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Culture Suspension and Cell Enumeration SOP

Standard Operating Procedure (SOP) for Microbial Culture Suspension Preparation, Cell Enumeration, Use, Storage, and Destruction in the microbiology department.

Microbial Culture Suspension & Cell Enumeration

1.0   Purpose:

    • The purpose of this SOP is to describe the procedure for the preparation of culture suspension and the enumeration of cells.

2.0   Scope:

    • This  SOP is applicable to Bacterial and Fungal cultures used for the Growth promotion test (GPT).

3.0   References:

    • British Pharmacopoeia (BP)
    • United States Pharmacopoeia (USP)

4.0   Responsibilities:

    • Microbiologist:

    • To perform the activity as per SOP
    • To maintain all the records as per SOP

    • QC Head or designee:

    • To check the SOP.
    • To give training to all concerned persons before the implementation of SOP

    • Quality Assurance:

    • To check the SOP.
    • To ensure the implementation of the system as per SOP.

5.0   Abbreviations & Definition of Terms :

    • Abbreviations:
TAC- Total Aerobic count. MLT-   Microbial Limit Test.
YMC- Yeast and mould count. SCDA- Soybean casein digest agar
SCDM- Soybean casein digest medium SDA- Sabouraud dextrose agar
PA – Pseudomonas aeruginosa. SL- Salmonella spp.
SA- S aureus. BS- Bacillus subtilis
EC- E. coli. CA- Candida albicans. 
AN- A. niger AET- Antimicrobial effectiveness testing.
USP- United States Pharmacopeia BP- British Pharmacopeia
    • Definition of Terms
    • Growth promotion test: Also referred to as fertility or nutritive properties test, which is performed on the media used during different tests like sterility test, microbial limit test, preservative efficacy test, etc to demonstrate that it is capable of supporting the growth of microorganisms.
    • Colony-forming unit (CFU): Visible outcome of the growth of micro-organisms arising from single or multiple cells.

6.0   Procedure for Culture Suspension (Cell):

    • Preparation of culture suspension, dilutions, enumeration of cells, and record:

    • Follow the procedure described in Microbial Culture Suspension Preparation, and Cell enumeration as per Attachment-1.

    • Preparation of stock culture suspension & it’s usage:

    • Use appropriate quantity and dilution for the preparation of stock culture suspension.
    • Use the appropriate quantity of the stock culture suspension as per the requirement of the test.
    • In case of intermittent use of the culture dilutions, remove them from the refrigerator at least half an hour before use.
    • Transfer back the culture dilutions in the refrigerator within two hours.

    • Storage of stock culture suspensions (cell):

    • Store the culture suspension in the refrigerator for up to one month from the date of preparation.

    • Validation of the hold time of stock culture suspension (cell):

    • Validate the hold (storage) time by enumerating the count in culture suspension on the 30th day and compare count with initial count as per Attachment-3.
    • Acceptance criteria: The count of the stored culture suspension should be within 50-200% (factor 2) of the initial count.

    • Destruction of culture suspensions (cell):

    • Follow the procedure defined in Procurement, transfer, preservation, and disposal of microbiological culture SOP and maintain the record.

7.0   Attachments- Culture Suspensions (cell) :

Attachment–1: Template for microbial culture suspension preparation & cell enumeration

Note: The name and type of organisms mentioned in the below template are only examples. As per the testing
need, one can remove or add the column of organisms to be used. But at the same time, one has to define the
name & type of organism to be used during GPT and MLT/Sterility/AET validation/container closure integrity
test in protocol/template/SOP as per the recommendation of respective pharmacopeia. Also one has to make sure
that the organisms & type recommended in pharmacopeia have been used.

Sr. No.

 Activity

Sign/Date (Performed by)
1.0 Bacterial culture suspension preparation, dilution, and cell density enumeration.
1.1 Bacterial culture suspension preparation.
1.1.1 Organisms to be used Working culture ID Organisms used
a)    Pseudomonas aeruginosa ATCC 9027
b)   Staphylococcus aureus ATCC 6538
c)    Escherichia colt ATCC 8739
d)   Salmonella spp. NCTC 6017
e)   B. subtilis ATCC 6633
1.1.2 Identify five containers having 10 ml sterile Soybean Casein Digest Brothas

PAB : Broth culture for P. aeruginosa.

SAB : Broth culture for S. aureus.

ECB: Broth culture for E. Coli.

SLB: Broth culture for Salmonella spp.

BSB : Broth culture for B. subtilis.
1.1.3 Inoculate a loopful of the respective bacterial strains separately in the above broth media containers.
1.1.4 Incubate the containers at 30-35°C for 18-24 hours.
1.1.5 Observe the containers for growth (Turbidity). Record the details in the below table.
Culture suspension preparation details Ps. aeruginosa S. aureus E. coli Salmonella spp. B. sub filis
Identification of media containers
Lot no. of SCDM broth used.
Incubation in date/time
Incubator no.
Incubation out date/time
Growth observed /not observed
Note- After growth, if the culture is not used within 2 hrs, store culture up to 24 hrs at 2-8°C

1.2

Preparation of serial dilutions of the bacterial cultures.
1.2.1 Aseptically transfer 1 ml of (NMT 24 hrs age) broth culture into 9 ml of sterile n-saline.
1.2.2 Vortex the test tubes for about 30 seconds to homogenate the contents.
1.2.3 Make serial dilution up to 10-8 using sterile n-saline. Homogenate the contents by vortexing the test tubes for about 30 sec while preparing each dilution.
1.2.4 Identify dilutions as follows.
Dilution Identification of culture dilutions
  P. aeruginosa S. aureus E. coli Salmonella spp. B. subtilis
10-1 PA-1 SA-1 EC-1 SL-1 BS-1
10-2 PA-2 SA-2 EC-1 SL-1 BS-1
10-3 PA-3 SA-3 EC-1 SL-1 BS-1
10-4 PA-4 SA-4 EC-1 SL-1 BS-1
10-5 PA-5 SA-5 EC-1 SL-1 BS-1
10-6 PA-6 SA-6 EC-1 SL-1 BS-1
10-7 PA-7 SA-1 EC-1 SL-1 BS-1
10-8 PA-8 SA-1 EC-1 SL-1 BS-1

1.3

Enumeration of culture suspension (cell).

(To identify the dilution containing <250 cfu/ml)
Note: Use the different dilution of the culture suspensions (cell) prepared within 2 hrs from the time of preparation. If not used, store at 2-8°C.
1.3.1 Transfer aseptically 1 ml of (10-4) dilution of  P. aeruginosa into empty sterile Petri plates in duplicate.
1.3.2 Add about 15 to 20 ml of previously melted and cooled to approximately 45°C Soybean Casein Digest Agar medium, mix the culture suspension with the agar, and allow it to solidify at room temperature.
1.3.3 Repeat the above step for the remaining four dilutions i.e 10– 5, 10-6, 10-7, 10-8as well.
1.3.4 Repeat the above procedure for the remaining four organisms i.e S. aureus, E. eon, Salmonella spp, and B. subtilis.
1.3.5 Immediately store the culture suspensions (cell) in the freeze.
1.3.6 Invert the Petri plates and incubate the plates at 30-35°C for 18 to 24 hrs.
1.3.7 Observe the plates and report the number of cfu/plate.
1.3.8 Record the details in the below table.
Note: Select the plates corresponding to a given dilution and showing the highest number of colonies less than 250 for TAMC. Take the arithmetic mean per culture medium of the counts to calculate the number of cfu per ml.

1.4

Stock culture suspension preparation and confirmation of count per ml:
1.4.1 Select the previous dilution of the identified dilution (having <250 cfu/ml) of culture suspension and dilute it further to have a sufficient amount of the culture suspensions (cell).
1.4.2 Take 5 ml of selected dilution and add to 45 ml of n-saline.
1.4.3 Transfer aseptically 1 ml of stock culture suspension of P aeruginosa into empty sterile Petri plates in duplicate. Add about 15 to 20 ml of previously melted and cooled to approximately 45°C Soybean Casein Digest Agar medium, mix the culture suspension with the agar, and allow it to solidify at room temperature.
1.4.4 Repeat the above procedure for the remaining four organisms i.e S. aureus, E. coli, Salmonella spp, and B. subtilis.
1.4.5 Immediately store the stock culture suspensions (cell) in the freeze.
1.4.6 Invert the Petri plates and incubate the plates at 30-35°C for 18 to 24hrs.
1.4.7 Observe the plates and report the number of cfu/plate.
1.4.8 Record the details

2.0

Fungal culture suspension preparation, dilution, and cell density enumeration.
2.1 Fungal culture suspension preparation.
2.1.1 Organisms to be used Working culture ID Organisms used
a)     C. albicans ATCC 10231    
b)     A. niger ATCC 16404    
2.1.2 Identify two slants of Sabouraud Dextrose Agar (SDA) slants as

CAC: Culture of C. albicans

ANC : Culture obi. niger
2.1.3 Streak a loopful of the recently grown working culture of fungal strains separately on the above slants.
2.1.4 Incubate the slants of C. Albicans at 20-25°C for 44-52 hours.
2.1.5 Incubate the slants of A. niger at 20-25°C for 6-7 days.
2.1.6 Observe the slants for growth. Record the details in the below table.
Culture suspension preparation details. C. Albicans A. niger
Identification of culture tubes.    
Lot no. of SDA medium used.    
Incubation in date/time.    
Incubator no.    
Incubation out date/time.    
Growth observed /not observed.    
Note- After growth, if the culture is not used within 2 hrs, store culture up to 24 hrs at 2-8°C

2.2

Preparation of serial dilutions of the fungal cultures.
2.2.1 Take a loopful of the slant culture and transfer into 9 ml of sterile n-saline aseptically.
2.2.2 Use sterile n-saline containing 0.05% polysorbate 80 for making dilutions of fungal culture.
2.2.3 Vortex the test tubes for about 30 seconds to homogenate the contents.
2.2.4 Make serial dilution up to 10′ using sterile n-saline Homogenate the contents by vortexing the test tubes for about 30 sec while preparing each dilution.
2.2.5 Identify dilutions as follows.
Dilution Identification of culture dilutions
C. albicans A. niger
10-1 CA-1 AN-1
10-2 CA-2 AN -2
10-3 CA-3 AN -3
10-4 CA-4 AN -4
10-5 CA-5 AN -5
10-6 CA-6 AN -6
10-7 CA-7 AN -1
10-8 CA-8 AN -1

2.3

Enumeration of culture suspension (cell)
Note: Select the plates corresponding to a given dilution and showing the highest number of colonies less than 50 for TYMC. Take the arithmetic mean per culture medium of the counts to calculate the number of cfu per ml.
Note: Use the different dilution of the culture suspensions prepared, within 2 hrs from the time of preparation. If not used, store at 2-8°C.
2.3.1 Transfer aseptically 1 ml of (10– 4) dilution of C. Albicans into empty sterile Petri plates in duplicate.
2.3.2 Add about 15 to 20 ml of previously melted and cooled to approximately 45°C Sabouraud Dextrose Agar medium, mix the culture suspension with the agar, and allow it to solidify at room temperature.
2.3.3 Repeat the above step for the remaining four dilutions i.e 10– 5, 10-6, 10-7, 10-8 as well.
2.3.4 Repeat the above procedure for dilutions of A. niger
2.3.5 Incubate plates of C. albicans at 20-25°C for 44 to 52hrs.
2.3.6 Incubate plates of A. niger at 20-25°C for 6 to7 days
2.3.7 Observe the plates and report the number of cfu/plate.
2.3.8 Record the details
Note: Select the plates corresponding to a given dilution and showing the highest number of colonies less than 50 for TYMC Take the arithmetic mean per culture medium of the counts to calculate the number of cfu per ml.

2.4

Stock culture suspension preparation and confirmation of count per ml.
2.4.1 Select the previous dilution of the identified dilution (having <50 cfu/ml) of culture suspension and dilute it further to have a sufficient amount of the suspensions.
2.4.2 Take 5 ml of selected dilution and add to 45 ml of n-saline.
2.4.3 Transfer aseptically 1 ml of stock culture suspension of C.albicans into empty sterile Petri plates in duplicate. Add about 15 to 20 ml of previously melted and cooled to approximately 45°C Sabarouds Dextrose Agar medium, mix the culture suspension with the agar, and allow it to solidify at room temperature.
2.4.4 Repeat the above procedure for A.niger (ATCC 16404)
2.4.5 Immediately store the stock culture suspensions in the freeze.
2.4.6 Invert the Petri plates and incubate the plates of C.albicans at 20-25°C for 44 to 52 hrs.& for A.niger at 6 to 7 days.
2.4.7 Observe the plates and report the number of cfu/plate.
2.4.8 Record the details

Stock culture suspension storage and destruction record

Date of culture suspension preparation Type of organism under storage Withdrawn  from a freeze at /Date Withdrawn by Stored back in a freeze at/Date Stored by Destroyed by/date Autoclave run no. Remarks

Attachment–2: Flow chart for the preparation of stock culture suspensions and cell enumeration

Transfer microbial cultures from working culture  tubes to broth or agar media and incubate

⇓

Observe the growth and harvest the cells (in case of bacterial cultures, use 24 hr broth cultures  for preparation of further dilution, and in case of fungal culture take a loopful from slant for preparation of further dilution

⇓

Serial dilutions of microbial cultures.

⇓

Cell count of last 4-5 dilutions of culture suspension by pour plate method for identification of dilution containing <250 cfu per ml for bacteria and <50 cfu per ml for fungi

⇓

Select previous dilution of above dilution  for stock culture preparation

⇓

Confirmation of the count in stock culture suspension and usage

⇓

Storage of the stock culture suspension  in the refrigerator when not in use

Attachment–3: Incubation conditions comparison for preparation of test strains as per different chapters of USP

Organism Chapter <61>  Table 1 (Enumeration test) Chapter <51> Table 2    (AET) Common condition derived for test strain preparation Remarks
Media Incubation condition
Bacteria SCDA / SCDM 30-35°C for 18-24 hrs SCDA / SCDM 30-35°C for 18-24 hrs 30-35°C for 18-24 hrs Nil.
C. albicans SDA / SD broth 20-25°C for 2-3 days (48-72 hrs) SDA / SD broth 20-25°C for 44-52 hrs 20-25°C for 44-52 hrs The incubation period selected considering it is sufficient for yeast growth.
A. niger SDA / PDA 20-25°C for 5-7 days or until good sporulation is achieved. SDA / SD broth 20-25°C for 6-10  days. 20-25°C for 6-7 days. The incubation period selected considering it is sufficient for mold growth.

Incubation conditions comparison for microbial recovery as per different chapters of USP

Organism Chapter <61>  Table 1 (Enumeration test)

Taken from the column: suitability of counting method in presence of the product. Not given specifically for enumeration of orgs.

 Chapter <51> Table 2 (AET)

Taken from microbial recovery incubation time.

 Common condition derived for cell enumeration Remarks
Bacteria 30-35°C , < 3 days 30-35°C for 3-5 days. 30-35°C for NMT 3 days.
  • Plate reading observations of 3rd, 4th, and 5th day should be recorded for at least three analyses to demonstrate no increase in the count on the 5th day.
  • In AET, the incubation period for enumeration for the initial count and after incubation of the test sample should be the same.  Hence NMT 3 days period is selected but in AET protocol a note shall be included to extend incubation time up to 5 days for enumeration of test sample if colonies are not well developed.
C. albicans 20-25°C , < 5 days 20-25°C for 3-5 days. 20-25°C for NMT 5 days. Nil.
A. niger 20-25°C , < 5 days 20-25°C for 3-7 days. 20-25°C for NMT 5 days.
  • Plate reading observations of the 6th and 7th day should be recorded for at least three analyses to demonstrate no increase in the count on the 7th day.
  • In AET, the incubation period for enumeration for the initial count and after incubation of the test sample should be the same. Hence NMT 5 days period is selected but in AET protocol a note shall be included to extend incubation time up to 7 days for enumeration of test sample if colonies are not well developed.

 

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