Phosphate Buffered saline

• Phosphate Buffered saline is a buffer solution.
• It is a water based salt solution.
• PBS solution used in rinsing (washing cells) in microbial analysis because it is isotonic and non-toxic to most cells.
• PBS solution used in serial dilution (diluting cells) for cell counting.  
• PBS solution used in reagent preparation.

    Composition                                     grams/liter

Sodium Chloride--------------- ---------------8.50

Di Sodium hydrogen phosphate----------1.910

Potassium di hyrogen phosphate---------0.380

          

pH= 7.2 ±0.2 at 25⁰C

• Heat if necessary to dissolve the medium completely.
• Sterilize by autoclaving 10 lbs pressure (115⁰C) for

Osmotic Pressure

• Osmotic pressure depends on the amount of solute that is present in the solvent (such as water).
• It also depends on the temperature of the solution.
• The movement of water from an area of low concentration of solute to an area of higher concentration of solute.
• The movement of water molecules through a selectively permeable from a region of high water concentration to low water concentration is called osmosis.
• The pressure that needs to be applied to a solution to stop the movement of a solvent into it, when the solution and solvent (such as the water) are separated by a semipermeable membrane that only allows the solvents to pass through.
• Osmotic pressure can prevent osmosis, which is the movement of a solvent (such as water) through a semipermeable membrane from a solution that has a lower concentration of solutes to one that has a higher concentration of solutes.

What is meaning of Solution?

• A solution is a homogeneous mixture of two or more substances. 
• One of the substance is called a solvent (a substance in which other substance or substances are dissolved).
• A solution can exit in a solid, liquid or gas from depending on mixed substances and external condition such as temperature and pressure.
• A liquid composed of two or more homogeneous substances.
• A liquid mixture in which the minor component (the solute) is uniformly distributed within the major component (the solvent).
  

Difference between Isotonic, Hypertonic,Hpotonic solutions?

• Cell is the basic unit of all life, there is nothing basic about it.
• Cells require very specific conditions to be able to function properly.
• Temperature and the amount of water and nutrients must all be just right in order for a cell to be healthy and these optimal conditions vary depending on the organism.
• The amount of fluid both inside and outside a cell is one condition that is very important and this fluid amount is often determined by the amount of solutes outside of the cell.
• Terms isotonic, Hypertonic and Hypotonic are usually used when a reference to cell of a living organism is made but it can be generally used to compare concentration of a solute in two solutions.

Isotonic Solution:

• It maintain equal osmotic pressure.
• Isotonic solutions are two solutions that have the same concentration of a solute.

Hypertonic Solution:

• Hypertonic solutions is one of two solutions that has a higher concentration of a solute.

Hypotonic Solutions:

• Hypotonic solution is one of two solutions that has a lower concentration of a solute.
  

Why 0.9% saline solution is used for the preparation of serial dilution or culture suspension.

• 0.9%  Saline solution is the best dilution for cell count.
• Saline water isotonic solution (no swelling or shrinking of cell) same as cell protoplasm, minimizes the chances of cell death.
• Phosphate buffer saline (PBS) solution also suitable for serial dilution preparation.
• Buffered sodium chloride peptone (BSCP) solution or peptone solution is not suitable for serial dilution because it supports growth and after utilizing the nutrients (peptone) bacterial cell die.
• Saline is 0.9% sodium chloride(Nacl) solution.
• Serial dilution or culture suspension used saline solution (0.9% Nacl) because 0.9% sodium chloride (Nacl) solution is isotonic in nature.
• In isotonic solution the concentration of solutes remains the same both inside and outside of the microbial cell and cells remains at their usual osmotic pressure.
• But water is hypo tonic in nature in nature in which the concentration of solute is grater inside the cell than outside and due to this water try to enter inside the bacterial cell.

Culture suspension preparation

• Microbiology laboratory culture suspension used for decreasing the culture concentration.
• This technique widely used and simple technique.
• Serial dilution purpose used 0.9 % sterile saline.
• 0.9 % sterile saline is best diluent for cell count.
• Cell numbers need to be reduced, which is done by repeatedly the amount of bacteria you have in your sample.
• A sample amount of bacteria sample is mixed with a diluent solution.
• First prepare inoculum is called stock solution or initial suspension..
• Next continue the dilution as per picture.
  
• Every dilution gently vortex.
• Prepared culture plating within 30 minutes time period.
• Incubate the bacterial culture plates at 32.5 ± 2.5⁰C for 18-24 hours (for except anaerobic bacteria).
• Incubate anaerobic organism plates in anaerobic jar or anaerobic incubator at 32.5 ± 2.5⁰C for 48-72 hours.
• Incubate fungal culture plates at 22.5 ± 2.5⁰C for 3-5 days.
• After the completion of the incubation period.
• Observed the colonies with the help of colony counter.
• Select the dilutions which are having cells between 10-100 CFU/mL.
• Count every colony on the plate.
• Preserve the 10-100 cells refrigerator 2-8⁰C for further test.
• By working backwards using multiplication with the " Dilution factor". you will be able to make a determination of the numbers of bacteria in your original sample.

Calculation :

CFU/mL = observed CFU  /  Volume to be analysed  X Dilution factor

Example:

Say you counted 38 CFU on the 10-5 plate with an inoculum of 0.1 mL

= 38 / 0.1  x 10-5 

= 380 x 10-5 

S.No	Serial Dilution	Plate-1	Plate-2	Avg
1	10-1	TNTC	TNTC	TNTC
2	10-2	TNTC	TNTC	TNTC
3	10-3	TNTC	TNTC	TNTC
4	10-4	TNTC	TNTC	TNTC
5	10-5	37	39	38
6	10-6	5	3	4
7	10-7	Nil	Nil	Nil

Fresh Deodorizing Pearls

Oval shaped, gel-like, emerald colored capsules having rose fragrance.

Use:

·  Pearls smelling deodorizing for masking oppressive odors autoclaving exposed plates and contaminated material.

·       These deodorizes will remove unwanted odors from your facility by deodorizing technique in autoclave, incubators, refrigerators, and similar closed apparatus where tissue culture ware may be used or stored.

Application:

·         Microbiology laboratory, Molecular laboratory, chemical laboratory, plant tissue culture work, and various other laboratory.

Types of Fragrance:

1.    Citrus fragrance

2.    Rose Fragrance

Composition : Fragrant oil

Fragrant oil: also known as aroma oils, aromatic oils and flavor oils blended synthetic aroma compounds or nature essential oils.

Bowie-Dick Test

• Bowie-Dick test has been widely used in pharmaceutical industries for the vacuum sterilizers.
• This test developed by J.H. Bowie and J.Dick.
• Bowie-Dick test is also called dynamic air removal test.
• This test is performing to verify heat penetration inside the chamber.
• Bowie –Dick test applied that the pressure and vacuum pulses.
• Sufficient to remove the entrapped air so as to facilitate
• The purpose of pressure and vacuum pulse is to effectively remove air (condensation gases) from the chamber because air is a bad conductor of heat.
• Bowie-Dick test demonstrates proper air removal from the chamber of a pre-vacuum autoclaves.
• If air pockets are present inside the chamber the it would be verify difficult to sterilize the load.
• Pockets of a cool air act as a barrier that prevents steam from penetrating the load.
• Therefore, the air must be removed by a vacuum.
• Heat penetration can be checked by observing EN ISO 11140 Class-2 chemical indicator (Specific test indicator or Bowie-Dick Test Indicator) sheet which is used for this test.
• If colour change of the indicator sheet is uniform then it means air has effectively removed from the chamber and no air pockets left inside the chamber during sterilization.
• But if colour change is not uniform then it means these pulses are not effective to remove all air and air pockets from the chamber.     
• In the past time, Bowie-Dick test was performed by using layer of towels having an indicator sheet in between the towels.
• These towels were placed near the door and over the drain point, which would be considered as cold spot of the autoclave.
• But now a days this towel concept has been replaced by the Bowie-Dick pack.
• Different companies provide different Bowie-Dick test kit but the purpose of all these kits it to check heat penetration inside the load.
• As per HTM 2010 guideline, it is also called standard test pack. Total weight of the pack should be 7.0 ± 0.7 kg and height of the pack should be 25 cm or 250 mm.
• This test has to be performed on daily basis as per HTM 2010 guideline.
• The basic principle of this test is that when we run the Bowie-Dick cycle air is removed from the chamber and all air trapped inside the Bowie dick pack which we place near the over the drain point of the autoclave.
• The test pack is place in an empty chamber on the lowest spot (cold spot) above the drain.
• This sterilization cycle typically consists of three to four pre vacuum pulses. Each of which involve injecting steam in to the autoclave and then pulling on the air and steam through a vacuum,reaching the set point.
• Manufactures recommend running a Bowie –Dick test before you sterilize  your first load of the day.
• If air removal is effective than heat penetration will be proper and there will be uniform colour change of the indicator sheet.
• If air removal is not effective then colour change will not be uniform.
• There are different type of Bowie-Dick test packs available in the market.
  
• For example one of them is a set of paper sheets in pack and middle of these sheets there would be a chemical indicator sheet.
  
• To use this type of Bowie-Dick pack just place the pack as such over the drain point, no need to maintain the weight and height of the pack.
• Another one is having a S.S holder which can hold a bunch of sheet and in the middle, indicator sheet would be there and that pack has to be placed over the drain point.
• This one provide the required pressure on the pack (weight) and required height is maintained by the SS holder.
  

Common causes for failure of a Bowie-Dick test.

• It’s the colour change that tells you the steam is penetrating the test pack and pulling the air out of the autoclave.
• A successful Bowie-Dick test is very evident based on the colour change of a thermochromatic paper within the test pack.
• Thermochromatic ink was used in Bowie-Dick indicator. Thermochromatic ink is a type of a dye that changes colour when temperature increase or decrease.
• If the thermochromatic paper turns completely black,then the steam has penetrated the load and the autoclave is operating correctly.
• If the Bowie-Dick should fail, autoclave and the laboratory facility utilities should be checked. Any pre-vacuum cycle should not be used with any confidence until the test is repeated with a passing result.
• Lack of  or  a partial colour change indicates an unsuccessful Bowie-Dick test cycle. If the colour hasn’t completely change, it means something hasn’t worked correctly.

Related: Classification of Chemical Indicators

S. No	Cause of Failure	Description	Possible Solution
1	Air Leak	If air is able to leak into the autoclave chamber, the steam will be unable to penetrate the load to the point of total sterilization.	Run a  vacuum Leak Test  to further determine if an air leak exists or not.
2	Unwanted Condensation	Occasionally condensation will get trapped in the jacket of the autoclave, which can lead to cold spots at the base of the autoclave.   This could also indicate a wet-steam issue.	Check the steam traps on the autoclave. Check steam quality and wetness.
3	Faulty test Pack	From time to time, a Bowie-Dick Test pack can be faulty.	Check the expiration date and make sure the packs are being stored in the proper environment.
4	No Warm-Up Cycle	A warm-up cycle allows the sterilizer chamber and jacket to reach temperature.	Run a 5 minute sterilization cycle prior to running the Bowie-Dick Cycle.
5	Incorrect Procedures	Test packs work under very specific conditions.	Test packs should be placed in an empty chamber directly over the drain on the bottom rack or shelf. They are designed for use at 250°-273°F (121°-134°C).

Related : Vacuum leak test

What is the importance of Vacuum leak Test

• Vacuum leak test is also called chamber integrity test.
• Vacuum leak test verify the any leakage in autoclave.
• Performed in daily once (first cycle). As per guideline HTM -2010 Vacuum leak test should be performed  on weekly once.
• Vacuum leak test is a critical test because verify the chamber leakage.
• Vacuum leak test applied the vacuum inside the chamber and wait for reach the vacuum as per set point (NLT -600 bar).
• After reach vacuum as per the set point hold the vacuum ten minutes.
• After ten minutes release the vacuum .
• Calculate leakage as per formula.
• After that check the results whether autoclave chamber leakage is within the limit or not.
• If there is any leakage in the autoclave chamber or leakage is exceeding the limits it means there might be chance of entering non-sterile air inside the chamber.
• When vacuum break happens, air enter inside the chamber through the 0.22 micron filter which is located on the unloading side of the autoclave.
• There is no chance of contamination because sterile air enters inside the chamber after filtering through 0.22 micron filter.

Acceptance criteria :

• As per HTM 2010 Vacuum leak test should be not more than 1.3 mbar per minute. But in autoclave printouts (machin fabric) acceptance criteria mentioned is 0.013 bar and not showing whether this vacuum leakage limit is per minute or per 10 minute.

1.  Acceptable leakage = -0.0013 bar/1 mins (1.3 mbar) (1bar =1000 mbar)
2. Acceptable leakage = -0.013 bar/10 mins (13 mbar)

  P= P3-P2/10

P3  =  Before hold the vacuum

P2 = After ten minutes hold

  

Example:

• Pre vacuum =-0.750 bar
•  Delay Before Hold = 5 min
•  Hold Time =10min
• After ten  minutes Hold =-0.745 bar

 P= P3-P2/10

=0.750-0.745 / 10

= 0.005bar  per 10 minutes.

Process End pressure= -0.050 bar

Related : Bowie-Dick Test

How to calculate the Accuracy ?

Accuracy:  

• Is also called "Precision".
• It means the quality or state of being correct or precise.
• It is calculated as follows.

                  Accuracy or Precision = Measured value / Theoretical value   X 100%

Example  :

   20 µl  = 20 mg  (or) 20000ug ( or) 0.02g

           

         1.       19.8 µl

         2.       19.6 µl

         3.       19.7 µl

         4.       19.9 µl

Average (Mean )  = 19.75 µl

19.75 µl = 19.75 mg

=20-19.75/20 X 100

= 20-19.75 = 0.25     0.25/20  X 100 = 1.25

=100-1.25 =98.75

Accuracy  value must be   99 to 101 %

Classification of Chemical Indicators

• Different types of sterilization methods used in pharmaceutical industries.
• Moist heat, dry heat and membrane filtration methods are used in majority of companies.
•  For example in case of moist heat sterilization there are three basic parameters we need to check during sterilization process.
• These are temperature, pressure and time.
• Any process would be called successful if all these parameters are met.
• So, to monitor these parameters in sterilization process we require chemical indicators.
• Chemical Indicator classifications to understand performance parameters and tolerances of various Chemical Indicators.
• The appropriate Chemical Indicator can then be used to obtain the information needed to determine the effectiveness of the sterilization process.
• Chemical Indicator for the sterilization process being monitored.
• ISO 11140 are classified in to six groups.

Ø  Class -1     Process indicators (tape roll, label) use  with individual indicative.

Ø  Class -2    Specific test indicators (Bowie –Dick test).

Ø  Class-3     Single variable indicators.

Ø  Class-4     Multi variable indicators.

Ø  Class -5    Integrating indicators.

Ø  Class-6     Emulating indicators.

1.       Class-1 ( Process Indicators):

• Process Indicators are intended for use with individual units (example. Packs, containers, bottles…etc.) to indicate that the unit has been directly exposed to the sterilization process and to distinguish between processed and unprocessed units.
• They shall be designed to react to one or more of the critical process variables.
• These tapes or labels are pasted on the each articles of the load which we sterilize.
• After completion of the sterilization cycles we can check that whether the load has attained required temperature or not by observing the change in colour of the indicator tape like some indicator tapes changes colour from light yellow to black or from light green to dark green.
• By these indicator tapes we can also differentiate between processed and unprocessed load.
  

2.       Class-2 (Specific test indicators):

• Class 2 indicators are intended for use in specific test procedure as defined in relevent sterilizer / sterilization standards.
• Bowie-Dick type tests are specific tests used for equipment control to evauate the sterilizer performance.
• These indicators are used in specific test. Bowie dick test is the example of this class of chemical indicator.
• In Bowie dick test we check the heat penetration.
• If steam properly penetrate in the load then there will be uniform colour change of the chemical
• Indicator sheet which is used in this process otherwise if colour change is not uniform it means air pockets are present inside the load or autoclave chamber.
• So by this chemical indicator, we can assure that heat penetration is properly talking place within the load.
  

3.       Class-3 (Single variable indicators):

•  A single variable indicator shall be designed to react to one of the critical variable and is intended to indicate exposure to a sterilization process at a stated values (SVs) of the chosen variable.
• Single variable indicators may also be used for exposure control monitoring.
• Determine the a specific temperature was reached at a specific location in the sterilizer chamber.

4.       Class -4 (Multi variable indicators):

• A multi- variable indicator shall be designed to react to two or more of the critical variable and is intended to indicate exposure to a sterilization process at the stated value (SVs) of the chosen variables.
  

5.       Class-5 (Integrating indicators):

• Integrating indicators are the most accurate of the internal chemical indicators.
• Integrating indicators are used for pack control monitoring.
• They can also be used as an additional monitoring tool

6.       Class-6 (Emulating indicators):

• Emulating indicators are cycle verification indicators which shall be designed to react to all critical variables for specified sterilization cycle.
• The sated values (SVs) are generated from the critical variables of the specified sterilization process.
• This is the most accurate class of chemical indicators which met all the three critical parameters of the sterilization process.
  
• For more information on  the ISO 11140 - 1:2005 and USP general chapter 1209.
• All these indicators are very important tool for the monitoring of the sterilization process so have a continue watch over each sterilization cycle with these chemical indicators.

How to ensure autoclave effectiveness ?

• Autoclaves mainly used for sterilization process.
• Autoclave Sterilization temperature at 121⁰C for 15 to 20 minutes depending on the size of the load and the contents.
• Sterilization in an autoclave is most effective when the organisms are either contacted by the steam directly.
• Under these conditions, steam at a pressure about  15 PSI. attaining temperature (121⁰C) will kill all organisms.
• The autoclave was invented by  Charles Chamberland in 1879.
•  This process ensure the 3 parameters

Related: Classification of Chemical Indicators

           1.       Physical  : Pressure, temperature and time recording devices.

           2.       Chemical :  Indicators that change color after being exposed to specific temperatures, such as              temperature sensitive tape.

           3.       Biological : Geo bacillus stereo  thermophilus  spores are used and due to its resistance to                    heat, for the testing that measures the biological performance of the autoclave process.

        Related : Types of Biological Indicators

Micropipette calibration

• Micropipette  requires to  calibration, this process is known as pipette calibration.
• Pipette calibration mainly two types

            1.       Gravimetric method

            2.       Colorimetric method

1.       Gravimetric Method.

• Gravimetric method based upon water gravity.
• Under a constant temperature and atmospheric pressure and relative humidity, the density of distilled water is constant.
• When determining the volume of water, the accuracy of measurements is effected by ambient temperature, atmospheric pressure and relative humidity.
• These factors are usually combined to give the Z factor, used in calculation of volume of water.
  

 Calibration Process:

• Calibrate the micropipette in a vibration free room at a constant temperature of distilled water between 20 to 25⁰C.
• Use analytical balance with a readability of 0.1 mg.
• First determine the density factor of water and set the desired volume.
• Take a clean, dry glass beaker or vial (based upon water evaporation). Keep it on the weighing pan of analytical balance and tare the volume.
• Adjust the pipette volume using adjustment setting knob.
• Carefully fit the tip.
• Flush  (rinse ) the tip with distilled water at least 3 times.
• Aspirate the distilled water and tare the volume .
• Repeat the at least ten times and record the results( the weight display of balance should be adjusted to zero every time).

All the ranges of micropipettes shall be calibrated as per the table.

Pipette range
10-100 ml	20-200ml	100-1000 ml
10 ml	20 ml	100 ml
25 ml	50 ml	250 ml
50 ml	100 ml	500 ml
100 ml	200 ml	1000 ml

Micropipette ID No :

Micropipette Range :  10-100 ml

Analytical balance ID No:

Mean Volume ( ml or ml ) =  Mean Weight ( mg or mg ) x Z

Z value : conversion factor, which is conversion of density

Set  Volume	10 ml	25 ml	50 ml	100 ml
Weight 1
Weight 2
Weight 3
Weight 4
Weight 5
Weight 6
Weight 7
Weight 8
Weight 9
Weight 10
Mean
Mean Volume
Standard Deviation (SD)
Coefficient of variation (CV) or Related Standard Deviation (RSD)
Accuracy (A)

• Calculate the average ( Mean )
• Calculate the standard deviation (S.D.)
• Calculate the imprecision or Coefficient of viaration  ( C.V. ) or RSD
• Calculate the accuracy

Acceptance Criteria:

• Accuracy value must be 99-101 % and C.V. value must be less than 1 %.

2.       Colorimetric method :

• The more expensive method for micropipette calibration.
• Colormetric method in colored water is used & dispensed.
•  The spectrophotometer is used for measuring the color and weight can be derived on the basis of those readings.
• Based on result of either of the method, Micro pipette should be recalibrated with tool provided with each pipette by manufacturer to ensure readings are within tolerance range as recommended in ISO-8655 standards.