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.