Microbiological tests related to industries of medical devices..........

1. BACTERIAL ENDOTOXIN TEST BY GEL CLOT METHOD:
1.
a. b. c. d. e. f. g. h.

PROCEDURE:
MATERIALS REQUIRED:
Limulus Amoebocyte Lystate (LAL) Reagent. Control Standard Endotoxin (CSE). Lal Reagent Water (LRW). Vortex shaker. Heating Block. Micropipette with sterile tip. Depyrogenated glass test tubes (15x100 & 10x75 mm). Aluminium foil and forceps. All materials coming in contact with the specimen or test reagents must be rendered endotoxin free by heating at 2500C for 60 minutes.

PREPARATION OF ENDOTOXIN:
a. b. c. d. e. Reconstitute CSE as per Certificate of Analysis (COA). Record the date of preparation on the vial. Vortex the vial for 15 minutes after rehydration. Vortex for at least 5 minutes immediate prior to each use or as specified in COA. Reconstituted CSE can be stored at 2-80C for up to 28 days in the refrigerator or as per manufacturer’s instructions. Dilute the endotoxin with LRW to a concentration of 0.5 EU/ml (4λ). Each dilution should be vortexed for 60 seconds prior to proceeding to the next dilution. Refer table – I: - If concentration of CSE is 40 EU/ml as per COA supplied by manufacturer. Using the 0.05 EU/ml endotoxin dilution, prepare the dilution of 0.125EU/ml (λ), where λ is Lysate’s sensitivity.

TABLE – I
Example: - If as per COA stock concentration of CSE is 40 EU/ml, then: S. Dilution ratio CSE LRW Dilution Concentration of Endotoxin No. 1. 1:80 0.1 ml 7.9 ml 4λ 0.5 EU/ml 2. 1:4 1.0 ml of 4 λ 3.0 ml λ 0.125 EU/ml

 Calculate the Maximum Valid Dilution (MVD) by the formula: MVD = Endotoxin limit as specified in Pharmacopoeia. / Lysate sensitivity as per COA.  ANTICOAGULANT SOLUTION: Sample size: - Three blood bag per batch and make a pooled sample. MVD calculation: MVD = 5.56EU/ml / 0.125EU/ml = 1:44. Sample dilution is prepared at MVD / 2 = 0.1 ml sample + 2.1 ml LRW.  MEDICAL DEVICES: I. Sample size: 10 pieces from each lot / batch.

PREPARATIO OF SAMPLE DILUTION:

a.  Calculation of MVD: The endotoxin limit for the extract is calculated by the formula: KxN / V. V = Total volume of the rinse. 2 3. MVD = 1:8.V. 3 way stop cock (10 pieces / batch) can be disassembled and immersed in 200 ml WFI. Where K = Amount of endotoxin allowed per device. This should be observed in the positive control vials (2λ.  A positive reaction is characterized by the formation of a firm gel that remains intact momentarily when the tube is inverted.6 ml LRW. Preparation of Extract: The process of preparing an extract {20ml water for injection (WFI) per device} for LAL test may vary for each device.II): - TABLE – II TUBE NO.II. The Lysate may show an increase in turbidity or viscosity. = 1 EU/ml / 0. Endotoxin limit = 20x10 / 200 = 1EU/ml. I. LRW and CSE in the assay tubes in duplicate and add 100µl of reconstituted LAL reagent to each tubes (as per table . λ/4) and in the positive product control (PPC) vial. INTERPRETATION OF RESULT: - .V. λ. Sample dilution is prepared at MVD / 2 = 0. 8 9. Cannula (10 pieces / batch) pass 200 ml of WFI through 10 pieces of I. 1. 10 11. Each tube is inverted at 1800.125 EU/ml.125 EU/ml = 8. c. Tubing products (10 pieces / batch) can be immersed in 200 ml WFI by cutting into pieces and d. Compare the sample vials to the control vials. LAL reagent sensitivity is 0.  Transfer the prepared sample dilution.  A negative test is characterized by the absence of solid clot after inversion. 14 Blank 2λ λ λ/2 λ/4 NPC PPC LRW 100 µl 50 µl 50 µl 75 µl 50 µl CSE 50 µl(4λ) 100 µl(λ) 50 µl(λ) 25 µl(λ) 50 µl(4λ) PRODUCT 50 µl 50 µl LYSATE 100 µl 100 µl 100 µl 100 µl 100 µl 100 µl 100 µl Incubate the assay tubes at 370C (±10C) in heating block for 60 minutes. 6 7. 12 13. Now MVD for medical devices = Endotoxin limit / Lysate’s sensitivity.4 ml of sample + 1. This extract is kept for 1 hour at controlled temperature (20 – 250C). λ/2. PREPARTAION OF LAL:  Reconstitute LAL Reagent as per manufacturer’s instruction. 4 5. N = Number of medical device tested. Syringes (10 pieces / batch) are flushed by 20 ml WFI (20ml WFI / device). Cannula (20 ml WFI / device). This should be observed in the negative control (blank) vial and in the negative product control (NPC) vial. b. this is considered as negative result.

similar to those used in previous test but weighing 20 ± 3 grams. All the mice survive. 48 and 72 hours. The sample complies with the test when a negative result is found for both tubes of NPC in the repeat result.  Assay tubes should not be removed from incubation or disturbed prior to the time specified for reading the test.  Test results are valid only when the PPC is positive at 2 λ endotoxin concentration and NPC is negative. The time of injection should normally be 15 – 30 seconds. INTERPRETATION OF RESULT: Observe the mice at the time of injection after 24. PREPARATION OF EXTRACT: Make 50 ml saline solution (0. PRECAUTION DURING TEST:  Careful techniques must be used to avoid microbial contamination. the sample passes the test for toxicity.  Samples to be tested must be stored in such a way that all bacteriological activity is stopped or the endotoxin level may increase with time. the test may be repeated at a dilution not greater than MVD. repeat the test using at least another 10 mice.Select 5 healthy mice weighing between 17-23 grams. If the test is positive for sample under test at a dilution less than the MVD.  Switch on the heating block 1 hour before starting the test to get the constant temperature.  Repeat the test when a positive result is found for 1 tube of NPC and a negative result for the other one. METHOD: Inject intravenously each of five mice 1. the sample passes the test for toxicity. Then autoclave this extract at 1210C for 15 minutes. The preparation of extract should be done under Laminar Air Flow.0.8 – 8.  All glassware and materials must be endotoxin free. .  pH of sample should be 6.9% w/v) in Depyrogenated conical flask then take 5 pieces of the batch and pass 10 ml of saline from each piece with 10 mldepyrogenated glass syringe slowly. ABNORMAL TOXICITY TEST: PROCEDURE: Test animal: . Monitor the temperature after every 15 minutes during the incubation period. After pass the saline from piece put that piece in passed saline. If one or more animals die. Record positive and negative results for the test sample vials.0 ml of test solution using 26 gauge needle of half inch. 2. If all mice survive for 72 hours.

Percentage recovery is calculated by using following factor: Percentage recovery = Average bio-burden X 100 / 83. Description Quantity required 1. Test procedure: Each sample is aseptically immersed separately in 100ml.0±0.III Name product of Medium Normal count (cfu/ml) I. of cfu observed / No.45µ and filter immediately. Preparation of Media: Prepare Soybean casein digest agar (SCDA) and Sabouraud Dextrose Agar (SDA) as per requirement and sterilize at 1210C for 15 minutes.2g 3.1 with 0.Product Bio-burden test Sample selection: The sample for bio-burden testing are collected on random basis after they are packed and are ready for sterilization but not sterilized. Sodium Chloride 4. of treatment solution.39 The result should complying limits shown in table – III. M.V. Syringe Alert level (cfu/ml) 35 25 Action level (cfu/ml) 40 30 . Di-Sodium hydrogen phosphate 7. Average bio-burden is calculated by following formula: Average Bio-burden = Total No. Cannula. Table – II S. No. the colonies of each plate are counted and average of all pates is calculated. Shake the flask for some time. D. Di-hydrogen potassium phosphate 3. Incubation: The SCDA plates are incubated at 300C-350C for 3 days and SDA agar plates are incubated at 200C-250C for 5 days. SCDA ˂ 30 Three way stop SDA ˂ 20 cock. previously sterilized at 1210C for 15 minutes. Interpretation of results: On completion of incubation. Table . Peptone 1. Treatment solution: Buffered sodium chloride peptone solution pH 7. Transfer to membrane filter of size 0. Three way stop cock. Syringes 10 pieces Other disposables 05 pieces Frequency: At least one batch in every 15 days is tested for microbial bio-burden. / Distilled water 1000 ml.3g 4.6g 2. Quantity: As given in table I: Table – I Name of product Quantity required I.5% tween – 80 made as given in table – II. of plates.V. Now. transfer 5 membrane filters to the surface of SCDA and transfer the other 5 membrane filters to the surface of SDA.0g 5. Cannula.

Other disposables SCDA SDA ˂ 100 ˂ 50 125 75 150 100 Steps taken on alert and action level:  During alert level the corrective action like increasing the frequency of spray. .  If total counts are crossing action level. mopping and fumigation of clean room is undertaken under the guidance of Q. microbiologist should advice the concerned department in writing about the preventive steps. C. Department.

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