Microbiology lecture

1. Water
 The microbial ecology of water has a great importance in
pharmaceutical industry because it has multiple uses such as:
Constituent of many products
Washing
Cooling
 Almost 98% contamination of water are caused by gram-
negative bacteria.
 Microorganisms of fresh water include E. coli, Streptococcus
faecalis, Clostridium spp., Pseudomonas spp., Flavobacterium
spp..

2. Sources of contamination in production zone
Microorganisms indigenous
to fresh water include
 Pseudomonas spp.,
 Alcaligenes spp.,
 Flavobacterium spp.,
 Chromobacter spp. and
 Serratia spp.
Contamination by sewage
results in the presence of
 Proteus spp.,
 Escherichia coli
and other enterobacteria,
 Streptococcus faecalis
and
 Clostridium spp.
Bacteria which are introduced
as a result of soil erosion, heavy
rainfall and decaying plant
matter include-
 Bacillus subtilis,
 B. megaterium,
 Enterobacter aerogenes and
 Enterobacter cloacae.
1. Indigenous
2. Introduced
3. Sewage contamination

3. The following types of water are used in pharmaceutical
industry:
1) Raw or Main water
2) Softened water
3) Deionized or Demineralized water
4) Distilled water
5) Water produced by reverse osmosis.
Types of Water

4. 1. Raw or Main Water:
 Natural water found in environment such as surface water, ground
water, tap water.
 Supplied by local authority through pipeline.
 Quality of raw water depends on the source from which it is
supplied.
Use: Used for washing equipment.
Limitation: Not used for pharmaceutical preparation.
2. Softened Water:
 Water that does not contain significant amount of minerals such as
compound of Ca⁺ and Mg⁺.
 Bacillus spp. And Staphylococcus aureus are usually found.
 Prepared by a base exchange method using:
Sodium Zeolite
Lime Soda Ash
Sodium Hexametaphosphate
Water

5. Use: Used for washing containers.
Limitation: Not used for pharmaceutical preparation.
3. Deionized or demineralized water:
 Water free from all charged atoms or molecules (ions).
 Prepared by anion and cation exchange resin bed to remove
ions.
Uses:
1) Used in pharmaceutical formulation
2) Used for washing containers and plant
3) Used for preparation of disinfectant solution
4) Used for preparation of reagents
Water

6. 4. Distilled Water
 Water that is free from microorganism.
 It is often stored at 80 ℃ to prevent bacterial growth and the
production of pyrogen.
 It can be contaminated by Gram-negative bacteria after
sterilization process.
Uses:
1) Used in the formulation of oral and topical
pharmaceutical preparation.
2) Used for the manufacture of parenteral preparation.
Water

7. 5. Water Produced by Reverse Osmosis:
 Reverse Osmosis (RO) is a process by which pyrogen,
microorganisms and solubles dissolved in water are removed.
 In this process water is passed through a semi-permeable
membrane, which acts as a molecular filter by an osmotic
pressure.
 Post-RO contamination may occur if plant is not free from
microorganisms.
Water

8. Distribution System
Water is contaminated during distribution.
Microorganism can colonize in different parts
of distribution system:
1) Storage vessel
2) Booster pumps
3) Water meters
4) Unused section of pipeline
5) Outlets such as cocks and taps
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9. Reduction of microbial count:
1) Ensuring constant recirculation of water at a
positive pressure through a ring-main without
‘dead-legs’.
2) Very short branches to the take off point.
3) Facility to re-sterilization of water by membrane
filtration or UV light.
4) Using PTFE and silicone-based compound for
jointing, packing.
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Distribution System

10. Three methods are used for treating water
Chemical
treatment
Filtration
UV Light
Disinfection of water

11. 1. Chemical Treatment
 Chemical treatment is applicable usually to raw, main and softened
water.
 It also can be used to treat the storage and distribution systems of
distilled and deionized water.
 Sodium hypochlorite and chlorine gas are the most common agents
for chemical treatment.
 The concentration of these chemical agents depend upon:
 the dwelling time
 the chlorine demand of the water.
Disinfection of water

12. 2. Microbial Filtration
 Membrane filtration is useful where a continuous circulation
of water is maintained.
 It is needed to install a 0.22µm pore-size membrane as water-
borne bacteria are small.
 Membrane filters require regular sterilization to prevent
microbial colonization and growth.
 They may be sterilized by chemically and moist heat.
Disinfection of water

13. 3. Light
• UV light is useful for the disinfection of water.
• It has advantage over chemical disinfections because it has no
odour or flavour problem.
• Unlike membrane filters, it is not subject to microbial
colonization.
• Because of its less penetrating power, it only kill surface
microorganism.
Disinfection of water

14. Water system design
1. Pipes sloped so water does not pool and can drain easily
2. Sanitary fittings & connections
3. Constructed of suitable materials such as stainless steel
4. Water should be kept circulated
5. Non-return valves (NRV) should be incorporated

15. Water system design

16. Water System Monitoring
Water monitoring:
– Total organic carbon (TOC)
– Conductivity
– Microbial Contaminants
– Endotoxin
Water for Injection (WFI) Systems:
– Microbial quality and endotoxin
• Daily system monitoring
• Each use point at least weekly
– TOC and Conductivity
• Weekly system monitoring
• can be taken from worst case point (end of loop, return to tank)

17. Water sampling
Sampling (1)
1. There must be a sampling procedure
2. Sample integrity must be assured
3. Sampler training
4. Sample point
5. Sample size
Sampling (2)
1. Sample container
2. Sample label
3. Sample storage and transport
4. Arrival at the laboratory
5. Start of test

18.  It is necessary to analyze the Purified Water and Water for Injection
(WFI) for microbial contamination
 There are two parts of microbial analysis of water
o Total Microbial Count
i. Filtration method
ii. Pour plate technique
o Pathogen Detection
i. Detection of E. coli
ii. Detection of Salmonella
iii. Detection of Pseudomonas aeruginosa
iv. Detection of Staphylococcus aureus
v. Detection of Enterobacteriaceae
vi. Detection of Shigella
vii. Detection of Clostridium
Microbial analysis of water

19. Suggested bacterial limits
Sampling location Target (cfu/ml) Alert (cfu/ml) Action (cfu/ml)
Raw water 200 300 500
Post multimedia filter 100 300 500
Post softener 100 300 500
Post activated carbon filter 50 300 500
Feed to RO 20 200 500
RO permeate 10 50 100
Points of Use 1 10 100
Action and alert limits
For the monitoring of water systems, appropriate alert and action levels should
be set for both bio-burden assessment and for levels of bacterial endotoxins.
Action levels, where appropriate, are typically drawn from the pharmacopoeia
or national water standards, whereas alert levels are assessed by
pharmaceutical organizations, based on a review of historical data.

20. Use and preparation techniques of various
grades of water