Microbial Analysis of Water(Pharma) (1).pptx.pdf

Microbial Analysis of Water For Injection (WFI)
The other grade of water used in pharma is water for injection (WFI) where microbial contamination, biofilms and endotoxins must be eradicated from the sample before use. Either drinking water or purified water should be considered as a feed for WFI production units.
Course Description:Pharmaceutical Water Systems must be appropriately maintained in order to produce high quality water. USP General Chapter Water for Pharmaceutical Purposes provides detailed information about nearly every aspect of maintaining, qualifying, and monitoring a pharmaceutical water system. Microbial control is unarguably the most challenging aspect of operating a pharmaceutical water system, which is why it is discussed extensively in USP along with how to monitor it. This webinar specifically addresses microbial monitoring issues, including biofilm development, microbial test methods, water sampling purposes and procedures, the “trigger levels” associated with the test results, and microbial identification. Other elements of the USP Chapter will be discussed in future webinars.

The live version of this webinar took place on July 21, 2015.

Includes English subtitles.

Access Duration:Access to this course expires 60 days from the date of registration or until you mark the course ‘Complete’ – whichever occurs first.

Objectives:

Understand the basics of water system biofilm growth
Understand the available test method options for determining the microbiome in your system
Learn the process control and quality control uses of the micro data, including Alert and Action Levels and Specifications
Understand the importance of proper and consistent sampling technique and locations
Learn when and why microbial identification is needed

1. MICROBIAL ANALYSIS OF WATER(PHARMA) &
STERILITY TESTING

PHARMACEUTICAL TECHNOLOGY

M.SC SEM-3 MICROBIOLOGY

PREPARED BY: VISHAL PATEL
LECTURER,
HVHP INSTITUTE OF PG STUDIES & RESEARCH

2. WHY WATER TO BE TESTED?
Water is widely used in Pharma company for
manufacturing of different products.
As water is most important ingredient in
manufacturing that’s why it is necessary to be
tested
It contains Microorganism that can be harmful or
pathogenic.

3. TYPES OF WATER
Purified Water
R.O Water
Water for Injection
Pure Steam

4. SAMPLING PROCEDURE
Sampling done in Sterile bottles along with 70%
Iso Propyl Alcohol.
All sampling points must be clean before taking
samples.
It must be drained Upto 1 minute to reduce the
chance of pathogens in water.
Analyst have to do this activity daily or in Shift
basis.

5. TYPE OF TESTING
Membrane Filtration method
Microbial Enumeration test for specified
organisms

6. MEMBRANE FILTRATION

7. 100 ml water have to take in grant for one
cycle of water testing.
With the help of Cellulose membrane (0.45
Micron) pore size we can trap microorganisms
from the water.
Vacuum can suck the water from container to
vacuum flask and after completion of 100 ml
water, membrane than placed on to R2A agar.
R2A Agar is Minimal nutrient medium which
is made to grow oligotrophic organisms.

8. INCUBATION & OBSERVATIONS
This tested plates placed in 30 to 35 Celsius for 5
days.
Plates are observed by Colony counter
Limitations for different water

Water Alert limit (CFU/ml) Action limit
(CFU/ml)
Potable Water 200 300
Purified Water 50 75
Water for Injection 0 1

9. MICROBIAL ENUMERATION FOR SPECIALIZED
ORGANISMS
This test is most important to check whether
Pathogens are present or not.
Test done for
E.coli
Pseudomonas aeruginosa
Staphylococcus aureus
Salmonella enterica

10. MEDIA
Soyabean Casein Digest Medium
MLBB (Macconkey Lactose Bile broth) E.coli
Macconkey Agar E.coli
Eosin Methylene Blue Agar E.coli
Mannitol Salt Agar S.aureus
Cetrimide agar Pseudomonas aeruginosa
Rappaport vassiliadis Salmonella enrichment broth
Xylose Lysine Deoxy-cholate Agar S.enterica

11. E.coli
For this organism first 1 ml of water sample add to
SCDM broth
Incubation for 24 Hrs at 30-35 Celsius
Loopful transfer to MLBB broth.
Incubation at 42 Celsius for 24 hrs
Loopful transfer on Macconkey agar
Incubate at 42 Celsius for 24 hrs.
If lactose ferment colony observed than transfer this
loopful colony to Eosin methylene blue agar to check
whether it is E.coli or Enterobacter

12. Staphylococcus aureus
For this organism first 1 ml of water sample add
to SCDM broth
Incubation for 24 Hrs at 30-35 Celsius
Loopful Transfer to Mannitol salt agar
Incubation for 24 Hrs at 30-35 Celsius
If red color colony observed than rapid test of
Rabbit plasma has to be done.

13. Pseudomonas aeruginosa
For this organism first 1 ml of water sample add
to SCDM broth
Incubation for 24 Hrs at 30-35 Celsius
Loopful Transfer to Cetrimide agar
Incubation for 24 Hrs at 30-35 Celsius
Observe the result

14. Salmonella enterica

For this organism first 1 ml of water sample
add to SCDM broth
Incubation for 24 Hrs at 30-35 Celsius
Loopful Transfer to Rappaport vassiliadis
Salmonella enrichment broth
Incubation for 24 Hrs at 30-35 Celsius
Medium color shows result
Transfer loopful on XLDA agar.
Observe next day result

15. CONCLUSION
Test for Pathogens must be negative
So water must be free from pathogens which will
be used for pharmaceutical manufacturing.

16. STERILITY TESTING
It is very important testing among all the
test in pharmaceutical microbiological
analysis.
This test is done for Parental products,
Medical devices.
Without the result compliance of sterility
testing no product can dispatch to the
market.

17. The test for sterility is carried out under aseptic conditions. In
order to achieve such conditions, the test environment has to
be adapted to the way in which the sterility test is performed.
The precautions taken to avoid contamination are such that
they do not affect any microorganisms which are to be
revealed in the test. The working conditions in which the tests
are performed are monitored regularly by appropriate
sampling of the working area and by carrying out appropriate
controls.
The following culture media have been found to be suitable
for the test for sterility. Fluid Thioglycollate Medium is
primarily intended for the culture of anaerobic bacteria.
However, it will also detect aerobic bacteria. Soybean–Casein
Digest Medium is suitable for the culture of both fungi and
aerobic bacteria.

18. Fluid Thioglycollate Medium is to be incubated at 30°
–35°. For products containing a mercurial preservative that
cannot the Growth Promotion Test and the Method
Suitability be tested by the membrane filtration method
Fluid Thioglycollate Medium incubated at 20°–25° may be
used instead of Soybean–Casein Digest Medium provided
that it has been validated as described in Growth
Promotion Test of Aerobes, Anaerobes, and Fungi.
Where prescribed or justified and authorized, the
following alternative Thioglycollate medium might be
used. Prepare a mixture having the same composition as
that of the Fluid Thioglycollate Medium but omitting the
agar and the resazurin sodium solution.

19. COMPOSITION OF BOTH MEDIA

20. GROWTH PROMOTION TEST

21. TYPES OF STERILITY TESTING
Membrane filtration
Direct Inoculation

Some diluents are used in membrane filtration method to
nullify the chances of contamination like peptone water,
Beta lactemase, Cephalosporin and polysorbate 80

24. INCUBATION
14 days total time for the incubation for
sterility testing
7 days for 20°–25° C and 7 days for 30°–35°

26. THANK
YOU