MICLAB 070 Sample

Standard Operating Procedure Title: Identification of Microorganisms to Genus and Species Level ______________________________________________________________________________________

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Table of Contents 1. Introduction ....................................................................................................................................... 3 2. Administration and Preparation of Isolates ......................................................................................... 3

2.1. Classification of Test Samples ....................................................................................................... 3 2.2. Unique Sample Identifier................................................................................................................ 3 2.3. Preparing Isolates for ID................................................................................................................. 4

3. ID of Isolates ..................................................................................................................................... 6 3.1. The Gram Stain ............................................................................................................................. 6 3.2. Technical Information..................................................................................................................... 7 3.3. The Potassium Hydroxide (KOH) test............................................................................................. 8 3.4. The Catalase Test.......................................................................................................................... 9 3.5. The Coagulase Test......................................................................................................................10 3.6. Sporulation and The Spore Stain...................................................................................................11

4. Quality Control (QC) .........................................................................................................................12 4.1. QC and Traceability of Reagents/Kits............................................................................................12

5. Documentation of Results.................................................................................................................13 5.1. Results Recording and Further ID .................................................................................................13

6. Monitoring Trends ............................................................................................................................13 6.1. Trending of Microbial ID Results....................................................................................................13

7. Appendices ......................................................................................................................................14 7.1. Colony Morphology .......................................................................................................................14 7.2. Cellular Shape and Arrangement ..................................................................................................14 7.3. ID Flowchart: Gram Positive Cocci ................................................................................................15 7.4. ID Flowchart: Gram Positive Rods.................................................................................................15 7.5. ID Flowchart: Gram Negative Rods ...............................................................................................15 7.6. ID Flowchart: Yeasts.....................................................................................................................15 7.7. Gram Positive Cocci......................................................................................................................15 7.8. Gram Positive Rods Sporing ......................................................................................................15 7.9. Gram Positive Rods Non-sporing ...............................................................................................15 7.10. Gram Negative Organisms (Non-glucose fermenters) ................................................................15 7.11. Gram Negative Organisms (Glucose fermenters).......................................................................15

8. Summary of Changes.......................................................................................................................15




010499/2

2.3. Preparing Isolates for ID

2.3.1. For example where the USI is for e.g. 010326/3 the number of isolate types is 3. These isolate types will be distinguished from one another using the following nomenclature; Category prefix, followed by the Isolate number, followed by the isolate type number i.e. 010326-1, 010326-2 and 010326-3. This is further illustrated in the following example:

The allocated USI for a given test sample is as follows:

010326/3 3 isolate types were recovered from this sample.

These isolate types will have a defined isolate type number as follows:

Isolate Type 1: 010326-1 Isolate Type 2: 010326-2 Isolate Type 3: 010326-3

2.3.2. Define the isolate types in the test sample using the given nomenclature.

2.3.3. Fill out an Isolate ID Record form appropriate to the ID Category of the test sample.

2.3.4. Record the details as follows:

Sample ID # - this is the USI Total CFU/Growth the total number of colonies recovered from the test sample OR

Growth/No Growth in broth Sample Details - including sample/test date, location, batch #, inspection lot number

(where applicable), associated Deviation Report (if any) etc. Sample Type/Machine/Environmental Grade/Water Type to be specified where

applicable Isolate ID # - isolate type number assigned to each individual isolate type Macroscopic Description morphological description of the isolate; size, colour,

shape, margin, lustre etc. (see Appendix 7.1). CFU number of colony forming units bearing the same macroscopic description.

2.3.5. Streak each isolate type onto the respective TSA plate labelled with the Isolate type marking. Use the following procedure to ensure growth of single, pure, well-isolated colonies.

The Prefix The sample is an air sample, which is classified as an Environmental sample, thus the prefix assigned is 01.

The Isolate Number This number is assigned as a next number in the Sample Identification Number Allocation spreadsheet.

Number of Isolate Types Two macroscopically different colonies were recovered from the test sample.




Note: You may wish to vortex a broth culture prior to creating a smear as this will ensure an even mix of cells in the culture. As the broth is a liquid culture, water is not required to create the smear.

3.1.2. Allow the smear to air dry and heat fix the cells to the slide by passing the slide through the hottest part of the flame three times. Allow to cool.

3.1.3. Flood the slide with Crystal Violet solution and stand for 30 seconds (Fig. 2A).

3.1.4. Rinse off Crystal Violet solution with water and tilt the slide to drain excess water.

3.1.5. Flood the slide with 1% Lugols Iodine and stand for 30 seconds (Fig. 2B).

3.1.6. Rinse off Iodine with water and tilt the slide to drain excess water.

3.1.7. Flood the slide with Decolourising solution and immediately rinse the slide with running water after 3 seconds (Fig. 2C). Tilt the slide to drain excess water.

Note: Prolonged contact with decolourising solution will result in over-decolourisation of cells and a false gram reaction. Ensure the contact time is limited to 3 seconds only.

3.1.8. Flood the slide with the desired counterstaining solution. Leave to act for up to 2 minutes (Fig. 2D).

3.1.9. Rinse with water and blot dry using a paper towel. Do not rub.

3.1.10. Examine the smears under Oil Immersion.

3.1.11. Record the cellular arrangement (see Appendix 7.2) and Gram stain result of the organism on the Isolate Identification Record form.

3.1.12. Interpretation

Gram-positive Gram-positive organisms stain purple Gram-negative Gram-negative organisms stain pink/red.

3.1.13. Quality Control

3.1.13.1. Gram-positive and Gram-negative reference cultures are to be used for quality control of the gram staining procedure and reagents.

Gram Positive Staphylococcus aureus ATCC 6538 Gram Negative Escherichia coli ATCC 8739

3.2. Technical Information

Some Gram-positive bacteria may appear Gram-negative in whole or in part eg. some strains of Gram-variable Bacillus and Clostridium. Yeasts will appear Gram-positive and will be visibly much larger than a prokaryotic bacterial cell. All Gram-variable and suspected over or under-decolourised organisms are to be subject to a KOH test (see section 3.3).




3.4.5. Interpretation

Positive result: Vigorous bubbling indicating the presence of catalase enzyme converting H202 to oxygen and water

Negative result: No bubbling

3.4.6. Quality Control

Control organisms are to be tested with each use, as H202 is unstable. Do not use the test if reactions with the control organisms are incorrect. QC organisms Positive Control Staphylococcus aureus ATCC 6538 Negative Control Enterococcus casseliflavus ATCC 700327

3.4.7. Technical Information

The enzyme is present in viable cultures only. Do not perform on cultures over 24 hours old as this may result in a false negative reaction. Anaerobic cultures should be exposed to air for 30 minutes prior to testing.

3.5. The Coagulase Test

Members of the genus Staphylococcus are differentiated by the ability to clot plasma by the action of the enzyme coagulase. Coagulase exists in two forms: Bound (Clumping Factor) - enzyme is bound to the cell wall. Enzyme absorbs fibrinogen from the plasma and alters it so it precipitates on the Staphylococci, causing them to clump resulting in cell agglutination. Free enzyme is liberated by the cell wall and reacts with a substance in plasma to form a fibrin clot. Staphytect Plus is a latex slide agglutination test for the detection of clumping factor, Protein A and certain polysaccharides found in methicillin resistant S.aureus (MRSA) from those staphylococci that do not possess these properties. It is much more sensitive than the traditional slide coagulase test due to its ability to detect Protein A and capsular polysaccharide. Note: The Staphytect Kit must be stored at 4C away from direct sunlight or heat sources. Ensure the caps are securely fitted after each use to prevent contamination and drying out of the reagent.

3.5.1. Bring the latex reagents to room temperature.

3.5.2. Vortex the latex reagent for 10 seconds and dispense one drop of test latex into one circle on the reaction card and one drop of control latex into another circle on the reaction card.

3.5.3. Using a sterile disposable loop, pick up 5 single isolated colonies and immerse the loop and the adhering colony mass into the Control latex reagent. Mix and spread to cover the entire circle.

Positive presence of bubbles is evidence of oxygen production from the conversion of H202.

Negative no reaction




Table 3: Quick Reference Table of QC Organisms for Regents/Kits

Test QC Organism(s) Frequency

Gram Stain S.aureus ATCC 6538 E.coli ATCC 8739

Reagents to be verified upon opening.

Spore Stain B.subtilis ATCC 6633 E.coli ATCC 8739

Reagents to be verified upon opening.

Potassium Hydroxide (3% KOH)

E.coli ATCC 8739 S.aureus ATCC 6538

Each week

Catalase (3% H2O2) S.aureus ATCC 6538 E.casseliflavus ATCC 700327

Each week

Coagulase/Staphytect S.aureus ATCC 6538 S.sciuri ATCC 29061

Each week

5. Documentation of Results

5.1. Results Recording and Further ID

5.1.1. When complete, the results of all characterisation tests conducted for each isolate are to be recorded on the Isolate ID Record forms. Record any additional information in the Additional Comments field.

5.1.2 Determine whether further ID of the isolate is required. This will be stated in the SOP relevant to the test sample from which the organism was recovered.

5.1.2. If the organism does not require any further ID, indicate this on the Isolate ID Record form and initial and date against all information recorded for the isolate. The subcultured isolate and original test sample may be discarded when all ID work and documentation is complete (check the retention requirements of the test sample and associated isolates before disposal see Table 2).

5.1.3. All completed Isolate ID Record forms are to be reviewed and approved upon completion. Once approved, all ID results are to be entered into the Sample ID Number Allocation spreadsheet.

5.1.4. The approved Isolate ID Record forms are to be filed in the Completed Identifications File. A separate file should exist for each ID category (see Table 1). File all forms in numerical sequence according to the assigned USI. This will ensure quick retrieval of ID records when required.

6. Monitoring Trends

6.1. Trending of Microbial ID Results

6.1.1. Where appropriate, trending of microbial isolates will be conducted on a routine basis in conjunction with trending of test results for the different sample types; including Environmental and Personnel monitoring samples, Settle plates, Disinfectant samples, Water, Non-sterile & Raw Material samples and Sterility test failures. The Microbial ID Sample ID Number Allocation spreadsheet can be used to filter data specific to different sample types to aid in trending.




7.3. ID Flowchart: Gram Positive Cocci

Conduct Gram Stain from pure, fresh culture

Record Cellular arrangement and Gram ID on the Isolate Identification Record

Conduct Additional Tests

Catalase Coagulase/Staphytect

Organism is Coagulase Positive?

(Presumptive S.aureus)

Record Catalase and Coagulase results on the Isolate Identification Record

Organism requires Further ID to species

level?

YES

Further identification needed.

NO

NO

YES

Finalise and Review Isolate Identification Record

ID result to be approved and entered where appropriate i.e. Sample Identification Number

Allocation Spreadsheet, MicroTrack.

Retention of Isolate required?

NO

Discard Isolate

YES Retain isolate for the specified

period (see Table 2).

If the organism is suspected to be a

Gram-negative coccus conduct a

KOH test

KOH Positive?

The organism is a GNC. Further identification is needed




7.6. ID Flowchart: Yeasts

Conduct Gram Stain from pure, fresh culture

Organism requires Further ID to species

level?

Further identification needed

Record Cellular arrangement and Gram ID on the Isolate Identification Record

YES NO

Finalise and Review Isolate Identification Record

ID result to be approved and entered where appropriate

i.e. Sample Identification Number Allocation Spreadsheet.

Retention of Isolate required?

YES NO

Retain isolate for the specified period (see Table 2).

Discard Isolate




7.7.3. Micrococcus species

Figure 09: Gram Stain of Micrococcus cells

7.7.4. General Information

Micrococcus species are strictly aerobic. Micrococcus luteus produces yellow colonies. Cells are large Gram-positive cocci arranged in tetrads. Catalase positive. Optimum growth temperature is between 30-37C. Sources - Environmental Soil, Water; Human Skin

7.7.5. Streptococcus species

Figure 10: Gram Stain of Streptococcus cells

Tetrads

Chains




Sources - Environmental Soil, Contaminated Water; Human Gastrointestinal tract, Female Genital Tract (C.perfringens)

7.9. Gram Positive Rods Non-sporing 7.9.1. Lactobacillus species

Figure 13: Gram Stain of Lactobacillus cells

7.9.2. General Information

Gram-positive large rods, non-spore forming, anaerobic or microaerophilic, occur singly or in pairs.

Convert Lactose and other sugars to Lactic Acid. Sources - Normal flora of the following sites of Humans and Animals: Mouth Oropharynx Gastrointestinal tract Female Genital Tract Food Dairy products

7.9.3. Corynebacterium species Figure 14: Gram Stain of Corynebacterium cells

Characteristic Palisade or V shape arrangement. Cells pictured here have tapered ends, however ends may also appear club shaped.




7.10.5. Other commonly isolated Non-fermenting Gram-negative rods include:

Achromobacter (Alcaligenes) xylosoxidans

Alcaligenes xylosoxidans was reclassified as Achromobacter xylosoxidans in 1998. It is both catalase- and oxidase positive.

Alcaligenes species Colonies have a thin, spreading irregular edge. It is catalase negative, oxidase positive and motile.

Brevundimonas species Brevundimonas vesicularis and Brevundimonas diminuta grow slowly on ordinary nutrient media. It forms a carotenoid pigment that produces yellow or orange colonies.

Elizabethkingia species Elizabethkingia (formerly Chryseobacterium) meningosepticum, is the species of Elizabethkingia most often associated with serious infection. E. meningosepticum is non-motile and oxidase positive. E. indologenes is also non-motile and oxidase positive.

Comamonas species It is motile, oxidase and catalase positive.

Methylobacterium species The organism is oxidase positive and motile, but both of these characteristics may be weak. Methylobacterium species are Gram-negative but may stain poorly or show variable results. It has a characteristic microscopic appearance because individual cells contain large, non-staining vacuoles.

Ochrobactrum species Colonies appear circular, low convex, smooth, and shining. Mucoid colonies may be produced on some media.

Ralstonia species Ralstonia pickettii (formerly Burkholderia pickettii) is non-pigmented, oxidase-positive, and will grow at 41C.

Shewanella species Shewanella putrefaciens is oxidase-positive and motile.

Sphingobacterium species They are oxidase-positive and non-motile. Colonies produce yellow pigment.

Stenotrophomonas species Catalase positive and Oxidase negative.

MICLAB 070 Sample

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