Laboratory Information File of
Microbiological Laboratory in Pharmaceutical Industry

1.  Microbial Testing flow diagram in microbiology lab

>The outline of sampling activities, sample storage, media preparation, test activities, Incubation, observation and reporting is represented in flow chart.

 

Sterilization of Sampling Vials, Spatula etc.(Dry heat sterilization,160°C for 2 hours).

  

Preparation of Sampling Kit (Vials, Gloves, Mask, Spatula) and transport to sampling area

 

Sampling of sample under RLAF (Sampling Booth) in sterile vials

Fig: Reverse Laminar air Flow(RLAF)

Transport the sample to Microbiology Lab and entry of sample in sample entry Log book

Storage of sample in Refrigerator (2-8ºC)

Media Preparation and Autoclaving in Autoclave

                 Sample preparation and inoculation under Laminar Air Flow (Class A area)

Fig: Laminar Air Flow

Incubation in Incubator (Bacterial Plate:35 ºC (3 days), Fungal Plate: 25 ºC (5-7 days)

                   Fig:Culture media plates after Incubation

 

Observation and colony counting under Colony Counter

Fig: Colony counter

Recording data in analytical work sheet and preparing final report

2. Preparation of Media 

The outline of media preparation activities is represented in flow chart.

 Preparation of Glasswares for media Preparation

 

Calculation of Required media for the test

 

Weighing of media on analytical balance using sterile aluminum foil and spatula

 

Transfer the media in conical flask and add required amount of Purified water as per calculation

Transfer conical flask containing media on water bath (100 ºC) to dissolve completely

 

Autoclave the media in Autoclave (121 ºC, 15 Psi for 15 minutes)

 

Fig:Autoclave

Control of Media

1.Stock of Culture Media shall be recorded in stock log book

2.Daily consumption of media shall recorded in Media Preparation Log Book and Monthly consumption record of each media shall be  recorded in monthly consumption log book.

 

Types of Media used

a. General purpose media

Used for Enumeration of Bacteria and Fungi in different test

a.       Microbial limit Test

b.      Environment Monitoring test

c.       Growth Promotion test etc

Bacteria: Soyabean casein Digest Agar, Nutrient Agar

Fungi: Sabouraud Dextrose agar

 

b. Transport Media /Diluent

a.       Buffered Sodium Chloride Peptone Water

b.      b. Normal saline

 

c. Enrichment Media

a. Mac conkey Broth              b. Rappaport Vassiliadis Enrichment broth

      c. Gram Negative Broth        d. Soyabean casein Digest medium

      e. Nutrient Broth

 

d. Selective Media

a. Mac conkey agar         b. Antibiotic assay Medium No.11

 c. XLD agar                    d. Cetrimide agar

e. Mannitol salt agar        f. Glucose Yeast Extract agar

g. ST agar                        g. Anaerobic agar

 

d. Biochemical Test media

a. SIM Medium               b. TSI agar

c.MR-VP Medium           d. Citrate agar

e. Pseudomonas Fluorescence agar

 

e. Reagent

a. Kovacs reagent                    b. Methyl red reagent  

c. Oxidase Paper                     d. Coagulase Plasma

e. VP reagent                           f. Gram staining

3.Procedure for Positive and Negative control

The outline of Positive and Negative Control Preparation activities is represented in flow chart.

 Preparation of Positive control by preparing culture dilution containing 10 -100 cfu/ml organism by serial dilution and preservation at 2-8 ºC (ATCC Cultures)

 

Use of positive control along with each test containing 10 -100 cfu/ml organism with test media

 

 Use of Negative control along with each test without inoculation of organism with test media

 

Incubation of positive control and Negative Control same as of test condition

 

Interpretation of result obtained in test as Pass or Fail on basis of limit (Quantitive Test) and comparison of organism if growth occurs on test media with positive control(Qualitative Test).

 

Recording result as per the interpretation.

4.Validation Policy

>Validation policy of Different test methods such as Verification of Microbial Limit Test Method, Validation of Microbial assay method, Cleaning Validation studies, Water Validation, preservative efficacy test should be employed in microbiology lab. 

 

5.Waste Disposal

The outline of Waste disposal activities is represented in flow chart.

 

 Collection of discard test samples, expired medias, organism in a container

 

 Autoclave the test samples, expired medias, organism in Autoclave (121 ºC, 15 Psi for 20 minutes)

                                                                       

Dilute the autoclaved discarded media, organism and drain into running tap water

 

 Further processing of Disposed waste in ETP Plant.

 

Fig : ETP Plant

Quality Control

Major focus area should be

•       Use of Calibrated equipments.
•       Use of Growth Promotion test pass media during Test.
•       Qualified reagents, chemicals use
•       Sops for all the process and tests employed in lab.
•      Validation/verification of test methods.
•      Qualified Technician/manpower.
•       Sops for Out of specification (OOS) /Out of trend (OOT) Test results.
•      Qualified Lab.
•      Approved Discard methods for Discarding test samples from lab.
•     Use of Good Documentation Practice (Alcoa/Alcoa⁺).

 

 

Reference: Annexure 2, WHO Technical Report Series No.961, 2011. “WHO good practice for pharmaceutical microbiology laboratories”.