Identification of Microorganisms and its SOP

Identification of Microorganisms; Purpose

To identify Microorganisms.

Identification of Microorganisms; Scope

This SOP applies for identification of Microorganisms in Microbiology Section at XX Pharmaceuticals Ltd.

Definitions

GN-ID System

The GN-ID system employs 12(GNA) or 24(GN A+B) standardized biochemical substrates in microwells to identify the family of Enterobacteriaceae & other non-fastidious Gram negative bacilli (Oxidase negative and positive). The kit is planned for professional laboratory use only.

[] CPG: Colony Pigmentation.
[] CAT : Coagulate test
[] LAT : Latex Agglutination Test
[] ONPG : Ortho-Nitrophenyl β-Galactoside
[] PYR : PYRROLIDONYL ARYLAMIDASE
[] TDA : Tryptophan Deaminase Agent( Indolepyruvic Acid)
[] VP : Voges-Proskauer

Identification of Microorganisms;Responsibilities:

Executive/ Sr. Executive, Microbiology

Selection of culture and identification of Microorganisms.

Assistant Manager, Microbiology

Ensure identification and application of sound technical information.

Head of Quality Assurance

Take initiative to Approval of SOP.

Identification of Microorganisms; Procedure:

Instructions

• Before handling of micro-organisms, wear sterile latex free gloves, mask, laboratory coat & eye protection (if required).
• Make sure that all personal ornaments, cell phone are left to prevent unauthorized contamination, before entrance into the test room. The use of Cell phone in the test area is firmly prohibited.
• Do not touch any identification materials directly because all are hazardous materials.
• Keep all used materials into specific designated container.
• The reagents kits are for in vitro use only.
• Discard all used items by immersion in an appropriate disinfectant e.g. 3% of sodium hypochlorite for 30minutes. Liquid waste containing acid must be neutralized before treatment.
• Care should be taken when handling additional reagents as they may contain corrosive or irritant materials.
• Read carefully the leaflet of all reagents before use.

General Requirements for the test

Glass Apparatus :

• Sterile Screw Capped Test Tube
• Sterile Pipette 1ml/2 ml/10 ml
• Glass slide

Media and Reagents :

• Casein Soyabean Digest Agar(CSDA)
• Casein Soyabean Digest Broth(CSDB)
• Cetrimide Agar
• Hydrogen Peroxide
• Kovac’s reagent
• Mac-Conkey Broth
• MacConkey Agar
• Mannitol Salt Agar
• Meat peptone
• Mineral Oil
• Neutralized Peptone
• Nitrate Reagent
• Oxidase strips
• PYR Reagent
• Rapport Vasiliadis Salmonella Broth
• Sabouraud Dextrose Agar
• Sterile 0.85% Saline
• TDA reagent
• VP I and VP II reagents
• Xylose Lysine Deoxycholate(XLD) Agar

These media and reagent can be purchased from commercially available manufacturer.

 Others Requirements

• 70% IPA or ethanol
• Forceps
• Micropipette
• Micropipette sterile Tips
• Surgical Gloves
• Surgical Cotton
• Scissors

Identification of E. coli/ Salmonella/ Others Enterobacteriaceae/ Pseudomonas aeruginosa :

Preparation of Specimens :

• Isolate bacterial culture by streaking on the slant initially on MacConkey Agar for coli or Xylose Lysine Deoxycholate (XLD) Agar for Salmonella species & Cetrimide Agar for Pseudomonas aeruginosa.

Identification of Staphylococcus aureus:

Preparation of Specimens

• Isolate the bacterial culture by streaking on the slant initially on Mannitol Salt Agar.
• Sub-culture on the slant of Casein Soyabean Digest Agar.
• Perform Staining as per SOP for Staining of Micro-organisms as per approved sop
• Use always 18 to 24 hours pure culture for identification.
• Ensure that the isolate bacteria is catalase positive and Gram Positive cocci in clausters in Gram staining test.

 Inoculation and Incubation :

• Confirm that the bacteria is the genus of Staphylococcus in slide Coagulate test (CAT)
• Emulsify a single colony from an 18-24 hors culture in the suspending medium supplied in the kit.
• Mix thoroughly.
• Carefully peel back the adhesive strip sealing the microwell strip.
• Do not discard sealing strip as they will be required later.
• Using a sterile pastuer pipette, add 100 µL of the bacterial suspension to each well of the strip.
• As a purity check, transfer 1 drop of the bacterial suspension on to the purity plate of Mannitol Salt Agar.
• Incubate the purity plate aerobically at 35-370C for 18-24 hours.
• After inoculation overlay wells 10 and 11 with 100 µL of mineral oil. This well is highlighted with a black circle around the well to assist in adding oil to the correct wells.
• Seal the top of the microwell strip with the adhesive strip removed earlier and incubate at 35-370C for 18-24 hours.

Reading and Addition of  Reagents

• Remove adhesive strip & record positive reactions the aid of the color chart.
• Record results on provided forms.
• Add 1 drop of PYR reagent to well 12. Read & record the results after 10 minutes.
• Perform nitrate reduction test on well 9 after reading & recording the ONPG result.
• Add 1 drop of Nitrate A reagent &1 drop of Nitrate B reagent to well and read after 60 seconds.
• Add small amount of zinc powder, If well 7 remains yellow or colorless after addition of nitrate reagents. After addition of zinc, colorless/yellow indicates positive & red color indicates negative.
• Record these additional results on the form provided.

 Identification

• Report in the form the substrates have been organized into triples (set of 3 reactions) with each substrate assigned a numerical value (1, 2 & 4). The sum of the positive reactions for each triplet forms a single digit of the profile number that is used to determine the identity.
• Enter the profile number into identification software which generates a report of the five likely organisms in the selected database.
• The software provides identification based on probability in % up to species level.
• Sub-culture on the slant of Casein Soyabean Digest Agar.
• Perform Staining as per SOP for Staining of Micro-organisms
• Use always 18-24 hours pure culture for identification.
• Confirm that the isolated bacteria is Gram Negative bacilli.

Inoculation and Incubation :

• Carry out an Oxidase test on the isolate. Oxidase positive organisms can only be identified by inoculating both GNA and GN B microwell strips.
• Emulsify a single colony from an 18-24 hour culture in 3 ml sterile 0.85% saline for the GN A microwell strip. If both GN A and GN B strips are to be inoculated, the colony should be emulsified in 3-5 ml sterile 0.85% sterile.
• Mix methodically.
• Carefully peel back the adhesive strip sealing the microwell strip.
• Do not discard sealing strip as they will be required later.
• Using a sterile pastuer pipette, add 100 µL of the bacterial suspension to each well of the strip.
• As a purity check, transfer 1 drop of the bacterial suspension on to the purity plate of MacConkey Agar/ Xylose Lysine Deoxycholate (XLD) Agar.
• Incubate the purity plate aerobically at (35 to 37)⁰C for 18 to 24 hours.
• After inoculation overlay wells 1,2 and 3 (GN A strip counting from the tabbed end) and well 20 and 24(GN B strip-well 13 is at eht tabbed end) with 100 µL drops of mineral oil.
• Do not overlay well 20 if isolate bacteria is Oxidase positive. These wells are highlighted with a black circle around the well to assist in adding oil to the correct wells.
• Seal the top of the microwell strip with the adhesive strip are over wells 7, 11 and 12 in the GN A strip and over well in the GN B strip.
• GN A and GN B microwell strips are read after 18 to 24 hours incubation for Enterobacteriaceae and after 48 hours for Oxidase positive bacteria.

Reading and Addition of  Reagents :

GN A Strip :

• Remove adhesive strip & record positive reactions the aid of the colour chart.
• Record results on the forms provided.
• Add 2 drops of Kovac’s reagent to well 8. Read & record the results after 60 seconds.
• Add 1 drop of VP I reagent and 1 drop of VP II reagent to well 10 and read after 15-30 minutes.
• Add 1 drop of TDA reagent to well 12 and read after 60 seconds.
• Perform the nitrate reduction test on well 7 after reading and recording the ONPG result.
• Add 1 drop of Nitrate A reagent and 1 drop of Nitrate B reagent to the well and read after 60 seconds.
• Add small amount of zinc powder, If well 7 remains yellow or colorless after addition of nitrate reagents, After addition of zinc, colorless/yellow indicates positive and red color indicates negative.
• Record these additional results on the form provided.

 GN B Strip

[] Remove the adhesive strip and record all positive reactions with the aid of the color chart.

Record the result

[] The gelatin well 13 must be read after 18-24 hours for Enterobacteriaceae and after 48 hours for Oxidase positive isolates. A positive gelatin liquefaction result is indicated by black particles visible throughout the well.
[] Arginine well  is interpreted differentially after 24 hours an 48 hours incubations as below :

After 24 hours:

[] Yellow indicates negative
[] Green/Blue indicated positive.

After 48 hours (Oxidase Positive organisms)

[] Yellow/Green : Negative
[] Blue : Positive

Identification :

[] Report form GN A+B, the substrates have been organized into triples (set of 3 reactions) with each substrate assigned a numerical value (1, 2 & 4). The sum of the positive reactions for each triplet forms a single digit of the profile number, that is used to determined the identity.
[] Enter the profile number into identification software which generates a report of the five likely organisms in the selected database.
[] The software provides identification based on probability in % up to species level.