Title: Identification of Microorganisms ATS-ATP-5925 Page: 1 of 30. Approval Block. Prepared by: Signature Date Margaret Crouse 18 JUN 2014

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1 Title: Identification of Microorganisms ATS-ATP-5925 Page: 1 of 30 Approval Block Prepared by: Signature Date Margaret Crouse 18 JUN 2014 Reviewed by: Signature Date Brian Flynn 18 JUN 2014 Approved by: Signature Date Kristal Jewell 18 JUN 2014 Historical Reference Section: Section Name Section Number (i.e., 5.2.1) Revision Affected Explanation of Change References 3 A Added references Materials 4 A Added materials Quality Control 7.5 A Added detail to Document Control Approval Block N/A A Added Approval Block 1. Purpose To describe the procedures for the preliminary identification of bacteria and fungi isolated from personnel, water, and manufacturing environment. 2. Scope This procedure shall be followed by personnel performing viable sample analysis. Additional testing and or deviation from this SOP shall be agreed upon prior to the start of testing.

2 Title: Identification of Microorganisms ATS-ATP-5925 Page: 2 of References 1. Medically Important Fungi, 5th edition, Davise Larone. 2. Koneman's Color Atlas and Textbook of Diagnostic Microbiology, 6th Edition, Washington C. Winn, Stephen D. Allen, William M. Janda, Elmer W. Koneman, Paul C. Schreckenberger. 3. USP<797> Pharmaceutical Compounding Sterile Preparations 4. USP<1116> Pharmaceutical Compounding Sterile Preparations 5. ISO , -2: ATS-SOI-5700 Rev (A) Standard Operating Procedure for the Operation of the 7. Model 3110 Forma Series II Water Jacket CO 2 Incubator 8. ATS-SOI-5705 Rev (A) Standard Operating Procedure for the Operation of the Dickson KT621 Remote Sensing Temperature Recorder 9. ATS-SOI-5715 Rev (A) Standard Operating Procedure for the Operation of the Thermo Scientific Revco Laboratory Refrigerator 10. ATS-SOI-5720 Rev (A) Standard Operating Procedure for the Operation of the NuAire Model NU Labgard Class II, Type A2 Laminar Flow Biological Safety Cabinet 11. ATS-SOI-5800 Rev (B) Standard Operating Procedure: Procedure for Tracking Viable Media 12. ATS-SOI-5820 Rev (A) Standard Operating Procedure: Viable Sample Workflow 13. ATS-ATP-5850 Rev (A) Standard Operating Procedure: USP <797> Compliant Sample Analysis 14. ATS-ATP-5855 Rev (A) Standard Operating Procedure: USP <1116> Compliant Sample Analysis 15. ATS-ATP-5856 Rev (A) Standard Operating Procedure: USP <1116> Compliant Sample Analysis WFI 16. ATS-ATP-5860 Rev (A) Standard Operating Procedure: ISO Compliant Sample Analysis 17. ATS-SOI-5880 Rev (A)Standard Operating Procedure: Gowning Procedures 18. ATS-SOI-5900 Rev (A) Logbook Entries 19. ATS-SOI-5901 Rev (-) Laboratory Results Documentation Review 20. ATS-SOI-5905 Rev (A) Standard Operating Procedure: Organization of plates in the incubator 21. ATS-ATP-5920 Rev (A) Standard Operating Procedure: Fungal Slide Preparation

3 Title: Identification of Microorganisms ATS-ATP-5925 Page: 3 of Equipment and Materials Equipment: All relevant equipment required Materials: 1. All required laboratory materials 2. All appropriate logbooks 3. All relevant forms 5. Safety 1. See ATS-SOI-5880 for proper gowning procedure. All work must be performed in a certified Biological Safety Cabinet. See ATS-SOI-5720 for proper BSC operating procedure. 2. Refer to manufacturer s safety precautions and Material Safety Data Sheet (MSDS) for appropriate protective equipment and safe handling procedures when using chemicals. 6. Responsibility 1. It is the responsibility of the President to maintain this document and oversee these activities. 2. It is the responsibility of the individual(s) performing this task to follow this procedure.

4 Title: Identification of Microorganisms ATS-ATP-5925 Page: 4 of Procedure Table of Contents 7.1. Introduction 7.2. Administration and Preparation of Isolates Classification of test Samples Unique Sample Identifier Preparing Isolates for ID 7.3. ID of Isolates The Gram Stain Technical Information The Potassium Hydroxide (KOH) test The Catalase Test The Coagulase Test Sporulation and the Spore Stain The Oxidase Test 7.4. Quality Control (QC) QC and Traceability of Reagents/Kits 7.5. Documentation of Results Results Recording and Further ID 7.6. Appendices Colony Morphology Cellular Shape and Arrangement ID Flowchart: Gram Positive Cocci ID Flowchart: Gram Positive Rods ID Flowchart: Gram Negative Rods Gram Positive Cocci Gram Positive Rods-Sporing Gram Positive Rods-Non-sporing Gram Negative Organisms (Non-glucose fermenters) Gram Negative Organisms (Glucose fermenters)

5 Title: Identification of Microorganisms ATS-ATP-5925 Page: 5 of Introduction Microrganisms will require identification (ID) to a general category, for example Gram negative fermenting rod, or a full Genus or Genus species ID dependent on the Quality Agreement between ATS and the client. The following sections detail the procedures for the preliminary ID of microorganisms. Further ID to species level is to be conducted for conformation Administration and Preparation of Isolates Classification of Test Samples Microbiological tests that have recovered organisms, which require ID, are to be classified into three ID categories according to the nature of the test sample. These categories are listed in Table 1. Table 1: ID Categories for Microbiological Tests Category Prefix ID Category Classification of Test Samples 01 Environmental Air and surface samples 02 Personal Glove samples of personnel 03 Settle Plates Agar plates exposed in the clean room 04 Water Water used for Unique Sample Identifier Each microbial isolate requiring ID will be assigned a Unique Sample Identifier (USI) relative to the category to which the sample belongs. The isolate identifier is a 6-digit number comprised of the Category Prefix (Table1), an Isolate Number and the number of differing isolate types recovered from the test sample. The USI distinguishes each microbial isolate requiring ID and provides a point of reference between the test result and the associated ID results of organisms recovered from the test. The following example describes how the USI is created.

6 Title: Identification of Microorganisms ATS-ATP-5925 Page: 6 of 30 Example: Following the specified incubation period, and air sample results in the growth of 2 colony-forming units (CFU). These CFU are of differing macroscopic morphology i.e. they are two different isolate types. The organisms isolated from the sample require ID. The USI assigned is as follows: Preparing Isolates for ID For example where the USI where the USI is for e.g /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 , and This is further illustrated in the following example: The allocated USI for a given test sample is as follows: /3 3 isolate types were recovered from this sample. These isolate types will have a defined isolate type number as follows: Isolate Type 1: Isolate Type 2: Isolate Type 3: Define the isolate types in the test sample using the given nomenclature Fill out an Isolate Identification Record form appropriate to the ID Category of the test sample.

7 Title: Identification of Microorganisms ATS-ATP-5925 Page: 7 of Record the details as follows: Sample ID # - this is the USI Total CFU/Growth - the total number of colonies recovered from the test sample Sample Details - including sample/test date, location, Chain of Custody #, etc. Sample Type - to be specified where applicable Isolate ID # - Macroscopic Description morphological description of the isolate; size, color, shape, margin, luster, etc. (see Appendix 6.1) CFU number of colony forming units bearing the same macroscopic description Streak each isolate type onto the respective TSA plate labeled with the Isolate type marking. Use the following procedure to ensure growth of single, pure, wellisolated colonies Flame a wire loop until red-hot by passing through the hottest part of a Bunsen burner flame OR take a sterile disposable loop. If using a wire loop, allow the loop to cool before proceeding (to cool the loop quickly touch the loop against the agar of the TSA plate to be used) With the sterile loop, lightly touch the colony to be streaked and immediately streak the growth onto the first quadrant of the TSA plate (Fig 1-1) Flame the loop and allow to cool OR take a fresh sterile disposable loop and streak from quadrant 1 to quadrant 2 (Fig 1-2). Be sure that you streak several times through quadrant 1 to pick up some organisms on the loop Repeat the same general procedure for quadrants 3 and 4 (Fig 1-3 and 1-4). Be sure to flame the loop OR take a fresh sterile disposable loop between each quadrant. Extend the last streak into the center of the plate. Note: A fresh sterile loop must be used for each new set of streak quadrants OR a wire loop must be flamed and cooled. Using a sterile loop facilitates the dilution of organisms with each set of streak lines enabling the growth single colonies.

8 Title: Identification of Microorganisms ATS-ATP-5925 Page: 8 of 30 Figure 1: Plate Streaking for Single Colonies Place the Isolate ID Record form for the test sample in the In Process folder in the laboratory ID of Isolates Note: Before proceeding with the following ID procedures visually inspect all streaked isolate cultures for purity and the presence of single, isolated colonies. If the culture appears mixed, re-streak the isolate for purity. Only single, isolated colonies are to be used for the following characterization tests. Purity cannot be visually confirmed in areas of heavy of confluent growth. Note: If a particular isolate cannot be cultured on solid media, inoculate the organism into 10 ml TSB to resuscitate and incubate at C for hrs. The resuscitated broth maybe gram stained and then plated onto TSA (if required). Note: Appendices 6.3 to 6.6 will assist in the identification of isolates. These should be used as a guide. Follow the flowcharts in to determine the appropriate tests to perform. For API kits follow the instructions listed in the package insert. At minimum all bacterial isolates should be Gram stained and have a catalase test performed. For all fungal isolates a fungal slide preparation should be done as per ATS- ATP-5920.

9 Title: Identification of Microorganisms ATS-ATP-5925 Page: 9 of The Gram Stain Organisms are classified according to their gram stain reaction. The gram stain is the primary and most critical step in the identification process. Gram-positive bacteria have thicker and denser peptioglycan layers in their cell wall. The CV-I (crystal violet-iodine) molecular complex is larger than the crystal violet or iodine molecules that initially entered the cell wall and cannot pass through the thick peptidoglycan. Gram-positive bacteria must have an intact cell wall to produce a positive reaction. The cell wall of gram-positive bacteria will deteriorate as the plate culture ages therefore cultures must be fresh when conducting the Gram Stain. Gram-negative cells do not retain the CV-I complex, due to a far less complex peptidoglycan layer in the cell wall are stained using a counter stain Prepare a smear: Using a sterile loop, lightly touch the colony to be examined and emulsify into a drop of sterile water on a glass slide. Note: Avoid picking up too much growth as this will result in a heavy smear, which will not stain effectively and cannot be examined with accuracy 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 Flood the slide with Crystal Violet solution and stand for 1 minute (Fig 2A) Rinse off Crystal Violet solution with water and tilt the slide to drain excess water Flood the slide with Iodine and stand for 1 minute (Fig 2B) Rinse off Iodine with water and tilt the slide to drain excess water Decolorize until solvent running from the slide is colorless, 3-60 seconds (Fig 2C).

10 Title: Identification of Microorganisms ATS-ATP-5925 Page: 10 of 30 Note: Prolonged contact with decolorizing solution will result in over-decolorization of cells Rinse off Decolorizer with water and tilt the slide to drain excess water Flood the slide with counterstain and stain for seconds Rinse off counterstain with water Blot with blotting paper or paper towel or allow to air dry. Do not rub Examine the smears under Oil Immersion Record the cellular arrangement (see Appendix 6.2) and Gram stain result of the organism on the Isolate Identification Record form Interpretation Gram-positive Gram-positive organisms stain purple. Gram-negative Gram-negative organisms stain pink/red Quality Control 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 Technical Information Some Gram-positive bacteria may appear Gram-negative in whole or in part e.g. some strains of Gram-variable Bacillus and Clostridium. Yeast will appear Gram-positive and will be visibly much larger than a prokaryotic bacterial cell. All Gram-variable and suspected over or under-decolorized organisms are to be subject to a KOH test (see section3.3).

11 Title: Identification of Microorganisms ATS-ATP-5925 Page: 11 of 30 Figure 2: The Gram Stain Prepare Smear Heat Fix Cells Gram Stain

12 Title: Identification of Microorganisms ATS-ATP-5925 Page: 12 of The Potassium Hydroxide (KOH) test Known Gram-variable organisms and organisms which have lost some of their cell wall integrity (due to aging culture), appear Gram-negative on staining, resulting in possible misidentification. The KOH test (also known as the String Test ) aids in the differentiation of Grampositive bacteria from Gram-negative organisms. In the presence of potassium hydroxide, Gram-negative cell walls are broken down, releasing viscid chromosomal material, which causes the bacterial suspension to become think and stringy. Most Gram-positive organisms remain unaffected. Note: The KOH test can only be performed using colonies grown on solid medium. The KOH test is to be performed on all non spore-forming rods and where the Gram reaction of any organism cannot be determined due to Gram variability or poor staining. The KOH test is positive for 100% of all Gram-negative organisms but only 97% negative for all gram-positive organisms. A negative result can only be obtained from a Gram-positive organism; a positive result can be obtained from all Gramnegative organisms and some Gram-positive organisms Place one drop of 3% KOH solution on a clean microscope slide Using a sterile loop pick up 2-3 colonies and emulsify in the KOH to make a dense suspension. The suspension should appear cloudy if a sufficient number of colonies have been picked Mix the suspension continuously for up to 60 seconds, and then gently pull the loop away from the suspension. If the organism is Gram-negative the suspension will form a string which adheres to the loop and stretches from the slide (Fig 3). Note: A positive result may be evident in as little as 5 seconds Record the KOH result of the organism on the Isolate ID Record form.

13 Title: Identification of Microorganisms ATS-ATP-5925 Page: 13 of 30 Figure 3: Positive KOH Test Interpretation Positive result: Gram-negative organisms become thick and stringy and form long strands Negative result: Organism is Gram-positive and remains unaltered The Catalase Test This tests detects the catalase enzyme present in most cytochrome-containing aerobic and anaerobic bacteria. The catalase enzyme decomposes hydrogen peroxide (H 2 O 2 ) to release oxygen and water. Note: Hydrogen peroxide solution should be stored at 2-8 C. The solution is light sensitive and should be stored appropriately avoiding any undue exposure to light. The Catalase test is to be conducted on all Gram-positive bacteria Place a drop of 3% H 2 O 2 solution on a clean microscope slide Using a sterile loop, pick up a single isolated colony and immerse the loop and the adhering colony mass into the drop of H 2 O 2 solution.

14 Title: Identification of Microorganisms ATS-ATP-5925 Page: 14 of Look for vigorous bubbling occurring within 10 seconds Record the catalase result of the organism on the Isolate ID Record form Interpretation Positive result: Vigorous bubbling indication the presence of catalase enzyme Negative result: No bubbling Quality Control Control organisms are to be tested with each use, as H 2 O 2 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 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 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. Sure-Vue Select Staph kit is a slide agglutination assay for the qualitative detection of coagulase (both clumping factor and protein A) to identify Staphylococcus aureus to the exclusion of other species of staphylococci. This test is for use on pure culture samples suspected of being S. aureus. The ASI Staphslide Latex Test does detect methicillin resistant S. aureus (MRSA) strains that produce clumping factor and protein A.

15 Title: Identification of Microorganisms ATS-ATP-5925 Page: 15 of 30 Note: Latex slide agglutination kits must be stored at 4 C away from direct sunlight or hear sources. Ensure the caps are securely fitted after each use to prevent contamination and drying out of the reagent Allow all reagents and samples to warm to room temperature (20-30 C) before use. Remove reagents from foam holders. Gently mix the reagents before use; avoid foaming Add a drop of the LATEX REAGENT to a well of the test card Using a disposable stirrer, collect a visible amount of an isolated colony about 2 mm in size from the overnight culture Emulsify the culture sample in the LATEX REAGENT on the card. Discard the stirrer into an appropriate biohazard container Add one free-falling drop of REACTIVE or NONREACTIVE CONTROL from the dropper vial supplied. Note the location of each sample by using the numbers located below and to the left of each circle Gently tilt and rotate the card in a complete circular motion for up to 45 seconds, or until agglutination is evident, whichever comes first. Positive reactions usually occur within seconds View the mixture on the card, using only a high intensity light source. Do not use a magnifying lens Record the results. Dispose of the card into an appropriate biohazard container Interpretation Positive result: Any degree of agglutination as compared to the negative control. Negative result: Smooth suspension with no visible agglutination after 45 seconds Quality Control Control organisms are to be tested with each use. Do not use the test if reactions with the control organisms are incorrect. QC organisms Positive control Staphylococcus aureus ATCC 6538

16 Title: Identification of Microorganisms ATS-ATP-5925 Page: 16 of 30 Negative control Staphylococcus epidermidis Technical Information All positive, presumptive S. aureus cultures are to be confirmed further Sporulation and The Spore Stain Member of the genus Bacillus have the ability to produce endospores under unfavorable growth conditions. The conditions under which sporulation takes place determine the spore characteristics in terms of chemical and/or heat resistance. Sporulation is not always evident in fresh cultures grown on general-purpose media, older cultures enhance sporulation Prepare a smear and heat fix the cells to the slide by passing the slide thorough the hottest part of the flame three times. Allow to cool Hold the slide level over a sink or staining tray. Place several drops of spore staining solution on the slide. Use enough spore stain to cover the entire smear on the surface of the slide Allow the stain 10 minutes to react with the cells on the slide Flame the slide twenty times by passing it through the flame of a Bunsen burner or similar flame source Allow the slide to cool Rinse the slide by dipping it in a large container of water several times. You may also rinse the slide under a running faucet in a sink. Be sure the flow of water is gentle so as not to wash the cells off the surface of the slide. Do not let the stream of water run directly on the smear. Let the water hit above the smear and flow across the surface of the slide Apply the counterstain, Safanin (0.25%), to the slide and allow the counterstain to react for approximately 30 seconds Rinse the slide as above and blow the slide dry with paper towels.

17 Title: Identification of Microorganisms ATS-ATP-5925 Page: 17 of Examine the stained cells under the oil-immersion objective. The deeply intense spore-staining will stain the endospores (if present) dark green while the counterstain, Safranin, will stain the rest of the cellular material pink to red, along with cells that do not contain any endospores Interpretation Positive Spores stain green Negative Vegetative cells stain pink/red Note: If sporulation is evident the organism is presumptively identified as a Bacillus spp Quality Control Spore forming and non-spore forming reference cultures are to be used for quality control of the spore staining procedure and reagents. QC organisms Positive Bacillius subtilis ATCC 6633 Negative Escherichia coli ATCC The Oxidase Test The oxidase test is a qualitative procedure for determining the presence or absence of cytochrome c oxidase activity in bacteria Open the BBL DrySlide Oxidase pouch and remove the required number of slides. After opening the remaining slides may be stored at room temperature for up to one week. Fold the top of the pouch over once and seal tightly with a self-adhesive sticker (provided). Discard unused slides 1 week after the package is opened. Do not place used or partially used BBL DrySlide Oxidase back into the pouch Using an appropriate inoculation device, pick isolated colonies or a sweep of confluent growth from the culture to be tested Smear the organism directly onto the reaction area. Each reaction area can accommodate up to 4 tests. To ensure a proper reaction, spread the inoculum on the reaction area to a 3-4 mm size Examine the reaction area for appearance of a dark purple color within 20 sec. Disregard color development after 20 sec.

18 Title: Identification of Microorganisms ATS-ATP-5925 Page: 18 of Alternately, oxidase dropper may be used. Using a sterile swab, remove approximately 1-2 isolated colonies from the culture to be tested. Add one free flowing drop of oxidase reagent. Examine as described above in Quality Control QC organisms Positive Pseudomonas aeruginosa ATCC 6633 Negative Escherichia coli ATCC Quality Control (QC) QC and Traceability of Reagents/Kits Reagents are to be challenged at each use (i.e. once daily) using the stated QC organisms (see Table 3). Staining reagents are to be challenged upon opening, using the stated QC organisms. They may then be verified for use The challenge organisms are to be no more than 24 hours old. Ensure a pure, fresh culture is always available for QC.

19 Title: Identification of Microorganisms ATS-ATP-5925 Page: 19 of 30 Table 2: Reference table of QC Organisms for Reagents/Kits Test QC Organism(s) Frequency Gram Stain S. aureus ATCC 6538 E. coli ATCC 8739 Reagents to be verified up on opening. Spore Stain B. subtilis ATCC 6633 E. coli ATCC 8739 Reagents to be verified up on opening. Potassium Hydroxide (3%) E. coli ATCC 8739 Each week KOH S. aureus ATCC 6538 Catalase (% H 2 O 2 ) S. aureus ATCC 6538 Each week E. casseliflavus ATCC Coagulase S. aureus ATCC 6538 Each week S. epidermidis Oxidase Pseudomonas aeruginosa ATCC E. coli ATCC 8739 Each week 7.5. Documentation of Results Results Recording and Further ID When complete, the results of all characterization tests conducted for each isolate are to be recorded on the Isolate ID Record forms and the Laboratory Chain of Custody forms. Record any additional information in the Additional Comments field Determine whether further ID of the isolate is required If the organism does not require any further ID, indicate this on the Isolate ID Record form and initial and date against all information recoded for the isolate. The subcultured isolate and original test sample will be held for two weeks after the report has been sent to the client. Bacterial isolate will be held in the bin at the bottom of the refrigerator. Fungal isolates will be held in the fungal incubator to prevent contamination of the refrigerator Transfer results to the Viable Results Excel spreadsheet. Completed Isolate ID Record forms are to be stored in the packet for the set of samples Give the completed packet including Laboratory Chain of Custody forms, Isolate ID records, Field Chain of Custody forms, and any associated API records to the Quality Manager. the Viable Results Excel spreadsheet to the Quality Manager for review as per ATS-SOI-5901.

20 Title: Identification of Microorganisms ATS-ATP-5925 Page: 20 of Appendices Colony Morphology Describe all of the following colony characteristics: SIZE OF COLONY- in mm CHROMOGENISES (Pigmentation) - white, off while, cream, pink, brown, etc. SHAPE OF COLONY (Fig. 6)- circular (if less than 1mm in diameter shape is punctiform), irregular, filamentous or rhizoid EDGE/MARGIN OF COLONY (Fig. 6) - entire, undulate, curled, lobate, etc. OPACITY OF COLONY- transparent, opaque, translucent or iridescent (changing colors in reflected light) ELEVATION OF COLONY (Fig. 6) - raised, convex, flat, umbonate, etc. SURFACE OF COLONY-smooth, glistening, rough, dull or rugose (wrinkled) CONSISTENCY- butyrous (buttery), viscid (sticks to loop), brittle/friable (dry, breaks apart), or mucoid Figure 6: Form, Elevation and Margin of Colonies

21 Title: Identification of Microorganisms ATS-ATP-5925 Page: 21 of Cellular Shape and Arrangement Most bacteria that will be isolated and examined in the laboratory will appear in one of two basic shapes- coccus or rod/bacillius. Coccus- The cocci are spherical or oval bacteria having one of several distinct arrangements based on their planes of division (Fig 7). Division in one plane produces either a diplococcus or streptococcus arrangement. Division in two planes produces a tetrad arrangement. Division in random planes produces a staphylococcus arrangement. Rod/Bacillus- Bacilli are rod-shaped bacteria. Bacilli all divide in one plane producing a bacillus, streptobacillus, or coccobacillus arrangement (Fig 7). Pleomorphic rods of the genus Corynbacterium also divide in one plane, however they possess a characteristic v-shaped arrangement known as a palisade (Fig 7). this is due to s snapping movement, which occurs immediately after cell division, which brings the cells into this arrangement.

22 Title: Identification of Microorganisms ATS-ATP-5925 Page: 22 of 30 Figure 7: Cellular Arrangements

23 Title: Identification of Microorganisms ATS-ATP-5925 Page: 23 of ID Flowchart: Gram Positive Cocci Gram positive cocci Catalase + - Staphylococci/ Micrococci Streptococci/ Enterococci Coagulase + - API Strep Presumptive S. aureus API Staph Modified Oxidase + - Micrococci Coag negative Staph

24 Title: Identification of Microorganisms ATS-ATP-5925 Page: 24 of ID Flowchart: Gram Positive Rods Gram positive rods Catalase + - Spores/ large rods + - Very tapered rods Bacillius API 50 CHB/E Small rods of characteristic shape Partial discoloring (Granulation) Corynebacterium API Coryne Lactobacillus API 50CHL

25 Title: Identification of Microorganisms ATS-ATP-5925 Page: 25 of ID Flowchart: Gram Negative Rods Gram negative rods Oxidase + - API 20 NE Enterobacteriaceae/ API 20 E

26 Title: Identification of Microorganisms ATS-ATP-5925 Page: 26 of Gram positive cocci Gram positive cocci Staphylococcus species Gram-positive, non-motile, non-sporing cocci occurring singly, in pairs and in irregular clusters Colonies are opaque and may be white, cream and occasionally yellow and orange Optimum growth temperature is between C They are facultative anaerobes and have a fermentative metabolism Catalase positive and oxidase negative Staphylococcus aureus is a primary pathogen o Coagulase positive* *(S. aureus is not the only coagulase positive Staphylococcus-see Table 5 below) Table 5: Commonly isolated Coagulase positive and negative Staphylococcus Species *Other non-pathogenic coagulase positive Staphylococci Coagulase negative Staphylococci (there are more than 30) S. hyicus S. epidermidis S. capitis S. schleiferi sub spp. coagulans S. saprophyticus S. cohnii S. intermedius S. simulans S. warneri S. haemolyticus S. hominis Gram positive cocci Micrococcus species 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 C Gram positive cocci Streptococcus species Streptococcus species are facultatively anaerobic Cells are Gram-positive non-motile cocci arranged in chains or pairs Catalase negative Optimum growth temperature is between C

27 Title: Identification of Microorganisms ATS-ATP-5925 Page: 27 of Gram Positive Rods-Sporing Bacillus species The genus Bacillus comprises in excess of 60 species, commonly found in the environment and as laboratory contaminants. Bacillus spp. are Gram-positive rods arranged in pairs or chains with rounded or square ends and usually have a single endospore Colonies are flat and irregular, often with numerous undulated outgrowths of long filamentous chain of bacilli Aerobic or facultatively anaerobic and most species are motile by peritrichous flagella Fermentative metabolism Oxidase positive (most species) Catalase positive (most species) Gram Positive Rods-Non-sporing Lactobacillus species Gram-positive large rods, non-spore forming, anaerobic or microaerophilic, occur singly or in pairs Convert lactose and other sugars to Lactic Acid Corynebacterium species Characteristic Palisade or v shaped arrangement (Chinese characters) Corynebacterium species are Gram-positive, pleomorphic, non-spore forming and slightly curved rods with tapered or clubbed ends. Cells may occur singly or in pairs, often in a V formation due to a snapping mechanism occurring after cell division. Cells will usually stain weakly and unevenly giving a beaded appearance. This is due to old cells storing inorganic phosphates in their cell wall, which appear as metachromatic granules when stained. Aerobic or Facultatively anaerobic Catalase positive

28 Title: Identification of Microorganisms ATS-ATP-5925 Page: 28 of Gram Negative Organisms (Non-glucose fermenters) Acinetobacter species Species of the genus Acinetobacter are strictly aerobic non-fermentative, nonmotie Gram negative bacilli. They show predominantly a coccobaccillary morphology on nonselective agar and appear in pairs under the microscope. They may not readily decolorize on Gram staining and can be pleomorphic, and sometimes appear as Gram-positive cocci. Colonies are normally smooth, sometimes mucoid, pale yellow to greyish-white and some environmental strains may produce a diffusible brown pigment. Most strains have an optimum growth temperature of C Catalase positive Oxidase negative Pseudomonas species Species of the genus Pseudomonas are strictly aerobic, glucose non-fermenting Gram negative rods. Motile by means of a single polar flagellum Growth temperatures range from 5 C-42 C. Optimum growth temperature is 37 C Oxidase positive Catalase negative P. aeruginosa is one of the only members of the species that can sustain growth at 42 C. The characteristic blue-green appearance of cultures is due to the production and mixture of pigments-pyrocyanin (blue) and pyroverdin (fluorescein, yellow). Some strains produce other pigments, such as pyrorubin (red) or pyromelanin (brown). P. putida and P. fluorescens are members of the fluorescent group of pseudomonads. Unlike P aeroginosa they are unable to grow at 42 C and do not produce pyrocyanin. P. stutzeri produces smooth, intermediate and rough colonies (sometimes yellow pigmented) non-selective agar. The latter can resemble colonies of Burkholderia pseudomallei or Bacillus species P. alcaligenes and P. psedoalcaligenes are both non-pigmented. Other commonly isolated Non-fermenting Gram-negative rods include: Achromobacter (Alcaligenes) xylosoxidans o Catalase and oxidase positive Alcaligenes species

29 Title: Identification of Microorganisms ATS-ATP-5925 Page: 29 of 30 o Colonies have a thin, spreading irregular edge. It is catalase negative, oxidase positive and motile Brevvundimonas species o Brevundimonas vesicularis and Brevundimonas diminuta grow slowly on ordinary nutrient media. It forms a carotenoid pigment that produces yellow or orange colonies. Elizabethkingia species o 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 o It is motile, oxidase and catalase positive. Methylobacterium species o The organism is oxidase positive and motile, but both of these characteristics may be weak. Methylobacterium species are Gramnegative but may stain poorly or show variable results. It has a characteristic microscopic appearance because individual cells contain large, non-staining vacuoles. Ochrobactrum species o Colonies appear circular, low convex, smooth, and shining. Mucoid colonies may be produced on some media. Ralstonia species o Ralstonia picketti is non-pigmented, oxidase positive, and will grow at 41 C. Shewanella species o Shewanella putrefaciens is oxidase positive and motile. Sphingobacterium species o They are oxidase positive and non-motile. Colonies produce yellow pigment. Stenotrophomonas species o Catalase positive and oxidase negative

30 Title: Identification of Microorganisms ATS-ATP-5925 Page: 30 of Gram Negative Organisms (Glucose fermenters) E. coli E. coli is the head of the large bacterial family, Enterobacteriaceae, the enteric bacteria, which are facultatively anaerobic Gram-negative rods. A number of genera within the family are human intestinal pathogens (e.g. Salmonella, Shigella, Yersenia). Several others are normal colonists of the human gastrointestinal tract (e.g. Escherichia, Enterobacter, Klebsiella), but these bacteria as well may occasionally be associated with diseases of humans. The enterics ferment glucose producing acid and gas Optimum growth temperature is 37 C Oxidase negative Produce peritrichous flagella