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 manufacturers instructions. Dilute the endotoxin with LRW to a concentration of 0.5 EU/ml (4). 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 (), where is Lysates 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 4 0.5 EU/ml 2. 1:4 1.0 ml of 4 3.0 ml 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:

II. Preparation of Extract: The process of preparing an extract {20ml water for injection (WFI) per device} for LAL test may vary for each device. This extract is kept for 1 hour at controlled temperature (20 250C). a. Syringes (10 pieces / batch) are flushed by 20 ml WFI (20ml WFI / device). b. 3 way stop cock (10 pieces / batch) can be disassembled and immersed in 200 ml WFI. c. Tubing products (10 pieces / batch) can be immersed in 200 ml WFI by cutting into pieces and d. I.V. Cannula (10 pieces / batch) pass 200 ml of WFI through 10 pieces of I.V. Cannula (20 ml WFI / device). Calculation of MVD: The endotoxin limit for the extract is calculated by the formula: KxN / V. Where K = Amount of endotoxin allowed per device. N = Number of medical device tested. V = Total volume of the rinse. Endotoxin limit = 20x10 / 200 = 1EU/ml. Now MVD for medical devices = Endotoxin limit / Lysates sensitivity. = 1 EU/ml / 0.125 EU/ml = 8. MVD = 1:8. Sample dilution is prepared at MVD / 2 = 0.4 ml of sample + 1.6 ml LRW.

PREPARTAION OF LAL:
Reconstitute LAL Reagent as per manufacturers instruction. LAL reagent sensitivity is 0.125 EU/ml. Transfer the prepared sample dilution, LRW and CSE in the assay tubes in duplicate and add 100l of reconstituted LAL reagent to each tubes (as per table - II): -

TABLE II
TUBE NO. 1, 2 3, 4 5, 6 7, 8 9, 10 11, 12 13, 14 Blank 2 /2 /4 NPC PPC LRW 100 l 50 l 50 l 75 l 50 l CSE 50 l(4) 100 l() 50 l() 25 l() 50 l(4) PRODUCT 50 l 50 l LYSATE 100 l 100 l 100 l 100 l 100 l 100 l 100 l

Incubate the assay tubes at 370C (10C) in heating block for 60 minutes. Each tube is inverted at 1800. Compare the sample vials to the control vials. A positive reaction is characterized by the formation of a firm gel that remains intact momentarily when the tube is inverted. This should be observed in the positive control vials (2, , /2, /4) and in the positive product control (PPC) vial. A negative test is characterized by the absence of solid clot after inversion. This should be observed in the negative control (blank) vial and in the negative product control (NPC) vial. The Lysate may show an increase in turbidity or viscosity; this is considered as negative result.

INTERPRETATION OF RESULT: -

 Record positive and negative results for the test sample vials. Test results are valid only when the PPC is positive at 2 endotoxin concentration and NPC is negative. Repeat the test when a positive result is found for 1 tube of NPC and a negative result for the other one. The sample complies with the test when a negative result is found for both tubes of NPC in the repeat result. If the test is positive for sample under test at a dilution less than the MVD, the test may be repeated at a dilution not greater than MVD.

PRECAUTION DURING TEST: Careful techniques must be used to avoid microbial contamination. All glassware and materials must be endotoxin free. pH of sample should be 6.8 8.0. 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. Assay tubes should not be removed from incubation or disturbed prior to the time specified for reading the test. Switch on the heating block 1 hour before starting the test to get the constant temperature. Monitor the temperature after every 15 minutes during the incubation period.

2. ABNORMAL TOXICITY TEST: PROCEDURE: Test animal: - Select 5 healthy mice weighing between 17-23 grams.

PREPARATION OF EXTRACT: Make 50 ml saline solution (0.9% 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. After pass the saline from piece put that piece in passed saline. The preparation of extract should be done under Laminar Air Flow. Then autoclave this extract at 1210C for 15 minutes.

METHOD: Inject intravenously each of five mice 1.0 ml of test solution using 26 gauge needle of half inch. The time of injection should normally be 15 30 seconds.

INTERPRETATION OF RESULT: Observe the mice at the time of injection after 24, 48 and 72 hours. All the mice survive; the sample passes the test for toxicity. If one or more animals die, repeat the test using at least another 10 mice, similar to those used in previous test but weighing 20 3 grams. If all mice survive for 72 hours, the sample passes the test for toxicity.

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. Quantity: As given in table I: Table I Name of product Quantity required I.V. Cannula, 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. Treatment solution: Buffered sodium chloride peptone solution pH 7.00.1 with 0.5% tween 80 made as given in table II, previously sterilized at 1210C for 15 minutes. Table II S. No. Description Quantity required 1. Di-hydrogen potassium phosphate 3.6g 2. Di-Sodium hydrogen phosphate 7.2g 3. Sodium Chloride 4.3g 4. Peptone 1.0g 5. D. M. / Distilled water 1000 ml. Preparation of Media: Prepare Soybean casein digest agar (SCDA) and Sabouraud Dextrose Agar (SDA) as per requirement and sterilize at 1210C for 15 minutes. Test procedure: Each sample is aseptically immersed separately in 100ml. of treatment solution. Shake the flask for some time. Transfer to membrane filter of size 0.45 and filter immediately. Now, transfer 5 membrane filters to the surface of SCDA and transfer the other 5 membrane filters to the surface of SDA. Incubation: The SCDA plates are incubated at 300C-350C for 3 days and SDA agar plates are incubated at 200C-250C for 5 days. Interpretation of results: On completion of incubation, the colonies of each plate are counted and average of all pates is calculated. Average bio-burden is calculated by following formula: Average Bio-burden = Total No. of cfu observed / No. of plates. Percentage recovery is calculated by using following factor: Percentage recovery = Average bio-burden X 100 / 83.39 The result should complying limits shown in table III. Table - III Name product of Medium Normal count (cfu/ml) I.V. Cannula, SCDA 30 Three way stop SDA 20 cock, Syringe Alert level (cfu/ml) 35 25 Action level (cfu/ml) 40 30

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, mopping and fumigation of clean room is undertaken under the guidance of Q. C. Department. If total counts are crossing action level, microbiologist should advice the concerned department in writing about the preventive steps.