SOP FOR MICROBIAL TESTING OF PRIMARY PACKING MATERIAL SOP FOR MAINTENANCE OF PURE CULTURE.

SOP FOR MICROBIAL TESTING OF PRIMARY PACKING MATERIAL



OBJECTIVE:The objective of this SOP is to define a procedure for testing microbial load on primary packing material in order to avoid microbial contamination of final product.



SCOPE:This SOP is applicable for monitoring microbial load on all primary packing material in microbiological laboratory.

RESPONSIBILITY: Microbiologist:  is to follow process or method  as per SOP.

Q.C Head: to ensure the proper execution of the SOP.

Q.A: to ensure that the SOP is being properly implemented.



ABBREVIATION:

SOP: Standard Operating Procedure

Cfu: Colony Forming Unit

PROCEDURE:

A. Sampling procedure:


A. For Bacteria:

1. Take the swab sample from the inside surface of the material to be tested of an area of 25 sq. mm from 10 different samples with the help of moist sterile saline swab.

2. Place the swab in a test tube containing 10 ml of sterile Soybean Casein Digest Medium & shake well. Pipette out 1 ml of this solution in two sterile Petri-plates each.

3.  In these plates, pour 15 ml of sterile molten Soybean Casein Digest Agar & incubate at 30-350 C for 48 to 72 hrs.

4.  Incubate the remaining 9 ml of solution of Soybean Casein Digest Medium at 30-350 C for 24 hrs.

5. Proceed further for pathogen testing i.e. E. coli, Salmonella, Pseudomonas and Staphylococcus as per SOP no. SOP/P/MB/14.

  B. For Yeast & Mould:

1. Follow step 1 & 2 from above.

2. In these plates, pour 15 ml of sterile molten Saboraud’s chloramphenicol agar and incubate the plates at 20-25°C for 48-72 hrs.

Limits:

Alert limit

100 cfu / 25 sq.cm

Action limit

500 cfu / 25 sq.cm.

FREQUENCY:

As and when required

ANNEXURE:

I. Swab testing report

II. Protocol for swab testing report





Annexure I: Swab testing report

Date of Sampling:  Date of Analysis:            Date of reporting:

Sample/Material:                                                         Autoclave load no:

Quantity sampled:                                                        Batch no:





SR. TEST SPECIFICATION OBSERVATION

1. Total viable count (TVC)

a. TBC

b. TFC NMT 100 cfu/ 25 sq. cm

2. Pathogens

A. Escherichia coli Absent

B. Salmonella typhi Absent

C. Pseudomonas aeruginosa Absent

D. Staphylococcus aureus Absent




Annexure II: Protocol for swab testing


Date of Sampling:                                           Date of Analysis:

Date of reporting:                                                      Batch no:

Sample/Material:

Quantity sampled:                                                     Autoclave load no:


Pathogen testing:-

II. After 48hrs:

Date-

Pathogen Medium used Batch no. Description Observation P.C. N.C. Analyzed by Checked by

E. coli MacConkey’s broth  Acid and gas production observed

S. typhi Rappaport Vassiliadis Salmonella enrichment broth  Broth observed is turbid

III. After 72-120hrs:

Date-

Medium used Batch no. TBC

(cfu/25 sq cm) Y/M

(cfu/ 25 sq cm) TVC

(cfu/ 25 sq cm) Control Analyzed by Checked by


Pathogen Medium used Batch no. Description Observation P.C N.C Analyzed by Checked by

E. coli MacConkey’s agar  Pink non-mucoid colonies observed

S. typhi Xylose lysine deoxycholate agar  Red colonies with or without black centers

S. aureus Mannitol salt agar  Yellow or white colonies with yellow zone

P. aeruginosa Cetrimide agar  Greenish color colonies.


SOP FOR MAINTENANCE OF PURE CULTURE.



OBJECTIVE:Objective of this SOP is to define a standard procedure for maintenance of pure culture.

SCOPE:This procedure is applicable for maintenance of pure culture in Microbiology department.


REFERENCE:

N.A.


RESPONSIBILITY:

Microbiologist: is to follow the process given as per SOP.

Q.C Head: to ensure proper execution of the SOP.

Q.A: to ensure that the SOP is being properly implemented.



DISTRIBUTION:

Sr. no Particular  Departments

  QA QC PD ST MB EHS PR MT

1 Master copy no. 01

2 Control copy no. 01

3 Total control copies 01



ABBREVATIONS:

N.A.: Not Applicable

Q.C: Quality control

Q.A: Quality assurance

MC: Master culture

WC: Working culture

RC: Routine culture



PROCEDURE:

A. Method of subculturing:

1. Operate LAF as per SOP No. SOP/P/MB/05.

2. Wipe out the outer surface of the culture slant tubes/ lyophilized ampoules as well as the sterile slant tubes with cotton soaked with 70% IPA.

3. Hold on the extreme end and heat the nichrome wire loop on the reducing flame till red hot.

4. Allow the wire loop to cool and slowly open the cotton plug of the old culture and gently warm the mouth of the tube.

5. Pick up the smear/growth with the help of loop after removing cotton plug of the slant tube.

6. Hold the nichrome wire loop with smear/culture growth, plug the culture slant tube, and lay aside the old slant culture.

7. Open the fresh slant tube, warm the mouth of the tube holding in other hand and carefully transfer the culture that is held on the loop.

8. Spread the culture spirally on the surface of the slant, warm the mouth of slant and plug it with cotton.

9. Label the culture tube, incubate at specified temperature and period, and then store it in refrigerator at 2-8°C

B. Maintenance of subculture:

1. Before subculturing, the cultures when received from the national laboratories are to be validated as per SOP No. SOP/P/MB/20.

2. After ensuring the purity of mother culture, prepare three master culture of mother culture by transferring loopful of growth from mother culture to fresh tube and name them as MCI, MCII and MC III.

3. Incubate both the tubes at specified temperature and period, as indicated in the following table and observe the growth after incubation.

4. Check the purity of culture by staining (SOP No. SOP/P/MB/18) and record their physical characteristics as per format.

5. If the cultures are not showing their physical characteristics, discard the culture as per SOP No. SOP/P/MB/09.

6. If the culture is showing their physical characteristics as stated in following table; transfer a loopful of growth from MCI to a fresh seven set of working slant i.e. W1 to W7 and label them as MCI-W1, MCI-W2, MCI-W3, MC1-W7. Each of these working cultures is to be used for six months.

7. Out of seven working cultures, one is intended to be held in reserve in the event of first working culture becoming contaminated.

8. Use other six working cultures, one culture per month for preparing routine culture (RC).

9. Prepare five routine cultures (R1-R5) every month from one working culture.

10. Use one routine culture every week.

11. All the culture must be stored at temperature 4°C to 8°C.

12. After six months use the second batch i.e. MC-II and proceed in the same manner.

13. The culture slant should be labeled with following details;

Name of the organism

Batch no.

Transfer date

Use before

Sub cultured by

14. Note down the results in the annexure-I.

15. Following is the table consisting of the culture details, its incubation temperature and its media.






Sr. No. Name of the culture Media slant to be used Incubation Temp. Physical characteristics Incubation Period

1 E.coli

Nutrient agar 30 – 350C Gram –ve, short rods 24 – 48 hrs

2 S. aureus

Nutrient agar 30 – 350C Gram +ve, cocci in clusters 24 – 48 hrs

3 Salmonella

Nutrient agar 30 – 350C Gram –ve rod shaped 24 – 48 hrs

4 P. aeruginosa

Nutrient agar 30 – 350C Gram –ve rod shaped 24 – 48 hrs

5 C. albicans

Sabouraud dextrose agar 20 – 250C Oval shaped 48 – 72 hrs

6 B. subtilis

Nutrient agar 30 – 350C Gram +ve, rods 24 – 48 hrs

7 A. niger

Sabouraud dextrose agar 20 – 250C Mycelial spores 48 – 96 hrs

8 S. boydii Nutrient agar 30-35°C Gram –ve short rods 24-48 hrs

9 B. cereus Nutrient agar 30-35°C Gram +ve rods 24-48 hrs

10 L. monoc

ytogenes Nutrient agar 30-35°C Gram +ve short rods 24-48 hrs

11 V. cholerae Nutrient agar 30-35°C Gram -ve short rods 24-48 hrs

12 Cl. sporogenes Columbia agar 30-35°C Gram +ve rods 24-48 hrs













Flow Diagram of Culture Transfer (Half Year)

                                                            Mother yearly

           Master culture  (MC-I) & (MC-II)                                        Master  culture (MC-III)

                                                                                     (Reserve)


                        Monthly


   Working  Working         Working     Working   Working    Working  Working

   culture  culture            culture        culture      culture      culture   culture

   MC-I-W 1      MC-I-W2       MC-I-W3   MC-I-W4 MC-I-W5  MC-I-W6   MC-I-W7

                                                                                                                (Reserve)

  Weekly


Routine        Routine    Routine     Routine     Routine

culture          culture     culture     culture       culture

   MC-I-W1-R1   MC-I-W1-R2  MC-I-W1-R3  MC-I-W1-R4   MC-I-W1-R5








Frequency:

First generation- Once in a year

Second generation- Once in six months

Third generation- Monthly

Fourth generation- Weekly

Annexure:

I. Microbiological culture maintenance record.

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