Microbiological best laboratory practices

Personnel’s

Education

Each person engaged in all phases of microbiology activity must have the basic educational background or experience and training to his or her job.

Training

Training curricular shall be established for his or her job function. They shall not be independently performing a microbial test unless and until they are qualified for the test. Training records shall be in line with the current version of SOP training.

Health and hygiene

Personal with open wounds, infectious diseases shall not be permitted in to the microbial Laboratory. Hygiene requirements such as trimmed hair / nails, general cleanliness shall be maintained by all the personnel working in Microbiological laboratory. Any person suffering from illness, infectious diseases and / or with open wounds must inform the concerned supervisor. Such personnel shall either be sent for treatment and / or shall be kept away from the analysis.

Wear clean laboratory uniform before entering in the laboratory. Follow procedure mentioned in relevant specific SOP for entry and exit procedure for microbiology department.

Keep hair covered (facial and scale) with a cap / mask while working in the microbiology testing area.

Microbiology Laboratory testing garment shall not be particle shedding, have no unnecessary tucks and edges of the garments are sealed and seams are enveloping.

The numbers of persons working in clean zones area are not more than the defined number.

Materials prone to be shedding the particles shall not be used in the laboratory. Such materials shall be handled with additional care.

Personnel shall not lean into the laminar air flow bench or place his /her hands on the bench. It shall be ensured that the defined working zones within the LAF are maintained and followed.

Only authorized personnel shall enter in the microbiology testing area. Unauthorized personnel / visitors shall be accompanied by concerned supervisor of the microbiological laboratory.

Maintenance staff and cleaning staff shall follow the same precaution of hygiene standards as followed by laboratory personnel.

Premises.

Cleaning and disinfection in microbiology lab

Maintain the laboratory and its premises clean.

Note: Approved laboratory layout should be available and displayed at the entrance.

Clean the microbiology lab and microbiology testing area with disinfectant as per procedure and Ensure all equipments and instruments are clean.

The laboratory Cleaning Status and Record shall be maintained.

Monitoring of microbiology lab

Ensure that the walls and floor of clean areas are devoid of cracks and crevices.

Daily monitor the microbiology lab for temperature, routine checks like pressure differentials, manometer reading of LAF and incubator temperature as mentioned in current version SOPs.

Perform Microbiological Monitoring as frequency mentioned in current version SOP of Monitoring of microbiology laboratory.

Material Storage

Materials such as glassware’s, Media Containers, Buffers, Reagents, Petri-plates and other consumable items should be properly identified with labels and shall be kept in dedicated area and defined storage condition.

Prepared Media, Sterile Material and accessories shall be properly identified with labels and kept in dedicated area.

Laboratory Practices

General Behavior:

Before entering in the Microbiology Laboratory, personnel shall understand the responsibilities of their position and know aseptic techniques.

The cosmetics, nail paint and jewellary are not allowed in microbiology laboratory.

Traffic into and within the laboratory should be minimum. Personnel should not walk around the clean room unnecessarily.

Eating, Drinking and chewing is not allowed in the microbiology laboratory. 

Movement in the laboratory should be slow and rhythmic.

Head snatching, rubbing hands, face or parts of the body or any other unnecessary movement of body must be consciously avoided.

Avoid loud, unnecessary talk, laughing, whistling, singing and shouting in the microbiology laboratory area as it increases the number of bacteria emitted from the mouth.

Any verbal communication with people outside the laboratory should be through telephone only.

All the doors of respective areas of laboratory should be kept in close condition.

At the end of the day close down the microbiology laboratory ensuring that required instruments / equipments are put off to the extent possible.

Aseptic Behavior in microbiology laboratory:

All aseptic practices should be done under laminar air flow, bio-safety cabinet and isolator.

During aseptic work, at periodic intervals, gloved hands shall be sanitized with approved sanitization solution.

LAF’s shall be kept “ON” continuously during working hours.

LAF shall be turn ON at least 30 minutes before, prior to start of microbiological analysis.

Disinfectant the working surface of LAF and biosafety cabinet after analysis.

Do not place the articles and glassware on top of the laminar air flow bench and Incubators.

Use pass box for transfer of any material from microbiology testing area.

Handle sterile disposable Petri plates with precautions.

All unwanted/waste materials must be properly removed from the concerned area at the end of each work day.

Calibration ,Validation, Qualification and Preventive Maintenance:

Ensure that equipments and instruments are always having calibration, validation or qualification status.

All Instruments/ equipment used in a microbiology laboratory should be calibrated, validated or qualified on a regular defined schedule and tested to verify performance on a routine basis as mentioned in relevant procedures.

New equipments, critical to the operation of the laboratory, shall be qualified according to a protocol approved by the head quality assurance.

Perform preventive maintenance of the instruments as per defined schedule.

Microbiological Culture Media and Microbial Culture  Handling:

Handle microbiological culture Media as per SOP for Procurement, Receipt, Preparation, Storage, Growth promotion, Usage and Disposal of Microbiological Media

Observe proper safety precautions while handling microbial cultures.

Do not handle live cultures with bare hands.

The number of transfers of working control cultures should be tracked to prevent excessive sub-culturing that increases the risk of phenotypic alteration or mutation. Only NMT (not more than) 5 Passage cultures shall be use for testing.

Appropriate sanitization/ disinfection procedures should be employed after handling the culture.

Handling of Reagent and Chemical:

Label the entire reagent container upon receipt with label and date of receipt.

Observe proper safety precautions while handling the reagent and chemical.

Verify availability MSDS of Reagent and chemicals. Keep MSDS in a dedicated file.

Sample Handling and Analysis:

Received sample through pass box only along with intimation slip.

Routine sampling (Like Water, Environment Monitoring, nitrogen and Compressed Gas) and analysis perform as per schedules.

Collect samples for microbial analysis in sterilized containers. Containers can be sterilized by dry heat sterilization or autoclaving. Alternatively use sterile containers or poly bags for sampling.

Store all samples at designated storage area and designated storage temperatures in the laboratory until their disposal.

Avoid exposure of media to the environment after analysis.

Note: Only one type of analysis to be performed at a time under laminar air flow to avoid cross contamination.

Report all incidents and failures if any, to the supervisor immediately.

Label all the media plates and flasks with the details such as media name (abbreviation) and lot number. Proper Labeling done during Testing i.e. Test Name or stage, Batch No or A.R. No. and done by sign and Date.

Take care during transfer of media plates /flasks/tube/container to or from the incubators after analysis.

Transfer of material shall be done with the help of trolley, tray or appropriate container.

Microbiological media incubation times consideration:

Incubation times for microbiological tests of less than 3 days duration should be expressed in hours: e.g., incubate at 30° to 35° for 18 to 72 hours. Tests longer than 72 hours duration should be expressed in days: e.g., incubate at 30° to 35° for 3 to 5 days.

For incubation times expressed in hours, incubate for the minimum specified time, and exercise good microbiological judgment when exceeding the incubation time. For incubation times expressed in days, incubations started in the morning or afternoon should generally be concluded at that same time of day.

Observation and Interpretation of test results:

Always Wear Gloves during observation of media plates /flasks/tube/container.

Perform the observation of plate either manually or by using colony counter.

Liquid media have turbidity or luxuriant growth shall be considered as Positive Result.

If observation falls on week off or holiday or after working hours, same shall be observed on next working day.

For Plate count observation follow below procedure:

·         When overlapped colony observed counting shall be done as per its defined point of origin. If there is single point of origin consider as single colony. If there is two point of origin consider as two colonies.

·         When two type of pigmentation observed in single colony or have sign of overlapping, counting shall be done as per point of origin.

·         When colony spread on plate having same phenotype consider as single colony.

·         In case of colony on periphery of media plate covering entire plate shall be conspired as single colony since there is no defined point of origin.

·         For 90 mm plate observation: plate having below 250 colonies shall be considered as a countable. Plate having more than 250 colonies shall be considered and reported as Too Numerous To Count (TNTC) in case of bacterial and yeast.

·         For 55 mm contact plate observation: plate having below 100 colonies shall be considered as a countable. Plate having more than 100 colonies shall be considered and reported as Too Numerous To Count (TNTC) in case of bacterial and yeast.

·         In case of mold only countable colony shall be considered.

·         During observation if media plate found missing than deviation shall be initiated and impact assessment & investigation shall be performed.

Handling and of glass ware, Reusable Material and Waste material:

Used glass ware, Reusable Material and accessories shall be transfer to dedicated place for cleaning and washing.

Waste Material shall be collected on dedicated Bin i.e. Hazardous Wastes(Hormonal,Cytotoxic etc), Biohazards Waste and General Waste.

Before Disposal neutralize the Hazardous Wastes.

Biohazard waste shall be decontaminated prior to disposal.

Handling of spillage of microbial cultures and media:

In general, the spillage in the microbiology laboratory can result either from the solid or liquid media with or without cultures.

Solid media includes dehydrated media powder, prepared media plates or slants with or without cultures. Liquid media include prepared media broth in tubes or bottles with or without cultures. Culture suspensions and biological indicators can also result in the spillage.

In case of any spillage* immediately notify the supervisor/ Head Microbiology about the spillage and initiate the cleanup activities under their supervision and Initiate the clean up activity as soon as possible to prevent the contamination of the areas / materials, if any in the proximity.

*Note: Slightly spillage during media Preparation shall be cleaned by 70% IPA or Validated disinfectant with help cotton or Mop.

In case if the spillage has resulted from the media, document the spillage details in the reconciliation record.

Wear the gloves and mask before handling the spillage.

Pour / spray the disinfectant solution on the spilled medium / culture.

Allow the disinfectant solution to act for validated time period.

Wipe the spillage area to remove traces of medium / culture using a sterile lint free mop soaked in disinfectant solution.

If required, use fresh lint free mops moistened with disinfectant. Wipe the areas till all the visible traces are completely removed.

Collect the media / culture traces in a disposable bag and subject for decontamination before disposal.

If glassware is broken (if it contains media / culture), pick up the broken glass ware pieces in appropriate container, soak in the disinfectant solution for validated time. Transfer the glass ware pieces in scrap yard .

In case of spillage of medium / microbial culture on the surface of laminar air flow bench, switch of the blower. If any of the adjacent samples are affected due to the spillage, raise an incident to evaluate the impact and for disposal.

Clean the spillage as per the procedure mentioned above.

In case of spillage of media in sterility testing areas, handle the spillage as per the procedure mentioned above. As a precaution, perform the cleaning of the areas.

In case of spillage of the media during environmental monitoring, follow the procedure detailed in the relevant environmental monitoring procedure.

Decontamination

Sterilize all the used media, cultures and glassware by using an autoclave cycle before disposal. Dispose the waste immediately after analysis. Don’t allow the waste to accumulate in the laboratory.

After autoclaving send the disposal items to incinerator or scrap yard. Ensure the appropriate documents are filled up.

Handling & Disposition for Hazardous and Toxic materials shall be done as per the MSDS or as per the manufacturer recommended procedure.

Documentation Practices

Maintenance of Laboratory Records

The laboratory Record should provided record of all critical details needed to reconstruct the details of the testing and confirm the integrity of the data. At a minimum, the laboratory Record should include the following:

·         Date

·         Material tested

·         Microbiologist's name

·         Procedure number(STP or SOP)

·         Document test results

·         Deviations (if any Capture in Remark)

·         Documented parameters (equipment used microbial stock cultures used,and media lots used)

·         Management/Second review signature.

Ensure that all relevant documents are available to demonstrate that the testing was performed in a laboratory using appropriate Quality Control procedures.

Enter all the analytical records concurrently when analysis is being carried out.

Ensure that all the laboratory documents are securely placed and kept at the designated place.

During documentation follow “Good Documentation Practices”.

Handling of Printouts:

Operator shall sign on the strip chart record and printouts. The reviewer / group leader or designee shall counter sign on the record as a proof of review. If required assign the page number on printouts.

The print outs of balance, pH meter and non viable particle shall be attached with respective report / log books. Fill the required identification details on printout and put overlapping signature with date on printout and respective report / log book.

If printout comes on thermal paper then take a photocopy of print. Attach original print with respective report or along with photocopy file separately with identification.

The reviewer / group leader or designees also do overlapping signature with date on printout and respective report / log book.

Handle the external party documents:

COA and Print out shall be verified by Responsible Person with sign and date.

Qualification Documents, Calibration and Validation certificate shall verified by Quality assurance.

REFERENCE:

Sr. No.	Reference
1.	Best Microbiological Laboratory practices	USP<1117>
2.	WHO good practices for pharmaceutical microbiology laboratories	Technical Report Series, No. 961, Annex 2
3.	In-house	NA

ABBREVIATIONS:

Sr. No.	Abbreviation Used	Full Form of Abbreviation
1.	COA	Certificate of analysis
2.	No.	Number
3.	LAF	Laminar Air Flow
4.	MSDS	Material safety data sheet
5.	NMT	Not more than
6.	SOP	Standard Operating Procedure
7.	STP	Standard Test Procedure
8.	QA	Quality Assurance
9.	QC	Quality Control
10.	°C	Degree Celsius
11.	UV	Ultra violet