GOOD MICROBIOLOGY LABORATORY PRACTICES

GOOD MICROBIOLOGY LABORATORY PRACTICES

To provide general guidance on Good laboratory practices in microbiology laboratory consisting of activities that depend on several principles, aseptic techniques, control of media, control of test strains, and control of equipment’s, personnel hygiene, health and garments.

SCOPE GOOD MICROBIOLOGY LABORATORY PRACTICES:

This SOP is applicable for Microbiology lab.

RESPONSIBILITY:

Concerned QC Microbiologist personnel shall be responsible to follow the laid down procedure and concerned head Microbiology / head quality control shall be responsible to ensure the compliance of laid down procedure.

DEFINITIONS:

Clean area:

A work place in which the air quality, temperature and Humidity (If required) are highly regulated in order to protect sensitive products from contamination.

Clean areas are required to reduce possibility of foreign particles entering in the product and ultimately in the patient.

PROCEDURE GOOD MICROBIOLOGY LABORATORY PRACTICES :

General Guidelines:

Health:

General health condition of personnel involved in microbiology laboratory must be Satisfactory.

Personnel with open wounds, any skin ailment or any respiratory tract infection shall not be permitted to work in clean zones.

Periodic health checks for the aforementioned conditions shall be carried out.

Personnel Hygiene:

Personnel involved in microbiology laboratory working shall follow good hygienic practices such as daily bath, regular trimming of nails and properly washed hairs.

Before entering in microbiology lab personnel shall wear clean laboratory coat. Hair shall be covered with cap, shall change footwear or wear shoe covers over street shoes and sanitize the hands.

Respective Procedure on personnel hygiene shall be followed while working in microbiology laboratory.

Activity of Personnel:

Number of persons working in microbiology laboratory shall be kept minimum up to possible extent i.e. microbiology lab shall not be over crowded. Movement in clean room should be slow and rhythmic.

Do not contact directly to products. Avoid direct contact with product contact Surface.

After initial gowning, gloves should be sanitized frequently.

In general, unnecessary personnel movement in microbiology lab shall be restricted to avoid accidental contamination.

Personnel involved in maintenance work shall also follow same precautions and hygiene standards as laboratory personnel.

Respective procedure of personnel hygiene shall be followed while working in microbiology laboratory.

Personal movement should be controlled between the clean and unclean area of the lab.

Personnel Garments:

Personnel of Microbiological testing areas should change into special garments, which are non-shedding, comfortable to wear and loose fitting.

Garments shall not have external pockets or unnecessary tucks. Edges of the garments shall be properly sealed and seams shall be all enveloping.

In case garments need to be sterilized, sterilize them in following manner.

Fold the garment neatly and put it in a bag of non- shedding material.

Garments shall be packed in such a manner, such that, surface which is being exposed in aseptic area remains untouched.

Pack head gear remains at top followed by boiler suit and lastly booties.

Pack the gloves in Rexam bag or bag of non- shedding material and label with date of sterilization and use before date. Alternatively, pre sterilized gloves can be used.

Wrap the non-shedding duster in Rexam bag and label with date of sterilization and use before date.

Sterilize the garments and non-shedding dusters in a validated autoclave load.

Sterilization of any object which are further required in testing area, shall be sterilized as per respective procedure and detail. Sterilized garments packets/articles shall be kept at designated place in microbiology lab.

The clean sterilized articles should not be used for more than validated time period.

In case non-sterile garments are to be used, after washing the garments shall be packed in polybag and shall be opened at the time of use.

For the entry in microbiology laboratory shall be followed as per respective procedure.

Maintain proper gown control. An adequate barrier should be created by the overlapping of gown components (e.g. gloves overlapping sleeves). If an element of a gown or gloves is found to be torn or defective, it should be changed immediately.

Good Media Preparation Practices:

Culture media are the basis for most of the microbiological tests. Safe guarding the quality of these media are therefore critical to the success of microbiology laboratory, if ensures proper media preparation, storage and quality control testing shall assure consistency of high quality media.

All dehydrated media/ready to use media container should be labelled properly with as per current version SOP.

Ensure during labelling no any vendor information should be hide by the label, it must be clearly visible.

While preparation of culture media, it is important to follow the manufactures instruction on media preparation, since different types of media may have different preparation requirements (e.g. heating, additives and pH adjustment).

Calibrated weighing balance having appropriate operating range, shall be used for weighing measurement of media.

Clean weighing containers and sampling tools (such as spatulas) shall be used while weighing to prevent foreign substances that may alter the composition of the finished media from entering the formulation. The weight of the components should be recorded.

Media box weight vary lot to lot or vendor to vendor, the entire weight of box with media consider for the reconciliation and reconcile the media up to 500 gm, remaining media if available more than 500 gm shall be discarded with box. For e.g. 500 gm media pack container weight is 625 gm (filled box) reconciliation shall start with 625 gm and remaining weight at last 125 g will be the empty box weight or some quantity of media may be also shall be discarded with box.

Equipment used in the preparation of media shall have appropriate facility to allow for controlled heating, constant agitation, and mixing of the media as applicable.

While addition of supplements to media, adequate mixing shall be carried out to assure proper mixing of supplements.

Preparation of media shall be carried out in cleaned glassware, since poorly cleaned glass may allow inhibitory substances to enter the media.

Be sure that the cleaning process removes debris and foreign matter, and that the detergent is thoroughly rinsed out with Water.

Autoclave, cycle should be validated to ensure proper heat distribution for selected loads and volumes and validated load patterns shall be executed during routine testing (autoclave cycle).

The pH of media shall be within the range of value indicated by the manufacturer, unless otherwise, a wider range is acceptable under the validated method.

Ready to use media container/ In house prepared plates and tubes of media shall be appropriately inspected before usage every time for but is not limited to, i.e. Cracked containers or lids, Unequal filling of containers, Dehydration resulting in cracks or dimpled surfaces on solid medium, Excessive darkening or colour change, Crystal formation from possible freezing, Excessive number of bubbles, Microbial contamination, Lot number and expiry date checked and recorded, Sterility of the media , cleanliness of plates (Lid should not stick to dish).

Standard procedure shall be followed while media preparation.

In case transfer the stock of media from one media stock record to another media stock record an appropriate remark for the stock transfer should be mention in both log book.

In case receiving of large quantity of the media (ready to use media Plates), further usage and reconciliation shall be perform in small quantity. For e.g. in case SCDA media plates received quantity is 200 box (50 plates in each box) total plates are 10,000 in number. So reconciliation of the media plates shall be started with 1000 no.

While maintenance of the media stock, verify the media expiry every time before use and segregate the near expiry material for easy identification and discard after expiry as per vendor instruction.

In case of provisional release of any media stock (prepared media) against the predefined parameter of pre-incubation and GPT, a clear remark should mention with testing detail in media record.

Good Media Storage Practices:

Protect stored media from exposure to light and excessive temperature.

Before prolonged storage, agar plates should be placed into a sealed package or container to retard moisture loss.

Agar media do not store at or below zero degree centigrade as freezing could damage the gel structure.

Good Microbiological Culture Maintenance Practices:

Microbiologist during receiving of the culture ensure the culture detail i.e. Name, ATCC No., Passage, with COA/COQ or other traceable document.

Analyst check the integrity of the culture container and in case of any breakage, damage inform to department head and notify to the vendor also.

After receiving of the culture, immediate store the culture as per procedure and vendor recommendation.

Person follow the all safety precaution including the applicable entry /exit procedure and gowning practices while handling the culture in culture area.

For control the contamination, better to restrict the usage of the culture area gown in to the other testing areas.

Standardizing the handling and storage of cultures by the user laboratory should be done in a way that will minimize the opportunity for contamination or alteration of growth characteristics.

The careful and consistent treatment of stock cultures is critically important to the consistency of microbiological test results.

Each batch / lot of culture confirm for purity, viability, identity prior to start the use in quality control testing.

Ready-to-use cultures i.e. Bio ball or any other also require to confirm for purity, identity, and inoculum size.

The number of transfers of working control cultures should be tracked to prevent excessive sub culturing that increases the risk of phenotypic alteration.

Any form of sub culturing shall be considered as transfer/passage.

COA of received culture shall be maintained by the person involved in the culture related activity for the reference.

In case of any spillage during the culture handling shall cover with routine disinfectant /70 % IPA immediate and clean after transfer activity.

During activity the working area must be sanitized with the 70 % IPA frequently to avoid any cross contamination.

Biosafety cabinet door should be adjust to close in such a way, analyst can work through hand only, no other body part expose to the working area.

After culture activity used accessories (Ready to use article) put in to the routine disinfectant solution immediate and destruct by decontamination autoclave, and after decontamination send to EHS for further process.

Method of colony isolation:

The streak plate method shall be used for obtaining isolated colonies from mixed culture. The streak plate technique is essentially a method to dilute the number of organisms, decreasing the density. This allows for individual colonies to be isolated from other colonies. Each colony is considered “pure,” since theoretically, the colony began with an individual cell. In order to obtain well isolated discrete colonies, the quadrant streak technique should be used.

Procedure for quadrant streaking method:

Remove a small amount of bacterial growth. (Either a loop full from a broth culture or a single colony from a plate or slant) with the help of sterile inoculating loop.

Immediately streak the inoculating loop very gently over a quarter of the plate using a back and forth motion (Area-1 in the figure below).

Take a fresh sterile inoculating loop again and go back to the edge of area 1 that is just streaked, extend the streak into the second quarter of the plate (Area-2).

Take a fresh sterile inoculating loop again and go back to the edge of area 2 that is just streaked, extend the streak into the third quarter of the plate (Area-3).

Take a fresh sterile inoculating loop again and go back to the edge of area 3 that is just streaked, extend the streak into the fourth quarter of the plate (Area-4)

If required double purification of isolate will be done by quadrant method. Small amount of bacterial growth (either a loop full from a broth culture or a single colony from a plate or slant) is removed with the help of sterile inoculating loop and proceed as above procedure.

Good Laboratory Equipment Maintenance Practices:

Equipment used in microbiology laboratory shall be qualified for their installation, operational and performance qualification prior to use.

In case of any abnormality observe during the uses of the Equipment /Instruments, a maintenance request shall be generated with labelled the Equipment / Instruments under maintenance and use after only satisfactory rectification.

Equipment/ Instruments used in micro lab shall be appropriately handled, calibrated as per procedure / frequency as detailed under

respective instrument/ equipment operation & calibration procedure.

Before in and out, maintenance person should ensure to take their tools, nut bolts and other required accessories.

No maintenance work should be done without permission and supervision of operating persons. Any damage during work should be informed to operating person.

Good Laboratory Operations:

Microbiological samples shall be handled in an environment that ensures no contamination.

Areas in which environmental or product samples are handled and incubated should be maintained and free from risk of cross contamination.

Sub culturing, staining, microbial identification, or other investigational operations should be undertaken in the culture handling area.

Sample found to contain growing colonies should not be opened in the clean area of the laboratory.

Careful segregation of contaminated samples and materials should be done to reduce false-positive results.

Personnel engaged in sampling activities should not enter or work in the culture handling area.

Good Microbiological Testing Practices:

Select the correct quality control standard microorganisms, following the manufacturer’s instructions, and perform the testing prior to unknown sample diagnostic testing.

Check the calibration status of instrument / equipment before using for testing.

Ensure the sample detail, storage condition as per test request, and sample container integrity during sample receiving.

Sample shall be store at specified temperature after receiving and record the detail in dedicated inward register.

Analyst shall refer the current specification prior to start the testing activity.

Analyst before start the analysis check the readiness of the area including cleaning, differential pressure, temperature, humidity etc.

In case any parameter is not within range, analyst shall not start any activity in the area.

In case any parameter sudden fluctuate and observe out of range during activity in area, person communicate to lab in charge / engineering for immediate action.

During analysis if in case observe sample condition is not proper, do not perform the analysis and inform to supervisor immediate.

In case of sample spillage occur during the sample analysis shall be clean immediate after completed the analysis.

Above mentioned precautions shall be taken while performing particular test.

Endotoxin Testing:

Verify the details of LAL reagent kit Lot no. expiry as per the vendor COA, and after satisfactory verification label the all material.

Check the compatibility of the lysate with the CSE and qualify the same for further use in analysis.

Ready to use sterile, depyrogenated container use in analysis shall be checked properly and store as per vendor recommendation.

While endotoxin testing in microbiology lab follow respective procedure, however, some of the general precautions to be taken during endotoxin testing are summarized as under, but is not limited to,

Wear hand gloves during endotoxin testing.

pH of testing solution should be in range of 6.0 to 8.0.

Adjust the pH between 6.0 to 8.0 by using endotoxin free Hydrochloric acid or Sodium hydroxide.

The pH of sample can be checked by applying drop of the solution to pH indicator paper with endotoxin free pipettes.

Ensure that there is no air bubbling during pipetting.

Avoid touch to the active surface of apparatus with finger.

Before prepare the dilution or testing, sample should mix proper (vortex mixer can use) to homogenize the Endotoxin molecule throughout the sample for better accuracy.

Ensure the use of only de-pyrogeneted glassware of recommended size.

Volume CSE solution should contain about 2.0 ml volume at each dilution.

Prepare dilution in following order, (i) Take LRW in dilution tubes and then add test solution/CSE solution for serial dilution preparation.

During test, add the solutions in following order. (i) LRW (ii) Test solution (iii) CSE dilution (4X for 50:50 methods and 20A, for Hot spike) (iv) LAL reagent.

Always perform the analysis of water for injection and the product sample separately to avoid the probability of cross contamination.

In case of WFI/PSC sample, do not perform the testing immediately because of high temperature and avoid the possibilities of false result.

Precautions to be taken while sterility testing:

While sterility testing in microbiology lab proceed as per respective procedure, however, some of the general precautions to be taken during sterility testing are summarized as under, but are not limited to:

Persons entering the sterility testing area must be qualified for aseptic gowning procedure.

A list of qualified person for entry in sterility test area shall be prepare and approved by QA.

Disqualified person’s entry should be restricted and their name should not be present in the list of authorised person.

Persons suffering from any illness (e.g. cold, fever, dysentery, etc.) or open wounds or lesions must not enter in the sterility testing area and he should inform the illness to the head of microbiology laboratory.

Only the microbiologists qualified through analyst qualification procedure should perform the sterility test.

Personnel entering the sterility testing area must follow the entry and exit procedure and should change inner and sterilized over garments every time.

Gloves shall be disinfected regularly during the any operation and check their integrity.

All equipment or vessels which come in contact with the sterile test media, or the materials to be tested, in the course of the sterility testing should be appropriately packaged or closed and sterilized prior to use.

All sample container should be immersing in the Sporicidal disinfectant and if cannot immerse sanitize with the help of sterile lint free cloth wetted with the Sporicidal agent followed by 70 % IPA especially at top of the container to avoid the possibilities of contamination during insertion of the needle.

All substances added to the sterile media or introduced into sterile membrane filtration units, other than the preparations under test, should be sterile.

Validation tests involving the inoculation of media with live micro-organisms should be carried out in laminar airflow in culture handling area and further transfer to the sterility area for testing.

Each tube/canister of inoculated media should be clearly identified for the sample being tested (batch no.), batch no of medium, and date of incubation.

If any unforeseen observations e.g., sneezing, coughing, power failure, Other break down etc., noticed during the performance of sterility test, abandon the testing and declare the tests carried out during this incident as invalid. Inform the head of department immediately about the incident and record the incidence in the report as per the respective procedure through Handling of laboratory incidents.

Take appropriate action like cleaning, fogging, disinfection, re-sampling before resuming the test.

Precautions to be taken during Water Sampling:

While water sampling proceeds as per respective procedure, however, some of the general precautions to be taken during water sampling are summarized as under, but is not limited to,

Do not sample water, if solvent fumes are present in area.

Do not perform sampling, if washing activity is going on in the area.

Do not contaminate inner surface of stopper or cap and neck of bottle.

Do not touch the edge of the sample container.

Check the condition of water sampling point before sampling and shall not perform sampling if condition is not good like inappropriate cleaning, rusting etc. further perform the sampling after appropriate action.

Precautions to be taken during Microbial Limit Test:

While Microbial Limit testing proceed as delineate under GTP titled as “Microbial enumeration test and test for specified microorganisms” shall be followed, however, some of the general precautions to be taken during microbial limit test are summarized as under, but is not limited to, Media pouring shall not be done at high temperature. (Preferably at 45°C unless otherwise specified).

Samples shall be opened only under LAF and never expose to UV light either during the transfer through the Pass box or prior to start the testing in LAF/BSC.

During testing care should be taken not to break the laminarity of air.

In case of Membrane filtration method, ensure no bubbles or air space remain back side of filter paper during put the filter on agar media.

During testing spirit lamp shall be handled carefully to avoid burning and spillage of spirit on LAF bench if required.

If spillage of sample occurs during test, repeat the analysis with fresh sample.

Precautions to be taken during Aseptic area activities:

While working in aseptic area proceed as per respective procedure.

Personnel Hygiene and behavior in critical area & working under LAF shall be followed, however, some of the general precautions to be taken during aseptic area activities are summarized as under, but is not limited to.

Walk slowly during transportation of sterile materials.

Gloves shall be disinfected prior to perform any operation in aseptic area and at every intervals of approx. 15 minutes and check the glove integrity.

Movement in clean room should be slow.

Sitting or leaning on surface and equipment shall be avoided.

Writing on equipment’s, hands and garments shall not be done.

Precautions to be taken during Sampling of sterile API/RM:

While Sampling of sterile API/RM proceed as respective procedure however, some of the general precautions to be taken during Sampling of sterile API/RM are summarized as under, but is not limited to,

During Sampling of sterile API / RM person should avoid contact of hands to any of the surface. After touching the hands to any surface, disinfect the gloves again with the hand disinfectant provided in the aseptic processing area.

Gloves shall be disinfected after about every approx. 10 minutes, and check the glove integrity.

For sampling disinfectant gloves and allow drying 1-2 minutes before touching the sampling container.

Remove the rubber stopper and keep it inverted so that the inner surface of the rubber stopper does not touch any surface.

Always hold the sampling bowl from sides and not from the top, to protect the rim of sampling bowl so that it does not get contaminated.

During sterile API / RM sampling activities avoid movement or hands bending over the open sterile materials and take precautions that handling of sterile vials and stoppers are under laminar airflow.

Training of Personnel:

Personnel working in microbiology laboratory should have education, training and experience to perform his activity.

Training curricula should be established for each member specific for his job function.

Analyst shall be qualified prior to conduct any microbial test. Training records should be current, documenting the microbiologist’s training in the proper revision to the particular SOP.

Performance testing should provide evidence of competency in core activities if Microbiology laboratory such as hygiene, plating, ascetic

technique, documentation and other as suggested by the microbiologist’s job functions.

Also, read – VALIDATION OF ASEPTIC PROCESSING AS PER USFDA GUIDELINE

Good Documentation Practices:

Always Follow ALCOA principle when generate documents.

Attributable: All data generated or collected must be attributable to the person generating the document. This should include who performed an action and when. This can be recorded manually by initialing and dating a paper record or by audit trail in an electronic system.

Legible: All data recorded must be legible (readable) and permanent. Ensuring records are readable and permanent assists with its accessibility throughout the data lifecycle. This includes the storage of human-readable metadata that may be recorded to support an electronic record.

Contemporaneous: Contemporaneous means to record the result, measurement or data at the time the work is performed. Date and time stamps should flow in order of execution for the data to be credible. Data should never be back dated.

Original: Original data, sometimes referred to as source data or primary data, is the medium in which the data point is recorded for the first time.

This could be a database, an approved protocol or form, or a dedicated notebook. It is important to understand where your original data will be generated so that its content and meaning are preserved.

Accurate: For data and records to be accurate, they should be free from errors, complete, truthful and reflective of the observation. Editing should not be performed without documenting and annotating the amendments.

Following documentation shall be ensured and maintained in microbiology Laboratory but not limited to.

  • Microbiologist training and verification of proficiency.
  • Equipment validation. Calibration, and maintenance.
  • Equipment performance during test (e.g., 24-hour-n-day chart recorders).
  • Media preparation, sterility checks, and growth-promotion and selectivity capabilities, Media inventory and control testing.
  • Critical components of test conducted as specified by a procedure Data and calculations verified.
  • Reports reviewed by QA or a qualified responsible manager.
  • Investigation of data deviations.

Record Maintenance:

Every critical piece of equipment should be noted in the write-up, and all should be on a calibration schedule documented by SOP and maintenance records.

Where appropriate, logbooks or forms should be available and supportive of the laboratory notebook records. Equipment temperatures should be recorded and traceable.

Changes in the data should be crossed off with a single line and initialled.

Original data should not be erased or covered over.

General Care to be taken while working in Microbiology Laboratory:

Personnel should not lean into the laminar airflow bench or place his/her hands on the bench.

No articles / glassware should be placed on top of the laminar airflow bench.

Sterile disposable plastic petri plates should be handled with care. Petri plates should be opened only in the laminar air flow bench.

Nervous relief type mannerisms such as head scratching or rubbing hands face or parts of body must be consciously avoided.

Do not use any accessories if fell down on the floor during testing same shall be discard, and further used after proper sterilization or sanitation.

Sitting or leaning on equipment or work surface shall be avoided.

Avoid standing with help of doors or wall or equipment.

Writing on hands, garments or equipment is strictly prohibited.

Paper in any form, except one designed for clean rooms (autoclaved) or covered with plastic lamination, is not allowed in clean facility.

Always open (By Push or Pull) the doors with the help of handle provided on Door and not by pushing on glass or door surface.

After completion of operation. Material handling items to be clean, dry and kept to its dedicated place with status tag.

Do not keep any material in front of return duct that may disturb differential pressure of area.

Good observation practices:

Precautions should be taken while taking out the observations of plates from incubator.

Test sample plates/ tubes always shall be released based on the following.

If in case incubation period mentioned in the “hours” sample shall be released after completed the given period. For e.g. If incubation period mentioned for a sample as 24 hrs. The sample must be release after completed 24 hrs.

In case incubation period mentioned in the “days” started in the morning or afternoon should generally be concluded at that same time of day. For e.g. If incubation period mentioned for a sample as 3 days and samples incubated at the morning time of the day further same shall be release at same time after completion of the 3 days’ incubation.

Plate’s lid should not open while taking observations.

Always use colony counter and magnifying glass for taking observations as applicable.

Do not speak unnecessarily while taking observations.

Colonies should be clearly marked at the back of plate with marker pen.

Plates shall be checked from both sides at the time of observations to ensure detection of embedded colony (if any).

Plates shall be checked from sides by rotating the plate in circular motion to ensure that peripheral colony, if any between plate surface and media is counted.

In case if media is coloured e.g. Dey-Engley agar, observe the plate after incubation against white light to detect embedded colony.

For the clarification of any doubtful result related to presence or absence of colony any further action should be take based on the justification.

Plates/Sterility canisters after completion of incubation period shall be properly discarded in their respective bins/ Autoclaveble discard polybags made for packing of hazardous materials & disposed as per respective procedure.

Keep the Colony counter background clean. If found unclean, ensure that it is cleaned so that dust particles do not interfere observation.

For spreader colonies combine the spreader count and the colony count to compute the total colony count, following procedure for “COUNTING OF SPREADER” shall be followed, but is not limited to.

Spreading colonies are usually of various distinct types:

Chain of colonies, not too distinctly separated, that appears to be caused by disintegration of a bacterial clump.

One that develops in film of water between agar and bottom of dish.

One that forms in film of water at edge or on surface of agar. If plates prepared from sample have excessive spreader growth so that Area covered by spreaders, including total area of repressed growth, exceeds 50% of plate area.

Do not count each individual growth in such chains as a separate colony.

After observation and reporting the result discard the plates in the Autoclavable disposal polybag and transfer to the decontamination area for further destruction.

ABBREVIATIONS:

TDS – Test Data Sheet

ATCC – American Type Culture Collection

NCTC – National Collection of Type Cultures

NCIB – National Collection of Industrial Bacteria

COA – Certificate of Analysis

COQ – Certificate of Quality.

CSE – Control standard endotoxin

REFERENCES:

About Pharmaguidanaces Channel

Ms. Abha Maurya is the Author and founder of pharmaceutical guidance, he is a pharmaceutical Professional from India having more than 18 years of rich experience in pharmaceutical field. During his career, he work in quality assurance department with multinational company’s i.e Zydus Cadila Ltd, Unichem Laboratories Ltd, Indoco remedies Ltd, Panacea Biotec Ltd, Nectar life Science Ltd. During his experience, he face may regulatory Audit i.e. USFDA, MHRA, ANVISA, MCC, TGA, EU –GMP, WHO –Geneva, ISO 9001-2008 and many ROW Regularities Audit i.e.Uganda,Kenya, Tanzania, Zimbabwe. He is currently leading a regulatory pharmaceutical company as a head Quality. You can join him by Email, Facebook, Google+, Twitter and YouTube

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