Document

STANDARD OPERATING PROCEDURE ON MAC KONKEY AGAR PREPARATION

Approved by: Date

Approved:

Prepared Effective Supersedes Document:

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

Date Reviewed: Date Revised: Comments Initials:

Distribution:
1.Master file
2.Bacteriology unit

Date of
Discontinuation:

Principle

RHLB/BAC/011 Version 1 Page 1 of 8

MacConkey agar is a differential medium to distinguish lactose fermenting gram negative bacteria from
non-lactose fermenters; lactose fermenters are coloured pink, while non-lactose fermenters are pale. It
is appropriate for stool, urine, uro-genital specimens, throat swabs, ear and eye swabs, wound swabs,
sputum, aspirates, cerebrospinal fluid

Purpose : This procedure provide instruction for the preparation of MacConkey Agar.

Materials Reagents
MacConkey agar

Reagents preparation: Ready to use

Reagents stability and storage: Store at 2-8OC till expiration date

Supplies
1. Distilled water
2. Autoclave bottle
3. Gloves

Equipment
1. Incubator
2. Weighing balance
3. Bunsen burner
4. Autoclave
5. Refrigerator
6. Measuring cylinder
7. Petri dish
8. Beaker

Sample
Sample type Amount required Transport and Storage Stability
N/A N/A N/A N/A
RHLB/BAC/011 Version 1 Page 2 of 8
Limitations: N/A

Sample retention: N/A

Special Safety Precautions:

1. Adhere to universal safety precaution
2. Adhere 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)
Escherich 1 New lot N/A
ia coli
Proteus
spp.

Control preparation: Isolate and inoculate in peptone water and store at 2-8 OC.

Note:

RHLB/BAC/011 Version 1 Page 3 of 8

Procedure

A. Mac Conkey Agar

1. Suspend 50.0g of powder in 1 litre of distilled or deionized water
2. Heat until completely dissolved
3. Sterilize in autoclave at 1210C for 15 minutes
4. Cool to 45-50 0C
5. Pour 20-25 ml of the ready media on to petri dishes
6. Leave to stand for thirty minutes to solidify
7. Perform sterility testing
8. Label the bottom of each plate with date of preparation and batch
number
9. Store the culture media plates upside down at 2-8 0C sealed in plastic
bags to reduce contamination
10. Check pH
B. Checking the pH of MacConkey agar
1.
Pour a sample of the MacConkey agar into a small beaker or petri dish.
2.
Lay a narrow range pH paper on its surface when the MacConkey agar
is solidified.
3. Compare the colour of the paper against the pH colour chart provided.
C. How to dispense MacConkey 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-
25ml 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.
5.
6. Store at 2-8˚C.
7. D. Sterility testing of MacConkey agar media
8. Incubate 5 % of the batch at 35-37˚C overnight.
9. Examine for growth. Contamination by microorganisms capable of
overnight growth will be shown on the MacConkey agar. MacConkey
agar media are best examined for contamination immediately before
use.

RHLB/BAC/011 Version 1 Page 4 of 8

Note:

 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 deterioration.
 Do not leave the plates exposed to bright light especially sunlight.

Calculation: N/A

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. 398.
2. Garcia L.S et al2007; Clinical Microbiology Procedures Handbook, 2 nd Edition, , ASM Press, Pg 18.

RHLB/BAC/011 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………………..........

RHLB/BAC/011 Version 1 Page 6 of 8

 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………

RHLB/BAC/011 Version 1 Page 7 of 8

 PROCEDURE ON MAC KONKEY AGAR PREPARATION

B. Mac Conkey Agar

1. Suspend 50.0g of powder in 1 litre of distilled or deionized water
2. Heat until completely dissolved
3. Sterilize in autoclave at 1210C for 15 minutes
4. Cool to 45-50 0C
5. Pour 20-25 ml of the ready media on to petri dishes
6. Leave to stand for thirty minutes to solidify
7. Perform sterility testing
8. Label the bottom of each plate with date of preparation and batch
number
9. Store the culture media plates upside down at 2-8 0C sealed in plastic
bags to reduce contamination
10. Check pH
B. Checking the pH of MacConkey agar
1.
Pour a sample of the MacConkey agar into a small beaker or petri dish.
2.
Lay a narrow range pH paper on its surface when the MacConkey agar
is solidified.
3. Compare the colour of the paper against the pH colour chart provided.
C. How to dispense MacConkey 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-
25ml 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.
5.
Store at 2-8˚C.
1. D. Sterility testing of MacConkey agar media
2. Incubate 5 % of the batch at 35-37˚C overnight.
3. Examine for growth. Contamination by microorganisms capable of
overnight growth will be shown on the MacConkey agar. MacConkey
agar media are best examined for contamination immediately before
use.

RHLB/BAC/011 Version 1 Page 8 of 8