Document

STANDARD OPERATING PROCEDURE FOR NUTRIENT AGAR PREPARATION

Approved by: Date

Approved:

Prepared Effective Supersedes Document:

by(Name/sign): Date:
Fosu Kwabena
Reviewed by:
(Name/sign)

Date Reviewed: Date Revised: Comments Initials:

Distribution:
1. Master file
2. Bacteriology unit

Date of
Discontinuation:

Principle:

RHLB/BAC/009 Version 1 Page 1 of 8

Nutrient Agar consists of peptone, beef extract and agar. This relatively simple formulation provides the
nutrients necessary for the replication of a large number of microorganisms which are not excessively
fastidious. The beef extract contains water soluble substances including carbohydrates, vitamins,
organic nitrogen compounds and salts. Peptones are the principle sources of organic nitrogen,
particularly amino acids and long-chained peptides. Agar is the solidifying agent.

Purpose: This procedure provides instructions for the preparation of nutrient culture medium.

Materials

Reagents
Nutrient medium powder

Reagents preparation: Ready to use

Reagents stability and storage: Store at 10-25OC and is stable till expiration date.

Supplies
1. Distilled water
2. Petri dish
3. Blood
4. Beaker
5. Glove

Equipment
1. Incubator
2. Weighing balance
3. Burner
4. Autoclave
5. Beaker

RHLB/BAC/009 Version 1 Page 2 of 8

Sample
Sample type Amount required Transport and Storage Stability
N/A N/A N/A N/A

Limitations: N/A

Sample retention: N/A

Special Safety Precautions:

1. Adhere to universal safety precaution
2. Adhere to aseptic technique

Calibrator Level Stability Frequency Preparation

(y/n)
N/A N/A N/A N/A N/A
Calibration

Calibrator preparation: N/A

Note: N/A

Quality
Control
Control Level Stability Frequency Preparation
(y/n)
Known 1 2 weeks New lot N/A
colonies (
streptoco
ccus
spp.,)

Control preparation: Isolate and store in peptone water 2-8 oC

Note: N/A

RHLB/BAC/009 Version 1 Page 3 of 8

 Step Nutrient agar
1. Suspend 23 g of the powder in 1 litre of distilled water
2. Heat with frequent agitation and boil for 1 minute to completely
dissolve the powder
3. Autoclave at 121˚C for 15 minutes
4. Cool to 45-500C
5. Pour 20-25 ml of the ready media into sterile petri dishes
6. Leave to stand for thirty minutes to solidify
7. Perform sterility testing
8. Label the bottom of each petri dishes with date of preparation and
batch number
9. Store the media at 2-80C sealed in plastic bags to reduce
contamination
10. Check pH
B. Checking the pH of Nutrient agar
1. Pour a sample of the Nutrient agar into a small beaker or petri dish.
2. Lay a narrow range pH paper on its surface when the Nutrient agar is
solidified.
3. Compare the colour of the paper against the pH colour chart provided.
C. How to dispense Nutrient agar
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 20-
25 ml of medium into each dish
3. If air bubbles enter while pouring, Then
4. 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 than 4 mm) and of a
firm gel.
D. Sterility testing of Nutrient agar media
1. Incubate 5 % of the batch at 35-37˚C overnight.
2. Examine for growth. Contamination by microorganisms capable of
overnight growth will be shown on the Nutrient agar. Nutrient agar
media are best examined for contamination immediately before use.

Note: Do not leave the plates exposed to bright light especially sunlight.

Calculation: N/A

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Results interpretation: N/A

Critical values: N/A

Expected values Parameter Male Female Children (< or = 12 Units

Reference Reference yrs) Reference
Interval Interval Interval
N/A N/A N/A N/A N/A

Result reporting: N/A

Related Procedures and Documents: N/A

Reference:

1. Monica Cheesbrough (2006); District Laboratory Practice in Tropical countries, Cambridge

University Press, 2nd Edition, pg. 400.
2. Garcia L.S et al2007; Clinical Microbiology Procedures Handbook, 2 nd Edition, , ASM Press, Pg 18.

RHLB/BAC/009 Version 1 Page 5 of 8

 Appendix A

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

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

Date………………………… Date………………..........

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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 Results of STD Gram –ve Results of STD Gram pH Initials Comments
medium set on each plate organism (Growth / No +ve organism
checked growth) (Growth / No growth)

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

Date………………………… Date………....................

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 PROCEDURE FOR NUTRIENT AGAR PREPARATION

Step Nutrient agar

11. Suspend 23 g of the powder in 1 litre of distilled water
12. Heat with frequent agitation and boil for 1 minute to completely
dissolve the powder
13. Autoclave at 121˚C for 15 minutes
14. Cool to 45-500C
15. Pour 20-25 ml of the ready media into sterile petri dishes
16. Leave to stand for thirty minutes to solidify
17. Perform sterility testing
18. Label the bottom of each petri dishes with date of preparation and
batch number
19. Store the media at 2-80C sealed in plastic bags to reduce
contamination
20. Check pH
B. Checking the pH of Nutrient agar
4. Pour a sample of the Nutrient agar into a small beaker or petri dish.
5. Lay a narrow range pH paper on its surface when the Nutrient agar is
solidified.
6. Compare the colour of the paper against the pH colour chart provided.
C. How to dispense Nutrient agar
5. Lay out the sterile petri dishes on a level, clean (disinfected) bench
surface.
6. 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 20-
25 ml of medium into each dish
7. If air bubbles enter while pouring, Then
8. 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 than 4 mm) and of a
firm gel.
D. Sterility testing of Nutrient agar media
3. Incubate 5 % of the batch at 35-37˚C overnight.
4. Examine for growth. Contamination by microorganisms capable of
overnight growth will be shown on the Nutrient agar. Nutrient agar
media are best examined for contamination immediately before use.

RHLB/BAC/009 Version 1 Page 8 of 8