3
Mac Conkey Agar
Source: Orgenics Ltd, Israel
Purpose: Mac Conkey agar is a differential medium to distinguish lactose fermenting gram negative
bacteria from non lactose fermentors; lactose fermentors are coloured pink, while non lactose
fermentors are pale. It is appropriate for stool, urine, urogenital specimens, throat swabs, ear and eye
swabs, wound swabs, sputum, aspirates, cerebrospinal fluid.
Procedure for preparation:
i)
Suspend 51.1g of powder in 1 litre of distilled or deionized water.
ii)
Heat until completely dissolved.
iii)
Sterilize in autoclave at 1210C for 15 minutes.
iv)
Cool to 45-50 0C.
v)
Pour 15-20 ml of the ready media on to petri dishes.
vi)
Leave standing for thirty minutes to solidify.
vii)
Perform sterility testing as described in Section 7.
viii) Label the bottom of each plate with date of preparation and batch number.
ix)
Store the culture media plates upside down at 2-80C sealed in plastic bags to reduce chances
of contamination.
x)
Test Samples for performance, using stable, typical control cultures.

Shelf life: up to sixteen weeks provided there is no change in the
appearance of the medium to suggest contamination or deteroriation.
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
4.4
pH of the medium 7.2-7.6 at room temperature.
Sorbitol MacConkey Agar
Source: Oxoid UK
Purpose: This MacConkey medium contains the carbohydrate sorbitol instead of lactose. E.coli 0157
produces colourless colonies on the medium because it does not ferment sorbitol. Most other E.coli
strains and other enterobacteria ferment sorbitol, producing pink colonies. Sorbitol MacConkey agar
is therefore a useful way of screening for E.coli 0157 (specificity 85%, sensitivity 100%).
Procedure for preparation:
This medium is best prepared from ready to use dehydrated powder.
(i) Use at a concentration of 5.15 g in every 100ml distilled water. Concentration may vary
depending on manufacturer.
(ii) Prepare, sterilize, dispense and store the medium as described above for MacConkey agar.
 Shelf life: up to sixteen weeks provided there is no change in the appearance of the
medium to suggest contamination or deteroriation.
 pH of the medium 7.2-7.6 at room temperature.
4.5
Selenite-F Broth
Source: Difco
Purpose: Selenite-F broth is used as an enrichment medium for the isolation of Salmonella from
faeces, urine, water, foods and any material of sanitary importance. Selenite is inhibitory to coliforms
and certain other microbial species in faecal specimens. The isolation of Salmonella is possible if
subcultures were made from the enrichment broth after 8-12 hrs of incubation. For detection of
Shigella, GN Broth is a satisfactory enrichment medium. The formula may vary slightly from
manufacturer to manufacturer. Enrichment broths should not be used as the sole isolation medium.
They are to be used in conjunction with selective and nonselective plating media to increase the
probability of isolating pathogens, especially when they may be present in small numbers.
Procedure for preparation:
(i)
Suspend 23 g of the dehydrated powder in 1 litre of purified water.
(ii) Heat to boiling. Avoid over heating. Do not autoclave.
(iii) Dispense into 20ml universal tubes with screw caps, filling the tubes to about three-quarters.
(iv)
Label the side of each tube with date of preparation and batch number.
(v)
Perform sterility testing as described in Section 7.
(vi)
Store at 2-80C.
(vi) Test samples of the finished product for performance using stable, typical control cultures.
 Shelf life: up to eighteen months provided there is no change in the appearance
of the medium to suggest contamination or deteroriation.
 pH of the culture medium is 5.0- 6.0 at room temperature.
4.6
S.S Agar (Modified)
Source: Orgenics Ltd, Israel
Purpose: A selective medium for the isolation of Salmonellae and Shigellae from faeces, foodstuffs
and other materials. Brilliant green, ox bile and high concentrations of thiosulfate and citrate largely
inhibit the accompanying microbial flora. Sulfide production is detected by using thiosulfate and iron
ions, the colonies turn black. The presence of coliform bacteria is established by detecting
degradation of lactose to acid with the pH indicator neutral red. Non-lactose fermenting colonies are
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colourless / pale, lactose fermenting colonies are pink to red. Colonies of microorganisms
(Salmonellae and Shigellae) producing hydrogen sulfide have a black centre.
Procedure for preparation:
i)
Suspend 52.0g of powder in 1 litre of distilled or deionized water.
ii)
Mix well.
iii)
Heat to boiling for 1 minute to completely dissolve the powder. DO NOT AUTOCLAVE and
avoid over heating.
iv)
Cool to 45-500C and dispense into petri dishes.
v)
Pour 15-20 ml of the ready media on to petri dishes.
vi)
Leave standing for thirty minutes to solidify.
vii)
Perform sterility testing as described in Section 7.
viii) Label the bottom of each plate with date of preparation and batch number.
ix)
Store the culture media plates upside down at 2-80C sealed in plastic bags to reduce chances
of contamination.
x)
Test Samples for performance, using stable, typical control cultures.
 Shelf life: up to sixteen weeks provided there is no change in the appearance of the medium
to suggest contamination or deteroriation.
 pH of the culture medium is 5.0- 6.0 at room temperature.
4.7
Mueller Hinton II Agar
Source: Becton Dickinson, U.S.A
Purpose: It is the medium of choice for antibiotics sensitivity testing. Fastidious bacteria like
Streptococci do not grow well on Mueller Hinton.
Procedure for preparation:
i)
Suspend 38g of the powder in 1 litre of purified water.
ii)
Mix thoroughly.
iii)
Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
Autoclave at 1210C for 15 minutes.
iv)
Do not over heat. Cool to 45-500C and dispense into petri dishes.
v)
Pour 15-20 ml of the ready media on to petri dishes.
vi)
Leave standing for thirty minutes to solidify.
vii)
Perform sterility testing as described in Section 7.
viii) Label the bottom of each plate with date of preparation and batch number.
ix)
Store the culture media plates upside down at 2-80C sealed in plastic bags to reduce chances
of contamination.
x)
Test Samples for performance, using stable, typical control cultures.

Shelf life: up to sixteen weeks provided there is no change in the appearance of the
medium to suggest contamination or deteroriation.

pH of the medium 7.2-7.6 at room temperature.
4.8
CLED Agar (Cystine-Lactose-Electrolyte-Deficient Agar)
Source: Becton Dickinson, U.S.A
Purpose: Used for isolating a wide variety of Gram positive and Gram negative bacteria from urine.
In 1960, Sandys reported on the development of a new method of preventing the swarming of
Proteus on solid media by restricting the electrolytes in the culture medium, to enable colony counts
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of Proteus. Lactose and sucrose in the medium was substituted for mannitol and the concentration of
bromothymol blue indicator in the agar increased. Mackey and Sandys further modified the medium
by incorporating cystine in order to enhance the growth of cyctine-dependent “dwarf colony”
coliforms and by deletion of sucrose. Hence the name Cystine-Lactose-Electrolyte-Deficient Agar,
and was found to be ideal for urinary bacteriology and for colony enumeration.
Procedure for preparation (CLED):
i)
Suspend 36g of the powder in 1 litre of purified water.
ii)
Mix thoroughly.
iii)
Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
Autoclave at 1210C for 15 minutes.
iv)
Cool to 45-500C and dispense into petri dishes.
v)
Pour 15-20 ml of the ready media on to petri dishes.
vi)
Leave standing for thirty minutes to solidify.
vii)
Perform sterility testing as described in Section 7.
viii) Label the bottom of each plate with date of preparation and batch number.
ix)
Store the culture media plates upside down at 2-80C sealed in plastic bags to reduce chances
of contamination.
x)
Test Samples of the finished product for performance, using stable, typical control cultures.
 Shelf life: up to sixteen weeks provided there is no change in the appearance of the medium to
suggest contamination or deteroriation.
 pH of the medium is between 7.3 – 7.5 at room temperature.
4.9
Sabouraud Dextrose Agar
Source: Becton Dickinson, U.S.A, or Oxoid UK
Purpose: A culture medium for fungi, containing neopeptone or polypeptone agar and glucose; it is
the standard, most universally used medium in mycology and is the international reference.
Appropriate for skin scrapping, sputum, genital swabs and almost all specimens for fungal culture.
The medium is often used with antibiotics for the isolation of pathogenic fungi from material
containing large numbers of other fungi or bacteria.
Procedure for preparation
i)
Suspend 65.0g of the powder in 1 liter of purified water.
ii)
Mix thoroughly.
iii)
Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
Autoclave at 1210C for 15 minutes.
iv)
Avoid over heating. Cool to 45-500C.
v)
Aseptically add 0.5g cycloheximide, 20,000 units penicillin and 40,000 units streptomycin to
each litre of autoclaved Sabouraud agar.
vi)
Pour 15-20 ml of the ready media on to petri dishes.
vii)
Leave standing for thirty minutes to solidify.
viii) Perform sterility testing as described in Section 7.
ix)
Label the bottom of each plate with date of preparation and batch number.
x)
Store the culture media plates upside down at 2-80C sealed in plastic bags to reduce chances
of contamination.
xi)
Test Samples of the finished product for performance, using stable, typical control cultures.

Shelf life: up to twenty weeks provided there is no change in the appearance of the
medium to suggest contamination or deteroriation.

pH of the medium is between 5.5 -5.7 at room temperature.
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Further notes on antibiotics addition to Sabouraud): Georg et al aseptically added 0.5g
cycloheximide, 20,000 units penicillin and 40,000 units streptomycin to each litre of autoclaved,
cooled medium. Cryptococcus neoformans, Aspergillus fumigatus and Allescheria boydii are
sensitive to cycloheximide; Actinomyces bovis and Norcardia asteroids are sensitive to penicillin and
streptomycin. Alternatively, one may add 0.4g chloramphenicol and 0.05 cycloheximide to each litre
of reconstituted medium before autoclaving (Ref: Ajello). Dolan used gentamycin, chloramphenicol
and cycloheximide for the selective isolation of pathogenic fungi.
References for sabouraud:



Georg Lucille K., Ajello L. and Papageorge Calomira (1954) J. Lab. Clin. Med. 44. 422-428.
Ajello Libero (1957) J. Chron. Dis. 5. 545-551.
Dolan C. T. (1971) Appl. Microbiol. 21. 195-197.
4.10 Brain Heart Infusion Broth
Source: Hiveg – Himedia, India
Purpose: Brain Heart Infusion is a general-purpose liquid medium used in the cultivation of
fastidious and non-fastidious microorganisms, including aerobic and anaerobic bacteria, from a
variety of clinical and non-clinical specimens. It serves as a base for supplemented media containing
0.1% agar, Fildes enrichment or 6.5 % sodium chloride. A supplemented pre-reduced formulation in
tubes is especially recommended for the cultivation of anaerobes. Rosenow (1919, Journal of Dental
Research 1:205) described brain-heart infusion broth prepared by adding pieces of brain tissue to
meat infusion or beef extract-dextrose broth. It is appropriate for blood culture and culturing
specimens likely to contain anaerobes.
Procedure for preparation (BHI Broth):
i)
Suspend 37.0g in 1 litre of distilled water.
ii)
Heat if necessary to dissolve the medium completely.
iii)
Dispense into bottles or tubes as desired. Sterilize by autoclaving at 15 Ibs (1210C) for 15
minutes.
iv)
Cool to 45-500C.
v)
Dispense into 20ml universal tubes.
vi)
Perform sterility testing as described in Section 7.
vii)
Label the side of each tube with date of preparation and batch number.
viii) Store the culture medium at 2-80C sealed in plastic bags to reduce chances of contamination.
ix)
Test Samples of the finished product for performance, using stable, typical control cultures.

Shelf life: up to eighteen months provided there is no change in the appearance of the
medium to suggest contamination or deteroriation.
 pH of the medium 7.2-7.6 at room temperature.
4.11 Nutrient agar
Source: Difco
Purpose: Nutrient agar is used for the cultivation of a wide variety of non-fastidious bacteria. It was
originally developed in recognition of the need for a standardized medium for use in the examination
of water and waste water, dairy products and various foods. Currently it is used as a maintenance
medium for Staphylococcus aureus, Proteus mirabilis, and Escherichia coli. Tube slants are used
primarily for the cultivation and maintenance of pure cultures.
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Procedure for preparation:
(i) Suspend 23 g of the powder in 1 litre of purified water.
(ii) Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
(iii) Autoclave at 121˚C for 15 minutes.
(iv) Cool to 45-500C.
(v) Pour 15-20 ml of the ready media into sterile 20ml glass universal tubes.
(vi) Leave standing for thirty minutes to solidify, resting the tubes leaning at 30˚- 60˚ to produce the
slope effects in the tubes..
(vii) Perform sterility testing as described in Section 7.
(viii) Label the side of each tube with date of preparation and batch number.
(ix) Store the tubes at 2-80C sealed in plastic bags to reduce chances of contamination.
(x) Test Samples of the finished product for performance, using stable, typical control cultures.

Shelf life: up to eighteen months provided there is no change in the appearance of the
medium to suggest contamination or deteroriation.
 pH of the medium 7.2-7.6 at room temperature.
4.12 Nutrient Broth
Source: Difco
Purpose: Maintenance medium and enrichment medium for non fastidious bacteria.
Procedure for preparation:
Prepare from ready to use dehydrated nutrient broth powder. The contents of nutrient broth are the
same as those for nutrient agar except that the agar is omitted. Its preparation and storage are the
same as described previously for nutrient agar.

Shelf life: up to twelve months provided there is no change in the appearance of the
medium to suggest contamination or deteroriation.
4.13 Semi-solid nutrient agar
Source: Same as for nutrient agar and nutrient broth.
Purpose: Maintenance medium for non fastidious bacteria.
Procedure for preparation:
(To make about 20 bottles)
(i)
Mix 0.75g nutrient agar and 1.3g nutrient broth in 100 ml distilled water, and heat to
100˚C to dissolve the ingredients (place the flask in a boiling water bath).
(ii)
Dispense the medium in 5-7 ml amounts in screw-cap bottles. Sterilize by autoclaving
(with caps loosened) at 121˚C for 15 minutes. When cool, tighten the bottle caps.
(iii) Perform sterility testing as described in Section 7.
(iv)
Date the medium and give it a batch number.
(v)
Sore at 2-8 ˚C.

Shelf life: up to twelve months provided there is no change in the appearance of the
medium to suggest contamination or deteroriation.
 pH of the medium is 7.2-7.6 at room temperature.
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4.14 Cooked meat medium
Source: Difco
Purpose: It is used as an enrichment broth for the cultivation of anaerobes and as a maintenance
medium (stock culture) for many anaerobes (Clostridia) and fastidious organisms including
streptococci. Cooked Meat medium provides a favourable environment for the growth of anaerobes,
since the muscle protein in the heart tissue granules is a source of amino acids and other nutrients.
The muscle tissue also provides reducing substances (eg glutathione) which permits the growth of
strict anaerobes. The sulfhydryl groups, which exert the reducing effect, are more available in
denatured protein; therefore, the meat particles are cooked for use in the medium. Growth is indicated
by turbidity and, with some organisms, by the presence of gas bubbles in the medium. Disintegration
and blackening of the meat particles indicates proteolysis.
Procedure for preparation:
(vi)
Suspend 12.5 g of the particles in 100 ml purified water (1.25g/10ml).
(vii) Let stand until all particles are thoroughly wetted and form an even suspension.
(viii) Autoclave at 121˚C for 15 minutes. Reduce pressure slowly and cool without agitation, to
45-500C.
(ix)
Dispense into 20ml universal tubes with screw caps, filling the tubes to about
three-quarters. If not used within 24 hrs, reheat (100˚C) prior to use to drive off absorbed
oxygen.
(x)
Perform sterility testing as described in Section 7.
(xi)
Date the medium and give it a batch number.
(xii) Test samples of the finished product for performance using stable, typical control cultures.

Shelf life: up to eighteen months provided there is no change in the appearance of the
medium to suggest contamination or deteroriation.

pH of the medium is 7.0 to 7.4 at room temperature.
4.15 Dorset egg medium
Souce: made from nutrient broth, and eggs.
Purpose: Maintenance medium for many organisms including Salmonella typhimurium.
Procedure for preparation:
(i)
Prepare and sterilize the nutrient broth as described above.
(ii)
Wash the eggs by scrubbing them carefully with soap and water followed by rinsing in
clean running water. Immerse the eggs in 70 % v/v ethanol (alcohol) for ten minutes.
(iii) As aseptically as possible, break the eggs into a sterile flask (pre-marked to hold 80 ml)
which contains a few sterile beads. Mix well until the egg yolks and whites are completely
homogenized.
(iv)
Strain the egg mixture through sterile gauze or muslin into a sterile bottle.
(v)
When the nutrient broth has cooled to 45-500C, add it aseptically to the eggs mixture, and
mix well.
(vi)
Dispense aseptically the medium in 3ml amounts in sterile Bijou bottles or screw cap
tubes.
(vii) Inspissate at 75-800C with the bottles in a sloped position until the medium has solidified.
(viii) Perform sterility testing as described in Section 7.
(ix)
Date the medium and give it a batch number.
(x)
Store the slopes with the bottle tops tightly screwed at 2-80C.
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
Shelf life: up to 2 years providing there is no change in the appearance of
the medium to suggest contamination or deteroriation.
 pH of medium 7.2-7.6 at room temperature.
5.0
Checking the pH of a culture medium
The pH of most culture media is near neutral. An exception is alkaline peptone water. The simplest
way of testing the pH of a culture medium is to use narrow range pH papers or a pH meter.
A fluid medium can be tested by dipping a narrow range pH paper into a sample of the medium when
it is at room temperature and comparing the colour of the paper against the pH colour chart provided.
An agar medium can be tested by pouring a sample of the molten medium into a small beaker or petri
dish and when it has solidified, laying a narrow range pH paper on its surface. The colour of the
paper is then compared against the pH colour chart provided.
The pH of a dehydrated medium should not require adjustment providing it has been prepared
correctly using pure water and clean equipment, and it has not been over-autoclaved. The
manufacturer’s instructions must be followed strictly.
The pH of other media should be adjusted as directed in the method of preparation. Minor
adjustments should be carried out using 0.1mol/l (N/10) sodium hydroxide when the medium is too
acidic, and 0.1 mol/l (N/10) hydrochloric acid when too alkaline. When adjusting the pH of a large
volume of medium it is best to measure the amount of acid that needs to be added to adjust 10 ml of
the medium, and then calculate the amount required to adjust the remaining volume.
6.0
How to dispense culture media
Media should be dispensed in a clean draught-free room. Most fluid media are dispensed into screwcapped bottles or tubes, and then sterilized by autoclaving. Sterile media must be dispensed into
sterile petri dishes, tubes, or bottles using an aseptic technique.
Dispensing sterile media into petri dishes.
1. Lay out the sterile petri dishes on a level, clean (disinfected) bench surface.
2. Mix the medium gently by rotating the flask or bottle. Avoid forming air bubbles. Flamesterilize the neck of the flask or bottle and pour 15-20 ml of medium into each dish (90-100
mm diameter). If air bubbles enter while pouring, rapidly flame the surface of the medium
before gelling occurs. Rotate the dish on the surface of the bench to ensure an even layer of
agar. Agar plates should be of an even depth (not less that 4 mm) and of a firm gel. Procedure
for dispensing and solidifying high protein media (inspissation) such as Dorset egg medium is
given above.
3. When the medium has gelled and cooled, stack the plates and seal them in plastic bags to
prevent loss of moisture and reduce the risk of contamination. Do not leave the plates exposed
to bright light especially sunlight.
4. Store at 2-8˚C.
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7.0
Sterility testing of Culture media
Sterility test routinely media to which blood or other substances have been added after autoclaving.
For sterile media in screw-cap tubes or bottles, the simplest way to test for contamination is to
incubate 5 % of the batch at 35-37˚C overnight. Contamination by microorganisms capable of
overnight growth will be shown by turbidity in a fluid medium and growth on or in a solid medium.
Media in petri dishes are best examined for contamination immediately before use.
All media, even those that have been sterility tested at the time of preparation, should always be
checked visually immediately before being inoculated for any change in appearance that could
indicate contamination or deterioration.
8.0
Performance testing
Quality control testing of prepared media is performed by inoculating, say 2% of each batch with
control organisms whose growth should be supported on the tested media. For the following media
and organisms, testing is performed at 35-37˚C incubation at 5-10 % CO2.
Quality control of commonly used culture media
Culture medium
Recommended
control Maintenance medium
species
Blood agar
Streptococcus pyogenes
Cooked meat medium
Streptococcus pneumoniae
Haemophilus influenzae
Chocolate agar slope
(with S.aureus streak)
(35-37˚C)
Chocolate agar slope
Chocolate agar
Haemophilus influenzae
(35-37˚C)
Cytine
Lactose Staphylococcus aureus
Electrolyte
Deficient Medium
Proteus mirabilis
(CLED)
Escherichia coli
Mac Conkey agar
Proteus mirabilis
Shigella spp.
SS agar
Salmonella typhimurium
(Salmonell-Shigella
Escherichia coli
agar)
Sabouraud agar
Candida albicans
Aspergillus brasiliensis
Nutrient agar semi
solid or deep
Subculturing
interval
3 months
1 month
1 month
6 months
Nutrient agar semi
solid or deep
6 months
Dorset egg medium
12 months
Nutrient agar semi
solid or deep
Sabouraud agar plate
6 months
2 months
References
1) Basic information was obtained from manufacturer’s labels of dehydrated media.
2) District Laboratory Practice in Tropical countries, Cambridge University Press, 2nd Edition
(2006) by Monica Cheesbrough.
3) Clinical Microbiology Procedures Handbook, 2nd Edition 2007, Garcia L.S et al, ASM Press,
Pg 18.
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Quality Control notes.
(i)
The lab personnel who makes the media must fill in a log (Media Making Log) indicating:
date, media made, batch number, and name of the personnel.
(ii)
QC failures must be documented after sterility testing, performance testing, and pH
testing, using the log “QC of Culture Media”.
(iii) The media should not be used beyond the expiration date. Prepared media should be used
within the allowed / stated duration of weeks or months, or within the manufacturer’s
reagent expiration date, whichever comes first. Expired media should be discarded in the
biohazard bin.
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APPENDIX
Joint Clinical Research Centre
QC Sheet
Culture media making log
Month…………………………………………
Year…………………………………….
This form should be used each time culture media is prepared (with each batch). Its purpose is to maintain a record of Technologists who make
media, plus associated QA/QC.
Date
Technologist
Initials
Media made
Reviewed by Section Head…………………………………
Date…………………………
NB:
Manufacturer
Batch number
Batch expiry
date
Checked by QA/QC Officer……………………………………
Date………………..........
1. This sheet is auditable and therefore must be filled and archived on file all the time.
2. A list indicating the names and initials of all staff in the section should be available.
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QC of Culture Media
This form should be used each time media is prepared (with each batch) and used to grow organisms to check its quality and ability to support
growth.
Name of medium:……………………………...Prepared by:……………………………..
Date prepared:…………………………………Expiry Date:………………….Lot:………………………..
Date
Sterility of Standard organisms
medium
set on each plate
checked
Results of STD Gram –
ve organism (Growth /
No growth)
Reviewed by :…………………………….(Section head)
Date…………………………
Results of STD Gram
+ve organism
pH
(Growth / No growth)
Comments
Checked by:………………………………….(QA/QC Officer)
Date…………………………
N.B: Instructions: 1- This sheet is auditable and therefore must be filed and archived on file all the time.
2- A list indicating the initials of all staff in the section should be available.
END
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Initials
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