Application Notes

125

TRIO.BAS Air Sampler - Care, Maintenance, Battery Charging

The TRIO.BAS microbial air samplers should be cleaned at regular intervals. The body of the unit should be treated with mild detergent or ethanol. The stainless steel aspirating head should be cleaned with different type of detergents (exclude chlorine detergent).

123

Indoor Air Bioaerosol Sampling from Building according NIOSH

This Application Note reports the original method from the technical paper 0800 published on 1998 by NIOSH USA (NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND HEALTH). After 20 years the method is still a valid and well done document. The new generation of air samplers simpli es the reported sampling process.

122

Microbial Air Monitoring suggestions for reference graphic preparation

A speci c graphic to be implemented should be the basis for the interpretation of the microbiological results in non “standardied closed environment”. The graphic is then used as a guide for the environment to be monitored at regular programmed intervals. It is important to consider in a closed space the occupant density, the building characteristics, the humidity for a correct evaluation of the results. The graphic is the most effective method to involve the personell in the Hygiene care.

119

Expulsion of Droplets during a Sneeze

During a sneeze, millions of tiny droplets of water and mucus are expelled at about 200 miles per hour (100 metres per second). The droplets initially are about 10-100 micrometers in diameter, but they dry rapidly to droplet nuclei of 1-4 micrometres, containing virus particles or bacteria. This is a major means of transmission of several diseases of humans, shown in the table.

118

TRIO.BAS Aspiration Cycle “Multimode”

The microbial air sampling can be programmed by sub-samples to obtain results during longer time. All TRIO.BAS air samplers can be programmed for “Delay time”, “Interval time”, “Sampling time”- It is therefore possible to obtain sequential sampling spaced out by several standby times: -Delay time from 1 to 120 minutes. -Sampling intervals up to 100. -Sampling time up to 60 minutes. These performances are very useful for sampling “AT REST” and “IN OPERATION”.

116

MUSEUM OF MICROBIAL AIR SAMPLERS Villa Cella – Milan

The Museum of Microbial Air Samplers is located in Villa Cella in Milan and presents the instrumentation starting from the prototype of SAS in 1970’ together with the subsequent copies made by competitors up today.

115

Protocol of Dressing in Cleanroom

The correct dressing of operators in controlled contamination environment is a key point to guarantee the lowering of the particle and microbial count to requested values. This aim is achieved by adopting a well-de ned procedure. One example is suggested by Philip Austin, resumed in the publication “Modular Cleanroom "Terra Universal Vol. 10. N. 3. It illustrates proper donning step-by-step itinerary.

114

The Gamma Irradiation

Gamma rays are a form of electromagnetic radiation-like x-rays, but with higher energy. The primary industrial sources of gamma rays are radionuclide elements such as Cobalt 60, which emit gamma rays during radioactive decay. Gamma rays pass readily through plastics and kill bacteria by breaking the covalent bonds of bacterial DNA. They are measured in units called kiloGrays (kGy). Gamma irradiation provides a number of bene ts in cost and sterility assurance. It can be applied under safe, wellde ned, and controlled operating parameters, and is not a heat- or moisturegenerating process. Consequently, there is no heat stress and condensate drainage or outgassing is not required. Most importantly, there is no residual radioactivity after irradiation.

113

Do’s and Dont’ts in aseptic practices

Cleanroom environments are not sterile as long as people populate them. The only viable approach is to have people gowned properly and strictly adhere to good aseptic practices and techniques.

112

Use of “Daily Shift” Sterile Aspirating Head for TRIO.BAS Air Samplers in Cleanroom

The “Daily Shift” head sterile aspirating head avoids the sterilization of the stainless steel head of the air sampler. It saves time and it certi es the sterilization with an of cial document. This document is requested by the regulatory authorities. The double irradiated sterile packaging allows you to have always available aspirating head ready for use. A single head is used during a complete daily working cycle in clean room. The transparency of “Daily Shift” head is useful to verify that th culture plate is inserted into che aspirating chamber. The plastic of the “Daily Shift” is reciclable.

111

Applied microbiology in Cleanroom

Air samples are quantitatively accurate. Microbiology is an exact science. Contamination detect by an active air samplers means that the product made at the same time is contaminated. Air sampling devices are generally equal in their ability to detect contamination. Statistic + sampling plan analysis are suf cient to enable the manager to create a formula for determining “accept/reject” of a product lot. Microbes will survive forever in a cleanroom unless killed by a disinfectant.

110

Test questions related to working activity in Clean Room

Essential Basic Vocabulary: Assurance, aseptic, aseptic environment, aseptic processing, bacteria, bioburden, cleanroom. Class, contamination, disinfectant rotation, disinfection, endotoxin, HEPA lter, Laminar ow, media ll, mold, pyrogen, sanitation, sterility assurance, terminal sterilization, unidirectional flow. Identify the criteria required for facilities and type of equipment used in an aseptic environment.

109

Agar moisture loss in Petri dish using an impact air sampler system

The optimal growing of colonies on a culture plate is obtained with an agar medium that has the correct volume, the correct level of moisture and is freshly prepared. The agar plates can lose moisture for high temperature during the storage and during the air sampling due to the impact of air on the agar surface. According to the literature the loss should be lower than 10%. It is here reported an example of SOP (Standard Operative procedure) to evaluate the loss of humidity on Petri dish during the air sampling by an active air sampler.

107

SOP (Standard Operating Procedure) for TRIO.BAS ISOLATOR Microbial Air Sampler

The TRIO.BAS ISOLATOR Microbial Air Sampler has been speci cally developed to be used in Isolator and in Multi Clean Rooms. The aspirating heads of the satellite units (1, 2 or 3) can be positioned in different location and commanded via cable from outside by a single comand unit. The main advantage is the fact that vacuum is not involved and all the potential problems due to the vacuum are not present. The connecting electrical cable may have different lenghts and it is easily connected and disconnected from the satellite units. The satellite units are compatible with the most commonly used sterilizing agents. 90 mm Petri dish or 55 mm Contact plates may be used.

106

Suggested procedures for microbial air sampling

In environments with constant characteristics it is easier to provide “Acceptability Levels” to be used for routine work. It is not possible to set the same standards for all applications and the “Acceptability Limits” will vary considerably in different environments.

105

Microbial Air sampler positioning

A microbial air sampler should be positioned with different orientations according to the purpose of the test in the considered area (e.g. risk evaluation). Are here reported several applications for the TRIO.BAS microbial air samplers.

102

Evaluation of two microbial active air samplers

The aim of this research was to evaluate and compare the results of two microbial air samplers present on the market: the non portable Anderson one stage model and the portable TRIO.BAS MONO system. The air samples were collected in 3 different environments with the two air samplers positioned 1 meter from the other, in absence of personell during the aspiration. The results showed that the variability between the two instruments was not signi cative and was comparable.

101

The “physical laws” that regulate the diffusion and deposition of particles in the air

The here repor ted de nitions of few fundamental physical laws can help to understand the mechanism of particle behavior and consider the consequent air sampling difficulties. Brownian motion: As the par ticles migrate through a body of air, random impacts from individual molecules will cause them to veer from course. Drag coefficient: It is the ratio of the force of gravity to the iner tial force on a par ticle in uid. It indicates how a par ticle will resist any force that could cause a change in the par ticle velocity. Smaller par ticles have smaller drag coef cients due to their lesser masses.

99

Microbial Air Sampling Collection Efficiency – The number of Stokes

The efficacy of microbial air sampling collection by impact on agar samplers is characterized by a number without dimensions, called the “number of Stokes”. This number, called (d50), indicates the value that refers to the aerodynamic diameter of particulate that are collected by 50%. Hinds (1982) considers the value (d50) as the diameter of particulates that have a bigger diameter and theoretically are all collected.

98

The Micro-Organisms in the Air

Of all environmental, air is the simplest one and it occurs in a single phase, gas. Air is not a natural environment for the growth and reproduction of microorganisms. It does not contain necessary amount of moisture and utilizable form of nutrient. In addition to gasses, dust particles and water vapour, air also contains microorganisms. There are vegetative cells and spores of bacteria, fungi and algae, viruses and protozoan cysts.

96

Subdivision of hospital operating department in different areas depending on the biological risk for staff and patients

The most advanced areas for the control of particulate and microbial contamination in the " contamination controlled " environments are undoubtedly space industry, electronics and pharmaceutical industries. The pharmaceutical industry is the one that comes closest to the hospital sector, at least from a microbiological point of view, and may therefore be useful to draw on its technological innovations in the field of contamination control and in particular to the Good Manufacturing Practice.

94

Instruction for Daily Shift Aspirating Head

Standard Operating Procedure (POS) Use of DAILY SHIFT aspirating head for TRIO.BAS air samplers OBJECT Procedure for the correct use of microbial air sampler in Cleanroom, Isolator, RABS PURPOSE Reduction of the risk of microbial contamination during air sampler handling

91

Standard Operating Procedure for TRIO.BAS ISOLATOR

Three stainless steel satellite aspirating chambers, with long connecting cable, can be positioned in different locations and commanded from outside by a single unit. The satellite aspirating chamber is compatible with the most commonly used sterilizing agents. Contact plates (RODAC) or Petri dish can be used.

90

The interval time programmed air sampling in isolator and RABS by TRIO.BAS DUO and TRIO

The sampling aspirating chamber of “TRIO.BAS Isolator” is compact, manufactured in stainless steel; the command – control unit is separated and positioned outside from the isolator. The compact design save space inside the isolator. The connection is only for power and doesn’t compromise the isolator integrity. No vacuum, valves and s/s tubing are involved. The use of “Shift Head” sterile certified aspirating head simplify the daily activity and reduce contamination risk. Three independent separated aspirating chambers are connected to the central command unit.

89

TRIO.BAS ISOLATOR microbial air sampler technical details

THE TRIO.BAS ISOLATOR MICROBIAL AIR SAMPLERS TO BE USED IN ISOLATORS OR RABS HAS INNOVATIVE FEATURES: a. Up to three independent aspirating heads (called satellite units) can be used for three sampling cycles in three different isolators or in the same isolator with different culture medium. b. The connection between the comand unit and the satellite units is via a simple electric cable. c. No conduits or valves to interfere with the daily activity. d. Bluetooth capability for sampling data transfer. e. The use of sterile “Daily Shift Head” reduces the risk of contamination.

88

Useful information about Pharmaceutical Environmental Microbiology

Most companies have chosen a harmonized approach using the recommendations from the EU and the FDA, to include applying the most stringent criteria where applicable, although some companies stated that they use the most stringent criteria across the board. However, the regulators prefer to see Alert and Action Levels based on historical data once the EM program has been established for a minimum of 12 months.

87

Useful information about Pharmaceutical Environmental Microbiology

How do you set Alert and Action Levels in Environmemtal Microbiology? Most companies have chosen a harmonized approach using the recommendations from the EU and the FDA, to include applying the most stringent criteria where applicable, although some companies stated that they use the most stringent criteria across the board. However, the regulators prefer to see Alert and Action Levels based on historical data once the EM program has been established for a minimum of 12 months.

86

Indoor air filtration system microbial monitoring

The filtration system of building should be regularly monitored to be sure they are not broken or uncorrectly fixed or contaminated. The HVAC (Heating Ventilation, Air Conditioning) may be responsible for the production and spread of airborne micro-organisms in buildings. The reason of this test is to guarantee the health of rooms, laboratory, hospital and building occupants. The reported “SOP” (Standard Operating Procedure) can help the building servicing engineers in their task. The principle of the test is the comparation of the outdoor microbial population (upstream) with the indoor of the building after filtration (downstream).

85

Compressed gas in pharma facilities. Microbial monitoring

Compressed gasses are used at different stages of the pharmaceutical manufacturing process for numerous applications. Compressed gas sampling for microorganisms is an important part of contamination control assessment. Compressed gases such as air, nitrogen and carbon dioxide are deployed in operations involving purging or overlaying. Aside from air, nitrogen is the most commonly used gas in the pharma sector.

84

USP 1116 for Microbial Air Monitoring – OOS (Out Of Specifications) and OOT (Out Of Trends)

GLOSSARY Bioburden, alert limit, action limit, corrective action, CFU, Environmental isolates, Environmental Monitoring, genus, microbial identification, OOS, OOT, species, trend. INTRODUCTION The USP Pharmacopea Inspectors request that the trend variation in a Cleanroom or RABS or Isolators in Pharma production should be seriously considered when "alert" and "action" levels are detected.

83

TRIO.BAS MINI BLUETOOTH Microbial air sampler – Barcode Brief instructions

BARCODE 1D, 2D USE for TRIO.BAS microbial air samplers The use of barcode ID in microbiological air monitoring procedures can help to save time, to better control the activity of the operator and to achieve a complete traceability of the test. The microbial air sampler preparation The air sampling instrument should be completed with a Bluetooth barcode reader and users and places should be identified by barcode tag / labels. Instrument itself is identified by a s/n written into the microcontroller memory.

82

Sampling viable organisms by microbial active air samplers

IMPORTANCE OF THE IMPACTION ON AGAR SURFACE. Effect of jet velocity, nozzle size, nozzle distance from the agar surface, quality of medium, volume of medium inside the culture, expiration date of the plate, moisture content and sterility of the medium, temperature should be considered to obtain the optimization of these variables. These facts are determinative to obtain an efficient design of a microbial air sampler. ISO-14698 standard mentions that impaction velocity should be less than 20 metres per second.

80

Glossary according USP 38

GLOSSARY FOR MICROBIOLOGICAL AIR ENVIRONMENTAL MONITORING Microbiology according USP 38 – The United States Pharmacopeial – Document (1116) Aseptic Processing Environments - Page 1201, 1202. Airborne Particulate Count – The total number of particles of a given size per unit volume of air. Airborne Viable Particulate Count – The recovered number of CFU per unit volume of air. Air Changes – The frequency per unit of time that the air within a controlled environment is replaced. The air can be recirculated or totally replaced.

79

Culture medium and diluent USP 38

CULTURE MEDIA AND DILUENTS – SELECTION OF GROWTH MEDIA IN CLEANROOMS, RABS, ISOLATOR Microbiology according USP 38 – The United States Pharmacopeial – Document (1116) Aseptic Processing Environments - Page 1201, 1196. – Culture media and diluents. The type of medium, liquid or solid, used for sampling or plating microorganisms depends on the procedures and equipment used. Any medium used should be evaluated for suitability for the intended purpose. The most commonly used all purpose solid microbiological growth medium is soybean casein digest agar. As previously noted, the medium can be supplemented with chemicals that counteract the effect of various antimicrobials.

78

Microbial isolates identification accordingUSP 38

IDENTIFICATION OF MICROBIAL ISOLATES Identification according USP 38 – The United States Pharmacopeial – Document (1116) Aseptic Processing Environments - Page 1201. A successful environmental control includes an appropriate level of identification of the flora obtained in sampling. A knowledge of the flora in controlled environments aids in determining the usual microflora anticipated for the facility and in evaluating the effectiveness of the cleaning and sanitation procedures, methods, agents and recovery methods. The information gathered by and identification program can be useful in the investigation of the source of contamination, especially when recommended detection frequency are exceeded.

77

Isolators, glove boxes, RABS, C-RABS: what are they and what are the differences?

Isolators, glove boxes, RABS, C-RABS: what are they and what are the differences? In this article we try to shed light on the different containment systems for highly active substances Glove boxes: To avoid contamination of the product, the environment or the operator They are the simplest containment systems and serve to prevent contamination of the material to be manipulated, the surrounding environment and the operator.

75

Sequential sampling

SEQUENTIAL SAMPLING WITH TRIO.BAS DUO AND TRIO IN CLEAN ROOM, ISOLATORS, RABS With TRIO.BAS DUO microbial air sampler (with 2 separated aspirating chambers) and TRIO.BAS TRIO microbial air sampler (with 3 separated aspirating chambers) it is possible to monitor two or three different environments or spots of the same environment, simultaneously.

74

Routine monitoring of air quality required in areas used for compounding sterile preparations

On January 1, 2004 Chapter <797>, of the United States Pharmacopeia/National Formulary (USP27/NF22) entitled “Pharmaceutical Compounding Sterile Preparations”, became effective. USP Chapter <797> details the procedures and requirements for compounding sterile preparations and sets standards that are applicable to all practice settings in which sterile preparations are compounded. Since USP Chapter <797> is considered a requirement, pharmacies may be subject to inspection for compliance with these standards by state boards of pharmacy, the FDA, and accreditation organizations, such as the Joint Commission on Accreditation of Healthcare Organizations (JCAHO), Accreditation Commission for Health Care, Inc. (ACHA) and the Community Health Accreditation Program (CHAP).

73

Air sampler disinfection

CLEANING AND DISINFECTION OF MICROBIAL ACTIVE AIR SAMPLERS IN CLEAN ROOM INTRODUCTION All the materials that are used with a microbial air sampler (protective aspirating head, aspirating head, body of the air sampler, bags, etc) should be cleaned and disinfected before the sampling cycles.

72

The choise and use of an active Microbial Air Sampler

THE CHOISE AND USE OF AN ACTIVE MICROBIAL AIR SAMPLER The microbial air sampler must be carefully chosen because the characteristics of the instrument may impact heavily on usability, on implementing deadlines and consequently the final cost of such activities in terms of buying and managing the facility, man hours and efficiency of operations The air sampler should be easily cleaned and sanitized and be constructed of materials compatible with the most common detergents and biocides such as isopropyl alcohol, hydrogen peroxide and sodium hypochlorite.

71

TRIO.BAS GAS Microbial content in compressed air and gas – SOP Document

MICROBIAL CONTENT IN COMPRESSED AIR AND GAS – SOP DOCUMENT Introduction The following SOP is a guide line to prepare a local SOP for the test of compressed gas or air used in Clean Room. Standard Operating Procedure OBJECT Quantitative bioburden collection in compressed gas lines in Clean Room.

70

Test of spontaneous generation

The history For many centuries many people believed in the concept of spontaneous generation, the creation of life from organic matter. Spontaneous generation is the hypothesis that some vital force contained in or given to organic matter can create living organisms from inanimate objects. Spontaneous generation was a widely held belief throughout the middle ages and into the latter half of the 19th century. The Italian Francesco Redi disproved spontaneous generation for large organisms by showing that maggots arose from meat only when ies laid eggs in the meat.

69

TRIO.BAS Air Sampler Biological ef ciency test according ISO 14698-1

CMA – CENTRO DI MICROBIOLOGIA APPLICATA Via Cusago 154 20019 Settimo Milanese Laboratorio accreditato ACCREDIA n. 1087 Direttrice Dr Miriam Magrì. Biological efficiency testing of several active microbiological air samplers according ISO14698-1 in a dedicated microbial wind tunnel.

68

The ideal m/s speed of the impact air on agar surface of active air sampler

The ideal m/s speed of the impact air on agar surface of active air sampler. The indications of the ISO 14698 about the ideal m/s speed of the air that impacts on the agar surface of an active microbial air sampler are quite general. Flow rate: sufficiently high to ensure the detection of low levels of contamination but weak enough to not dehydrate excessively agar medium; Flow velocity and impact: sufficiently high to be sampled microorganisms of size less than 1 micron and sufficiently weak to avoid damage during the impact.

67

Air sampling in food premises

Safe food with microbial air monitoring in production premises The microbial air monitoring in food, beverage and dairy production premises is an important factor to be considered. In fact clean air in production and storage building means better quality of food, longer self-life and better customer satisfaction. Cleanrooms and Controlled environments play an important role in the prevention of contamination during the production of food and food supplements. They are most often built around food packaging processes, where there is a high potential for exposure of the finished food product to the atmosphere or people. Ensuring that the environment within the room remains uncontaminated requires continuous monitoring of several variables including microbial air monitoring.

66

Air sampling in Hospital

Microbial contamination monitoring in operating theatre of hospital The microbiological contamination in operating theatre is an important parameter to be considered to reduce / eliminate the risks of infections in hospital during surgery. The microbiological monitoring of this critical environment has two specific goals: A. to confirm the Heating Ventilation Air Conditioning (HVAC) system and the sterile unidirectional air flow are working regularly; B. to confirm the staff is following a correct aseptic protocol.

65

Microbial air monitoring in Isolators

Monitoring of atmosphere inside isolators is very critical in maintaining high quality standards. In fact there are risks in using portable environmental samplers because they must be introduced each time you sample. “TRIO.BAS Isolator” from ORUM International in Milan is a dedicated instrument for this application. The sampling aspirating chamber of “TRIO.BAS Isolator” is compact, manufactured in stainless steel; the command – control unit is separated and positioned outside from the isolator. The compact design save space inside the isolator. The connection is only for power and doesn’t compromise the isolator integrity. No vacuum, valves and s/s tubing are involved. The use of “Shift Head” sterile certified aspirating head simplify the daily activity and reduce contamination risk. Three independent separated aspirating chambers are connected to the central command unit.

64

Full traceability

GAP (Good Air-Monitoring Practice) in Clean Room: Full Traceability The Good Manufacturing Practice and Pharmacopeial request that all the data collected during the microbial air monitoring are registered with full traceability. The TRIO.BAS air samplers include the Barcode 1D or 2D or the Radio Frequency Identification System (RFID) to collect air volumes, date, hours, operator name, location name, plate identification. All these data cannot be modified according to the official inspectors request.

63

Good Air Monitoring Practice

The “GAP” (Good Air-Monitoring Practice) requests that all the material used for the microbiological monitoring of the environment is sterile and the inspector authorities ask to document the sterilization compliance. The “Shift-Head” sterile disposable aspirating head for microbiological air samplers of the TRIO.BAS air sampler family has been produced for this purpose. Each “Shift-Head” from ORUM International in Milan is individually packaged and includes a sterilization certificate. All the preparations connected to cleaning, sterilization, packaging, documentation of the metal aspirating heads are eliminated saving time and in conformity with the requests of the inspectors.

62

Position of samplers in Cleanroom

The microbiological active air sampler should, in some particular situations, vertically positioned. This situation could be specifically problematic in Clean Room. If the air sampler is on top of a trolley and is transferred to the more critical points, it is necessary to fix the air sampler to obtain a complete stability. It is for these reasons that a new holder has been developed and produced by ORUM International in Milan. The new “Stand Up” is quickly and easily attached to any portable TRIO.BAS air samplers. It provides a sturdy and robust platform that greatly improves the stability of the air samplers, whatever their location, from Clean Room and Isolator to operating theatres. It is also useful in dairy, food premises and even composting sites.

61

Choose and comparation of samplers

Active Microbial Air Sampler idea The portable active microbial air sampler collects a pre-determined volume of air that impacts against agar-based growth culture medium contained in a Petri dish or contact plate. The aspiration is produced by a motor-fan. The plate is then incubated at appropriate temperature and the number of colonies (Colony Forming Unit = CFU) per plate are typically reported to 1.000 litres of air.

60

Microrganism stress

Effect of Impact stress on microbial recovery on an agar surface of an active air sampler. Microbial stress due to impaction of microorganisms onto an agar collection surface is dif cult to experimentally demonstrate. Recovery and injury due to collection depends not only on the magnitude of the impaction velocity, but also on the degree to which the microorganisms may be embedded in the collection medium.

59

Hole impact distribution

Even distribution of air flow on the agar of Petri and Contact dish. The purpose of the test was to verify the regular distribution of the air flow on the surface of agar of a 90 mm Petri dish. The evaluation is performed counting the number of the impact holes produced on the gar surface, after aspiration. AIR SAMPLER TRIO.BAS MINI (100 litres/minute) with s/s aspirating head (Petri dish containing SDA medium)

58

Fumigation

The aspiration of bio-aerosol by the TRIO-BAS MINI and TRIO.BAS MONO. The picture shows the aspiration of the aerosol by the smoke test.

57

SOP Microbial Compressed Air/Gas Sampling according ISO 8573-7

Object The purpose of this document is to describe the procedure for compressed air / gas sampling, and reporting of the viable airborne particulates as part of environmental program, using TRIO.BAS GAS Microbial Air Sampler. Purpose This procedure applies for sampling and evaluation of compressed air quality for viable particulates using a Microbiological Air Sampler in Clean Room. The purpose is to certify the absence of microorganisms in 1.000 litres of compressed gas/air.

54

Application of MPN in microbial air environmental tests

When the air is aspirated through the aspirating head of the air samplers indicated in the USP 1116 (Slit to agar samplers, Sieve impactors, Centrifugal samplers, Surface Air System) and micro-organisms impact upon the agar surface there is a statistical probability that the micro-organisms may land on top of each other and be visually interpreted as one single colony (CFU).

51

Training of personnel in Cleanroom, Isolator, RABS according USP 38 – The United States Pharmacopeial – Document (1116) Aseptic Processing Environments – Page 1195

The personnel performances Good personnel performance plays an essential role in the control of contamination, proper training and supervision are central to contamination control. Aseptic processing is the most critical activity conducted in Microbiological Controlled Environments, and manufacturers must pay close attention to details in all aspects of the endeavor. Rigorous discipline and strict supervision of personnel are essential in order to ensure a level of Environmental quality appropriate for aseptic processing.

50

Ten suggestions to apply the Best Micro-Pipette Practice in microbiology

Equipment Care – Service every 8-12 months (Calibration, greasing parts, seals replacement, cleaning, disinfection, etc.). Tips – Replace tips every transfer (reused tips will carry extra organisms over the next sample). Aspiration Depth – Place tip just below the surface of the liquid (Too deep results in extra organism carry over on the outside of the tip). Slow, fluid motions – Do not allow pipette to “snap” up liquid into the tip (This creates air bubbles in the tip, and risk liquid entering the pipette shaft).

49

Simple tips to facilitate the phenotypic identification of aerobic microorganisms after collection and incubation in Petri dish.

SCOPE OF IDENTIFICATION The purpose of the bacterial air monitoring is not only to count the total number of microorganisms for cubic meter (CFU/cm3), but also to identify the microorganism. STARTING PROTOCOL A. The best phase to pick up a colony from the agar surface for identification is the “LOG” phase (not the “LAG” and “Stationary”), because they are behavior properly being at the top of the health.

47

Glossary for Microbiological Air Environmental Monitoring

Microbiology according USP 38 – The United States Pharmacopeial – Document (1116) Aseptic Processing Environments – Page 1201, 1202. -Airborne Particulate Count – The total number of particles of a given size per unit volume of air. -Airborne Viable Particulate Count – The recovered number of CFU per unit volume of air. -Air Changes – The frequency per unit of time that the air within a controlled environment is replaced. The air can be recirculated or totally replaced.

46

Culture media and diluents – Selection of Growth Media in Cleanrooms, RABS, Isolator

Microbiology according USP 38 – The United States Pharmacopeial – Document (1116) Aseptic Processing Environments -Page 1201, 1196. - Culture media and diluents The type of medium, liquid or solid, used for sampling or plating microorganisms depends on the procedures and equipment used. Any medium used should be evaluated for suitability for the intended purpose. The most commonly used all purpose solid microbiological growth medium is soybean casein digest agar. As previously noted, the medium can be supplemented with chemicals that counteract the effect of various antimicrobials.

45

Identification of microbial isolates

Identification according USP 38 – The United States Pharmacopeial – Document (1116) Aseptic Processing Environments – Page 1201 A successful environmental control includes an appropriate level of identification of the flora obtained in sampling. A knowledge of the flora in controlled environments aids in determining the usual microflora anticipated for the facility and in evaluating the effectiveness of the cleaning and sanitation procedures, methods, agents and recovery methods. The information gathered by and identification program can be useful in the investigation of the source of contamination, especially when recommended detection frequency are exceeded.

44

Environmental Microbial Data interpretation in aseptic processing.

We report what USP 38 – USP Pharmacopeial Conventions says at page1199 in the Chapter “Further considerations about data interpretation” of the document “Overall Management of a microbiological control program”. ZERO CONTAMINATION “In the high quality environments required for aseptic processing, detection frequency typically is low. As can be seen from the rates recommended in the enclosed table.

43

Available microbial air samplers for airborne microorganisms.

The USP 38 – The United States Pharmacopeial Document – Official from May 1, 2015 – presents on page 1200 the list of the most commonly used tools for monitoring aseptic environments. SLIT-TO-AGAR AIR (STA) The unit is powered by an attached source of controllable vacuum. The air intake is obtained through a standardized slit below which is placed a slowly revolving Petri dish that contains a nutrient agar. Airborne particles that have sufficient mass impact the agar surface, and viable microorganisms are allowed to grow. A remote air intake is often used to minimize disturbance of unidirectional airflow.

41

Evaluation of different microbial air samplers

To evaluate different microbial air samplers it is useful to read what is written in the USP 38 of the US Pharmacopeial – official from May 1, 2015: “There are no standard methods for air sampling, and available literature indicates that air sampling methods are highly variable. It should not be assumed that similar sample volumes taken by different methods will produce similar rates of recovery.

35

Certificate of conformity

The TRIO.BAS air samplers: fulfil the requirements of: · ISO Standard 14698-1 – 2004 Guidance for Industry on Sterile Drug Products by Aseptic Processing – Current Good Manufacturing Practice · FDA – 1987 Guideline on Sterile Drug Products by Aseptic Processing · ACGIH – Guideline for Assessment of Bio-aerosol in the Indoor Environment · ASTM – Draft Protocol – Committee D22.05.06 · USP Chapter <1116> Microbiological Evaluation of Clean Rooms and Controlled Environments · EU Guide for GMP – Manufacture of Sterile Medicinal Products Control Medicines and Inspection

34

Positioning of microbiological air samplers in Clean Room and Controlled Environment

The “TRIO.BAS” microbiological air samplers have the possibility to use different holders and tripods for different positioning (vertical, horizontal, oblique). The “TRIO.BAS” may also be used in horizontal or vertical positions without the use of holder or tripods, due to their shape. The environmental microbiologist should chose the system that best covers his needs in his specific environment.

33

Correct aseptic sampling during air sampling monitoring

The correct sampling is fundamental to avoid a microbial contamination with possible wrong results at the end of the analytical process. It is for this reason that all the staff involved in the sampling process should be trained. We are reporting some Guide Lines that should be distributed and explained to all involved personnel.

32

Chromogenic media for colorful air microbial detection - SOP

The chromogenic media can be used for the direct identi cation of some microrganisms present in the air. An example of chromogenic media to be adopted is the “CHROMagar Orientation” of the companyCHROMagar that is succesfully used in the clinical eld, as well as food/beverage/envirnoment microbial quality control. This is a non-selective agar, similar to a TSA, but with the addition of chromogens that will allow the bacteria to grow in different colors, depending on the species.

31

Performances of TRIO.BAS air sampler according ISO 14698

Airborne, Alert Level, As-built, AT-Rest, Bacteria, Bio-contamination, Cleaning, CleanRoom, Contact Plate, Controlled Environment, Culture Medium, Disinfection, Micro-organisms, Impact-Sampler, Risk Zone, Sampling Devices, Sampling Plan, Sterilization, Unidirectional Air Flow, Validation, Viable Particles.

30

TRIO.BAS mod. Trio air sampler: WHY?

There are several reasons to adopt an air sampler with 3 aspirating heads 1. Plate Count Agar plate on left, up, and right aspirating heads (1, 2, 3) to calculate an average result and to obtain a more reliable and realistic number of Colony Forming Unit (CFU) to produce a valid statistical evaluation of the results, according to GMP (Good Microbiological Practice)

29

SAS Surface Air System History

DATE NAME CHARACTERISTICS 1976 BAPD Slit to agar model 1977 BC SAS Anderson type air sampler 1978 SAS Prototype 1979 SAS Surface Air System Metal case with separate heavy battery metal case 1982 SAS Compact Metal body with incorporated heavy battery 1984 MTM-3 Stationary unit with 3 aspirating head 1992 SAS Super 90 Plastic.

28

USP Regulation 797 for sterile compounding

Issued by the non-pro t US Pharmacopoeia (USP) and endorsed by the Joint Commission on Accreditation of Healthcare Organizations (JCAHO), “USP Regulation 797” is the rst enforceable standard for sterile compounding. Originally enacted on January 1, 2004, the latest version became of cial on June 1, 2008. “USP 797” is a broad regulation that covers a variety of pharmacy policies and procedures. It is designed to reduce the number of patient infections due to contaminated pharmaceutical preparations.

27

Mapping for the Microbiological Environmental condition monitoring in food production

It has been recommended that food processing plants adopt air monitoring programs to detect possible contamination sources in their regular HACCP program. Juice, ready-to-eat foods, pasta, meat, dairy and other food processing facilities have been advised to establish a system for weekly air monitoring. Air sampling of various areas (finished product, protein, fresh produce areas, etc.) yield information as to possible microbial contamination. That is to say, knowing the microbial population, number of colony forming units per cubic meter of air (CFU/m3) of a given area can be predictive of possible food contamination possibilities, e.g., yeast, molds, and pathogens.

26

Maintenance and Calibration of air sampler according GMP and GLP – EN 14042

Chapter 12 “Quality Assurance” of the European Standard EN 14042 – “Workplace atmosphere – Guide for the application and use of procedures for the assessment of exposure to chemical and biological agents”. “It is good practice to set up a Quality Assurance scheme for the maintenance and calibration of the samplers.

25

European Standard for biological Agents Monitoring in workplace atmosphere

The European Standard EN 14042, April 2003 “Workplace atmospheres – Guide for application and use of procedures for the assessment of exposure to chemical and biological agents” 6.3 “Measurement of airborne micro-organisms and endotoxin”, 6.3.1. “Methods using an impactor as sampler” presents the Table.

24

Record of results for microbial air sampling

The dispersion and diffusion of micro-organisms in indoor air is very irregular and influenced by several factors like the presence of air currents, number of people present in the environment, movement of people in the area, electrostatic charges, humidity, temperature, etc. It is clear the correct sampling technique is important. It is imperative to make several tests in the same place, at same time to produce a statistical result that takes an accurate picture of the microbiological conditions of the considered environment. To obtain a statistical result, it is imperative to have a correct record of all data of the sampling cycles.

23

Food Glossary according EFSA (European Food Safety Authorities)

An estimate of the amount of a substance in food or drinking water that can be consumed over a lifetime without presenting an appreciable risk to health. It is usually expressed as milligrams of the substance per kilogram of body weight and applies to chemical substances such as food additives, pesticide residues and veterinary drugs.

22

Microbial monitoring in closed environment: general suggestions

Viable particles are bacteria, yeast and moulds living microscopic organisms present in the air and on surfaces of the environment. Their testing usually includes the monitoring of – air, – surfaces, – staff. Viable air sampling Gravitational or settling Petri or Contact plates containing sterile growth media are used for sampling.

21

Air Sampling Standard According ISO 14698

The FDA Process Analytical Technology (PAT) requires a more efficient and reliable microbiological air monitoring system. The ability to check and to identify possible microbial contamination during the production and process enables companies to take more quick appropriate and immediate measures to avoid economic losses. The new generation of microbiological air samplers and new monitoring procedures improve the efficiency of the measurement of the microbial air quality in clean rooms and controlled areas.

20

Legionella aerosol monitoring

Legionnaires’ disease was first recognized as a result of an outbreak of acute pneumonia that occurred at the Convention of the American Legion in Philadelphia USA in 1976. The route of infection has been recognized as the inhalation of small aerosol containing bacteria of the Legionella sp into the lungs of the host animal. The bacterium primarily responsible for this disease is Legionella pneumophila sero-group 1 Pontiac. It can result in typical Legionnaires’ disease an acute pneumonia with low attack rate and relatively high fatality rate or Pontiac fever, a mild non-pneumonia infection with a high attack rate.

19

Industrial air handlers microbiological monitoring

The microbiological efficiency of the filters used in buildings is an essential parameter that should be regularly monitored to be sure that the contamination is under control. This Application Note reports a simple method used at 3M Development Laboratory in St. Paul, Minnesota, USA to measure the filter efficiency for fungi of industrial air handlers. The test can help to determine Indoor Air Quality in office buildings.

18

Compressed air microbiological monitoring

The present Application Note intends to be a simple Guide Line for the operators involved in the compressed air monitoring applying the ISO 8573-7. The ISO 8573 consists of nine different parts: 1. Contaminants and purity classes. 2. Test methods for aerosol oil content. 3. Test methods for measurement of humidity. 4. Test methods for solid particle count. 5. Test methods for oil vapour and organic solvent content. 6. Test methods for gaseous contaminant content. 7. Test method for viable microbiological contaminant content. 8. Test methods for solid particle content by mass concentration. 9. Test methods for liquid water content.

17

Nosocomial Infections: Aspergillosis air sampling

Nosocomial-acquired aspergillosis typically occurs in the setting of treatment for leukaemia or other haematological malignancy. As Aspergillus species are easily found in the environment, it is accepted that aspergillosis disease can be a consequence of exogenous acquisition of the fungus.

16

Stainless Steel Aspirating head for microbiological air sampler

The stainless steel sampling heads for Contact Plates (RODAC) or Petri dishes are manufactured of AISI stainless steel and are drilled with holes 1 mm in size. The operators can interchange these heads with no difference in air flow on an air sampler regardless of which head is used, as long the recommended procedures below are followed.

15

Trouble shooting in environmental monitoring

The major routes of contamination can be broadly classified as surfaces, water, people and air. Each time that “ALERT LEVEL” and “ACTION LEVEL” are reached, the person responsible for environmental monitoring should know what action must be taken. The reported troubleshooting guide taken from the Document Guide Line No.20 – Effective Microbiological Sampling of Food Processing Environments – by C. & C. gives some useful hints.

14

Standard Operative Procedure for Microbial Air Sampling in Clean Room

It is important to organize a correct and clear sampling plan when several clean rooms or controlled contamination areas are to be monitored at the same time for microbial bioburden. To reach this goal it is necessary to have a suitable set of air samplers and a specific SOP that gives to the operators all the information to avoid mistakes and / or misunderstanding. The reported Standard Operating Procedure (SOP) can be used as a guide to be adopted to each individual case.

13

Microbiological Air Monitoring in Operating Room

The prime function of a “Ventilation System” or a “Heating Ventilation Air Conditioning System” (HVAC) in an operating theatre, intensive treatment units and isolation suites is to closely control the environment and air movement of the space that it serves in order to contain, control and reduce hazard to patients and staff from airborne contaminants, dust and harmful micro-organisms.

12

Number of location and measurement for a reliable microbiological air testing

The people involved in a daily microbiology air sampling activity know quite well the frequent difficulties to obtain reliable results even in the same closed environment where the conditions are constant. The reasons are several: uneven distribution of micro-organisms in the air, inconstant biological activity of micro-organisms, influence of operator, air sampler, media, etc. The goal is to obtain results that are comparable with the previous data.

11

Mycobacterium tuberculosis bioaerosol sampling

Mycobacterium tuberculosis is a very well known old enemy that after some decades of relative “sleeping” is now again a “new emerging” disease. The World Health Organisation (WHO) estimated more than 8 million new cases and 3 million deaths are occurring annually. The micro-organism is spread by the airborne transmission of droplets produced by infectious individuals while they are sneezing, speaking, laughing, coughing.

9

Fungal Spore Control Ventilation (FSCV) in Hospital

The breathing of ambient concentration of airborne fungi for a normal person has no adverse effect on his health. The effects are adverse for the hospitalized patient with immune suppression: he is in fact susceptible to infections caused by naturally occurring airborne fungi that may grow at body temperature. The incidence of infections caused by fungi that were a few years ago considered only saprophytic has risen dramatically during the last decade.

8

Legionella protocol according CDC USA

Legionnaires’ disease was first recognised as a result of an outbreak of acute pneumonia that occurred at the Convention of the American Legion in Philadelphia USA in 1976. The route of infection has been recognised as the inhalation of small aerosol containing bacteria of the Legionella sp into the lungs of the host animal. The bacterium primarily responsible for this disease is Legionella pneumophila serogroup 1 Pontiac. It can result in typical Legionnaires’ disease an acute pneumonia with low attack rate and relatively high fatality rate or Pontiac fever, a mild non-pneumonia infection with a high attack rate.

7

Hands Monitoring in aseptic filling premises

The microenvironment of the skin is very restrictive, with little water, high levels of salt and a pH of around 5.6. Nevertheless, bacteria with adaptive properties colonize the hands, where they grow and constitute the normal resident flora. A transient flora may be temporarily tranferred from the environment onto the hands, from person to person and from person to objects.

6

Sampling Plan

The air sampling plan in check-list format aids both the operator’s supervisor and the operator in remembering all the points required for correct microbiological air sampling.

5

Bioaerosol Glossary

A ACGIH – American Conference of Governmental Industrial Hygienists, Inc. (USA) Action level – Established level of viable particles requiring immediate follow-up and corrective action and measures if exceeded. Aerosol – Colloid dispersed solid or liquid particles (biological o particulate) in a gaseous environment presentingnegligible gravitational setting.

4

Staff training for food handlers

The importance of mantaining good hygiene is well known to dairy and food quality control personnel, but it can be difficult to impress on general plant workers the importance of good manufacturing practice (G.M.P.). Increasing emphasis on general hygiene conditions will allow manufacturing of products with longer shelf-life, lower rejection rates, fewer complaints from customers, and higher production rates.

2

Microbiological Validation of Biohazard Cabinet and Laminar Flow Bench

Each laminar flow bench and Biohazard cabinet must be regularly tested and validated to control their performance. The purpose of the test is to identify breakage or irregularity of filters and/or alteration of the laminar flow with consequent turbulence and contaminated air introduced into the working area. The validation can be performed by company staff or entrusted to an external service organisation.

1

Principle of microbial air sampler by impact method on agar plate

Air containing microbe-carrying particles is aspirated and accelerate through a hole and direct towards a nutrient agar surface of a plate. As the air turns away from the agar surface, the microbe-carrying particles that cannot follow the flow are impacted. The plate containing nutrient agar is then incubate at a suitable time and temperature, and the resulting Colony Forming Units (CFU) are counted to evaluate the number of microbe-containing particles collected from a specific volume of air.