Filter Integrity

• Filter integrity testing is an essential procedure to detect defective Sterilize filter cartridges.
• The filter Integrity test one of the most important test that are carried out on the final product. 
• A satisfactory result from this test gives assurance that the final product is sterile and therefore acceptable for human use.
• The membrane filter integrity test is not mentioned in the International Pharmacopoeia.
• However, it is a requirement of GMP guidelines on aseptic processing using final sterilizing filtration.
• It is highly recommended that the test is performed prior to release of the product.
• Different types of filter integrity machines available in market.

1. Pall Corporation Ltd
2. Merk-Millipore
3. Sartorious 

Different size of filters Integrity ;

a) 20 inch Sterilize filters  

b) 10 inch Sterilize filters

c) 5 inch Sterilize filters

d) 4 inch Sterilize filters (Vent filters or Cartridge filters)

e) 0.45μm Sterilize filters

f) 0.22μm Sterilize filters

Types of test 

1. Bobble point test (BPT)

2. Diffusion Test (DIFT)

3. Combined Test (BPT + DIFT)

4. Pressure Drop Test / System Leak Test

5. Water Flow Test (WFT)

Filter 2 types :

a) Hydrophobic Filter :

• will not pass in water but will wet in low surface tension liquids.
• Example organic solvents or alcohols.
• 0.45μm Sterilize filters
• This filters are best suited for gas filtration, low surface tension liquids and venting.
• wetting purpose used 60% Iso Propyl alcohol.

b) Hydrophilic Filters:

• Easily wet with water.
• This filters are best suited for aqueous solutions.
• filter integrity used cooled WFI (20°-25°C)

Filter wetting :

• Wet the filter with the appropriate fluid,typically water for hydrophilic membranes or an alcohol / water mixture for hydrophobic membranes.

1 millibar (mbar) = 0.01450 PSI (pounds per square inch)

1PSI = 68.9475 millibar

15 PSI= 68.9475 X 15 =1034.2131 millibar

1380 millibar = 20.01 PSI

Inoculation loop

• Is also called a smear loop, micro streaker and inoculation wand.
• The loop is used for cultivation of microorganisms on plates by transferring inoculum for streaking. 
• It is easy to sterilize reuse because nichrome wire resists deterioration with repeated heat/ cooling cycles(easy to heat and easy to cool).
• Flame sterilization is a very simple method of killing microorganisms on an inoculating loop.
• hold the wire loop end inside an alcohol burner or Bunsen burner flame for a few seconds to bring it.
• and 15-30 seconds to cool.
• when cooled, it is ready for culture inoculations.
• this tool can be reused in different experiments without fear of cross - contamination.
• The loop of wire at the tip may be made of 24 swg nichrome, platinum or tungsten.
• this is inexpensive way.

• The wire forms a small loop with a diameter about 5 mm.
• The length of loop and wire should be no more than 50 mm.

Two types of inoculation loops available in market.

1. With calibrated inoculation loop (calibrated).
2. With out calibrated inoculation loop (Non- calibrated).

1. Calibrated inoculation loops:

• 1µL and 10 µL size available.
• This is used for quantitative specimen cultures.
• Certified calibration.

2. Non- calibrated loops:

• Available in range of sizes with internal  diameter  (Ø) of 2, 3, 4 or 5 mm.

• now a days ready to use Inoculation loops available in market.
• this loop is Gama irradiated sterilizated loop.
• easy to handle  and discard.

Previ Color Gram

• This equipment used for staining purpose.
• Automated gram staining machine.
• Improved the consistency and quality of slides.
• Reducing potential variability among samples.
• This system utilizes patented spray technology to provide rapid, standardized results for all types of specimen.
• Staining process complete within 5 minutes.
• Assurance of quality - No worry about cross contamination.
• Reduce the reagents use and waste.
• Minimal hands- on time and clean up.
• Full traceability of reagents, users, maintenance and slides.
• Touchscreen interface for easy-handling of the system.
• two models available.

1. 12 -slide carousel.
2. 30 -slide carousel.

• Quick to load, push button carousel.

Fogging and Fumigation

• Both process used for controlling of microorganism.
• Fumigation activity banned by different regulatory guidelines because negative effect(causing of irritation to the eyes, nose and skin) .

Fogging

• Fogging is a technique.
• This technique used for killing of microorganisms.
• Which is directed by a blower and vaporized spray.
• This process require fogging machine and fogging solution (Fumigant).
• By these fogger machines solution is sprayed in the area in form of aerosol.
• The small particles of disinfectant solution suspend in the air for long time and kill all the air borne bacteria,fungus and their spores.
• This is very effective way to control the contamination.
• The recommended ULV (Ulta Low Volume)fogger used for fogging. 

• Fogging purpose Hydrogen peroxide vapor or Virosil is better.
• Fumigant valid 6% Hydrogen peroxide, 10% Hydrogen peroxide and 20% Hydrogen peroxide used for better controlling of microbes.
• Virosil (mixer of Hydrogen peroxide 10% and Silver Nitrate 0.01%Approximate pH 1.5).
• Market available different brand names.
• Fogging is safer activity.
• Defogging is not necessary.  

Fumigation:

• This method used for Formaldehyde solution and Potassium permangnate chemicals.
• These method generate the fumes and effectively kill the bacteria, fungi and spores.
• This is very effective method for controlling the microorganisms.
• These chemicals are carcinogenic because cause to irritation,to the eyes, nose and skin.
• carcinogenic means cancer causing or risk of cancer.
• It is not safer for the personnel who is handling this chemicals.
• After completion activity defumigation is required at least 6 hours.

operation :

• Before stating the Fogging/ Fumigation activity Keep the area Under Fumigation/ Fogging.
• Stop the AHU Up to Closing the Fumigation/ fogging activity.
• After completion of the activity continuously circulate the AHU (Air Handling Unit)at least 6 hours.
• Frequency : Fortnight  or whenever expose the cultures.

How to calculate the Area Volume:

• Area length x Width x Hight

Calculation total qty of liquid

=   liqiud (ml) = volume X applicatio rate (800ml/ 1000 cubic meter(M³)

=     1464 X 800/1000 = 1171 ( 1200 ml)

Calculate Foggining Time:

=    total liquid / flow rate

=   1200/ 70 = 17.14 minuts (20 minuts)

•   application rate based on the fogger/fumigator flow rate.

How to calculate the hydrogen peroxide percentage:

 1000/ 30 % H2O2  X 6% = 200

=1200/30 % H2O2  X 6%

=240

Streaking

• Streaking is a microbiology technique.
• Streaking technique used for single cell colonies identification.
• Bacteria and fungi to grow on a semi solid surface to produce decrease colonies.      
• Isolate the single species of microorganism. 
• Large concentration of bacteria to small concentration of bacteria. 
•  Robert koch is isolated this technique.

·         Types of streaking 

1.    T- streak (Three Sector Streak)

2.    Zigzag streak

3.    Quadrant streak

4.    Ecometry streak

T-Streaking:

Three Sector Streak (t streak):

 

1. Sterilize the wire loop.
2. Cool the loop by touching it on the edge of the sterile agar plate.
3. Dip the loop into the broth culture containing the mixture of bacteria.
4. Lift the lid of the plate just enough to insert the loop. Drag the loop over the surface of the top one-third of the plate back and forth in a "zig-zag" formation.
5. The loop has picked up thousands of bacteria which are spread out over the surface of the agar.
6. Sterilize the loop in the flame.
7. Turn the plate 90 degrees and drag the loop through the area you have just streaked two to three times and continue to drag the loop in a "zig-zag" formation in the remaining half of the plate without touching that area again.
8. Sterilize the loop in the flame.
9. Turn the plate 90 degrees. Repeat the procedure. Drag the loop two to three times through the area you just streaked, and fill in the remaining area of the plate (zig-zag formation), being very careful not to touch any of the areas you previously streaked.
10. Incubate the plate for 24 hours. If you streaked correctly, you will see isolated colonies in the third sector. The heaviest growth will be in the first sector. There will be less growth and some isolated colonies in the second sector. The third area should have the least growth with isolated colonies.

Four Quadrant Streak :

1. Loosen the cap of the bottle containing the inoculum.
2. Hold an inoculation loop in your right hand.
3. Flame the loop and allow it to cool.
4. Lift the test tube containing the inoculum with your left hand.
5. Remove the cap/ cotton wool plug of the test tube with the little finger of your right hand.
6. Flame the neck of the test tube.
7. Insert the loop into the culture broth and withdraw. At all times hold the loop as still as possible.
8. Flame the neck of the test tube again.
9. Replace the cap/ cotton wool plug of the test tube using the little finger of your right hand. Place the test tube in a rack. For a liquid culture, dip the loop into the broth, or for solid media, lightly touch a colony with the loop.
10. Partially lift the lid of the Petri dish containing the solid medium.
11. Place a loopful of the culture on the agar surface on the area 1. Flame the loop and cool it for 5 seconds by touching an unused part of the agar surface close to the periphery of the plate, and then drag it rapidly several times across the surface of area1.
12. Remove the loop and close the Petri dish.
13. Reflame and cool the loop, and turn the petri dish 90°C then touch the loop to a corner of the culture in area1 and drag it several times across the agar in area 2, hitting the original streak a few times. The loop should never enter area 1 again.
14. Remove the loop and close the Petri dish.
15. Reflame and cool the loop and again turn the dish 90°C anticlockwise. streak area 3 in the same manner as area 2, hitting last area several times.
16. Remove the loop and close the Petri dish.
17. Flame the loop, again turn the dish 90°C and then drag the culture from a corner of a area3 across area 4, contacting area 3 several times and drag out the culture as illustrated. Using a wider streak. Do not let the loop touch any of the previously streaked areas. The flaming of the loop at the points indicated is to effect the dilution of the culture so that fewer organisms are streaked in each area, resulting in the final desired separation.
18. Remove the loop and close the Petri dish.
19. Tape the plate closed and incubates the plate in an inverted position in an incubator for 24-48 hours.
20. Flame the loop before putting it aside.

Bio chemical tests

	· ·	Catalase Test Put the one drop 3 % H2O2(hydrogen peroxide) Visible bubble split formation
EX:
	1.	Stephylococci
	2.	Bacillus subtitils
	3.	Pseudomonas aeuroginosa
	4.	Candida Albicans
	5.	Aspergillus brasillensis
	6.	Micrococci
	7.	E.coli
	8.	Protious vlugartus 9. Salmonella enterica

 ·         Catalase is the Enzyme that breaks Hydrogen peroxide (H2O2) in to H2 , O2.

 ·    2H2O2----------------------------- 2H2 O + O2

·         3% hydrogen peroxide is topical disinfectant in wound. And the bubbling that is seen is due to the evolution of O2 gas.

·         H-O-O-H

Oxidase test:

·         Bacterium produced the Cytochrome oxidase enzyme production

·         This enzyme is indicative of aerobic respiration.

·         Oxidase disk/oxidase reagents.

Positive Test:

·         Purpule (dark purpule) due to free of oxigen. Negative Test:

·         Light pink (no colour change)

EX:

1.       Psedomonas auroginosa

2.       Vibrio cholerae

      ·         Organism is a aerobe or aerobic respiration.

·         Electron transport chain that the final phase normally oxygen is final electron transfer but

·         In this Oxidase test a artificial final electron acceptor (N,N,N1,N1 –tetra methyl 1-4 phenlenedimine di chloride) is used in the place of oxygen.

·         This process Artificial chemical that change colour to a dark blue/purple colour.

·         When it takes the electron from last element (Cytochorome Oxidase) in the electron transport chain.

                                          Coagulation Test

·         Blood clotting test.

·         0.5 ml Mammalian (rabbit or horse) plasma + 1ml culture

·         Incubate 30-35⁰C 1 day incubation after gel formation.

          Ex : Staphylococcus aureus.

Indole test

·         Indole test used to determine the ability of an organism to spilt amino acid tryptophan to from the Compound Indole.

·    Tryptophan/amino acids ---------------------covert ------------------------ Indole.

·         Indole test differentiate the Entero a teria’s or other genera.

  

Method:

·         Inoculate the tryptophan broth add 1 ml of young culture.

·         Incubate at 37°C for 24-28 hours in ambient air.

·         Add 0.5 ml or 5 drops of Kova ’s reagent or Ehrli h’s reagent to the broth culture

Expected results:

Positive: Pink or Red –Violet colored ring or layer after addition of appropriate reagent

Negative: No color change even after the addition of appropriate reagent. e.g. Klebsiella Pneumoniae

Indole positive organisms: Most strains of E.coli, P. vulgaris are indole positive.

Kovac’s reagent or Ehrlich’s  reagent

Isomyl Alcohol, Para- Dimethyl amino benzaldehyde, concentrated hydrochloric Acid.

Motility test

1. Hanging Drop motility test:

·         Place a drop of bacterial culture(young broth culture) in middle of a cover slip.

·         Take a slide.

·         Slide around the petrolium jelly or paraffin wax applied.

·         The slide is inverted (gently press the cover slip)

·         The bacterial drop hangs the cover slip.

·         Observe under microscope.

·         Use immersion oil in under 100X objective.

·         Use a lower power lens to find the bacteria.

2. Biochemical test:

·         Take the TTC motility agar tubes.

·         Inoculate the culture (young culture) with the help of the needle, all the way to the bottom.

·         Incubate the tube 30-35⁰C.

·         Observed the tube

·         Motile bacteria spread (red colored) the throughout tube.

·         Red colour dye –Tetrazolin which turn to red as a result of the bacterium metabolizing.

Expected Results /Response Motility

Eschericha coli -------------------Growth Positive

Salmonella ------------------------Growth Positive

Staphylococcus aureus ---------Growth Negative (non-motile)