How to perform the water BET?

Ø  Take the Depyrogenate glassware (250⁰ temperature at least 1 hour).

Ø  Be ensure all material are Pyrogen free.

Method :1

Ø  Water Endotoxin Release Limit (ERL) = 0.25 EU/ml

Ø  Water potency (C)=1 mg/1ml.

Ø  (LAL ) Lysate Sensitivity (λ)= 0.125 EU/ml

Ø  Select the MVD (Maximum Valid Dilution) method.

Ø  Calculate as below

Test Sample:

MVD = ERL X C/ λ

= 0.25 x 1/0.125

=2 (dilution factor)

Ø  You will do 2 dilution of water.(it means 1 dilution water sample and 1 dilution LRW).

Ø  Next take the 100 µl of sample and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ at 1 hour .

Ø  Observe the reading.

ERL Verification :

= Lambda X Dilution factor = ERL

=0.125 X 2 = 0.25

Negative Control:

Ø  Next take the 100 µl of LRW and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ at 1 hour .

Ø  Observe the reading.

Positive Control :

Ø  take the 100 µl (ERL X 2) CSE  and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ at 1 hour .

Ø  Observe the reading

  

Method :2

Ø  Water Endotoxin Release Limit (ERL) = 0.125 EU/ml

Ø  Water potency (C)=1 mg/1ml.

Ø  (LAL ) Lysate Sensitivity (λ)= 0.125 EU/ml

Ø  Select the MVD (Maximum Valid Dilution) method.

Ø  Calculate as below

Test Sample:

 MVD = ERL X C/ λ

= 0.125 x 1/0.125

= 1(dilution factor)

Ø  You will do 1 dilution of water. (not required dilution)

Ø  Next take the 100 µl of sample and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ at 1 hour .

Ø  Observe the reading.

ERL Verification :

= Lambda X Dilution factor = ERL

=0.125 X 1 = 0.125

Negative Control:

Ø  Next take the 100 µl of LRW and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ at 1 hour .

Ø  Observe the reading.

Positive Control :

Ø  take the 100 µl (ERL X 2) CSE and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ at 1 hour .

Ø  Observe the reading

Method :3

Ø  Water Endotoxin Release Limit (ERL) = 0.125 EU/ml

Ø  Water potency (C)=1 mg/1ml.

Ø  (LAL ) Lysate Sensitivity (λ)= 0.03125 EU/ml

Ø  Select the MVD (Maximum Valid Dilution) method.

Ø  Calculate as below

Test Sample:

 MVD = ERL X C/ λ

=0.125 x 1/0.03125

= 4 (dilution factor)

Ø  You will do 4 dilution of water.(it means 1 dilution water sample and 3 dilution LRW).

Ø  Next take the 100 µl of sample and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ at 1 hour .

Ø  Observe the reading.

ERL Verification :

= Lambda X Dilution factor = ERL

=0.03125 X 4 = 0.125

Negative Control:

Ø  Next take the 100 µl of LRW and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ at 1 hour .

Ø  Observe the reading.

Positive Control :

Ø  take the 100 µl (ERL X 2) CSE and 100µl of Lysate (LAL).

Ø  Incubate at 37⁰ at 1 hour .

Ø  Observe the reading

How to calibrate the Heating block ?

ü  Set the required temperature that is 37°C for calibration

ü  Take 5 endotoxin test tubes (10x 75 mm)

ü  add approximately 0.5 ml of purified water in each tube for performing the calibration

ü  Place the test tubes in the wells.

ü  about few minutes for stabilize the water within the tubes.

ü  Place a calibrated thermometer or Temperature RTD sensor or thermocouples in one of the tube for few seconds

ü  record the temperature. And also note down the display temperature of heating block.

ü  After note down the reading take out and wipe the thermometer or Temperature RTD sensor or Thermocouples with the help of a tissue paper

ü  Continue this process for all the remaining four tubes of LAL heating block

ü  Place the heating block on a hard vibration free surface

ü  When incubation is in progress do not disturb the incubator.

 ACCEPTANCE CRITERIA : 37 ± 1 º C.

How to calculate the log reduction?

·         "Log reduction" is a mathematical term (as is "log increase") used to show the relative number of live microbes eliminated from a surface by disinfecting or cleaning.

·         For example, a "5-log reduction" means lowering the number of microorganisms by 100,000-fold, that is, if a surface has 100,000 pathogenic microbes on it, a 5-log reduction would reduce the number of microorganisms to one.

Log Reductions

1 log reduction means the number of germs is 10 times smaller (10¹)

2 log reduction means the number of germs is 100 times smaller (10²)

3 log reduction means the number of germs is 1000 times smaller(10³)

4 log reduction means the number of germs is 10,000 times smaller(10⁴)

5 log reduction means the number of germs is 100,000 times smaller(10⁵)

6 log reduction means the number of germs is 1,000,000 times smaller(10⁶)

7 log reduction means the number of germs is 10,000,000 times smaller(10⁷)

v  Another way to look at it is: 1-Log Reduction would reduce the numberof bacteria 90%.

v   This means, for example, that 100 bacteria would bereduced to 10, or 10 reduced to 1.

Example:

1 log reduction = 100( bactreia) to 1log reduction = 100-10 =90 kill the bactria

 1000( bactreia)= 2log reduction = 1000-100 =900 kill the bactria

= Log =10 10 X10 =100

=log=10 10 X 100 =1000

EX:

Log Reduction (LR) = log10 (107) – log10 (105) =7-5 =2

Pyrogen Test (Rabbit test or Sham Test)

Ø  1912 : pyrogen test was done by Rabbit Test method.

Ø  USP XII –(Rabbit test ) approved method (1942 to 1980 )

Pyrogen :

Ø  A pyrogen is a foreign substance that causes a fever (temperature elevation) in an animal’s body. Typically, pyrogenic substances include endotoxin and other bacterial by products. Vaccines and other injectable drugs must be confirmed to be pyrogen free according to regulatory requirements of 21CFR, USP, and EP.

Pyrogen Testing :

1.       Rabbit test method (Sham test).

2.       LAL test  ( Bacterial Endotoxin Test).

Rabbit Test Method:

 3 rabbits is selected

 Endo toxin sample is inoculated (10mL/kg per body weight)

 3 days rabbit temperature is observed

 Rabbit temperature is with in 37^oC.(sample does not have endotoxins or toxic)

 Rabbit temperature out off 37^oC.(sample have endotoxins or toxic)

 37^oC =healthy human temperature.

 If <1.4⁰C increase the temperature  acceptable. (product is Pyrogen free).

 If >1.4⁰C increase the temperature  not acceptable. (product is Pyrogenic).

Pyrogen Test Procedure:

Ensure the test material all are pyrogen free.

 Check the pressure or absence of pyrogen in all aqueous parentenals.

 Rabbits are used to perform the test their body temperature increases when pyrogen is

    introduced by parentenals route.

Procedure:

 Three healthy rabbits are selected.

 Each weighing at least 1.5 kg

Note:

. Do not use any rabbit having a temperature higher than 39.8⁰C  and lower than 38⁰C.

. Inject the solution being examined slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes.

Method:

 Test performed in air condition room.

 The food and water is withheld to rabbit overnight.

 Clinical thermometer is in inserted in the rectum of each rabbit to a depth of not less than 7.5

        cm.

 Two readings of temperature of rabbit in normal conditions should be taken at the interval of

      half an hour before start the test.

 Record the “initial temperature” of each rabbit.

 Record the “initial temperature” of each rabbit.

  The equipments, injections and needles used in the test should be pyrogen free.

 The injection is warmed to 38⁰C before inject to the rabbits 0.5 to 1.0 ml per kg dose should be

      injected through the ear vein.

 Six reading of temperature are recorded at an initial of half an hour.

Pyrogen Test Results:

 Each rabbit is detected by difference of initial temperature and the highest temperature

     recorded.

 Three rabbits are calculated.

 If the sum of responses does not greater than 1.4⁰C and any rabbit shows the response less than

    0.6⁰C, the product passes the test.

 If sum of responses is greater than  1.4⁰C or any of rabbit shows the response 0.6 or greater,

     continue the test using 5 rabbits.

 If test is done using 5 rabbits then if sum responses of all 5 rabbits is greater than 3.7⁰C and the

individual responses of not more than three rabbits is greater than 0.6⁰C the product passes

the test.

Different types of sterilization process used microorganisms

Name of the Sterilization Process	Microorganisms Used
1.    Steam sterilization	Geo bacillus Stearothermophilus (7953)
2.    Hydrogen peroxide sterilization
3.    Formaldehyde Strilization
4.    Propylene Oxide Sterilization
5.    Ozone Sterilization
1.    Ethylene Oxide sterilization	Bacillus Atrophaeus (9372)
2.    Dry heat sterilization
3.    Chloroxidene sterilization
4.    Ozone Sterilization
1.    Low temperature or steam sterilization	Bacillus Subtilis (6633)
1.    Ionizing radiation	Bacillus pumpilus (27142)
2.    Ultra violet sterilization
1.    Food industry	Bacillus Cerues

Classification of microbial media

·         Culture media contains nutrients and  physical growth parameters necessary for microbial growth. All microorganisms cannot grow in a single culture medium and in fact many can’t grow in any known culture medium.

·         Organisms that cannot grow in artificial culture medium are known as obligate parasites. Mycobacterium  and  Chlamydias are obligate parasites. Bacterial culture media can be distinguished on the basis of composition, consistency and purpose.

1.    Solid medium (Agar medium):
solid medium contains agar at a concentration of 1.5-2.0% or some other, mostly inert solidifying agent. Solid medium has physical structure and  allows bacteria to grow in physically informative or useful ways (e.g. as colonies or in streaks). Solid medium is useful for isolating bacteria or for determining the colony characteristics of the isolate.

2.    Semisolid media:
They are prepared with agar at concentrations of 0.5% or less. They have soft custard like consistency and are useful for the cultivation of microaerophilic bacteria or for determination of bacterial motility.

3.    Liquid medium(Broth medium):
These media contains specific amounts of nutrients but don’t have trace of gelling agents such as gelatin or agar. Broth medium serves various purposes such as propagation of large number of organisms, fermentation studies, and various other tests. Example : SCDM & RVSEB.

Types of media:

1.    Complex media

2.    Defined media

3.    differential media

4.    Enrichment media

5.    Enriched Media

6.    Thermolaible media

7.    Selective media

8.    Basel media

9.    Storage media

Culture:  

The microbes can grow and Multiply. In or an culture medium.

Culture Medium:

This nutrient medium Prepared for a growth of microorganism in laboratory.

Nutrient Broth:

If the Complex media in liquid form.

Nutrient agar:

 If the complex media in solid Forms, agar is added.

Selective Media:

Suppression of unwanted microbes and encouraging the desired microbes.

Differential media (Indicative media):

Differentiation of colonies.

 

Enrichment Media:

Similar to selective but designed to increasing the number of colonies  of desired microbes to detectable levels.

Enrichment media is liquid forms that inhibited the unwanted microbes.

Enriched Media:

Additional source of blood are serum having.

Solid media like chocolate agar media and blood agar media.

Basel Media:

Used for growth (culture) of bacteria that do not need enrichment of the media.

Ex:

Nutrient Agar

Nutrient Broth

Peptone water.

Storage media:

Media used for long time storage purpose.

Ex :  Egg saline medium

Chalk cooked meat broth.

Bacterial growth curve

·         Bacterial Generation Time (or) Doubling Time

1. Lag Phase

2. Exponential (or) logarithomic Phase (or) Log phase

3. Stationary phase

4. Decline Phase

1. Lag Phase:

·         When microorganism is introduced in to the first medium.

·         It take some time to adjust which the new environment.

·         Cellular metabolism is accelerated.

·         Cells are increasing in size.

·         By bacteria replicate.

2. Exponential (or) Log phase:

·         The microorganism rapidly growing and divided state.

·         Organism DNA replicate begin by Binary fission at a constant rate.

·         The time taken bacteria to double in number a specified period is Known as the  generation time.

3. Stationary phase:

·         As bacteria Population continuous to growth .

·         All the nutrients in the growth medium are used up by the microorganism for the Rapid multiplication.

·         Then reproduction rate will slow down.

·         This phase divided cells and number of cell death rate is equal.

·         Finally stop the division completely.

4. Decline (or) Death Phase:

·         The bacteria completely loses its ability to reproduce.

Ex:

E.coli ---------------------- 20 minutes

S.enterica --------------20 minutes

S.aureus -------------------30 Minutes

B.Cerus----------------------30 Minutes

Formula:   2 Number of Generation X Intial Number of Bacteria = Total Cells

Ex :

“Bacillus subtilies “ Divided every 30 minutes,  you inoculated a culture with exactly 100 Bacterial cells. After 3 hours, how many bacteria are present?

3 hours, B.subtilies Cells will Divided 6 times

Therefore,

N= 6 =26 = 2 X2 X 2X 2X 2X 2 = 64

= 64 X 100= 6400 cells

Environmental monitoring (Viable monitoring) limits

·         WHO(World Health Organization) & EU (European Union) viable monitoring :

Class		Settling plates cfu/m2/4 hours	Active Air sampling  Cfu/m3	Surface Monitoring Cfu/plate (25 cm2)	Personal monitoring Cfu/glove
5	A	<1	<1	<1	<1
6	B	5	10	5	5
7	C	50	100	25	--
8	D	100	200	50	--

·         Schedule-M (Drug and Cosmotic Act 1940) Limits :

Class		Settling plates cfu/2 hours	Active Air sampling  Cfu/m3	Surface Monitoring Cfu/plate (25 cm2)	Personal monitoring Cfu/glove
5	A	<1	<1	<1	<1
6	B	5	10	5	5
7	C	50	100	25	--
8	D	100	500	50	--

·         Suggested Initial Contamination Recovery Rates in Aseptic Environments

(As per USP 40 general chapter 1116 ).

Class		Active Air Sample (%)	Settle Plate (9 cm) 4 h Exposure (%)	Contact Plate or Swab (%)	Glove or Garment (%)
Isolator/Closed RABS (ISO 5 or better)		<0.1	<0.1	<0.1	<0.1
5	A	<1	<1	<1	<1
6	B	<3	<3	<3	<3
7	C	<5	<5	<5	<5
8	D	<10	<10	<10	<10

Related : What is the role of Viable monitoring programme in pharmaceutical manufacturing facility?