Microbiology Test Methods for Pharmaceutical Laboratrory
Method 001: Preparation of Malt Extract Agar
1. Purpose
This operator instruction describes the method for the preparation of Malt Extract Agar used in the microbiology laboratory.
2. Scope and application
Malt Extract Agar is a medium used for detection, isolation and enumeration of yeasts and moulds.
3. Materials required list
i. Plastic spoon
ii. Beaker (microwaveable)
iii. Measuring cylinder
iv. RO water
v. Malt Extract Agar (Oxoid CM59)
vi. 1N NaOH, 1N HCl
vii. Plastic pipettes
viii. Clean glass bottles
4. Work instruction
4.1. Weigh 50g per litre of Malt Extract Agar powder into a beaker.
4.2. Add approximately half the required volume of RO water, mix, and boil to dissolve in the microwave.
4.3. Makeup to the required volume with RO water.
4.4. Adjust pH to 5.4 +2.
4.5. Dispense 150mL into clean glass bottles.
4.6. Sterilize by autoclaving at 115°C for 10 minutes.
4.7. Final pH after sterilization should be 5.4 + 0.2.
QUALITY CONTROL REQUIREMENTS |
STORAGE: BULK – 6 months. POURED PLATES – 2 weeks. |
ECO-METRIC EVALUATION / FERTILITY CONTROL ORGANISM: Positive: C.albicans A.T.C.C: 10231 Growth Index: >=3 Negative: N/A Growth Index: N/A |
INCUBATION CONDITION: Temperature: 25° ± 1°C Time: 3-5 days |
Method 002: Enumeration of Biological Indicators
1. SCOPE AND APPLICATION
2. REAGENTS AND MATERIALS REQUIRED
3. GENERAL TEST PROCEDURE
4. CALCULATION OF RESULTS
10-4 10-5 10-6
240 24 2
= 2.4 x 106 cfu/spore strip
Method 003: Membrane Filtration Techniques
1. Scope and application
This general test method sets out the procedure to be followed for estimating the number of colony forming units (cfu) using the membrane filtration technique.
This procedure is applicable only to liquids, which can be efficiently filtered without causing a build-up on the membrane (eg. Listerine, Ora-sed jel) and will be followed to test purified water, rinse water and alcohol.
2. REAGENTS AND MATERIALS REQUIRED
2.1 Membrane filtration assembly unit consisting of:
2.1.1 Manifold
2.1.2 Magnetic filter holder (funnel)
2.1.3 S/S collecting vessel and clamp
2.1.4 Vacuum hoses
2.2 Grid marked 0.45μm membrane filters of 47 mm to 50 mm diameter (sterile), individually wrapped. Hydrophobic edge membranes for products, hydrophilic edge for testing of water and Supor-200 47mm 0.2μm for alcohol.
2.3 A source of vacuum.
2.4 Forceps – flat tip.
2.5 Sterile graduated pipettes.
2.6 Appropriate media required for a particular product test.
2.7 Laminar flow unit.
2.8 Ear plugs (optional)
2.9 Gloves
3. GENERAL TEST METHOD
SAFETY NOTE: Do NOT use Bunsen Burner when filtering products containing alcohol.
3.1 Decontaminate laminar flow unit by turning on the UV lamp for 15 minutes. Open unit and decontaminate work bench using an approved decontaminating solution.
3.2 Place manifold on bench.
3.3 Aseptically remove filter funnel from the autoclave bag by holding the top of the funnel in one hand and opening the autoclave bag at the base of the funnel with the other hand and exposing only the filter funnel neck.
3.4 Place filter funnel in the manifold suction base.
3.5 Attach the vacuum hoses to the manifold and s/s collecting vessel. Ensure that the vacuum taps on the manifold are closed (horizontal position). If the collecting vessel is not a safety vessel (ie coated in wax) place vessel into steel canister and seal lid down with clips.
3.6 Remove sterile forceps from packaging.
Note: Ensure flat tip forceps are used, these will not puncture the membrane.
3.7 Hold forceps in one hand. With the other hand remove from a box a single envelope containing the filter membrane or use ezi pak dispenser.
3.8 Using both hands gently open envelope to expose membrane. Ensure that forceps do not touch any part of the envelope.
3.9 Hold envelope in one hand and remove membrane using forceps held in the other hand.
3.10 Lift the top of the filter funnel with one hand and place the membrane with the grid-marked side upwards onto the filter support screen.
3.11 Replace the top of the filter funnel.
3.12 Pour the required volume of the sample into the filter funnel.
3.13 Gently apply vacuum to draw sample through the membrane. (ie. turn the vacuum tap on the manifold to the vertical position – ON).
If membrane filtration technique is used to test the product, then the membrane and inside of the filter funnel must be rinsed with an appropriate rinse solution. If volume is to be filtered is less than 10 mls add about 50 mls of diluent to the funnel before addition of the sample to aid uniform dispersion of the bacteria over the entire surface of the membrane during filtration or alternatively, add the volume to be tested into the bottle containing sterile diluent – mix well and then pour the entire volume into the funnel. Some products require rinsing with Diluent and Tween to ensure that maximum filtration of the product has occurred. Refer to validation methods for the number of rinses for the product.
3.14 Lift the top of the filter funnel.
3.15 Remove membrane with sterile forceps, replace the top of the funnel. Ensure a new pair of sterile forceps are used for each transfer of the membrane.
3.16 Place membrane onto required agar plate by rolling the membrane, grid-marked side upwards, taking care to avoid entrapping air bubbles between the membrane and the agar surface.
When enrichment procedure is required, then roll the membrane into a tube using two pairs of forceps then hold the rolled membrane in one hand, pick up a bottle of media with the other hand. Unscrew the lid, pass the neck of the bottle through flame and then gently insert the membrane into the bottle.
Flame the neck of the bottle and replace lid.
3.17 If membrane filtration technique is used to test a product, a 100mL of rinse solution such as DIL+Tween must be filtered and placed on appropriate media.
NOTE: This will be diluent control plate.
SAFETY NOTE: Take care when lifting and emptying collecting vessel in the sink to avoid waste splash back.
Method 004: Presence or Absence Test of E.Coil
1. SCOPE AND APPLICATION
This General Test Method shall be followed in order to determine the presence or absence of E. coli. This procedure applies to all products manufactured at the GMP site and the raw materials used to manufacture those products.
2. REAGENTS AND MATERIALS REQUIRED
2.1 Lauryl Tryptone Broth (LTB) (OXOID CM451) in test tubes containing Durham tubes.
2.2 Double Strength Lauryl Tryptone Broth (D/S LTB) in test tubes containing Durham tubes.
2.3 E.C. Broth (OXOID CM853) – in test tubes containing Durham tube.
2.4 Eosin Methylene Blue (EMB) (OXOID CM69) agar.
2.5 Tryptone Water (TW) – (OXOID CM87)
2.6 Indole Reagent (KOVAC’s).
2.7 0.1% Peptone or other appropriate diluent.
2.8 Thermometer with 0.1oC graduations.
3. GENERAL TEST METHOD
3.1 For the presence or absence of E.coli/g weigh out 10g of sample into a stomacher bag.
3.2 Make up to 100g with 0.1% Peptone or chosen diluent. Mix well.
3.3 Inoculate 10mL of the above dilution into each of 3 tubes containing 10mLs of D/S LTB. If a higher sensitivity is expected inoculate an appropriate amount of the above dilution (ie. 1mL and 0.1mL) into 3 tubes containing 10 mLs of LTB.
3.4 Inoculate 2 additional tubes with reference cultures.
Negative control – Enterobacter aerogenes NCTC 10006 or ATCC 13048
Positive control – E. coli NCTC 9001 or ATCC 1175
3.5 Mix the tubes by gentle rotation.
3.6 Incubate the inoculated tubes of 37 + 1°C for up to 48 hours and examine for gas production after 24 hours and 48 hours.
Note: Approximately 30 minutes before each examination, gently tap all tubes to guard against false negative results due to gas supersaturation.
3.7 A tube is regarded as positive if sufficient gas is produced to fill the concavity of the Durham tube.
Note: If none or only one of the 3 tubes shows a positive reaction record the results as negative. If at least 2 of the tubes show gas production, proceed with the remainder of the test.
3.8 Using a sterile inoculating loop, subculture all cultures as they become positive at 24 hours and 48 hours into EC broth previously warmed to approximately 37°C; include both reference cultures.
3.9 Incubate in a water bath at 44.0°C to 44.5°C for up to 48 hour and examine at 24 hour and 48 hour. Tap the tubes gently after the initial 24 hour period. Those tubes showing gas production shall be deemed to be positive.
Note: Thermometer with 0.1°C graduations must be used to check water bath temperature.
3.10 Inoculate a dried EMB agar plate to obtain single colonies from each positive test obtained, and from both reference cultures.
3.11 Incubate the EMB plate at 37 + 1°C for 18 hours to 24 hours.
3.12 Select at least three typical colonies showing a green metallic sheen by reflected light and / or dark purple centres by transmitted light, from each plate, and one typical colony of each reference culture. Subculture into individual tubes of Tryptone Water (for Indole test) and incubate at 44.0°C to 44.5°C for 24 hours.
3.13 Test the cultures for indole production by using Kovac’s indole reagent. Add 1 drop into each tube. Tubes indicating the presence of indole (appearance of a pink ring) are recorded as positive for the presence of E. coli.
3.14 Record the number of positive tubes.
3.15 Calculation and report.
Record presence or absence of E. coli in the amount inoculated into each tube, provided that confirmed organisms are recorded in at least two of the three tubes. Report the method used and any circumstances which may have influenced results of the test.
3.16 Optional confirmatory tests.
Where further confirmation is required, the methyl red, Voges-Proskauer and citrate tests are recommended (Refer to Compendium of Methods for the Microbiological Examination of Foods – 2nd Edition 1984 American Public Health Association).
Note: Voges-Proskauer and citrate tests can be determined using Biomerieux API20E strip.
American Public Health Association – Washington.
Method 005: Preparation of Diluent plus 0.1% Tween 80
1. Definition
None
2. PURPOSE
This document describes the method for preparation of Diluent plus 0.1% Tween 80.
3. SCOPE
Diluent plus 0.1% Tween 80 is used as a high quality diluent for microbiology testing.
4. RESPONSIBILITY \ BUSINESS RULES
All microbiology staff at the GMP site.
5. PROCEDURE
5.1 Materials and Reagents required
5.1.1 Plastic spoon
5.1.2 Measuring Cylinder
5.1.3 RO Water
5.1.4 Beaker
5.1.5 Appropriate glassware
5.1.6 Automatic dispenser
5.1.7 pH meter
5.1.8 1N NaOH and 1N HCl
5.1.9 Bacteriological Peptone (Oxoid L34)
5.1.10 Tween 80. (Polysorbate BP 80 item 119A012)
5.2 Method
5.2.1 Weigh out 1g per litre of peptone into a beaker and add approximately 500mL of RO water. Mix well to dissolve.
5.2.2 Weigh out 1g per litre of Tween 80 into a beaker and add RO water.
5.2.3 Dissolve on high setting in the microwave with frequent stirring.
5.2.4 Combine dissolved peptone with dissolved Tween 80 and make up to required volume with RO water, mix well.
5.2.5 Adjust pH to 6.9.
5.2.6 Dispense into appropriate glassware.
5.2.7 Sterilise by autoclaving at 121°C for 15 minutes.
5.2.8 pH after autoclaving, 7.0 ± 0.2
5.2.9 Record all details of media preparation into appropriate form.
QUALITY CONTROL REQUIREMENTS |
STORAGE: BULK – 6 months in dark cupboard |
ECOMETRIC EVALUATION / FERTILITY |
CONTROL ORGANISMS: Positive: P.aeruginosa (A.T.C.C 9027) Growth Index: Growth Negative: S.aureus (N.C.T.C 6571) Growth Index: Growth INCUBATION CONDITIONS: Temperature: 30 ± 1°C Time: 24 hours |
Method 006: Preparation of Hard Water
1. Definition
None
2. Purpose
This document describes the method of preparation of Hard Water.
3. Scope
Hard water is used for preparation of test organisms in the TGA test.
4. Responsibility \ Business Rules
All microbiology staff at GMP Site.
5. Procedure
5.1 Materials and Reagents Required
5.1.1 Plastic spoon
5.1.2 Beaker
5.1.3 Measuring Cylinder
5.1.4 RO water
5.1.5 Automatic dispenser
5.1.6 Anhydrous Calcium Chloride (CaCl2)
5.1.7 Anhydrous Magnesium Chloride (MgCl2)
5.1.8 McCartney bottles
5.2 Method
5.2.1 Dissolve 0.304g/L of CaCl2 and 0.065g/L of MgCl2 in required volume of RO water, mix well.
5.2.2 Dispense required amount into McCartney bottles.
5.2.3 Sterilise by autoclaving at 121°C for 15 minutes.
5.2.4 Expiry date is 1 year from date of preparation.
5.2.5 Record all details of media preparation in SF150712.
5.2.6 NOTE: If anhydrous form not available use hydrated form but compensate for water.
e.g.
CaCl2 x 2H2O – 0.4g
MgCl2 x 6H2O – 0.14g
Distilled water – 1L
Method 007: Presence of Viable Spore on Spore Strips
1. SCOPE AND APPLICATION
This general test method applies to the determination of presence of viable spores on biological indicators (spore strips). This procedure applies to biological indicators which have been subjected to sterilisation.
2. REAGENTS AND MATERIALS REQUIRED
2.1 Tryptone Soya Broth (TSB) (Oxoid CM131) – 10 mls volume in MacCartney bottles.
2.2 Sterile forceps.
2.3 Laminar flow.
2.4 Bunsen Burner.
3. GENERAL TEST METHOD
3.1 Determine the number of TSB in MacCartney bottles required. One bottle per spore strip is needed, plus one for control.
3.2 Place bottles in Biohazard cabinet.
3.3 Label each bottle with a load number, job number (if applicable) and date.
3.4 Place spore strips in petri dish in Biohazard Cabinet.
3.5 Loosen lids on MacCartney bottles.
3.6 Using sterile forceps remove spore strip from envelope and aseptically transfer each spore strip into 10 mls of TSB. Replace and tighten lid on bottles.
Note: Flame the neck of the bottle immediately before and after transferring spore strip. Replace lid as soon as possible.
3.7 Repeat the above procedure for each spore strip.
3.8 Once all test spore strips have been transferred, set up a positive control in the same manner using an unautoclaved spore strip containing viable spores.
3.9 Incubate the broth containing spore strip using the following conditions:
Bacillus stearothermophilus – 55 ± 1°C / 7 days. (Steam sterilisation)
Bacillus subtilis var.niger – 37 ± 1°C / 7 days. (ETO sterilisation)
Bacillus pumilus – 37 ± 1°C / 7 days. (Gamma irradiation)
3.10 Check daily (each working day) for growth.
3.11 Streak any broths showing turbidity onto TSA plate and incubate overnight at an appropriate temperature, Ref. to 3.9. (Record number of days at which growth became evident).
3.12 Confirm growth for presence of the test organism by colony morphology, Gram stain and spore stain, and identification kit if necessary.
3.13 Record 7 day results for spore strips showing no growth.
Method 008: Preparation of Malt Extract Agar
1. PURPOSE
This operator instruction describes the method for the preparation of Malt Extract Agar used in the microbiology laboratory.
2. SCOPE AND APPLICATION
Malt Extract Agar is a medium used for detection, isolation and enumeration of yeasts and moulds.
3. MATERIALS REQUIRED LIST
3.1 Plastic spoon
3.2 Beaker (microwaveable)
3.3 Measuring cylinder
3.4 R O water
3.5 Malt Extract Agar (Oxoid CM59)
3.6 1N NaOH, 1N HCl
3.7 Plastic pipettes
3.8 Clean glass bottles
4. WORK INSTRUCTION
4.1 Weigh out 50g per litre of Malt Extract Agar powder into a beaker.
4.2 Add approximately half the required volume of RO water, mix, and bring to the boil to dissolve in the microwave.
4.3 Make up to required volume with RO water.
4.4 Adjust pH to 5.4 + 0.2.
4.5 Dispense 150mL into clean glass bottles.
4.6 Sterilize by autoclaving at 115°C for 10 minutes.
4.7 Final pH after sterilization should be 5.4 + 0.2.
QUALITY CONTROL REQUIREMENTS |
STORAGE: BULK – 6 months POURED PLATES – 2 weeks |
ECOMETRIC EVALUATION / FERTILITY CONTROL ORGANISM: Positive: C.albicans (A.T.C.C: 10231) Growth Index: ≥ 3 Negative: N/A Growth Index: N/A |
INCUBATION CONDITION: Temperature: 25° ± 1°C Time: 3-5 days |
Method 009: Preparation of Asparagine Broth
1. DEFINITION
None
2. PURPOSE
This document describes the method for preparation of Asparagine Broth.
3. SCOPE
Asparagine Broth is a medium used in the confirmation step for Pseudomonas aeruginosa.
4. RESPONSIBILITY \ BUSINESS RULES
All microbiology staff in the laboratory.
5. PROCEDURE
5.1 Materials and Reagents required
5.1.1 Plastic spoon
5.1.2 Measuring Cylinder
5.1.3 RO Water & RO water squeeze bottle
5.1.4 3 Glass beakers
5.1.5 Funnel
5.1.6 Flat medical bottles / Schott bottles
5.1.7 Automatic dispenser
5.1.8 DL-Asparagine Monohydrate
5.1.9 Di Potassium Hydrogen Phosphate K2HPO4
5.1.10 Magnesium Sulphate Heptahydrate MgSO47H2O
5.1.11 pH meter
5.1.12 1N NaOH and 1N HCl
5.2 Method
5.2.1 Weigh out 3.0g per litre of Asparagine DL.
5.2.2 Weigh out 1.0g per litre of Di Potassium Hydrogen Phosphate K2HPO4.
5.2.3 Weigh out 0.5g per litre of Magnesium Sulphate Heptahydrate MgSO47H2O
5.2.4 Dissolve the above ingredients in approximately 10mL of RO water.
5.2.5 Combine all 3 ingredients. Use the squeeze bottle to wash out all particles.
5.2.6 Make up to required volume with RO water.
5.2.7 Adjust pH to 7.1 using 1N NaOH or 1N HCl.
5.2.8 Dispense 95mL volumes into flat medical bottles / schott bottles.
5.2.9 Label all bottles with medium name, batch number and date of preparation.
5.2.10 Sterilise by autoclaving at 121oC for 15 minutes.
5.2.11 Final pH after sterilisation should be 7.1 + 0.2. If not within range, notify the microbiologist and discard the batch.
5.2.12 Record all details of media preparation on SF150712.
Method 010: Preparation of Bacillus SPP Spore Suspension
1. DEFINITION
None
2. PURPOSE
This procedure details instructions for the preparation of Bacillus spp spore suspension.
3. SCOPE
This procedure is to be used when preparing a Bacillus spp spore suspension for microbiological testing in the laboratory (eg. product inoculation, TMV, antibiotic assays, inhibition tests, sterilization biological indicators).
4. RESPONSIBILITY \ BUSINESS RULES
This procedure applies to all Microbiology Laboratory staff.
5. PROCEDURE
5.1 Materials Required List
5.1.1 Chosen Bacillus spp. strain
5.1.2 Tryptone Soya Broth (TSB) in MacCartney bottles
5.1.3 Tryptone Soya Agar – TSA (large slope and bulk for pouring plates)
5.1.4 Pipettes – sterile
5.1.5 Petri-dishes sterile
5.1.6 Inoculating loop 10μL
5.1.7 Incubator 30°C ± 1°C, 37°C ± 1°C
5.1.8 Water bath
5.1.9 Centrifuge and centrifuge tubes (sterile)
5.1.10 Phosphate buffer – pH 7.2 (prepared by QC laboratory)
5.2 Procedure
5.2.1 Open freeze dried ampoule (of chosen Bacillus spp. strain).
5.2.2 Incubate at 37 ± 1°C for 48 hours.
5.2.3 Streak onto TSA to check purity.
5.2.4 If pure, streak onto a large TSA slope and incubate at 37 ± 1°C for at least 7 days.
5.2.5 Check spore production every 3 days by performing spore stain until approximately 80% of the cells yield spores.
5.2.6 Wash off the growth with 30mls Phosphate Buffer pH 7.2 and dispense into sterile centrifuge tubes.
5.2.7 Centrifuge the tubes at 1000 r.p.m. for 10 minutes. Decant the supernatant liquid.
5.2.8 Wash pellet three times in fresh Phosphate Buffer and combine harvested spores.
5.2.9 Heat shock the suspension following procedure GM062932 Step 3.6 and 3.7.
5.2.10 Enumerate the number of viable spores. (Usually contain about 109 spores per mL)
5.2.11 Pour plates with TSA and incubate at appropriate temperature depending on species (GM062932 Step 3.10) for 2 days.
5.2.12 Dispense the spore suspension into a sterile plastic container and keep refrigerated at 2- 8oC.
5.2.13 Label container containing spore suspension with strain number, date of preparation, count/ml and date of expiry which is 6 months from date of preparation.
Method 011: Presence or Absence of Clostridium Perfringens
1. SCOPE AND APPLICATION
This general test method shall be followed in order to determine the presence or absence of Clostridium perfringens in raw materials and finished products.
2. REAGENTS AND MATERIALS REQUIRED
2.1 Cooked Meat Medium (CMM) (OXOID CM81)
2.2 Thioglycollate + 0.5% Tween 80 (THIO+T) (OXOID CM173)
2.3 Tryptose Sulfite Cycloserine (TSC) agar (OXOID CM 587, SR88, SR47)
2.4 Streaking loops – 10μL
2.5 Solution A – Sulfanilic Acid
2.6 Solution B – N-(1-naphthyl) ethylenediamine reagent
2.7 Lactose – Gelatin (LG) Medium
2.8 Nitrate Motility (NM) Medium
2.9 Reference culture C. perfringens N.C.T.C. 8237
2.10 Tryptone Soya Agar (TSA)
2.11 Lactose Egg Yolk (LEY) Agar
3. GENERAL TEST PROCEDURE
3.1 Weigh out 1.0g of sample into CMM or THIO+T as pre-determined by validation study.
Note: If CMM is not freshly made, or if THIO+T as oxidised, boil medium for 10 min. to drive out oxygen. Cool down media in water bath to approximately 45-46ºC.
Carry out a positive control by inoculating a 10μL loopful of C. perfringens into the appropriate enrichment broth.
3.2 Incubate at 37 ± 1°C for 48 hours.
3.3 Streak a 10μL loop full of enrichment and control onto a dried TSC agar plate.
3.4 When the inoculum has been absorbed into the agar, overlay plates with an additional 10 ml of TSC agar without egg yolk. Allow overlay to set.
3.5 Incubate plates anaerobically at 37 ± 1°C for 18-24 hours.
3.6 Examine plates for black colonies surrounded by opaque white haloes due to lecithinase activity.
NOTE: Routinely confirm suspect colonies by streaking onto 2 TSA plates and incubating one aerobically and one anaerobically. Typical colonies for C. perfringens will not grow aerobically. If only anaerobic growth is found set up a RAPID ID32A identification strip and follow the manufacturers instructions for anaerobic microorganisms.
3.7 Confirmation Tests:
– Subculture suspect colonies onto LEY agar (lactose egg yolk), incubate anaerobically for 24 hours at 37 ± 1°C. Check for purity.
– Examine culture by Gram stain. Gram positive – short, thick rods, occurring singularly or in pairs or as short chains.
– Inoculate tubes LG medium (Lactose Gelatin medium) NM (Nitrate Motility medium) by stabbing with a straight wire.
– Incubate at 37 ± 1°C. for 24 hours.
3.8 Examine LG Medium.
– Lactose fermentation – gas production and colour change from red to yellow.
– Gelatin liquefaction – chill tubes at 5°C. for 1 hour. If the medium gel, incubate at 37 ± 1°C. for another 24 hours and recheck for gelatin liquefaction.
perfringens – ferments lactose and liquefies gelatin.
3.9 Examine NM Medium.
Motility – diffuse growth out into the medium away from the stab line.
Nitrate detection – add 0.5 ml of solution A and 0.2 ml of Solution B.
Development of red colour indicates presence of nitrites. If no colour develops, test for residual nitrate by adding powdered zinc. If red colour does not develop after addition of zinc the organism does not reduce nitrate to nitrite.
perfringens is non-motile – reduces nitrate to nitrite.
Method 012: Precence or Absence Test of Indicator Organisms
1. SCOPE AND APPLICATION
This general test method shall be followed in order to determine the presence or absence of indicator organisms such as Pseudomonas spp., Coliforms (e.g. Klebsiella spp., Enterobacter spp.) and coagulase positive Staphylococcus spp. This general test method is also followed when screening for objectionable organisms in order to determine the presence or absence of any gram negative rod.
This procedure applies to products manufactured at GMP Manufacturing site as well as raw materials used to manufacture those products.
This procedure covers tests for objectionable and deleterious organisms.
2. REAGENTS AND MATERIALS REQUIRED
2.1 Tryptone Soya Broth containing 4% Tween 80 (TSB+T) or other enrichment broth as predetermined by validation study ie. Letheen Broth + 2% Lecithin + 4% Tween 80.
2.2 Stomacher bag or sterile jar
2.3 Streaking loops (10μL and 1 μL)
2.4 Pseudomonas Agar Plates (PAB)
2.5 CFC Agar Plates (CFC)
2.6 MacConkey No. 3 Agar plates (MAC)
2.7 Baird-Parker Agar plates (BP)
2.8 Lauryl Tryptose Broth (LTB)
2.9 EC Broth (EC)
2.10 Tryptone Soya Agar Plates (TSA)
2.11 Saboraud Dextrose Agar (SDA)
2.12 EMB agar plates (EMB)
2.13 Tryptone Water (TW)
2.14 Latex Gloves
3. GENERAL TEST PROCEDURE
Note: The analyst must wear latex gloves during all steps
3.1 Weigh out 10g of sample into the stomacher bag.
3.2 Make up to 100g with TSB+T (enrichment) or other enrichment broth.
3.3 Mix well.
Incubate the enrichment at 30 ± 1°C for 2 days.
NOTE: Enrichment broths of finished products and raw materials must be incubated in separate baskets to avoid cross contamination.
3.5 After the required incubation time has passed, remove broth from the incubator and streak a loopfull (10μL) onto relevant selective and non selective agar plates: CFC, MAC, BP, PAB, EMB, TSA and SDA.
PAB, CFC – Pseudomonas spp
MAC – Coliforms
BP – Coagulase positive Staphylococcus (S.aureus)
EMB – E.coli
SDA – Candida albicans
TSA – Gram negative rods
NOTE: Dispose each enrichment broth into the autoclave bag after streaking.
3.6 Incubate plates as follows:
CFC, PAB, TSA, SDA at 30 ± 1°C for 48 hours, checking after 24 hours.
BP, MAC at 37 ± 1°C for 48 hours, checking after 24 hours.
EMB at 37 ± 1°C for 24 hours.
Confirm any growth on agar plates for the presence or absence of indicator organisms as outlined below:
3.7 CONFIRMATORY TEST FOR COLIFORMS ex MAC
Coliforms that colonies are lactose fermenters that produce red-violet colonies, whilst non- lactose fermenters are colourless on MacConkey agar.
3.7.1 Confirm at least three suspect colonies. Subculture each colony into a tube of LTB.
3.7.2 Incubate tubes at 37± 1°C for up to 48 hours checking at 24 hours.
3.7.3 Any tubes producing sufficient gas at 24 hours are considered positive and need no further incubation. Tubes not producing gas at 24 hours must be re-incubated for a further 24 hours. Tubes at 48 hours showing insufficient gas production are considered negative.
A positive reaction is indicated by the production of sufficient gas to fill the concavity of the durham tube.
3.7.4. If any LTB tubes show a positive reaction then Coliforms/10g are DETECTED. Consult the Microbiology Team Leader to determine if further identification is required (eg API).
3.8 CONFIRMATORY TEST FOR E.coli ex EMB
Typical E.coli colonies appear 2-3 mm in diameter, with little tendency to confluent growth, exhibiting a greenish metallic sheen by reflected light and dark purple centres by transmitted light.
3.8.1 To determine whether E.coli is present or absent per 10g of sample proceed as follows.
3.8.2 Confirm at least three suspect colonies. Subculture each colony into a tube of EC broth.
3.8.3 Incubate tubes at 44.5°C+ 0.5°C in a waterbath for 48 hours checking at 24 hours.
3.8.4 Any tubes producing sufficient gas at 24 hours are considered positive and need no further incubation. Tubes not producing gas at 24 hours must be re-incubated for a further 24 hours. Tubes at 48 hours showing insufficient gas production are considered negative.
A positive reaction is indicated by the production of sufficient gas to fill the concavity of the durham tube.
3.8.5 Subculture each positive EC tube into a separate tube containing Tryptone Water using a 1μL loop.
3.8.6 Incubate tubes at 44.5°C+ 0.5°C in a waterbath 24 hours.
3.8.7 After incubation add 1-2 drops of Kovacs reagent to each Tryptone Water tube.
3.8.8 A positive result is indicated by the presence of a red-pink ring at the top of the tube.
3.8.9 If both EC tubes and Tryptone Water tubes show a positive reaction then E.coli/10g are DETECTED.
3.8.10 Consult the Microbiology Team Leader to determine if further identification is required (eg API).
3.9 CONFIRMATORY TEST FOR COAGULASE POSITIVE STAPHYLOCOCCUS
(S. AUREUS) x BP
Typical Staphylococcus species on BP appear as black to grey shiny or non- shiny convex colonies, which may or may not produce a clear zone around the colony.
3.9.1 Perform a wet mount, if cocci proceed with agglutination test.
3.9.2 Confirm at least 3 suspect colonies
3.9.3 Check for agglutination using latex agglutination kit as per manufacturer’s instruction running a control at the same time.
3.9.4 Formation of small particles is indicative of coagulase positive Staphylococcus.
3.9.5 Perform further identification tests (if necessary) “Identification of Contaminants”.
Note: This confirmatory test will be used routinely. If a full confirmatory test is required this shall be done according to AS 1766.24 – 1986.
3.10 CONFIRMATORY TEST FOR PSEUDOMONAS SPP x PAB and CFC
The presence of blue-green or brown pigmentation or fluorescence may be taken as presumptive Pseudomonas spp, but futher testing must be carried out to confirm the identify of the organism. Confirm all typical and atypical growth on PAB and CFC agar.
3.10.1 Streak all suspect colonies onto TSA plates. Incubate at 30 ± 1°C for 18 to 24 hours check purity.
3.10.2 If a pure culture is indicated, proceed with identification “Identification of Contaminants”.
3.11 CONFIRMATORY TEST FOR CANDIDA ALBICANS x SDA
Perform a wet mount, if yeast are present (ie large round to ellipsoidal cells with or without budding) proceed with API Identification using BioMerieux API Candida identification kit as per manufacturer’s instruction.
3.12 CONFIRMATORY TEST FOR GRAM NEGATIVE RODS x TSA
3.12.1 If a pure culture is indicated, perform a gram stain.
3.12.2 If gram negative rods are obtained, proceed with identification “Identification of Contaminants.”
3.13 REPORTING OF RESULTS
For specific organisms report as: Organism Not Detected per unit of sample tested Eg Coliforms ND / 10g-ml
For Objectionable or Deleterious organisms report as:
Objectionable or Deleterious organisms Not Detected per unit of sample tested. Sample is free of Gram negative rods, C.albicans, S.aureus.
Eg Objectionable or Deleterious organisms ND / 10g-ml. Sample is free of Gram negative rods, C.albicans, S.aureus.
Method 013: Presence or Absence Test of Salmonella SPP
1. SCOPE AND AND APPPLICATION
This general test method shall be followed in order to determine the presence or absence of Salmonella spp. This procedure applies to all products manufactured at a GMP site and the raw materials used to manufacture those products.
2. REAGENTS AND MATERIALS REQUIRED
2.1 Buffered peptone water (BPW) (OXOID CM 509)
2.2 Stomacher bags (Sterile)
2.3 Mannitol Selenite Cystine broth (MSC) (OXOID CM 399) or bio Merieux (42052)
2.4 Rappaport – Vassiliadis broth (RV) (OXOID CM 669)
2.5 Xylose Lysine Desoxycholate plates (XLD) (OXOID CM 469)
2.6 Bismuth Sulphite agar plates (BSA) (OXOID CM 201)
2.7 Streaking loops – 10L
2.8 Homogeniser
2.9 Reference culture S. salford I.M.V.S. 1710
2.10 Reference culture C. freundii N.C.T.C. 9750
3. GENERAL TEST PROCEDURE
3.1 Weigh out 10g of sample into a stomacher bag.
3.2 Make up to 100g with BPW, or appropriate volume as pre determined by validation.
3.3 Place in homogeniser to mix (Pre-set for 15 seconds)
3.4 Allow to stand for approximately 1 hour.
3.5 The pH of the primary enrichment must be between 6.5-7.5.
For new products and raw materials, check pH. If not within 6.5-7.5, adjust using sterile 1N HCl and 1N NaOH.
3.6 Set up controls parallel with the test.
Note: Set up controls in Biohazard cabinet.
Using 2 MaCartney bottles of BPW (10mL), inoculate with C. freundii (-ve control) and S.
salford (+ve control) respectively.
3.7 Incubate primary enrichment at 37 ± 1C for 16-20 hours.
3.8 Remove from incubator – mix well and subculture primary enrichment broth into selective enrichment broths.
– 1 mL into 10 mLs of MSC (Mannitol Selenite Cystine broth containing 0.1mL of 0.1% filter sterilised L-cystine solution) pre-warmed to room temperature.
NOTE: L-cystine solution is not added to MSC purchased from bio Merieux.
– 0.1 mL into 10 mL of RV (Rappaport-Vassiliadis) pre-warmed to room temperature.
– Vortex to mix.
3.9 Incubate MSC at 37 ± 1C for 18-24 hours.
Incubate RV at 42 ± 1C for 18-24 hours.
Note: Examine tubes for evidence of growth (turbidity and colour change) MSC from clear to red, RV from clear blue-green to turbid light blue-green.
3.10 Streak each broth onto selective agar plates XLD and BSA.
3.11 Incubate XLD plates in an inverted position for 24 hours at 37 ± 1C.
Incubate BSA plates in an inverted position for 48 hours at 37 ± 1C.
3.12 Remove plates from incubator and examine for typical colonies ie. red colonies with black centres on XLD and black (rabbit-eye or uniformly black) colonies with a black zone and metallic sheen on BSA.
NOTE: Routinely confirm suspect colonies by streaking onto TSA and following the appropriate steps outlines in OI150102.3.
The following describes the confirmatory steps outlined by the Australian Standards AS1766.2.5 – 1991.
3.13 Confirm at least 3 suspect colonies.
– Subculture each colony into a separate tube of peptone water (1%).
– Incubate at 37 ± 1C. for 3 to 4 hours or until growth is visible.
– From each peptone water culture, inoculate the following media.
i) Lysine decarboxylase broth
ii) Lysine decarboxylase broth base (control).
iii) ONPG broth
iv) CLED agar plate – to check purity
v) Nutrient agar slope.
3.14 Incubate all cultures at 37 ± 1C. for 18-24 hours.
3.15 Examine all cultures and proceed as follows –
i) The purple Lysine decarboxylase broth remains purple after 18 hours. This reaction is typical of Salmonella.
Lysine decarboxylase broth base turns yellow and remains yellow. This reaction is atypical of Salmonella.
ii) ONPG broth remains colourless due to a negative beta-D-galactoside reaction. This is typical of Salmonella.
ONPG broth turns yellow due to a positive beta-D-galactoside reaction. This is atypical of Salmonella.
3.16 Serological confirmation.
i) Wash off growth ex nutrient slope with approx. 1 ml of formalinised saline solution.
ii) Mark three sections onto a glass slide. Place one drop of this suspension onto each section of a glass slide.
iii) Add one small drop of polyvalent ‘O’ antiserum to the first section and of polyvalent “H” antiserum to the second section.
iv) Tilt the slide back and forth for 1 minute and examine for agglutination. Agglutination in sections containing antisera indicates a positive result.
Agglutination in the third section indicates auto-agglutination and invalidates the test.
3.17 Interpretation of test results
– Isolates which give a result typical of salmonellae in both biochemical tests and serological reactions, are considered to be salmonellae.
– Isolates which give a result typical of salmonellae in one biochemical test and give negative serological results are considered not be salmonellae.
– Isolates which give typical reactions in both biochemical test but negative serological reactions and isolates which give a typical result in one biochemical test and positive serological reactions require further testing.
3.18 Carry out negative and positive controls throughout the test using the following reference cultures:
Reference Cultures
Salmonella Salford – IMVS 1710
Citrobacter freundii – NCTC 9750
Method 014: Microbiological Method for the Testing of Disinfectants
1. SUMMARY OF CHANGES
Version # | Revision History |
MICLAB – METHOD 015 | New |
2. PURPOSE
This document describes the method for the testing of disinfectants.
3. SCOPE
Disinfectants are used by the microbiological laboratory to decontaminate work surfaces and spills.
4. RESPONSIBILITY \ BUSINESS RULES
All microbiology staff at the GMP Site.
5. PROCEDURE
5.1 Materials and Reagents required
5.1.1 Inactivating broth – Letheen broth + 2% Lecithin + 4% Tween 80
5.1.2 Nutrient Agar (NA) (OxoidCM3): pre-poured plates
5.1.3 Vortex
5.1.4 Sterile 1mL pipettes
5.1.5 Transfer pipette
5.1.6 Sterile pipette tips
5.1.7 Disinfectant testing report
5.1.8 SOP – Identification of Contaminants
5.2 Method
5.2.1 Pipette 1mL of the disinfectant sample into 9mL of inactivating broth and vortex. This is a 1 in 10 dilution.
5.2.2 Place ten drops each containing 20μL of the 1 in 10 dilution onto two NA plates i.e. 10 separate drops per plate.
5.2.3 Incubate one plate at 37 + 1°C for 3 days and the other plate at 25 + 1°C for 7 days. Incubate plates in an upright position.
5.2.4 Record results as Colony Forming Units (cfu) / plate on SF020273.
5.3 Acceptance Criteria
5.3.1 The acceptable limit for disinfectants is <5 colonies on either plate.
5.3.2 Identify contaminants as per Identification of Contaminants QO-S00103.
5.3.3 Raise a Microbiological Laboratory Investigation if the count is ≥ 5 colonies on either plate.
Method 015: Maintenance of Culture Collection for Aseptic Practices in the Laboratory
1. SUMMARY OF CHANGES
Version # | Revision History |
MICLAB – METHOD 016 | New |
2. PURPOSE
This document deals with the correct means of maintaining a culture collection with an emphasis on aseptic practices in the laboratory.
3. SCOPE
All laboratory staff must follow aseptic handling instructions to ensure continued purity of the culture collection.
4. RESPONSIBILITY \ BUSINESS RULES
All microbiology staff appointed to maintenance of the culture collection at the GMP site.
5. PROCEDURE
5.1 Materials and Reagents required
5.1.1 Tryptone Soya Agar (TSA) slopes
5.1.2 Sabouraud Dextrose Agar (SDA) slopes
5.1.3 White adhesive labels 3.5 cm2
5.1.4 White, green, blue and yellow adhesive dots
5.1.5 Diagnostic media plates
5.1.6 Storage baskets
5.1.7 TSA & SDA plates
5.1.8 Metal file/ diamond pen
5.1.9 10ml Tryptone Soya Broth (TSB) in test tubes / MacCartney bottles
SAFETY NOTE: All microbiological cultures used in the microbiology laboratory are classified as either Risk Group 1 (low individual & community risk) or Risk
Method 016: Method for Preparation of DNase Agar (Oxoid CM321)
1. SUMMARY OF CHANGES
Version # | Revision History |
MICLAB – METHOD 017 | New |
2. PURPOSE
This document describes the method for preparation of DNase Agar (Oxoid CM321).
3. SCOPE
DNase Agar is used for the detection of microbial deoxyribonuclease enzymes, particularly from Staphylococci. The DNase reaction for Staphylococci is an indication of pathogenicity; it cannot be used as the sole criteria for identification.
4. RESPONSIBILITY \ BUSINESS RULES
All microbiology staff at GMP site.
5. PROCEDURE
5.1 Materials and Reagents required
5.1.1 Plastic spoon
5.1.2 Measuring Cylinder
5.1.3 RO Water
5.1.4 Microwaveable beaker
5.1.5 Plastic Pipettes
5.1.6 DNase Agar powder (Oxoid CM321)
5.1.7 Clean Glassware (flat medical or 250mL schott bottles)
5.1.8 Sterile Petri dishes
5.1.9 1N NaOH and 1N HCl
5.2 Method
5.2.1 Weigh out 39g per litre of DNase Agar powder into a beaker.
5.2.2 Add required volume of RO water and mix well.
5.2.3 Dissolve on high setting in microwave with frequent stirring.
5.2.4 Adjust pH to 7.5 using 1N NaOH or 1N HCl.
5.2.5 Dispense 150mL or 300mL volumes into flat medical bottles or schott bottles respectively.
5.2.6 Sterilise by autoclaving at 121oC for 15 minutes.
5.2.7 Final pH after sterilisation should be 7.3 + 0.2.
5.2.8 Mix gently before pouring into sterile petri dishes.
5.2.9 Record all details of media preparation on SF150712.
5.3 Quality Control Requirements
QUALITY CONTROL REQUIREMENTS |
STORAGE: BULK – 6 months in dark cupboard POURED PLATES – 2 weeks in fridge (2-8oC) |
ECOMETRIC EVALUATION / FERTILITY |
CONTROL ORGANISMS: Positive: Staphylococcus aureus Negative: N/A N.C.T.C 6571 Growth Index: ≥ 3 Growth Index: N/A INCUBATION CONDITIONS: Temperature: 37 ± 1°C Time: 18 hours |
Method 017: Procedure for maintenance of IQ 2000 MKII Precision Peristaltic and Perimatic Premier
1. SUMMARY OF CHANGES
Version # | Revision History |
MICLAB – METHOD 018 | New |
2. PURPOSE
This procedure describes how to use and maintain the IQ 2000 MKII Precision Peristaltic Dispenser and the Perimatic Premier Dispenser.
3. SCOPE
This procedure is to be followed when dispensing large volumes of broth and molten media or diluent.
4. RESPONSIBILITY \ BUSINESS RULES
Microbiology staff at a GMP Laboratory.
5. PROCEDURE
5.1 IQ2000 PERISTALTIC DISPENSER
5.1.1 MATERIALS REQUIRED LIST
5.1.1.1 IQ 2000 Perimatic Premier Dispenser
5.1.1.2 Beaker of media.
5.1.1.3 Measuring cylinder.
5.1.1.4 Tubing.
5.1.1.5 Arrangement of containers and racks for dispensing runs.
5.1.2 WORK INSTRUCTION
5.1.2.1 Preparation
5.1.2.1.1 Prepare containers and racks for easy access with nozzle, prior to dispensing.
5.1.2.1.2 Ensure agar is fully molten and dissolved.
5.1.2.1.3 Select tubing according to required dose volume, e.g. 3 mm bore tube for 10 ml doses, 5 mm bore tube for 30-150 ml doses and 8mm bore size for larger volumes.
5.1.2.1.4 Fit tubing to Perifill IQ 2000 as per diagram below:
Figure 1
5.1.2.1.5 Ensure that the reservoir tubes are fully immersed in the fluid to be dispensed and the outlet nozzle is over the container.
5.1.2.1.6 Switch power on.
5.1.2.1.7 The Liquid Crystal Display (LCD) will alternate between
Prime Tubing and Switch Pump START to RUN
5.1.2.1.8 Prime tubing by pressing ‘START’ or foot switch.
5.1.2.1.9 Once tubing is fully primed and contains no air bubbles, press stop or foot switch.
5.1.2.1.10 The LCD will read Select nearest tube size –→ 3 5 8
5.1.2.2 Calibration
5.1.2.2.1 Select nearest tube size by pressing either 3mm, 5mm, or 8mm bore tube buttons on the “calibrate sample” display.
The tube size is displayed on the glass T-junction of the tubing.
5.1.2.2.2 Once the nearest tubing size has been entered, the instrument indicates an approximate calibration volume of 10 ml for 3mm bore, 30 ml for 5mm bore and 50 ml for 8mm bore.
5.1.2.2.3 Place nozzle over appropriate sized measuring cylinder and press start, to measure sample volume.
5.1.2.2.4 LCD alternates between,
When → x ml When→ x ml and Test → x ml
agreed program sample start
(x being approximate calibration volume)
5.1.2.2.5 Use DISPENSE VOLUME arrow keys to adjust display volume to agree with dispensed volume.
5.1.2.2.6 Press START to re-check calibration. When satisfied with calibration, press SLOW or FAST to continue.
5.1.2.3 Dispensing
5.1.2.3.1 If FAST is selected, LCD will read
sso 0.00ml sfo
fast manual P
LCD will read 0.00 ml slow manual P
5.1.2.3.2 Select required dispense volume using DISPENSE VOLUME arrow keys.
5.1.2.3.3 For single doses, press start key every time dose is required.
5.1.2.3.4 For multiple doses, press auto/delay.
5.1.2.3.5 Display changes to 0.00 ml
slow auto 0.5s M
if set on slow.
OR sso 0.00ml sfo
fast auto 0.5s M
5.1.2.3.6 Press auto/delay to increase delay between each fill in 0.5 second steps to 9.5 seconds maximum.
5.1.2.3.7 Press START and the dose will be repeated until STOP is pressed.
5.1.2.3.8 During dispensing display changes to
rec nDOSE
set OCOUNT.
and shows the number of doses delivered.
5.1.2.3.9 The number of doses can be set by using the arrow keys with the instrument on stand by. When the instrument has delivered the pre set number of doses it will stop.
5.1.2.3.10 Zero the recorded dose count by setting the instrument to stand by and pressing the zero record key.
5.1.2.3.11 Pump can be switched to STAND BY when temporarily not in use.
5.1.2.3.12 When finished, wash out tubing by priming with hot tap water followed by rinsing with purified water and dry in oven before storing.
5.2 PERIMATIC PREMIER DISPENSER
5.2.1 MATERIALS REQUIRED LIST
5.2.1.1 Jencons Perimatic Premier Dispenser
5.2.1.2 Beaker of media
5.2.1.3 Measuring cylinder
5.2.1.4 Tubing
5.2.1.5 Arrangement of containers and racks for dispensing runs
5.2.2 WORK INTRUCTION
5.2.2.1 Preparation
5.2.2.1.1 Loosen the red knob and lift off pipe clamp. Refer figure 2.
Figure 2
5.2.2.1.2 The silicone tubing is fitted into the arc of the rotor casting, facilitated by turning the rotor by hand.
5.2.2.1.3 Tighten the pipe clamp and red knob. Use a clockwise twisting action to release the flow valve tops, secure the tubing into place.
5.2.2.1.4 Twist the flow valve tops closed once tubes are in position.
5.2.2.1.5 Feed all three pipes through the slots on right side of case, with the entire mechanism enclosed by the front panel before operations begin.
5.2.2.1.6 Ensure that the reservoir tubes are fully immersed in the fluid to be dispensed and outlet nozzle is over the container.
5.2.2.1.7 Switch power on at the rear of machine.
5.2.2.2 Setting the Dispensing Volume
5.2.2.2.1 Display will show
5.2.2.2.2 Press “Set up” button.
5.2.2.2.3 Press “OPTIONS” button.
5.2.2.2.4 Press “VOLUME” button.
5.2.2.2.5 Enter the amount of fluid to be dispensed (ml) using the numeric keys.
5.2.2.2.6 Press “ENTER” to update display.
5.2.2.2.7 Press “ACCEPT” to store volume.
5.2.2.3 Setting the repeated dispensing count
5.2.2.3.1 Press “COUNT” button.
5.2.2.3.2 Enter the number of times dispenser is to repeat.
5.2.2.3.3 Press “ENTER”.
5.2.2.3.4 Press “ACCEPT”.
5.2.2.3.5 Press “NEXT”.
5.2.2.4 Setting delay between dispensing
5.2.2.4.1 Press “DELAY”.
5.2.2.4.2 Enter time delay using keypad, followed by “ACCEPT”.
5.2.2.5 Automatic and Manual Modes
5.2.2.5.1 Press “MODE” button –the current selected mode is displayed.
5.2.2.5.2 Press “NEXT” key to change mode.
5.2.2.5.3 Press “ACCEPT” to store selected set up.
5.2.2.5.4 Press “NEXT”.
5.2.2.6 Setting the pumping Rate
5.2.2.6.1 Press “PROFILE”.
5.2.2.6.2 Press “NEXT” until the desired combination is displayed FAST, FAST, FAST is most commonly used.
5.2.2.6.3 Press “ACCEPT”.
5.2.2.7 Set Tube size
5.2.2.7.1 Press “TUBE”.
5.2.2.7.2 Press “NEXT” button until the correct tube size appears.
5.2.2.7.3 Press “ACCEPT”.
5.2.2.7.4 Press NEXT”.
5.2.2.7.5 Press “ENTER”.
5.2.2.8 Prime the System
5.2.2.8.1 Press “PRIME”.
5.2.2.8.2 Press “ACCEPT”
5.2.2.8.3 Once tubing is fully primed and contains no air bubbles press “ACCEPT”.
5.2.2.9 Calibration
5.2.2.9.1 From main menu press ”SET UP”.
5.2.2.9.2 Press “CAL”.
5.2.2.9.3 Press “VOLUME”.
5.2.2.9.4 Enter the amount of fluid to calibrate to in mL.
5.2.2.9.5 Press “ENTER”.
5.2.2.9.6 Press “ACCEPT”.
5.2.2.9.7 Press “START” to dispense volume entered, into appropriate sized measuring cylinder.
5.2.2.9.8 Enter the actual volume dispensed in mL.
5.2.2.9.9 Press “ENTER”.
5.2.2.9.10 Press “ACCEPT”.
5.2.2.9.11 Press “QUIT” to store calibration.
5.2.2.9.12 If needed – Press “START” to pump a new sample to check calibration.
5.2.2.9.13 Press “ENTER”.
5.2.2.9.14 Press “PUMP” to start dispensing.
5.2.2.10 Memory Facilities
5.2.2.10.1 To store a setting, from set up menu press “MEMORY”.
5.2.2.10.2 Press “STORE”.
5.2.2.10.3 Use the numeric keypad to enter a memory number.
5.2.2.10.4 Press “ACCEPT”.
5.2.2.10.5 A comment may be entered. Use the 4 or 6 number keys to move the cursor left or right. The 2 and 8 keys change the character under the cursor 2 moves up the alphabet and 8 moves down. Pressing 5 will copy the character to the left of the cursor.
5.2.2.10.6 Press “ACCEPT”.
5.2.2.10.7 To retrieve a setting in memory press “REVIEW”.
5.2.2.10.8 Press “NEXT” until desired setting.
5.2.2.10.9 Press “RECALL”.
5.2.2.10.10 When finished, wash out tubing by priming with hot water followed by rinsing with purified water and dry in oven before storing.
6. DEFINITIONS / ACRONYMS
N/A
7. REFERENCES
N/A