Standard Operating Procedure for Preparation of Media



Joint Clinical Research Centre

Laboratory Policy and Procedure Manual

Preparation of Culture Media

|Non-Smile Resource |

|Author: JCRC |Document Number: |Pro67-A-11 |

| |Effective (or Post) Date: |13-Oct-09 |

|Document Origin |Company: |JCRC |

| |SMILE Approved by: |Peggy |

|Review by |Peggy Coulter |Review date |27 April 20 |

|pSMILE 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 encouraged to ensure compliance with local laws and study protocol |

|policies when considering the application of this document. If you have any questions contact your SMILE representative. |

| Index code |Name of policy/procedure |

|Media 402 |Preparation of Culture Media |

|Prepared by |Date to be adopted |Signature Laboratory Manager or Section Head |

|Juliet Kitabalwa | 01 Oct 2009 | |

|Reason for procedure (check one) |Signature Director |

|New procedure |Supercedes retired procedure index code | |

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|Annual review |REVIEWED BY | | |

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ACKNOWLEDGEMENT OF READING AND UNDERSTANDING SOP:

MEDIA 402

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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.

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.

• pH of the medium 7.2-7.6 at room temperature.

4.4 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 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 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.

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.

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.

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.

• 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 screw-capped 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. Flame-sterilize 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.

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 species |Maintenance medium |Subculturing interval |

|Blood agar |Streptococcus pyogenes |Cooked meat medium |3 months |

| |Streptococcus pneumoniae | | |

| | | | |

| |Haemophilus influenzae (with S.aureus |Chocolate agar slope (35-37˚C) | |

| |streak) | |1 month |

| | |Chocolate agar slope (35-37˚C) | |

|Chocolate agar |Haemophilus influenzae | |1 month |

| | | | |

|Cytine Lactose Electrolyte |Staphylococcus aureus |Nutrient agar semi solid or deep |6 months |

|Deficient Medium | | | |

|(CLED) |Proteus mirabilis | | |

| |Escherichia coli |Nutrient agar semi solid or deep | |

|Mac Conkey agar |Proteus mirabilis | |6 months |

| |Shigella spp. | | |

|SS agar |Salmonella typhimurium |Dorset egg medium |12 months |

|(Salmonell-Shigella agar) | | | |

| |Escherichia coli |Nutrient agar semi solid or deep |6 months |

|Sabouraud agar |Candida albicans |Sabouraud agar plate |2 months |

| |Aspergillus brasiliensis | | |

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.

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.

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 |Media made |Manufacturer |Batch number |Batch expiry date |

| |Initials | | | | |

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Reviewed by Section Head………………………………… Checked by QA/QC Officer……………………………………

Date………………………… Date………………..........

NB: 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.

Joint clinical Research Centre

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:………………………..

| |Sterility of |Standard organisms set on each |Results of STD Gram –ve organism |Results of STD Gram +ve organism | | | |

| |medium checked |plate |(Growth / No growth) |(Growth / No growth) |pH |Initials | |

|Date | | | | | | |Comments |

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Reviewed by :…………………………….(Section head) Checked by:………………………………….(QA/QC Officer)

Date………………………… 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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