Blog

Microbial Cultures – Isolation and Identification Guide

Procedure for Isolation and Identification of Microbial Cultures & Microbial Isolates using different Analytical Methods from the manufacturing environment of OSD, oral liquid, sampling & Dispensing area, Microbiology section and water isolates from water system, colony isolated from MLT positive results and Standard Microbial cultures.

Isolation and Identification of Microbial Cultures & Microbial Isolates

1.0   Objective :

    • To lay down a procedure for Isolation and Identification of Microbial Cultures and Microbial Isolates Using Different Analytical Methods.

2.0  Scope :

    • This SOP is applicable for isolation and identification of microorganism including environment isolates from the manufacturing environment of –
      • Oral Solid Dosages ,MICROBIAL CULTURE
      • Oral liquid,
      • Sampling & Dispensing area,
      • Microbiology section and water isolates from water system,
      • Colony isolated from MLT positive results and Standard Microbial cultures.

3.0   Procedure – Isolation and Identification of Microbial Cultures :

    • Criteria For isolation and Identification of Microbial Cultures:

    • All organisms shall be identified up to species level recovered through environmental monitoring including personal monitoring, settle plate exposure, swab sampling, air sampling from Grade A,B,C,D critical area of Microbiology & environmental monitoring Through settle plate exposure,  and air sampling of Grade D area of  OSD, oral liquid, sampling & Dispensing area,
    • The organisms shall be identified up to species level from the initial sterility positives.
    • All organisms shall be identified up to species level recovered from purified water and Potable water.
    • Whenever organisms is isolated from environment, Keep record for identification of Microorganisms in Annexure-I.
    • If the isolated colony simulates with already identified organism neglect the organism, if it is not matching with any one of the identified organism carry out the identification.
    • 100 % identification from Grade C and Grade D in case counts exceeds action level.
    • Perform identification up to species level by selecting the organisms based on morphological characteristics.
    • In case of fungi identification shall be done up to genus level (desirable up to species level) whenever fungi appears in Grade A and whenever counts exceeds the action level in any area.
    • Incase of yeast cells the organism should be identified up to genus level only.
    • In case of sterility testing failure colony should be isolated up to species level.
  •   Isolation of microbes shall be divided into two groups depending on the source of growth.

    • Isolation procedure from Liquid Media (Microbial Cultures)

    • Take a loopful of organism from the enriched growth medium and streak it on Soya bean casein digest agar plate.
    • Incubate the plate at 30-35°C for 24-48 hrs.
    • After the incubation period, observe the plate for growth and select the isolated colony.
    • Perform the Gram Staining by taking the isolated colony.
    • Note down the observations of colony morphology and gram staining in Annexure-I.
    • Preserve the Isolated organism on the SCDA slant at 2-8 0C for reference & other usages.

    • Isolation procedure from Solid Media (Microbial Cultures) :

    • Take a loop full of Microbial cultures from the selected plate.
    • Streak it on Soya bean casein Digest agar plate.
    • Incubate the plate at 30-35 °C for 24-48 hrs.
    • After the incubation period, observe the plate for growth and select the isolated colony.
    • Perform the Gram Staining by taking the isolated colony.
    • Note down the observations of colony morphology and gram staining in the Annexure-I.
    • Preserve the Isolated organism on the SCDA slant at 2-8 0C for reference & other usages.
  • Identification of microorganisms using different microbiological Analytical methods
    • Following are some of the microbiological analytical methods used commonly for identification of microbial cultures and environmental isolates.

    • Phenotypic analytical methods:

    • These are conventional microbiological techniques used for the identification of microbes which includes Microscopic and biochemical characterization, FTIR, MALDI- TOF and other rapid microbiological identification kit like API kit (Biomeriux), Biolog kit and Hi-bio kit and reader.

    • Genotypic analytical methods:

    • These are nucleic acid based which involves molecular techniques for identification like PCR and Genome Sequencing which normally includes 16s ribotyping and 5 s ribotyping.
  • Identification of Procedure of Microorganisms by using API Identification Kit :

    • Principle:

    • The API WEB identification system is a miniaturized identification method employing modified conventional and chromomeric substrates.
    • It is intended for identification of frequently isolated bacteria.
    • The API Strip contains dried biochemical and enzymatic substrates.
    • A bacterial suspension in the inoculums fluid is used for rehydration of the substrates.
    • The tests used in the system are based on microbial utilization and degradation of specific substrates detected by various indicator systems.
    • Chromomeric substrates upon hydrolysis produce colour changes that can be detected visually.
    • In addition there are tests that detect the ability of an organism to hydrolyze, degrade, reduce or otherwise utilize a substrate.
    • The conventional tests are inoculated with the saline bacterial suspension which reconstitutes the media.
    • During incubation, metabolism produces colour changes that are either spontaneous or revealed by the addition of reagents.
    • The assimilation tests are included with a minimal medium and the bacteria grow if they are capable of utilizing the corresponding substrate.
    • The reactions are read according to the reading table and the identification is obtained by referring to the analytical profile index or using the identification software.

The API identification kit should be used for the identification purpose and the kit contains:

    • API strip (API staph/API strep/API C aux/API 50CH/API 20 NH/API 20E/API 20 NE).
    • API strip incubation box
    • Inoculum fluid tube
    • Mc Farland standard
    • Additional reagents and
    • API Identification report pad
    • Other material required but not provide with the kit: Sterile cotton swabs (do not use polyester swabs) incubator (30 – 35°C), API web and appropriate Microbial cultures media.
    • Also required necessary glass wares used for preparation, storage and handling of specimens.

    • Storage, Handling and Shelf Life of API identification Kit:

    • API strips:
    • API Strips are individually packaged and must be stored unopened in refrigerator at 2-8°C (do not freeze).
    • Visually inspect the package for holes or cracks in the foil package.
    • Do not use if the packaging appears to be damaged.
    • Strips in the original packaging, if stored as recommended, will retain expected reactivity until the date of expiration.
    • Additional reagents: Store in a dust free environment below 25°C, until ready to use.

    • Inoculum Fluid:

    • Inoculum fluid is packaged in two sets of ten tubes.
    • Visually inspect the tubes for cracks, leaks, etc.
    • Do not use if appears to be leak, damaged tube or cap or visual evidence of contamination (i.e. haziness, turbidity).
    • Store the tubes Inoculum fluid at 2-8°C, and before expiry date as displayed on label.
    • Each of the material including API strip, API strip bases and Inoculum fluid must be brought to room temperature prior to use.

    • User Quality Control:

    • Whenever the new lot of API strips are received perform the testing of Strip with known Microbial cultures of microorganism as per below mentioned procedure to confirm that the lot received gives acceptable results as claimed by supplier and is suitable to use.

    • Specimen Collection And Processing:

    • Use isolates from media such as Trypticase Soy Agar with 5% sheep Blood (TSAII) or Columbia Agar with 5% sheep Blood or Soyabean casein Digest agar.
    • Media containing esculin should not be used.
    • The test isolate must be in a pure Microbial cultures form, no more than 18-24 hours old for most genera, for some slow growing organisms, test isolate can be used up to 48 hours.
    • Use swabs with cotton tips as polyester swabs may cause problems with inoculation of the panels.
    • Once the lids are removed from the sealed pouches they must be used within 1 hour to ensure adequate performance.

    • Choose the colony on the basis of the criteria mentioned above.

    • Preserve the plate at temperature 2-8°C if identification shall not be performed on the same day of the occurrence of the colony.
    • Select single well-isolated colony from the media plate and prepare pure Microbial cultures using streak plate method.
    • After achieving pure Microbial cultures prepare suspension in sterile 0.5ml normal saline solution.
    •  Streak loopful of suspension on pre-incubated, Tripticase soy agar with 5% sheep blood or Columbia agar with 5% sheep blood or Soya bean casein digest agar plate surface, in case of yeast and mold streak on potato dextrose agar or Sabouraud dextrose agar plate surface as appropriate.
    • Keep the plate(s) for incubation at 30 – 35° C for 24 – 48 hours for bacteria.
    • Select single well isolated colony and determine morphological characters of the same including Size, Shape, Colour, Margin, Opacity, Elevation (as mentioned in below diagram and Table) motility and report in Annexure –I.

Morphological Characteristics

SIZE SHAPE COLOUR OPACITY MARGIN ELEVATION
Pinpoint Round White Opaque Entire Flat
Small Oval Pink Transparent Undulate Raised
Medium Irregular Yellow Translucent Lobate Convex
Large Filamentous Pale yellow Erose Umbonate
Orange Crateriform
Brown Hilly
Red
Black
    • After identifying the gram nature perform the orientation test which includes Catalase test for Gram Positive Organism and Oxidase test for Gram Negative organism.
    • Based on Gram staining and Orientation tests, Choice of Strip is made (Refer the diagram given below).
    • Take an Inoculum fluid tube and label with the identification number or name.
    • Suspend suspected colony in the Inoculum fluid with the help of sterile Swab.
    • Recap the tube and vortex for 10 – 15 seconds.
    • Match the Turbidity with McFarland’s as per table given below.
    • If turbidity appears more than the McFarland’s standard, using aseptic techniques, dilute the Inoculum by adding minimum required volume (not to exceed 1.0 ml) of 0.9% sterile saline or Inoculum fluid.
    • Remove excess fluid with sterile pipette so that final volume of Inoculum fluid will be similar to that of original volume in the tube and if turbidity appears less than the McFarland’s standard then use fresh Inoculum fluid tube and proceed.

Table:

Mc Farland’s Std No. API Strip
0.5 API 20 NE (ref. no.20100)
0.5 API STAPH (ref. no. 20500)
4 API 20 STREP (ref. no. 20600)
2 API Candida I(ref. no. 105000)
One colony API 20 E
Not required API 20 CH
2 API 20 C AUX(ref. no. 20210)

    • Preparation of the Strip :

    • Prepare an incubation box, tray and lid and distribute about 5 ml of distilled water /sterile water for injection into the bottom of the tray to create humid atmosphere.
    • Record the specimen number on the elongated flap of the tray.
    • Remove the tray from the individual packaging.
    • Place the strip in the incubation box.
  • Preparation of inoculum and Inoculation of the Strip:

    • API 20 NE For Non fastidious, non enteric gram  negative rods

    • Preparation of the inoculum
    • Open an ampoule of API NaCl 0.85% medium 2 ml.
    • Using the pipette pick up 1-4 colonies of identical morphology from the agar plate.
    • Prepare a suspension with a turbidity equivalent to 0.5 Mc Farland.
    • Solution should be used immediately after preparation.

    • Inoculation of the strip

    • Inoculate into test NO3 to PNPG by distributing the saline solution into tubes and not the cupules using the same pipette.
    • To avoid the formation of bubbles at the base of the tubes, tilt the strip slightly forward and place the tip of the pipette against the side of the cupules.
    • Open an ampoule of API AUX medium and add 200 micro liter of remaining saline solution to the ampoule, homogenize well with the pipette avoiding the formation of bubbles.
    • Fill the tube and cupules of test GLU to PAC with the suspension.
    • Add mineral oil to the cupules of three under lined test GLU, ADH, URE until convex meniscus is formed.
    • Close the incubation box and incubate at 30-35°C for 24 hrs.

    • Reading the strip

    • After incubation period read the strip by referring to the reading table
    • Record all spontaneous reaction (GLU. ADH, URE, ESC, GEL and PNPG) on the result sheet
    • NO3 Test: add 1 drop of NIT 1 and 1 drop of NIT 2 reagent to NO3 After 5 mins.
    • A red colour indicates a positive reaction and should be recorded in the record sheet.
    • Negative reaction may be due to production of nitrogen, add 2-3 mg of Zn reagent to NO3 couple, after 5 mins.
    • Colour less indicate the positive reaction and should be recorded in the record sheet.
    • If the couple turns pink red the reaction is negative.
    • TRP Test: Add 1 drop of James reagent, immediately a pink colour is produced showing the positive reaction and should be recorded in the record sheet.
    • Assimilation test: Observe the bacterial growth. An opaque cupule indicates the positive reaction. In case Cupule shows weak growth record the result as ±.
  • API Staph for Staphylococci, micrococcus and related genera 

    • Preparation of the inoculum

    • Microbial sub-cultures the organism onto Columbia blood agar or P agar for 18-24 hrs at 35° C
    • Check that the strain belongs to micrococcaceae family (morphology, grams nature, catalase)
    • Check that the Microbial cultures is pure
    • Open the ampoule of API Staph medium and prepare a homogenous bacterial suspension with a turbidity equivalent to 0.5 Mc Farland.
    • Suspension must be used immediately after preparation.

    • Inoculation of the strip

    • Using a pipette fill the micro tubes with the inoculated API Staph medium.
    • Only fill the tube portion of the micro tubes, not the cupules.
    • To avoid the formation of the bubbles at the base of the tubes, tilt the strip slightly forward and place the tip of the pipette against the side of the cupule.
    • Ensure the anerobiosis in the ADH and URE test by filling the cupules with mineral oil to form the convex meniscus.
    • Close the incubation Box.
    • Incubate at 30-35°C for 18-24 hours.

    • Reading the strip

    • After the incubation period develop the reaction by adding 1 drop of each of the following reagents and read the reaction by referring the reading table.
    • VP Test: VP 1 and VP 2 reagents, wait 10 mins. A violet-pink colour indicates a positive reaction, pale pink colour after 10 mins should be considered negative.
    • NIT Test: NIT 1 and NIT 2 reagents, wait 10 min. A red colour indicates a positive reaction.
    • PAL test: ZYM A and ZYM B reagents, wait 10 min. A violet colour indicates a positive reaction.
    • Record the results on the result sheet.

    • Interpretation:

    • On the result sheet the test are separated into groups of 3 and are numbered 1,2,4, by adding the values corresponding to positive reaction within each group will provide a 7 digit number.
    • Identification is obtained with this 7 digit numerical
    • This is performed using the database V7.0.
    • With the analytical profile index look up the numerical profile in the list of profile.
    • With the apiwebTM identification software enter the 7 digit numerical profile via key board.

    • API 20 C AUX for yeast identification

    • Preparation of the inoculum
    • Open an ampoule of API NaCl 0.85% medium or API suspension medium 2 ml.
    • Using the pipette pick up a portion of a yeast colony of identical morphology from the agar plate.
    • Prepare a suspension with a turbidity equivalent to 2 Mc Farland. Solution should be used immediately after preparation.
    • Open an ampoule of API C medium and transfer about 100 µl of the previous suspension.
    • Gently homogenize with the pipette to avoid the bubbles.
    • Inoculation of the strip
    • Fill the Cupule with the suspension obtained in the ampoule of API C medium.
    • Avoid formation of bubbles by placing the tip of the pipette against the side of cupule.
    • Close the box and incubate at 30 °C for 48-72 hrs.
    • Reading of strip
    • After 48 hours of incubation, or 72 hours (If the tests, in particular glucose, are not clear cut after 48 hours), compare growth in each cupule to the 0 cupule, which is used as negative control.
    • A cupule more turbid than the control indicates the positive reaction to be recorded to the results sheet.
    • In order to minimize the risk of contamination when re incubation is necessary, remove the lid only when reading the strip and replace immediately.
    • Morphology test: Determine the presence of hyphae (mycelium) or pseudo hyphae (pseudo mycelium) using RAT medium.
    • Dispense 1 drop of suspension obtained in the ampoule of API suspension medium or API NaCl 0.85% medium onto RAT medium.
    • This test constitutes the 21st test of the strip.
    • It is considered positive if hyphae or pseudo hyphae are detected.
    • Interpretation shall be done using API web.

    • Interpretation:

    • On the result sheet the test are separated into groups of 3 and are numbered 1,2,4, by adding the values corresponding to positive reaction within each group will provide a 7 digit number.
    • Identification is obtained with this 7 digit numerical
    • This is performed using the database V7.0.
    • With the analytical profile index look up the numerical profile in the list of profile.
    • With the apiwebTM identification software enter the 7 digit numerical profile via key board.
  • API 20 E for identification of Enterobacteriaceae and other non fastidious gram negative bacteria

    • Preparation of the inoculum

    • Open an ampoule of API NaCl 0.85% medium or API suspension medium 5 ml or use any tube containing 5 ml of sterile saline without any additive.
    • Using the pipette pick up a single well isolated colony from the agar plate.
    • Carefully emulsify to achieve a homogeneous bacterial suspension.
    • This suspension must be used immediately after preparation.
    • Note: Most Vibrio species are halophillus; if vibrio is suspected suspend the bacteria in API NaCl 0.85% medium

    • Inoculation of the strip

    • With the same pipette distribute the bacterial suspension into the micro tubes of the strip.
    • Avoid formation of bubbles by placing the tip of the pipette against the side of tubes.
    • For the CIT, VP and GEL test fill both tube and cupule.
    • The other test fill only the tubes and not the cupule.
    • For the test of ADH, LDC, ODC, H2S and URE create an anerobiosis by over laying with mineral oil.
    • Care should be taken not to over fill or under fill the cupule otherwise incorrect result may be obtained.
    • Close the box and incubate at 30 -35°C for 18-24 hrs.

    • Reading of Strip

    • After the incubation period, read the strip by referring to the Reading table.
    • If 3 or more tests (GLU test + or -) are positive, record the result on the report sheet, and then reveal the tests which require the addition of the following reagents.
    • TDA Test: add 1 drop of TDA Reagent a reddish brown colour indicates the positive reaction.
    • IND Test: Add 1 drop of James reagent, a pink colour indicates the positive reaction.
    • VP Test: Add 1 drop of VP 1 and VP 2 reagent and wait for 10 mins.
    • A pink or red colour indicates the positive reaction, a slight pink colour indicates the negative reaction.
    • Determination of the numerical profile: on the result sheet test are separated into groups of 3 and a value 1,2 or 4 is indicated for each, by adding the values corresponding to positive reaction within each group, a 7 digit profile no. is obtained for 20 tests of API 20E strip, the Oxidase test constitute the 21st test and has the value of 4 if it is positive.

    • Interpretation:

    • On the result sheet the test are separated into groups of 3 and are numbered 1,2,4, by adding the values corresponding to positive reaction within each group will provide a 7 digit number.
    • Identification is obtained with this 7 digit numerical
    • This is performed using the database V7.0.
    • With the analytical profile index look up the numerical profile in the list of profile.
    • With the api web TM identification software enter the 7 digit numerical profile via key board.
  • API 20 Strep for Streptococcaceae and other related organism
    • Preparation of the inoculum
    • Open an ampoule of API suspension medium 2 ml.
    • Using the swab harvest all the Microbial cultures from the previously prepared sub- cultures plates.
    • Prepare a dense suspension with a turbidity greater than 4 Mc Far land.
    • Solution should be used immediately after preparation.
    • Gently homogenize with the pipette to avoid the bubbles.

    • Inoculation of the strip

    • In the first half of the strip Test VP to ADH distribute this suspension avoiding the formation of bubbles.
    • For the tests VP to LAP distribute approx. 100 µl into each cupule.
    • For ADH test fill the tube only.
    • In the second half of the strip (Test RIB to GLYG), open an ampoule of API GP medium and transfer the rest of the suspension into it (approx. 0.5 ml) Mix well.
    • Distribute this new suspension into the tubes of RIB to GLYG.
    • Fill the cupule of the underlined tests ADH to GLYG with mineral oil to form the convex meniscus.
    • Close the box and incubate at 30-35 °C in aerobic condition for 4-4 ½ hrs to obtain the first reading and for 24 hrs to obtain the second reading if required.

    • Reading of Strip:

    • After 4 hrs of incubation add the reagents.
    • VP test: add 1 drop of each VP 1 and VP 2.
    • HIP test: 2 drop of NIN.
    • PYRA, alpha GAL, beta GUR, beta GAL, PAL, LAP, test: add 1 drop of each of ZYMA and
    • Wait 10 mins. And read the reaction by referring the reading table.

    • Interpretation:

    • On the result sheet the test are separated into groups of 3 and are numbered 1,2,4, by adding the values corresponding to positive reaction within each group will provide a 7 digit number.
    • Identification is obtained with this 7 digit numerical
    • This is performed using the database V7.0.
    • With the analytical profile index look up the numerical profile in the list of profile.
    • With the apiwebTM identification software enter the 7 digit numerical profile via key
  • API Candida

    • Preparation of the inoculum

    • Open an ampoule of API NaCl 0.85% medium 2 ml.
    • Using the pipette or swab pick up one or several well isolated and identically colonies .
    • Prepare a suspension with a turbidity equivalent to 3 Mc Farland.
    • Solution should be used immediately after preparation.
    • Gently homogenize with the pipette to avoid the bubbles.

    • Inoculation of the strip

    • Distribute the prepared yeast suspension into the tubes only avoiding the formation of bubbles, tilt the incubation box slightly forward and position the pipette on the edge of the cupule.
    • Cover the first 5 tests GLU to RAF and the last test URE with mineral oil immediately after inoculating the strip.
    • Note quality of filling is important tubes which are insufficient or excessively full may cause false positive or false negative results.
    • Close the box and incubate at 30 -35 °C for 18-24 hrs in aerobic condition.

    • Reading of Strip:

    • After 18-24 hrs incubation read the reaction by referring the reading table in the package insert and record them as + or – on the record sheet.
    • Note: Tube 8 or 9 are bi functional and enable 2 reaction to be performed in the same tube, Tube 8: Beta XYL(test no. 8) / beta NAG(test no. 11) Tube No.9: beta GUR(test no. 9) / beta GAL(test no. 12).

    • Interpretation:

    • On the result sheet the test are separated into groups of 3 and are numbered 1,2,4, by adding the values corresponding to positive reaction within each group will provide a 4 digit number.
    • Identification is obtained with this 4 digit numerical
    • This is performed using the database V7.0.
    • With the analytical profile index look up the numerical profile in the list of profile.
    • With the apiwebTM identification software enter the 4 digit numerical profile via key board.

  • API 50 CH for the study of carbohydrate metabolism of microorganism.

    • (API 50CH in conjunction with API 50 CHL medium is used for identification of lactobacillus and related genera and API 50CH in conjunction with API 50 CHB/E medium is used for identification of Bacillus and related genera, Enterobacteriaceae and vibrionaceae).
    • Depending on which medium is used API 50CHL medium or API 50CHB/E refer the corresponding package insert.

    • Preparation of the Strip :

    • Each strip is made up of 5 smaller strips each containing 10 numbered tubes.
    • Prepare an incubation box (Tray and Lid).
    • Record the specimen number on the elongated flap of the tray.
    • Distribute about 10 ml of distilled water into honey combed wells of the tray to create the humid atmosphere.
    • Remove two strips(0-19 and 20-39) from their packaging, and separate into 4 smaller strips (0-9, 10-19, 20-29,30-39) and place all 4 in the incubation tray.
    • Take the remaining smaller strip (40-49) out of the packaging and place it next to other in the incubation tray to complete the strip.

    • Preparation of the inoculum

    • Microbial Cultures the microorganism on soya bean casein digest agar plate.
    • Check the purity of the strain.
    • Harvest all the bacteria from the solid medium using the swab.
    • Prepare the inoculum in appropriate medium i.e. API 50CHL or API 50CHB/E.
    • Gently homogenize with the pipette to avoid the bubbles.
    • Use the suspension immediately after preparation.

    • Inoculation of the strip

    • Distribute the bacterial suspension using sterile pipette into 50 tubes.
    • Tilt the incubation box slightly forward.
    • Avoid the formation of bubbles by placing the tip of the pipette against the side of the cupule.
    • When only the tube is to be inoculated do not exceed the top of the tube so as to maintain the anaerobic condition.
    • When the tubes and the cupule are to be completely filled avoid the formation of a concave or convex meniscus.
    • Incubate the strips at the optimum temperature for growth of group of micro organism being tested e. 30°C, 37°C, or 55°C
    • Close the box and incubate for 24-48 hrs.

    • Reading and interpretation

    • The strips are read after the stipulated incubation period e.g. 24 hrs or 48 hrs depending on the microorganism and the type of reaction studied.
    • Interpret each test positive, negative or doubtful and record the result on the result sheet.
    • The results from the biochemical profile which when entered in the identification software provide the identification of lactobacillus and related genera or bacillus and related genera, Enterobacteriaceae vibrionaceae.
    • Purity plate:
    • Streak loopful of inoculum from inoculum fluid tube (before or after) inoculating the base on the appropriate medium for purity check.
    • Discard the inoculum fluid tube and cap.
    • Incubate the plate at 30 – 35° C for 24 – 48 hours may be used for any supplementary tests, if required.

    • Interpretation of Result Using API WEB

    • Log on using the user ID and exclusive Password.
    • A web page with the list of all types of API Strips appears, select the API Strip, for which Identification result has to be interpreted.
    • Either Enter the Bio number or the Biochemical Profile and Confirm.
    • Result is displayed and contains a system generated ID Comment (Excellent Identification, Very Good Identification, etc) % ID, T Index, Test Against, Significant Taxa and Next Taxon.
    • Recheck all the procedures from the beginning, if comment like, Low Discrimination, Doubtful Profile or Unidentified is displayed.
    • Take a printout or Export your Identification Results to a location on your system and save and report as per Annexure I.

  • Identification Procedure of Microorganisms by using biochemical testing

    • Biochemical tests are the tests used for the identification of bacteria species based on the differences in the biochemical activities of different bacteria.
    • Perform the Gram Staining by taking the isolated colony as per the SOP.
    • Perform other biochemical analysis by taking the isolated colony as per the SOP.

  • Identification of Fungi:

    • Identification of fungi up to Genus level only.
    • For identification of fungi is based on surface color, hypal structure and types of spores.
    • Conclusive identification shall made only with microscopic observation which is determined by type of hyphae and spores present.
    • Preserve the plate at temperature 2-8°C if identification not performed on the same day of the occurrence of the colony.
    • Transfer small quantity of Inoculum on a fresh Media plate of SDA / PDA plate and incubate at 20-25°C for 3-5 days.
    • Take a clean, scratch less glass slide and put a drop of lacto phenol cotton blue (Methylene blue) dye on it.
    • Place one drop of 95% ethanol to the glass slide.
    • Open the plate having fungal colony and scratch a small quantity of the fungal colony with the help of a needle cleaned with 70% v/v IPA. Fix the smear on glass slide.
    • Place this Inoculum from the glass needle on the dye already placed on the slide and moves the needle gently so that the mycelia of the fungus should not get toned off.
    • Put a glass cover slip on the dye and focus the slide first at 10X and then at 40X magnification under the microscope.
    • Observe the characteristics of the fungal cultures (Microbial) and report as per Annexure II.

4.0   Reference

    • API manual
    • Bergey’s Manual of Systematic Bacteriology

5.0  Annexure – Microbial Culture :         

Annexure –I : Record for Identification of Microorganism (Bacterial isolates)

I.              HISTORY: Isolate Reference No.
Date of Isolation Date of  Subculture
Isolated From Area
Media used Location
Incubation Temp. &Time 30-350C for 24 hours /3 0-350C for 48 hours
Type of Organism Aerobic Anaerobic Facultative
I.              COLONY  MORPHOLOGY
Shape of the colony Margin of the Colony
Shape Standard Observation Edge Standard Observation
Round Entire
Oval Undulate
Irregular Lobate
Filamentous Erose
Elevation of the Colony
Elevation Standard Observations Colour of the colony
Flat
Raised
Convex Opacity of the colony
Umbonat Opaque
Createriform Transparent
Hilly Translucent
     III.MICROSCOPIC EXAMINATION Cell Morphology
Staining Procedure Structure Standard Observation
Gram positive Gram negative Rods
Bacilli
Motility test Diplobacilli
Motile Non Motile Streptobacilli
Spirilum
Spore staining Micrococci
Spore Forming Non Spore Forming Diplococci
Tetrads
Size of the Colony Streptococci
Staphylococci

Annexure –II : Record for Identification of Microorganism (Fungus isolates)

I.              HISTORY: Isolate Reference No.
Date of Isolation Date of  Subculture
Isolated From Area
Media used Location
Incubation Temp. & time 20–250C for 3 days /20–250C for 5 days
Color of the culture Type of spores/

Hypal structure

 Name of the organisms
Bluish green with sulfur yellow area on the surface Black, Phialospores
White Large ,thick walled ,round or irregular structures formed within or terminally on a hypa Chlamydospores
Bluish Brush arrangement of  Phialospores
Buff coloured surface Hypae break up into thin walled rectangular arthrospores
Pinkish brown Elliptical micro conidia
Light green to grayish surface with gray to black Blastoconidia

 

 

Janki Singh

Mrs. Janki Singh is the professional pharmaceuticals Blogger. She has already posted more than #1000 articles on varrious topics at different blogging plateforms. Contact : guideline.sop@gmail.com