Title: Aseptic Technique 

1.0  

PURPOSE

 

1.1   

To establish guidelines on Aseptic techniques to be followed in Microbiology Lab.

2.0   

SCOPE:

 

2.1   

This SOP is applicable for the operations and practices in Microbiology Lab.

 

2.2   

To prevent contamination of test samples and personal protection from hazardous microorganisms handled in Microbiology laboratory.

3.0   

RESPONSIBILITIES

 

3.1   

It is the responsibility of authorized QC Officer for the implementation of SOP

 

3.2   

It is the responsibility of HOD,QC  to monitor  the proper  implementation of SOP

4.0   

MATERIAL AND EQUIPMENT

 

4.1   

70% Isopropyl Alcohol  or 70% Ethanol

 

4.2   

Disinfectants 

 

4.3   

Autoclave

 

4.4   

Bunsen Burner

 

4.5   

Nichrome loop

 

4.6   

Laminar Air Flow/ Microbiological Safety Cabinet

 

4.7   

Hot Air Oven

 

4.8   

Sterile Surgical gloves

 

4.9   

Personal Protective equipment such as head cap, mask, surgical gloves,  goggles and aprons etc.

5.0   

PROCEDURE

 

5.1   

Aseptic techniques shall comprise of, but no limited, to the following

 

 

5.1.1         

The Microbiology Lab shall be cleaned and sanitized as per the SOP “Cleaning of Microbiology Laboratory”.

 

 

5.1.2         

Enter the Microbiology laboratory following the SOP “Entry/Exit Procedure for Microbiology Laboratory”.

 

 

5.1.3         

Sanitize hands and gloves with 70% IPA before carrying out any microbiological operation to prevent contamination and for safety of self.

 

 

5.1.4         

Do not touch the samples and/or glassware, sterilized solution with bare hand to prevent contamination.

 

 

5.1.5         

Monitor carefully the temperature of sterilization as specified in the corresponding SOPs.

 

 

5.1.6         

Perform the following operations under Laminar Air Flow (LAF) / Microbiological Safety Cabinet 

 

 

 

5.1.6.1   

Opening of test samples.

 

 

 

5.1.6.2   

Holding and opening the sterilized glassware, petri-dishes, pipettes, 

Tip box, Nichrome wire loops, etc.   

 

 

 

5.1.6.3   

All containers used for sterilized test media

 

 

 

5.1.6.4   

Test tubes containing cultures, subcultures, and diluted cultures.

 

 

 

5.1.6.5   

Sample Testing Procedure

 

 

5.1.7         

Following practices should be followed while using LAF/ Microbiological Safety Cabinet

 

 

 

5.1.7.1   

After switching on LAF/ Microbiological Safety Cabinet, the internal surfaces shall be sanitized using  70% IPA.

 

 

 

5.1.7.2   

The UV light should be switched ON for 30 minutes before using LAF/Microbiological Safety Cabinet for performing tests.

 

 

 

5.1.7.3   

Ensure that the pressure differential of LAF/ Microbiological Safety Cabinet is working properly before performing the tests.

 

 

 

5.1.7.4   

Do not disturb the flow of LAF/ Microbiological Safety Cabinet by placing large objects in the pathway of the Machine or by fast movements of objects or hands that create turbulence thereby increasing the chances of contamination in the test samples and or media/cultures etc.

 

 

 

5.1.7.5   

Do not expose sterilized items to external environment that could contaminate them.

 

 

 

5.1.7.6   

Periodically, sanitize gloved hands with 70% IPA during testing and every time if  touching  the table top of the LAF/ Microbiological Safety Cabinet or any object/surface of suspect microbial status.

 

 

 

5.1.7.7   

The test samples shall be opened under LAF/ Microbiological Safety Cabinet only.

 

 

 

5.1.7.8   

If there is any deficiency that compromises the basic principles of aseptic techniques, stop the work; rectify the deficiency and proceed.

 

 

 

5.1.7.9   

The internal surfaces of LAF/ Microbiological Safety Cabinet must be frequently sanitized using 70 % IPA and all time after removal of spills if any.

 

 

5.1.8         

The temperature and pressure of Autoclave must be maintained at minimum of 121°C and 15 psi for 15 minutes during sterilization cycle.

 

 

5.1.9         

The temperature of Dry Heat Sterilization (Hot Air Oven) must be maintained at minimum of 160°C for 2 hours or 180°C for 30 minutes during sterilization cycle.

 

 

5.1.10     

Adequate care must be taken to prevent contamination of sterilized objects and media by transferring them through pass box and moved further to LAF/ Microbiological Safety Cabinet in working condition as soon as possible.

 

 

5.1.11     

Tests using Plate methods:

 

 

 

5.1.11.1                        

Glass Petri plates of 90 mm or 100 mm diameter, autoclaved at 121 to 124 Degree centigrade for 15 minutes or Presterilized  petriplates shall be  used for testing purpose 

 

 

 

5.1.11.2                        

Autoclave should be run at 121 to 124 Degree centigrade for 20 minutes for discarding purpose.

 

 

 

5.1.11.3                        

20 to 25 ml. of previously sterilized and cooled up to 45 °C (approximately) media are poured in the sterilized plates under LAF / Microbiological Safety Cabinet using aseptic techniques. 

 

 

 

5.1.11.4                        

Pour Plate Method:

  • In this method, sample to be tested is aseptically added to the previously sterilized petri plate under LAF/ Microbiological Safety Cabinet and swirled along with previously sterilized and cooled up to 45 °C media.  
  • These plates are allowed to solidify under LAF/ Microbiological Safety Cabinet.
  • They are closed with lid and incubated in the corresponding incubators.

 

 

 

5.1.11.5                        

Spread Plate Method:

  • In this method, test sample (e.g. culture dilution) is transferred on the pre-incubated plate containing media under LAF/ Microbiological Safety Cabinet.
  • This sample is spread well on the surface of media by using previously dipped in 70% IPA and held on burner flame and cooled glass spreader.
  • They are closed with lid and incubated in the corresponding incubators.

 

 

 

5.1.11.6                        

Streak Plate Method:

 

  • In this method Nichrome wire loop previously sterilized by dipping in 70% IPA and held on burner flame is used.
  • This is dipped in the microbial culture and it is streaked once horizontally and once vertically on the media surface in the petri plate as shown below:

 

  

 

 

 

 

 

 

5.1.12     

Filtration Technique:

 

 

 

5.1.12.1                        

This technique is used for aseptic filtration of the test samples; for example, filtration of Purified Water for testing microbial quality, Cleaning Validation, Hold time study samples.

 

 

 

5.1.12.2                        

For this purpose, SS filtration assembly is autoclaved and cooled to room temperature under LAF/ Microbiological Safety Cabinet.

 

 

 

5.1.12.3                        

A sterile 47-mm Diameter 0.45 ยต membrane filter is aseptically placed on the holder of the filtration assembly under LAF/ Microbiological Safety Cabinet. 

 

 

 

5.1.12.4                        

The assembly is attached to the pre sterilized plastic cup and locked.

 

 

 

5.1.12.5                        

To this assemble, which is now ready for filtration, test sample (e.g. water) is added in the cup and vacuum is applied to filter the test sample through sterile 0.45 ยต membrane filter.

 

 

 

5.1.12.6                        

Next, the filtration assembly is opened and the membrane filter with Microorganism captured from the test sample on the top of the filter is removed aseptically by previously sterilized forcep (using 70 % IPA and flame) under LAF/ Microbiological Safety Cabinet and placed on corresponding media for testing microbial quality of the test sample.

6.0   

REFERENCE:

 

6.1   

IP 2018

 

6.2   

USP-NF (Chapter-1116) Microbiological Control and Monitoring of Aseptic Processing Environments.

 

 

END OF DOCUMENT

Converted to HTML with WordToHTML.net | Document Converter for Windows

Comments

Post a Comment

Popular posts from this blog

Microbial Mapping of Personnel in Class D Areas: A Risk-Based Approach to Glove Monitoring

Image
  Microbial Mapping of Personnel in Class D Areas: A Risk-Based Approach to Glove Monitoring  The “finger-dab test” is a critical, routine environmental assessment tool utilized in the Class D Manufacturing Environment (typically GMP Grade D or ISO-8), which assists in gauging the efficacy of personnel hygiene and cleaning methodologies. While there are no defined maximums by WHO TRS No. 961 for finger-dabs collected from Class D Manufacturing Facilities, most in-house action limits are established at 100 CFU (Colony Forming Units) from 5 fingerprints collected per operator. 1.        Objectives: To determine the risk of an increase in microbial load within finished products through manufacturing personnel during the formulation process. 2.        Study Design: a. Design and Implementation: 1-day study completed during processing time in Class D Areas. b. Media used: Soybean casein digest agar c. Incubation temperat...
Read more

Urinary tract infection

Image
  Urinary tract infection    Introduction A urinary tract infection (UTI) is an infection in any part of the urinary system, which   includes the kidneys, ureters, bladder, and urethra . Most infections involve the lower urinary tract— specifically the bladder (cystitis) and the urethra (urethritis). UTIs are common, especially in women, and are typically caused by bacteria like E. coli entering the urethra.     Causative Agents The primary causative agent for Urinary tract infection (UTIs) is bacteria, most commanly Escherichia coli (E.coli) ,and other bacteria  like Klebsella pneumonae, proteus miralibis and Enterococcus. Common fungi involved in UTI Include Candida albicans. Organism involved in UTIs  generally originates from the gut and enter the urinary tract .       Escherichia coli (E.coli) is the most common which originates from gut and accounts for 75-95% of cases world wide.   ...
Read more

Water Activity: A Faster Alternative to Traditional Testing of Non-Sterile Products in the Pharmaceutical Industry

Image
  Water Activity: A Faster Alternative to Traditional Testing of Non-Sterile Products in the Pharmaceutical Industry T he microbial limit test (also known as the traditional testing method) is a time-consuming test adopted by most of the pharmaceutical industry for the release of non-sterile commercial products to market after completing their manufacturing processes. The test is generally done as per USP <61> (Microbial Enumeration Tests) for total aerobic microbial count (TAMC) and total yeast/mold count (TYMC). USP <62> (Tests for Specified Microorganisms) for the presence or absence of specific objectionable pathogens. Total time consumed to complete the test is between 5 and 7 days. The cost of the test is high because of consumption time, manpower, culture media, and the utilization of different equipment for a single test. Water activity (aw) is the ratio of vapor pressure of H₂O in product (P) to vapor pressure of pure H₂O (Po) at the same temperature. It is num...
Read more