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MLT Method Validation Procedure & Protocol

The MLT method must be validated before it will be applied when testing a product for resistance to bacteria in order to ensure that the product has no microbial inhibitory characteristics that could lead to false negative results. The MLT Method Suitability Test is known as the title of this validation test.

Validation Protocol for MLT Method

PROTOCOL NO.
SUPERSEDES NO.
FFECTIVE DATE

TABLE OF CONTENTS – MLT METHOD VALIDATION

S. No Topics Page No
1.0 Protocol Preparation and Approval
2.0 Purpose
3.0 Scope
4.0 Responsibility
5.0 References
6.0 Safety Considerations
7.0 Test Pre Requisites
8.0 Media And Test Organisms
9.0 Test Procedure
10.0 Acceptance Criteria
11.0 Report
12.0 Reconduct Of Test
13.0 Abbreviations
14.0 Annexures

1.0   PROTOCOL – APPROVAL:

    • This is a protocol to demonstrate the MLT Method Validation of products.
    • The protocol has been prepared, reviewed and approved for execution by personnel from the following departments:
      • MicrobiologyMLT Method Validation

      • Department Head

      • Head Regulatory Affairs

      • Head Quality Assurance

      • Quality Head

2.0   PURPOSE:

    • This MLT method validation protocol is designed to establish the method for demonstration that the test specimens to which the test for Microbiological Examination of Nonsterile Products: Microbiological Enumeration and Tests for Specified Organisms are applied, do not of themselves inhibit the multiplication, under the test conditions of microorganisms that may be present.

3.0   SCOPE:

    • This MLT method validation protocol is applicable for conducting the Microbiological Examination of Nonsterile Products: Microbiological Enumeration and Tests for Specified Organisms for Finished Products as per Harmonized method recommended by Ph. Eur., BP, USP and IP, at Microbiology department.

4.0   RESPONSIBILITY:

    • Microbiology Executive/Designee- Preparation of MLT method validation protocol, Execution of the validation studies and Completion of the MLT method validation report.
    • Head Q.C./Designee – Responsible for review of the protocol and its summary report for execution of experimental validation study and arranging resources for the validation program and review of validation results and summary report.
    • Head Q.A./R.A. or Designee – Responsible for review of the protocol and summary report, after completion of qualification summary report shall be checked and reviewed.
    • Head Quality: Responsible for the final approval of the MLT method protocol and summary report, after completion of qualification summary report shall be checked, reviewed and approved.

5.0   REFERENCES:

    • European Pharmacopoeia 7.0, volume 1, including chapters 2.6.12, Microbiological Examination of Non-Sterile Products: Microbiological enumeration test & 2.6.13 Microbiological Examination of Non-Sterile Products: Test for Specified Microorganisms.
    • British Pharmacopoeia Volume-V, Appendix XVI B.
    • Indian Pharmacopeia 2014: Para no. 2.2.9 Microbial Contamination in non-sterile product.

6.0   SAFETY CONSIDERATIONS – 

    • Procedures should be carried out aseptically.
    • Glassware and materials that are to be used should be sterile.
    • Glassware and materials used for handling the microorganisms should be decontaminated.

7.0   TEST PRE REQUISITES – MLT METHOD VALIDATION

    • Microbial Strains
    • Culture Mediums
    • Test Tubes, calibrated micro pipette and sterilized pipette tips
    • Incubators ( for 22.5 +5°C, 32.5 + 2.5°C and 43 + 1°C)
    • Water Bath
    • Pre validated autoclave
    • Colony Counter
    • Test Samples
    • Petri dishes (90 mm)
    • Compound microscope
    • Pre validated LAF Units
    • Sterilized inoculation loops

8.0   MEDIA AND TEST ORGANISMS

    • Organism: Use the test organisms as mentioned below:
Name Organisms Strain No.
Candida albicans ATCC10231
Aspergillus brasiliensis ATCC16404
Escherichia coli ATCC8739
Staphylococcus aureus ATCC6538
Pseudomonas aeruginosa ATCC9027
Bacillus subtilis ATCC6633
Salmonella abony NCTC 6017
    • Refer Annexure-1 titled ‘Acceptance Criteria for the Growth Promotion Test for Microbiological Culture Media’.

9.0   TEST PROCEDURE –MLT METHOD VALIDATION

    • Procedure for Microbiological Enumeration Test

Note: All tests shall be done in duplicate, including negative controls. These tests shall be performed for each of the mentioned organisms separately as per point no. 8.2 (In negative controls no inoculation is done in the sterile media dispensed as per the requirements of the experiments).

    • Prepare serial dilutions of specified organisms and select the dilution which is giving approximate 1x 104 CFU/ml. and record in Annexure-10.
    • Record the value of enumerated number of cells in the standardized cell suspension in Annexure-2, 3, 4 and 5 titled ‘Observation sheet of microbial enumeration test for coli, Salmonella abony, P. aeruginosa, S. aureus, Bacillus subtilis, Aspergillus brasiliensis & Candida albicans’.

    • Preparation of positive product control (PPC):

    • Dilute the product to be examined (1:10) in Soyabean Casein Digest Broth with 0.5% Polysorbate 80 (if required).
    • Further dilutions, where necessary, are prepared with the same diluent.
    • Add the sample prepared above a sufficient volume of microbial suspension to obtain an inoculum of not more than 100 cfu.

    • Preparation of Positive control (PC):

    • To 100 ml of Soyabean Casein Digest Broth with 0.5% Polysorbate 80 (if required).
    • Further dilutions, where necessary, are prepared with the same diluent. add a sufficient volume of microbial suspension to obtain an inoculum of not more than 100 cfu.

    • Preparation of test sample (NPC):

    • Dilute the product to be examined (1:10) in Soyabean Casein Digest Broth with 0.5% Polysorbate 80 (if required).
    • Further dilutions, where necessary, are prepared with the same diluent.
    • Preparation of negative control (NC):
    • Put an uninoculated flask of 100 ml Soyabean Casein Digest Broth with 0.5% Polysorbate 80 (if required) as a negative control to confirm the sterility of the diluent.
    • Take 1 ml of the positive product control at “0” hrs. and put in to 90 mm sterile in duplicate petri plate.
    • Pour 15-20ml of Soyabean Casein Digest Agar for bacteria and Sabouraud dextrose Agar for fungi.
    • Repeat the same process for positive control, for test sample and for negative control.
    • Incubate at 30-35°C up to 120 hrs. for bacteria and 20-25°C up to 168 hrs. for fungi.
    • Record the observations in Annexure-2 for bacteria titled ‘Observation sheet of microbial enumeration test for bacteria for 0 hr.’ and in Annexure-4 for Fungi titled ‘Observation sheet of microbial enumeration test for fungi for 0 hr.’
    • Incubate positive product control, test sample, positive control and negative control for 2 hrs.
    • proceed and Record the observations in Annexure-3 for bacteria titled ‘Observation sheet of microbial enumeration test for bacteria for 2 hrs.’ and in Annexure-5 for fungi titled ‘Observation sheet of microbial enumeration test for fungi for 2 hrs.’
    • If the results are not satisfactory at 1:10 dilutions of the sample with diluents like Soyabean Casein Digest Broth then dilution of the sample with increased quantity of the diluent (sample quantity remaining the same) shall be tried like at1:15; at1:20; at 1:25, etc. and if the dilution options do not work, then option of addition of neutralizing agents like Lecithin at 0.3% concentration in the media shall be used.

    • Test for Specified Micro-Organisms

    • Standardized cell suspension shall be prepared as per SOP, titled ‘Preparation of Microbial Culture Suspension’ .
    • Record the value of enumerated number of cells in the standardized cell suspension in Annexure-6, 7, 8 & 9 titled ‘Observation sheet of microbial enumeration test for coli’ , Salmonella abony, P. aeruginosa & S. aureus’.

    • Preparation of positive product control (PPC):

    • Dilute the product to be examined (1:10) in Soyabean Casein Digest Broth with 0.5% Polysorbate 80 (if required).
    • Further dilutions, where necessary, are prepared with the same diluent.
    • At the time of mixing, add each test strain (coli, Salmonella abony, P.aeruginosa and Staphylococcus aureus) in Soyabean Casein Digest Broth with 0.5% Polysorbate 80 (if required).
    • Inoculate the test strain individually. Use a number of microorganisms equivalent to not more than 100 cfu in the inoculated test preparation.

    • Preparation of Positive control (PC):

    • To 100 ml of Soyabean Casein Digest Broth with 0.5% Polysorbate 80 (if required).
    • Further dilutions, where necessary, are prepared with the same diluent.
    • Add a sufficient volume of microbial suspension (coli, Salmonella abony, P.aeruginosa and Staphylococcus aureus). Inoculate the test strain individually.
    • Use a number of microorganisms equivalent to not more than 100 cfu.

    • Preparation of test sample (NPC):

    • Dilute the product to be examined (1:10) in Soyabean Casein Digest Broth with 0.5% Polysorbate 80 (if required).
    • Further dilutions, where necessary, are prepared with the same diluent.

    • Preparation of negative control (NC):

    • Put an uninoculated flask of 100 ml Soyabean Casein Digest Broth with 0.5% Polysorbate 80 (if required) as a negative control to confirm the sterility of the media.
    • Proceed with the positive product control, positive control, test sample and negative control, incubated for 18-24 hrs. at 30-35°C in enumeration test and follow the procedure for detection of coli, Salmonella abony, P. aeruginosa, and S. aureus as per standard test procedure.
    • Observe the growth and record the observations in Annexure-6, 7, 8 & 9 respectively for coli, Salmonella abony, P.aeruginosa and S.aureus titled ‘Observation sheet of microbial enumeration test’.
    • During testing if any deviation found, that shall be recorded as per SOP  “Handling of Out of Specification (OOS) Result during Microbiological Testing”

10.0   ACCEPTANCE CRITERIA – MLT METHOD VALIDATION

    • The estimated number of cells in positive control (Media + Organisms) shall be less than 100 cfu for the Microbial Limit Test.
    • The counts from the Positive Product Control shall not less than 70% of that recovered from the inoculum control at 72 hrs. (Checked up to 120 hrs.) incubation period for ‘0’ & ‘2’ hrs. dilution of PPC calculated with respect to CFU obtained for positive control under same conditions for the bacteria.
    • The counts from the Positive Product Control shall not less than 70% of that recovered from the inoculum control at 120 hrs. (Checked up to 168 hrs.) incubation period for ‘0’ & ‘2’ hrs. dilution of PPC calculated with respect to CFU obtained for positive control under same conditions for the fungus.
    • The bactericidal activity of the product is demonstrated when no growth of the inoculated organisms take place in PPC, hence the product under test is not likely to be contaminated with the given species of the microorganism.
    • For growth characteristic on medium refer to Annexure-1 titled ‘Acceptance Criteria for the Growth Promotion Test for Microbiological Culture Media’.
    • The negative control should not show any growth.

11.0   REPORT

A detailed report on Microbial limit test shall be prepared, comprising:

    • Data Sheets of product under evaluation. (Refer Annexures 2, 3, 4, 5, 6, 7, 8 & 9)
    • For Summary of the report (Refer Annexure-11)
    • Certificate of the Validation of procedure (Refer Annexures-12)

12.0   RECONDUCT OF TEST

The test shall be re-conducted in the following cases:

    • Any major change in manufacturing procedure of the formulation.
    • Major change in the specifications of the drug product.
    • Change in the procedure of Microbiological Examination of Non-Sterile Products.

13.0   ABBREVIATIONS:

    • ATCC : American Type Culture Collection
    • IPA  :   Iso Propyl Alcohol
    • NMT :   Not More Than
    • NLT :   Not Less Than
    • PPC    :   Positive Product Control
    • PC       :   Positive Control
    • RA : Regulatory affairs
    • SCDA : Soyabean Casein Digest Agar
    • SCDM : Soyabean Casein Digest Medium
    • SCS :   Standardized Cell Suspension
    • SDA :  Sabouraud Dextrose Agar

14.0   ANNEXURES:

Annexure1 : Acceptance Criteria For The Growth Promotion Test for Microbiological Culture Media

Name of Media Name of Micro organism Incubation Conditions Method of inoculation Growth characteristics on media/Microscopic examination
Temp. (°C) Time (Hrs.)
Soyabean Casein Digest Broth Escherichia coli 30-35 24-48 Direct Turbidity, -Ve rods
Pseudomonas aeruginosa 30-35 24-48 Direct Turbidity, -Ve rods
Staphylococcus aureus 30-35 24-48 Direct Turbidity, +Ve Cocci
Bacillus subtilis 30-35 24-48 Direct Turbidity, +Ve rods
Salmonella 30-35 24-48 Direct Turbidity, -Ve rods
Candida albicans 20-25 48 Direct Turbidity
Aspergillus  brasiliensis 20-25 72 Direct Turbidity, Spores appear as pale to bright red
Soyabean Casein digest Agar Pseudomonas aeruginosa 30-35 24-72 Pour Plate/Streaking Colourless to yellowish colonies, -Ve rods
Staphylococcus. aureus 30-35 24-72 Pour Plate/Streaking Yellow shining colonies, +Ve Cocci
Bacillus subtilis 30-35 24-72 Pour Plate/Streaking Irregular whitish colonies, +Ve rods
Candida albicans 20-25 48-72 Pour Plate/Streaking Small colonies, Spores appear as pale to bright red
Aspergillus  brasiliensis 20-25 48-72 Pour Plate Formation of mycelia, Spores appear as pale to bright red
Sabouraud Dextrose agar Candida albicans 20-25 48-72 Pour Plate Rounded shining whitish colonies, gram+ve yeast cell
Aspergillus  brasiliensis 20-25 48-72 Pour Plate Cottony growth with mycelia, spores appear as pale to bright red
MacConkey Broth Escherichia coli 42-44 24-48 Direct Turbidity, -Ve rods
MacConkey agar Escherichia coli 30-35 18-72 Streaking Brick red colonies, -Ve rods
Rappaport Vassiliadis Salmonella enrichment broth Salmonella 30-35 18-24 Direct Turbidity, colour change
Xylose, lysine, deoxycholate agar Salmonella 30-35 18-48 Streaking Well-developed red colour  colonies with or without black center
Cetrimide agar Pseudomonas aeruginosa 30-35 18-72 Streaking Gram negative rods, Greenish colony
Mannitol salt agar Staphylococcus aureus 30-35 18-72 Streaking Yellow/white  colonies surrounded by clear zone.

Annexure 2 : Observation Sheet of Microbial Enumeration Test  for Bacteria For 0 Hr .

Protocol No.: 

Date of initiation of experiment:                                                            Media lot no.:

Name of microorganism:                                                                          Product:

Incubation temperature: 30-35°C                                                           Batch No.:

For 0 hr. observations                                                                      Incubator ID.:

Negative control (A)(Diluent) Positive product control (B)(Diluent + Product + SCS)
Observation from the dilution bottle
Time intervals of incubation of plates (hrs.) Plate-1 Plate-2 Avg. cfu/ml Dilution Plate-1 Plate-2 Dilution Factor Avg. cfu/g
24
48
72
96
120

 

Test sample (C)(Diluent + Product) Positive control (D)(Diluent + SCS) Observed bySign & Date
Dilution Plate-1 Plate-2 Dilution Factor Avg. cfu/g Dilution Plate-1 Plate-2 Avg. cfu/ml

# Enumerated number of Cells in Standardized Cell suspension (SCS) __________ cells/ml inoculated to obtain final inoculum of not more than 100 cfu/ml.

Percent recovery = (B-C)/D x 100 (at 72 h) (Acceptance criteria: Not less than 70% of that recovered from the inoculum control)

Remarks:                                                                                                                              

 Recorded by:                                                                                                            Reviewed by:               

Sign/ Date                                                                                                              Sign/ Date

Annexure 3 : Observation Sheet of Microbial Enumeration Test for Bacteria For 2 Hrs.

Protocol No.: 

Date of initiation of experiment:                                                            Media lot no.:

Name of microorganism:                                                                          Product:

Incubation temperature: 30-35°C                                                           Batch No.:

For 2 hrs. observations                                                                      Incubator ID.:

Negative control (A)(Diluent) Positive product control (B)(Diluent + Product + SCS)
Observation from the dilution bottle
Time intervals of incubation of plates (hrs.) Plate-1 Plate-2 Avg. cfu/ml Dilution Plate-1 Plate-2 Dilution Factor Avg. cfu/g
24
48
72
96
120

 

Test sample (C)(Diluent + Product) Positive control (D)(Diluent + SCS) Observed bySign & Date
Dilution Plate-1 Plate-2 Dilution Factor Avg. cfu/g Dilution Plate-1 Plate-2 Avg. cfu/ml

# Enumerated number of Cells in Standardized Cell suspension (SCS) __________ cells/ml inoculated to obtain final inoculum of not more than 100 cfu/ml.

Percent recovery = (B-C)/D x 100 (at 72 h) (Acceptance criteria: Not less than 70% of that recovered from the inoculum control)

Remarks:                                                                                                                              

 Recorded by:                                                                                                            Reviewed by:               

Sign/ Date                                                                                                              Sign/ Date

Annexure 4 : Observation Sheet of Microbial Enumeration Test  for  Fungi For 0 Hr.

Protocol No.: 

Date of initiation of experiment:                                                                    Media lot no.:

Name of microorganism:                                                                                  Product:

Incubation Temperature: 20-25°C                                                                 Batch No.:

For 0 h. observations                                                                              Incubator ID.:

Negative control (A)(Diluent) Positive product control (B)(Diluent + Product + SCS)
Observation from the dilution bottle
Time intervals of incubation of plates (hrs.) Plate-1 Plate-2 Avg. cfu/ml Dilution Plate-1 Plate-2 Dilution Factor Avg. cfu/g
24
48
72
96
120
144
168

 

Test sample (C)(Diluent + Product) Positive control (D)(Diluent + SCS) Observed bySign & Date
Dilution Plate-1 Plate-2 Dilution Factor Avg. cfu/g Dilution Plate-1 Plate-2 Avg. cfu/ml

# Enumerated number of Cells in Standardized Cell suspension (SCS) __________ cells/ml inoculated to obtain final inoculum of not more than 100 cfu/ml.

Percent recovery = (B-C)/D x 100 (at 120 h) (Acceptance criteria: Not less than 70% of that recovered from the inoculum control).

Remarks:                                                                                                                                                          

Recorded by:                                                                              Reviewed by:         

Sign/Date                                                                                 Sign/ Date

Annexure 5 : Observation Sheet of Microbial Enumeration Test for Fungi For 2 Hrs.

Protocol No.: 

Date of initiation of experiment:                                                                    Media lot no.:

Name of microorganism:                                                                                  Product:

Incubation Temperature: 20-25°C                                                                 Batch No.:

For 2 hrs. observations                                                                          Incubator ID.:

Negative control (A)(Diluent) Positive product control (B)(Diluent + Product + SCS)
Observation from the dilution bottle
Time intervals of incubation of plates (hrs.) Plate-1 Plate-2 Avg. cfu/ml Dilution Plate-1 Plate-2 Dilution Factor Avg. cfu/g
24
48
72
96
120
144
168

 

Test sample (C)(Diluent + Product) Positive control (D)(Diluent + SCS) Observed by Sign & Date
Dilution Plate-1 Plate-2 Dilution Factor Avg. cfu/g Dilution Plate-1 Plate-2 Avg. cfu/ml

# Enumerated number of Cells in Standardized Cell suspension (SCS) __________ cells/ml inoculated to obtain final inoculum of not more than 100 cfu/ml.

Percent recovery = (B-C)/D x 100 (at 120 h) (Acceptance criteria: Not less than 70% of that recovered from the inoculum control).

Remarks:                                                                                                                                                          

Recorded by:                                                                              Reviewed by:         

Sign/Date                                                                                   Sign/ Date

Annexure 6 : Observation sheet of Microbial Enumeration test for coli

Date of Initiation of Experiment:                                                                Product:

Name of Microorganism: E.coli                                                                  Batch No.:

Media lot no.:                                                                                                 Incubator ID.:

Time intervals (hrs.) Negative control (A) (Media) Positive product control (B) (Media + Product + SCS) Test sample (C)(Media + Product) Positive control (D)(Media + SCS) Observed By

Sign & Date
Observation from the dilution bottle at 18-24 hrs.
After 18 -24 hrs. of incubation transfer 1 ml of Soyabean casein digest medium to 100 ml of  McConkey broth and  incubate at 42-44°C for 24-48 hrs.
Observation on MacConkey Broth Medium after 24-48 hrs
After 24-48 hrs. of incubation streak on petri plates of MacConkey agar and incubate at 30-35°C for 18-72 hrs..
Observation on MacConkey agar Medium after18-72 hrs.
Transfer suspected colony to tube containing 5 ml of peptone water and incubate at 42-44°C for 18-24 hrs, add 0.5ml of Kovac’s reagent after completion of incubation.
Observation for Indole

After 18-24 hrs
Gram staining

Acceptance Criteria for Growth Characteristics of E. coli:

MacConkey Broth:         Colour change from Orange to yellow or pink.

MacConkey agar:           Brick red mucoid colonies.

Indole:                               Presence of red coloured ring in the upper layer of medium.

Gram Stain:                      Gram -ve rods.

Inference:                                                                                                                                  

 Annexure 7 : Observation Sheet of Microbial Enumeration Test for  Salmonella Abony

Date of Initiation of Experiment:                                                          Product:

Name of Microorganism: Salmonella abony                                      Batch No.:

Media lot no.:                                                                                             Incubator ID.:

Time intervals (hrs.) Negative control (A)(Media) Positive product control (B)(Media + Product + SCS) Test sample (C)(Media + Product) Positive control (D)(Media + SCS) Observed BySign & Date
Observation from the dilution bottle at 18-24 hrs.
After 18-24 hrs. of incubation, transfer 0.1 ml of  Soyabean casein digest broth  to 10 ml of Rappaport Vassiliadis Salmonella enrichment broth and incubate at 30-35° C for 18-24 hrs.
Observation in Broth Medium after18-24 hrs.
After 18-24 hrs. subculture on plate of Xylose, lysine, deoxycholate agar and incubate at 30-35°C for 18-48 hrs.
Observation in Agar medium after 18-48 hrs.
Streak suspected colony on Triple sugar iron agar slant and incubate at 30-35°C for 18-72 hrs
Observation in TSI after 18-72 hrs
Gram Staining

 # Enumerated number of cells in Standardized Cell suspension of Salmonella……………….. cells /ml 

Acceptance Criteria for Growth Characteristics of Salmonella:

Xylose, lysine, deoxycholate agar:   Well developed, red colonies with or without black centers

TSI agar : Colour change red to yellow due to formation of acid.

Gram Stain: Gram –ve rods

Inference:                                                                                                                                                                                

Annexure 8 : Observation sheet of Microbial Enumeration test for  Aeruginosa

Date of Initiation of Experiment:                                                                        Product:

Name of Microorganism: P. aeruginosa                                                            Batch No.:

Media lot no.:                                                                                                          Incubator ID.:

Time intervals (hrs.) Negative control (A) (Media) Positive product control (B) (Media + Product + SCS) Test sample (C) (Media + Product) Positive control (D)(Media + SCS) Observed by Sign & Date
Observation from the dilution bottle at 18-24 hrs.
After 18-24 h. of incubation, subculture on the plate of Cetrimide agar medium and incubate at 30-35°C for 18-72 hrs.
Observation on Cetrimide agar Medium after 18-72 hrs.
After 18-72 hrs. place oxidase test disc on  the suspected colonies observed on cetrimide agar.
Observation in for Oxidase
Gram Staining

# Enumerated number of cells in Standardized Cell suspension of P.aeruginosa……….. cells/ml. 

Acceptance Criteria for Growth Characteristics of P. aeruginosa:

Cetrimide Agar: Greenish colony

Oxidase Test:     Development of Pink colour, change to Purple.

Gram Staining:   Gram-ve rods

Inference:                                                                                                                                                     

Annexure 9 : Observation sheet of Microbial Enumeration test for  S. aureus

Date of Initiation of Experiment:                                                                       Product:

Name of Microorganism: S. aureus                                                                    Batch No.:

Media lot no.:                                                                                                           Incubator ID.:

Time intervals (hrs.) Negative control (A)(Media) Positive product control (B)(Media + Product + SCS) Test sample (C)(Media + Product) Positive control (D)(Media + SCS) Observed by Sign & Date
Observation from the dilution bottle at 18-24 hrs.
After 18 – 24 hrs. of incubation, subculture on the plate of Mannitol Salt Agar medium and incubate at 30-35°C for 18-72 hrs.
Observation on MSA medium after 18-72 hrs
After 18 – 72 hrs add few of the suspect colonies to plasma of rabbit or horse, incubate for 24 hrs.at 37°C
Observation for coagulation from the tubes up to 24 hrs.
Gram Staining

# Enumerated number of cells in Standardized Cell suspension of S.aureus……………….. cells/ml    

 Acceptance Criteria for Growth Characteristics of S.aureus:

Mannitol Salt Agar:        Yellow/White colonies, surrounded by a yellow zone.

Plasma tubes:                   Presence of coagulation.

Gram Staining:                 +ve cocci

Inference:                                                                                                                                                                           

Annexure 10: Inoculum Preparation Record

Name Organism ATCC No.
Date of Preparation Date of Reporting
Media Used Media Lot. No.
Incubation Temperature Incubator ID
From To
Reference: Protocol No.:
Dilution Volume Tested Count /Plate Final Inoculum Population Observed By (Sign / Date)
Plate 1 Plate 2 Average

Remarks:

Annexure 11: Summary Sheet

SUMMARY:                                                                                                                                                                                  

Annexure 12: Certificate – MLT METHOD VALIDATION

CERTIFICATE

Microbiological Examination of  Non-Sterile Products

Product: ………………………………………………………………………..

Stands validated with the requirements of Microbiological Examination of Non-Sterile products as per the following detail:

Protocol No.:

The above mentioned test as per the standard test procedure shall be used for routine microbiological testing of the product.

Date of Issuance of Certificate:

Microbiologist-QC Head QC Head QA Head Quality
Signature
Name

 

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pharmabeginers

Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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