HANDLING OF ISOLATES
This procedure elaborates the systematic approach for the isolation and identification of organism from the routine microbiological testing and monitoring followed by Preparation of cryo vials for isolates.
SCOPE HANDLING OF ISOLATES :
This SOP is applicable for the characterization of the environmental isolates, Water isolates / product isolates in the microbiology Laboratory.
RESPONSIBILITY:
QC Microbiologist personnel shall be responsible to follow the laid down procedure for characterization and isolation of the microbial flora for the identification.
QC Microbiologist shall prepare the cryo vials of culture as per the procedure.
Head Microbiology /head quality control/ his designee shall be responsible to ensure the compliance of laid down procedure.
DEFINITIONS:
Isolates: Microorganisms which are recovered from the environment monitoring, water, and from product is called isolates, to separate (a pure strain) from a mixed bacterial or fungal culture.
Microbial Identification: Identification of recovered organism from various microbiology tests, up to the genus level or species level.
Microbial characterization: The use of colony growth, cellular morphology, differential staining, and key diagnostic features to characterize a laboratory isolate for trending and investigative purposes without identification, e.g. nonpathogenic Staphylococci.
Genus: A taxonomic category ranking below a family and above a species and generally consisting of a group of species exhibiting similar characteristics. In taxonomic nomenclature the genus name is used, either alone or followed by a Latin adjective or epithet, to form the name of a species.
Species: A group of organisms having many characteristics is common and ranking below a genus. Organisms that reproduce sexually and belong to the same species interbreed and produce fertile offspring. Species names are usually written lowercase and in italics, as epidermis in staphylococci epidermidis.
Gram’s Stain: A staining technique used to classify bacteria; bacteria are stained with crystal violet and then treated with Gram’s solution; after being decolorized with alcohol and treated with safranin and washed in water, those that retain the crystal violet are Gram positive and those do not retain are Gram negative.
Spore: A small, usually single-celled reproductive body that is highly resistant to desiccation and heat and is capable of growing into a new organism, produced especially by certain bacteria, fungi, algae, and nonflowering plants. A dormant non-reproductive body formed by certain bacteria in response to adverse environmental conditions.
Colony: A visible growth of microorganisms, usually in a solid or semisolid nutrient medium.
Fungi: The taxonomic kingdom including yeast, molds, smuts, mushrooms, and toadstools; distinct from the green plants.
Pathogen: The microorganism capable to produce disease.
Objectionable Organism: The organism which can cause illness/ injury.
Source: It is the home/ foundation the organism from it got detected.
Representative colonies: Morphologically different colonies on the plates.
PROCEDURE:
SAFETY PRECAUTIONS:
Cultures should always handle under LAF / Biosafety Cabinet in dedicated culture handling area.
Always gown as per respective entry/gowning procedure and disinfect the working area before start the activity and after completed the activity.
After completion of work dip the spreader, loops, Micropipette tips broken glass pieces, forceps, and used ampoule cutter in routine disinfectant solution.
Clean laminar airflow bench / Bio Safety Cabinet bench with scheduled disinfectant solution after completion of work.
EQUIPMENT USED
- Laminar Air Flow bench
- Biosafety cabinet
- Loop
- Incubators
- Colony counter
- Water bath
- Vortex mixture
- Microscope
- Identification system (wherever applicable)
General Instruction for Identification:
Identification of isolates provides the knowledge of flora present in controlled environments, which helps to determine the other usual microbial flora anticipated in the facility and their possible origins.
Isolates identification also helps in evaluating the effectiveness of the cleaning, sanitization procedure and suitability of methods use for recovery.
Isolates related information is also useful in the investigation, to identify the source of contamination followed by root cause.
For the genus level identification, observe the colony morphology on the general purpose /selective media, biochemical test and further observe the cellular morphology under microscope for recording of result.
For the proper characterization of the observed colonies on the media follow the relevant literature and guidance.
Perform the staining of the target organism for microscopic study, Gram staining for the bacterial culture, staining of fungi shall be performed with the Lacto phenol cotton blue for microscopic study.
Procedure of Gram Staining:
Fixation of bacterial cell:
For the preparation of the smear, take a loop full culture on the slide and one drop of water to spread the culture on the slide. Further for the fixation of bacterial cell dry with air followed by heating.
Note: Bacterial smear shall dry to keep the slide aside of the flame, do not keep in direct contact of the flame.
Application of the primary stain (crystal violet). Crystal violet stains shall pour on the smear ensure complete smear comes in contact for 1 min, Wash slide in a gentle and indirect stream of water.
Application of mordant: The iodine solution (mordant) is added to the smear form a crystal violet-iodine (CV-I) complex for 1 min.
De-colorization step: For the de-colorization add the organic solvent such as acetone or ethanol to wash’ out the extracts of blue dye complex completely.
Application of counterstain (safranin): The red dye safranin stains add on the smear and wait for 30 second, Wash slide in a gentle and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper.
Microscopic observation: Gram positive bacteria observe blue to Violet and Gram-negative bacteria observe pink to red.
Procedure of Staining of fungi (Lacto phenol cotton blue):
Place a drop of ethanol on a clean microscopic glass slide and Immerse the specimen in the drop of alcohol.
Add one or at most two drops of the Lacto phenol cotton blue (LPCB) stain before the alcohol dries out.
Holding the coverslip between the index finger and thumb, touch one edge of the drop of mountain with a coverslip edge and lower gently avoiding air bubbles.
This preparation is now ready for examination.
Make the initial examination using low power objective. Switch to higher power (40X) objective for more detailed examination of spores and other structures.
Procedure for Staining of Yeast.
Take a clean grease free slide and take a loop full culture on the slide and one drop of water to spread the culture on the slide.
Crystal violet stains shall pour on the smear ensure complete smear comes in contact for 1 min, Wash slide in a gentle and indirect stream of water.
Examine the slide under microscope, and observe the purple / violet color budding cells.
Procedure for Spore staiping:
Take a clean grease free slide and make smear of Spore forming bacteria.
Air dry and heat fix .the organism on a glass slide and cover with a square of blotting paper or toweling cut to fit the slide.
Saturate the blotting paper with malachite green stain solution and steam for 5 minutes, keeping the paper moist and adding more dye as required.
Alternatively, the slide may be steamed over a container of boiling water.
Wash the slide in tap water.
Counterstain with 0.5% safranin for 30 seconds. Wash with water; blot dry.
Examine the slide under microscope for the presence of endospores.
Endospores are bright green and vegetative cells are brownish red to pink.
During staining of the bacterial / fungal isolates take appropriate precaution to avoid false positive and negative result.
Because wherever use the biochemical method base techniques like in Vitek-2 select the card on the basis of appropriate colony morphology study and microscopic observation i.e. for gram positive bacteria select the Gram positive card and for the gram negative bacteria select the Gram negative card. Wrong card selection gives incorrect result.
To evaluate the impact of identified isolates in the product, raw material, environment etc. for example: If gram negative bacteria identified in the water sample (Potable /Purified/WFI), and identified bacteria declared as objectionable (Should not allow in the Water system), immediate evaluate the water system with consideration of manufactured batches (Purified /Water for injection) for the impact assessment.
In case of identification by Vitek-2, only consider the identification NL T 85 % confidence, in case of low discrimination, re-identification or identification by other method should be performed with appropriate justification as per site procedure.
Procedure for Identification:
In case of environmental monitoring result of product processing area Grade A and Grade B area (Immediate surrounding of product processing zone) each colony recovered in Active air sampling / Passive air sampling / Surface Monitoring / Personnel monitoring shall be identified up to species level.
In case Grade B area other than product processing area like Areas of microbial lab, process area where actual product/product related components are not exposed/handled or area which is not directly contact with product processing zone each colony or at least representative colonies (Colony of different morphology) shall be identified up to species Level based on the assessment.
For other area Grade Cl Grade DI UDAF (Non-aseptic area) identification shall perform in case of excursion i.e. out of Alert /Action /Specification limit select the representative colonies only for identification. Whenever any atypical colony is observed in Grade C Grade D / UDAF (Non-aseptic area), it shall be identified up to species level.
Each observed colony on Membrane filter of total aerobic viable count test of water for injection / pure steam sample need to identify up to species level.
In case of Purified water sample identify the representative colonies up to species level once in a quarter and in case of excursion, verify the recovered isolates with established data base and identify subsequently in case of new isolates record.
Recovered isolates shall be compare with the existing database with respect to colony morphology and gram staining property.
For the Potable / Raw water, identification of representative colonies shall be performed in case of Excursion.
Recovered organism from the sample failure either from sterility test or from the Microbial limit test / Bio burden test / media fill system simulation study shall be identified up to the species Level.
Other than characteristic growth observes on the selective media plates in case of the specified microorganism test also need to be isolated and identify up to species level to confirm whether identified organism is objectionable or not.
Routine assessment for recovered microbial flora:
For the routine assessment recovered organism morphology compare with the established data base (Photo and Data library) and identify if any new morphology observed in the monitoring /testing plates.
Routine assessment shall be performed for the recovered organism from EM and water especially for Grade A/ B area and WFI / PSG.
In case of other grade area (C and D) recovered isolates shall be identifying in case of excursion and verify with existing database.
In case of purified water (routine monitoring), at the time of identification verify the presence of organism in the database and identify if new isolates.
For purified water isolates also shall be identifying in case of excursion and check their presence in the Database.
In case of potable water /raw water/ treated water recovered isolates identify in case of excursion and check their presence in the database.
Sub culturing:
Observed flora on the monitoring/ testing plates i.e. analyzed/exposed plates, Environmental monitoring plates / sterility failure etc. shall be considered the first passage of that isolates and ensure further not more than five passage including broth enrichment will use for any analytical purpose.
Assign the Isolate No. to all isolates, prior to identify in lab and even if isolates send to outside for the identification; same isolate no. share to vendor for tracking and also generated report shall with same isolate no. for allotment of isolate no.
Isolates no. should not be allotted until growth observed on the fresh medium after sub culturing from the analysis or exposed plates.
If there is observed more than one colony type on the plates after sub-culturing during the preparation of the pure culture, allot the isolates no. to all isolates and further proceed for the identification.
For the identification of isolates, prepare the pure culture on the same media, first enrich the colonies in the liquid broth SCDM (2nd Passage) then further subculture on the same agar media on which it was found i.e. Water isolates on R2A Agar and EM isolates on SCDA agar (3rd Passage) and ensure further not more than five passage including broth enrichment will use for any analytical purpose.
In case observe growth in liquid media/ or colony difficult to distinguish on the solid media, transfer to the fresh agar media for the morphological study.
Identification of environmental isolates shall be initiated within 15 working days from the date of release in case of the routine testing and monitoring.
After identification of the isolates, preserve the culture in the library for further evaluation.
Newly identified isolates shall maintained with the photographs for the reference.
A list of all atypical observed isolates shall be prepared, which observed as part of routine identification and shall updated in case of any new isolates observed.
Media Growth promotion test and Disinfectant challenge test.
Recovered environmental isolates shall be challenged to the existing disinfectant within the 90 days from the date of identification.
Each new organism recovered from routine environmental monitoring (Grade A /Grade B /Grade C and D area) or from any excursion shall be challenged to the existing disinfectant and GPT of the monitoring media.
Note: For Grade A/B consider the routine identification with excursion if any and for Grade CID area only in case of excursion.
Re-challenge of organisms shall be perform based on the annual assessment of occurrence as per Annexure-6 (in all area i.e. grade A, B, C and D area).
Table
| Activity | Routine Assessment | |||
| Area | Grade A | Grade B | Grade C | Grade D |
| Initial Identification | Each isolates | Each isolates | Representative isolates in case of excursion, | Representative isolates in case of excursion, |
| Routine Identification | Each isolates | Each isolates / Representative isolates/ in case of excursion, | ||
| DET Challenge | New isolates irrespective of different grade areas.
Example: Any isolates challenge in Grade A area, shall not be needed to re-challenge at repetitive occurrence in any grade area. |
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| GPT Challenge | New isolates irrespective of different grade areas.
Example: Any isolates challenge in Grade A area, shall not be needed to re-challenge at repetitive occurrence in any grade area. • Selection of organism based on the rational quarterly basis and challenge with higher occurrence for routine GPT of the Testing and monitoring media |
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| Re-challenge criteria of DET | Re-challenge of organism shall be performed based on the annual assessment of higher occurrence. | |||
Note: Re-challenge of organism shall be performed based on the annual assessment on the surfaces selected based on scientific rationale.
For the establishment of scientific rational (all grade area) to select the organism for the disinfectant challenge test /GPT (EM / Water), evaluate the organism for following parameter but not limited to: organism type, Source, Gram nature, specific character for e.g. spore former I Non spore former, endospore former etc. refer the Annexure-4 for the documentation of the established scientific rational to select the organism.
In case if observes challenged organism shows resistance against disinfectant revalidate the disinfectant at higher concentration or discontinued with new one.
For the disinfectant challenge test, use the same contact time, concentration of the disinfectant, media and method as originally validated, for other requirement consider the worst case.
In case of mold isolates, if sample send to outside lab for the genotypic identification, culture should be pure and sporulation should not be there.
Selected isolates (based on rational) shall be used for the growth promotion test of the EM / sterility media and water isolates in case of R2A /PCA/ media on rotational basis, after completed the activity and generation of enough data discard the isolates, further re-evaluation shall be started for next year, for the traceability of isolates selected isolates shall be re- challenge for DET in next year subjected to availability of cryo vials or as and when isolated.
Perform the trending of the isolates as per site-specific procedure considering the following points.
- Repeat occurrence at same location and different locations.
- Related to the excursion.
- New flora / Resident flora.
Prepare the summary report for the environmental monitoring and mention the disinfectant challenge test result detail against the recovered isolates, including any changes in the .existing validated disinfectants etc.
Similarly, summery report for water shall be prepared with the detail of recovered isolates from the particular water type.
Isolates recovered from the sterility failure/ Media fill vial, shall be challenged to the sterility media for the growth promotion check.
If DET study for an isolate has been performed in one block of a unit, then no need to perform the DET study for the same isolate identified in other block of same unit provided the same disinfectant and surfaces are used in both blocks.
Preparation of Isolates:
In case of bacterial isolates found, shall be subculture on SCDA media plate and fungus isolate subculture on SDA media plate.
For the increasing of the inoculum size use the Roux bottle with the mentioned media SCDA and SDA.
Incubate SCDA plates/Roux bottle at 30 to 35°C for 24 to 48 Hrs. and SDA plates/Roux bottle at 20 to 25°C for 3 to 5 days for further isolation and purification as per respective SOP.
After completion of incubation period, observe the subculture plates and identification shall be done as per the respective SOP.
After isolation and identification of new, isolates store in form of cryo vial as per the (procurement and maintenance of the microbial culture).
All the cryo vials shall be stored for future reference at below – 30°C up to its consumption.
ABBREVIATIONS:
IPA : Isa Propyl Alcohol
SS : Stainless steel
LAF : Laminar Air flow.
ML T : Microbial limit test
BET : Bacterial endotoxin test
LBPC : Liquid borne particulate count
DET : Disinfectant efficacy testing
SCDA : Soyabean Casein Digest Agar
SDA : Sabouraud Dextrose Agar
SCDM : Soyabean Casein Digest Medium
PCA Plate Count Agar
EM Environmental Monitoring
UDAF Unidirectional air flow
GPT Growth promotion test
PSG Pure steam generator
