BASIC MICROBIOLOGY

AIN MEDICARE SDN. BHD.

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BASIC TOPICS
 Introduction To Microorganisms  Growth & Its Control  Analysis in Sterile Product Manufacturing

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1. Introduction To Microorganisms
A Brief History of Microbiology 1.1 Phylogenetic tree :
Eubacteria Prokaryotes Archaebacteria Proctotista, e.g algae, sea weeds Eukaryotes Fungi Animalia Planta
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Bacteria

1.2 The birth of modern microbiological theory in the later 19th century heralded by Louise Pasteur, Robert Koch, Joseph Lister & others began the quest to clarify the bacterial causation & mechanisms of infection. 1.3 The first person to see microorganisms in any detail was Anton van Leeuwenhoek, who used simple microscope of his own construction. 1.4 In the late 1870s, Robert Koch established that individual types of microbes were associated with specific diseases.

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Properties of Bacteria & Fungi

Size : Less than 1 micron to macroscopic (1.0 micron = 1 millionth of a meter) Shape : Rod, oval, coccoid, spherical, spiral, filamentous Types : Vegetative (living cells) and spores (seeds) Distribution : Everywhere Growth Requirements : Aerobic or anaerobic, moisture, limited nutrients Reproduction : Binary fission (doubling time 20-30 min) Asexual and sexual reproduction Toxicity : Some can produce endotoxins and pyrogens
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What Do Microscopic Bacteria Look Like?

Streptococcus Staphylococcus

Bacilli / Rod
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Penicillium sp.

Aspergillus niger

Bacillus sp.

Penicillium sp.

Aspergillus niger

Candida albicans

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Classification of Bacteria :
1. Requirement for oxygen * Aerobes ( need O2 to grow ) * Anaerobes ( cannot grow in air ) * Facultative anaerobes ( prefer to grow without O2, but can grow in the presence of it ) 2. Cell structure * Gram positive or Gram negative stained via Gram stain * Shape cocci or bacilli 3. Temperature dependence to grow * Psychrophilles : 7 - 15rC * Mesophilles : 25 - 40rC * Thermophilles : > 45 rC

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Classification of fungi
1. 2.

Yeast non sporing fungi, colourless, asexual production Mold colourful, filamentous, sexual & asexual production

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2. Growth & Its Control

Cell growth :
1. 2. 3.

Bacterial cell able to duplicate itself, divides into 2 new cells, called binary fission. Under best nutritional conditions, E.coli can complete the cycle in about 20 minutes. Measurement of growth through plate count : - spread plate - pour plate - membrane filtration

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Effect of Environmental Factors on Growth : 1. Temperature 2. Acidity & Alkalinity 3. Water Availability 4. Oxygen 5. Nutrient

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Temperature 1. One of the most important factor influencing the growth & survival of organism 2. Growth becomes faster at higher temperature but denatured at certain temperature 3. Four groups of organisms : - psychrophiles - mesophiles - thermophiles - extreme thermophiles

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Acidity & Alkalinity 1. Acidophiles organisms that live at low pH, fungi are more acid tolerant 2. Alkalinophilic high pH optima

Water Availability 1. Desiccation kills the bacteria Nutrient 1. Two main groups autotrophs, heterotrophs 2. Autotrophs obtain carbon from carbon dioxide, nitrogen from ammonia, nitrites, nitrates 3. Heterotrophs obtain their carbon, nitrogen & energy from organic compounds such as carbohydrates & amino acids

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Oxygen 1. Terms used to describe O2 relations of microorganisms : - Aerobes Obligate O2 required Facultative not required, but growth better with O2 - Anaerobes Obligate harmful or lethal Control of microbial growth 1. Heat sterilization 2. Filter sterilization 3. Chemical control

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Composition of media 1. The media usually contain peptone, meat extract, salt 2. Glucose and glycerol may be added to increase the nutritive value 3. Media in solid form are usually prepared by adding agar to the desired nutrient medium 4. Types of media : - selective media - differential media

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REVIEW OF CRITICAL MICROBIAL CONTROL ISSUES

 Starting materials control  Process & cleaning ( pharmaceutical )  Water & other critical services  Personnel  Environment & production equipment  Finished product release & QC

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STARTING MATERIALS CONTROL

 QC Monitoring Program based on the origin of material,

intended usage and probability of microbial contamination

 Storage Conditions dry, sealed and if necessary cold  Retest Period consider : water content / support  Supplier Audits may be site inspection or extended QC  Treatment further treatment to reduce incoming bioload

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PHARMACEUTICAL WATER
 Purified Water ( BP ) intended for the formulation of non

sterile products and final rinsing of equipment after cleaning

 Water for Injection ( WFI BP ) intended for the formulation of

parenteral, dialysis or inhalant products

 Highly Purified Water ( HPW ) - intended for use in the

preparation of products where water of high biological quality is needed, except where WFI is required.

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PERSONNEL ( BIOLOADS ) AND MICROBIAL CONTROL

 Skin microbial bioload 20,000/cm2  Faeces 1,000,000,000,000/g  Organisms of concern :

* Staphylococcus aureus food poisoning * Streptococcus faecalis diarrhea * E.coli foodborne illness * Salmonella typhi typhoid fever

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EQUIPMENT AND SURFACES ( 1 )

 Equipment should be installed in such a way as to prevent any

risk of error or contamination

PIC/S Guide to GMP for Medicinal Products

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EQUIPMENT AND SURFACES ( 2 )

 Equipment should be demountable for periodic cleaning &

sanitation  Tanks should have an air break to drains  Equipment should be stored protected from the environment and in dry condition

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EQUIPMENT MICROBIAL MONITORING PRODUCT CONTACT SURFACES

 Focus monitoring on equipment at risk, e.g. liquids lines and

product contact surfaces  Conduct after cleaning or before reuse  Periodically identify bioload to check for objectionable microbes-build up profile

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Microbiological Contamination Hazard Analysis In Sterile Product Manufacturing

Microbiological Function refers to all coordinated & integrated microbiological activities that are needed to assure that harmful microbes do not contaminate non sterile drug products & that all microbes are excluded from sterile drug products through environmental monitoring, sterility test, bioburden test & test for specific microorganisms. Microbiological Function in Drug Product Manufacturing : 1. To protect drug users from harmful microorganisms 2. To be in compliance with government regulations in microbial issues 3. To be in step with industry trends in microbial control policies & technologies
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