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Thursday, May 28, 2009
Saturday, May 2, 2009
The Importance of Microbiology in the Contamination Control Plan for Aseptic, Terminally Sterilized and Non-sterile Manufacturing
Introduction
The development of a contamination control program is critical to the effort to get a new facility qualified, and to maintain the facility in a state of control once qualified. The design and successful execution of a contamination control program requires a plan. The creation of a specific document allows the company philosophy, goals, and expectations to be formalized and agreed to by all parties. It also provides the goals and metrics by which the state of control for the facility can be measured in the annual review. The business reasons for this are obvious in terms of reduced regulatory risk and reduction of rejected/recalled batches (Lowry 2001).
This plan is important no matter what type of facility is being developed. Although it is most frequently used in the Quality plan for commissioning an aseptic facility, this is also important and should be used for commissioning and controlling facilities using terminal sterilization, and for non-sterile manufacturing facilities.
Why be concerned with contamination control in a nonsterile manufacturing facility? In many ways contamination control is more of a concern in a non-sterile facility than in sterile product production facilities. The sterile production facility knows there is a problem with contamination and cross-contamination of batches, the non-sterile facility has a great temptation to belief they are not touched by these issues. This can lead to an extremely cavalier attitude about contamination control by the operators and management. The non-sterile manufacturer is responsible for all aspects of his product, including any objectionable organisms present (Sutton, 2006) as described in a recent newsletter (PMF Newsletter v12 n7).
The API manufacturer is also concerned with contamination control. The FDA has explicit instruction on this score (FDA 1998) out of CBER. The EMEA guidance on API manufacture also includes guidance on control of bioburden and cross-contamination of batches (EMEA 2000).
This essay will not be able to provide more than an overview of issues in the space available this month. However, it is hoped that the need for an adequate contamination control plan for a facility will be made clear, and the beginnings of the content of such a plan explained. The interested reader is referred to the articles listed in the “References” and the “Further Readings” sections.
Scope
The Contamination Control Plan should be developed as part of the facility commissioning effort. As such, there will be four distinct phases of the facility operations that will need to be addressed:
Commissioning and initial start-up
Ongoing Operations
Shut-down for regular maintenance
Start-up after scheduled shut-down.
These phases will not have the same level of contamination control. In fact, the third and fourth phases may well have different levels of control to be addressed. A good plan will discuss the concerns specific to each of these phases.
This program, and the protocol governing the program, are essential documents useful in documenting the rationale and methods used to accomplish three tasks:
Minimizing the bioburden throughout the manufacturing processes
Minimizing the level of batch residual cross-over contamination
Minimizing the level of cleaning material residual contamination
As the SME (Subject Matter Expert) in microbiology, we will be most heavily involved in the first of these three tasks, minimizing bioburden. However, all three will be discussed (at least briefly) in this essay for context.
Minimizing Bioburden
Validated methods
All measures of bioburden in a facility will be indirect. We cannot count bacterial cells on a surface or in the air. We must transfer the microorganisms to an agar plate (or some other mechanism) and count colony forming units. If we make the assumption that the transfer of microorganisms from the air or from a surface to agar is consistent, then we can use these numbers to estimate trends over time. This assumes that the nutrient agar is capable of growing the microorganisms to visible colonies. As residual disinfectant on a surface may impede the growth of microorganisms, neutralizers are frequently incorporated into the growth media (Dey-Engley agar, MCTA, etc.). All sampling methods must be validated for the conditions of use.
The facility should be disinfected regularly using validated sanitizers and sporicides. The contamination control plan should describe the methods for testing and rationale for acceptance of materials to be used in the ongoing program of disinfection. The plan should ideally describe the in vitro or laboratory tests to evaluate the sanitizers, including the identification of the most resistant microorganisms found in the facility as well as the most difficult-to-disinfect materials in the facility. This is also where the method for on-going evaluation of the sanitizers based on environmental monitoring data will be recorded. The choice of disinfection regimens should be reevaluated annually, and the contamination control plan should describe how this evaluation will occur.
Know the enemy
A successful contamination control program is geared to providing the most useful information on the microorganisms present while at the same time showing some fiscal responsibility. The FDA aseptic processing guidance document recommends genetic identification of all organisms isolated from the manufacturing environment on a regular basis. (FDA, 2004) This is a laudable goal, but few of us have anything near the required budget to accomplish this task, and in all honesty it is reasonable to wonder if it is really necessary. The numbers of CFU from validated sites (viable air and surface, non-viable) is sufficient to provide a measure of the state of control of the facility. However, periodic cataloging of the resident microflora will provide you with a good check on the continued effectiveness of the disinfectants in use. Shifts of bioburden to spore forming microorganisms will be strong evidence of the need for use of a sporicidal agent. Occasionally, this effort will also pick up shifts among non-spore-forming organisms – this is not due to “resistance” but rather ecological shifts towards species more naturally resistant to the disinfectant in use.
Control incoming bioburden
The first step in any control program is to control contamination at the very beginning of the process. This includes raw materials (excipients, API, water, etc) and the primary containers. All materials should be tested for incoming bioburden against documented acceptance criteria. Part of the incoming bioburden will also be any water used as an excipient to the process. A good guide for the water bioburden is the EMEA guidance on the subject (EMEA 2002).
Appropriate gowning
The gowning methods and materials are of critical importance to minimization of contamination. Although most attention is placed on aseptic gowning procedures, the appropriate use of gowning precautions will be a great boon to most non-sterile manufacturing facilities as well. All personnel should be well-trained in appropriate gowning practice and behavior. The contamination control plan should describe the rationale for the level of gowning chosen, the frequency of gown cleaning, behavior and the acceptable gown materials for the type of manufacturing process.
Training
Operator training is critical to contamination control. No supervisor can be present at all locations at all times. Each operator must be aware of his or her role in contamination control and how to minimize the risk to batch integrity. The PDA has published a technical report that speaks to some of these training requirements from the microbiological perspective (PDA 2001).
Controlled Environments
Control and monitoring of the environment is another critical element of the contamination control plan. Large portions of this can be addressed by the corporate Environmental Monitoring Master Plan (which provides rationale and consistency for a single EM philosophy across the different facilities of the corporation) or the site Environmental Master Plan (which provides consistency and detailed instruction for the various manufacturing buildings at a given site). However, the Contamination Control Plan should cite the relevant documents and their role in contamination control. Those interested in more on environmental monitoring should refer to the PDA’s treatment of the subject for a good overview (PDA 2001).
The appropriate Environmental Monitoring (EM) plan for non-sterile manufactures and for API manufacturers is not well-defined from a regulatory sense. There are no strong recommendations such as those seen for the environmental monitoring of aseptic facilities; however the absence of regulatory guidance is not the same thing as the absence of need for the activity. EM is useful for determining the state of control of the facility (not so much, perhaps an indicator of the finished product quality) and so is an important part of the monitoring program for all manufacturers.
Well-defined and Understood Manufacturing Processes
The manufacturing process should be evaluated for its potential to limit or eliminate bioburden. The two common methods for performing this is either a HACCP-type (Jahnke and Kuhn 2003) or a FMEA approach. The use of organic solvents, heat, or other inhospitable activities can greatly reduce bioburden of a process. The contribution of compression (and associated shear), for example, should be evaluated for a potential reduction in risk of excessive microbial contamination (Blair 1991). The contribution of the finished product water activity should also contribute to this analysis (USP 2007).
Of particular importance in this evaluation for the potential for microbial contamination of the process are cleaning steps, equipment hold times, HVAC, control level of environments for critical tasks, open-system vs closed-system operations, and bioburden monitoring (among others specific to your process). As an example of the importance of the bioburden control point issue, there is a strong regulatory expectation in Europe that products sterilized by filtration should have a pre-filtration bioburden of not more than 10 CFU/100 mL immediately before the sterilizing filter.
Finally the Contamination Control Plan should cite the need clear SOPs on all aspects of manufacturing, monitoring and control. These SOPs are critical for training, documentation and batch release.
Minimization of Batch Residual Cross-over Contamination
The contamination control plan should also address the potential for a batch to be contaminated by material from the previous batch manufactured using that equipment. Obviously, the contamination control plan should describe the methods by which this likelihood is minimized.
The concern over batch residual cross-over is most relevant when there is more than one product manufactured at a site. This concern has little to do with the sterility of the finished product, and is relevant to sterile and non-sterile manufacture alike.
Minimization of Cleaning Material Residual Contamination
Validation of cleaning procedures is essential to demonstrate not only that the cleaning procedure effectively cleans and sanitizes the manufacturing equipment, but also that residual cleaning material is removed to prevent contamination of the next batch manufactured.
Conclusions
The Contamination Control Plan is an important document designed to formalize the rationale, methods and validation of contamination control procedures in a manufacturing facility. This plan is a valuable tool for pharmaceutical, medical device and personal product manufactures and should be written to address all phases of the facilities life cycle. The Contamination Control Plan should specifically address:
Minimizing the bioburden throughout the manufacturing processes
Minimizing the level of batch residual cross-over contamination
Minimizing the level of cleaning material residual contamination
The microbiologist, as SME, has a critical role to play in the first of these three primary goals, and this essay has therefore been directed at that first topic. Minimization of bioburden in the manufacturing process occurs through (but is not limited to):
Minimizing bioburden in the process
Control incoming bioburden
Appropriate Gowning
Controlled Environments
Well-defined Standard Operating Procedures; and
Well-defined and understood manufacturing processes.
The development of a contamination control program is critical to the effort to get a new facility qualified, and to maintain the facility in a state of control once qualified. The design and successful execution of a contamination control program requires a plan. The creation of a specific document allows the company philosophy, goals, and expectations to be formalized and agreed to by all parties. It also provides the goals and metrics by which the state of control for the facility can be measured in the annual review. The business reasons for this are obvious in terms of reduced regulatory risk and reduction of rejected/recalled batches (Lowry 2001).
This plan is important no matter what type of facility is being developed. Although it is most frequently used in the Quality plan for commissioning an aseptic facility, this is also important and should be used for commissioning and controlling facilities using terminal sterilization, and for non-sterile manufacturing facilities.
Why be concerned with contamination control in a nonsterile manufacturing facility? In many ways contamination control is more of a concern in a non-sterile facility than in sterile product production facilities. The sterile production facility knows there is a problem with contamination and cross-contamination of batches, the non-sterile facility has a great temptation to belief they are not touched by these issues. This can lead to an extremely cavalier attitude about contamination control by the operators and management. The non-sterile manufacturer is responsible for all aspects of his product, including any objectionable organisms present (Sutton, 2006) as described in a recent newsletter (PMF Newsletter v12 n7).
The API manufacturer is also concerned with contamination control. The FDA has explicit instruction on this score (FDA 1998) out of CBER. The EMEA guidance on API manufacture also includes guidance on control of bioburden and cross-contamination of batches (EMEA 2000).
This essay will not be able to provide more than an overview of issues in the space available this month. However, it is hoped that the need for an adequate contamination control plan for a facility will be made clear, and the beginnings of the content of such a plan explained. The interested reader is referred to the articles listed in the “References” and the “Further Readings” sections.
Scope
The Contamination Control Plan should be developed as part of the facility commissioning effort. As such, there will be four distinct phases of the facility operations that will need to be addressed:
Commissioning and initial start-up
Ongoing Operations
Shut-down for regular maintenance
Start-up after scheduled shut-down.
These phases will not have the same level of contamination control. In fact, the third and fourth phases may well have different levels of control to be addressed. A good plan will discuss the concerns specific to each of these phases.
This program, and the protocol governing the program, are essential documents useful in documenting the rationale and methods used to accomplish three tasks:
Minimizing the bioburden throughout the manufacturing processes
Minimizing the level of batch residual cross-over contamination
Minimizing the level of cleaning material residual contamination
As the SME (Subject Matter Expert) in microbiology, we will be most heavily involved in the first of these three tasks, minimizing bioburden. However, all three will be discussed (at least briefly) in this essay for context.
Minimizing Bioburden
Validated methods
All measures of bioburden in a facility will be indirect. We cannot count bacterial cells on a surface or in the air. We must transfer the microorganisms to an agar plate (or some other mechanism) and count colony forming units. If we make the assumption that the transfer of microorganisms from the air or from a surface to agar is consistent, then we can use these numbers to estimate trends over time. This assumes that the nutrient agar is capable of growing the microorganisms to visible colonies. As residual disinfectant on a surface may impede the growth of microorganisms, neutralizers are frequently incorporated into the growth media (Dey-Engley agar, MCTA, etc.). All sampling methods must be validated for the conditions of use.
The facility should be disinfected regularly using validated sanitizers and sporicides. The contamination control plan should describe the methods for testing and rationale for acceptance of materials to be used in the ongoing program of disinfection. The plan should ideally describe the in vitro or laboratory tests to evaluate the sanitizers, including the identification of the most resistant microorganisms found in the facility as well as the most difficult-to-disinfect materials in the facility. This is also where the method for on-going evaluation of the sanitizers based on environmental monitoring data will be recorded. The choice of disinfection regimens should be reevaluated annually, and the contamination control plan should describe how this evaluation will occur.
Know the enemy
A successful contamination control program is geared to providing the most useful information on the microorganisms present while at the same time showing some fiscal responsibility. The FDA aseptic processing guidance document recommends genetic identification of all organisms isolated from the manufacturing environment on a regular basis. (FDA, 2004) This is a laudable goal, but few of us have anything near the required budget to accomplish this task, and in all honesty it is reasonable to wonder if it is really necessary. The numbers of CFU from validated sites (viable air and surface, non-viable) is sufficient to provide a measure of the state of control of the facility. However, periodic cataloging of the resident microflora will provide you with a good check on the continued effectiveness of the disinfectants in use. Shifts of bioburden to spore forming microorganisms will be strong evidence of the need for use of a sporicidal agent. Occasionally, this effort will also pick up shifts among non-spore-forming organisms – this is not due to “resistance” but rather ecological shifts towards species more naturally resistant to the disinfectant in use.
Control incoming bioburden
The first step in any control program is to control contamination at the very beginning of the process. This includes raw materials (excipients, API, water, etc) and the primary containers. All materials should be tested for incoming bioburden against documented acceptance criteria. Part of the incoming bioburden will also be any water used as an excipient to the process. A good guide for the water bioburden is the EMEA guidance on the subject (EMEA 2002).
Appropriate gowning
The gowning methods and materials are of critical importance to minimization of contamination. Although most attention is placed on aseptic gowning procedures, the appropriate use of gowning precautions will be a great boon to most non-sterile manufacturing facilities as well. All personnel should be well-trained in appropriate gowning practice and behavior. The contamination control plan should describe the rationale for the level of gowning chosen, the frequency of gown cleaning, behavior and the acceptable gown materials for the type of manufacturing process.
Training
Operator training is critical to contamination control. No supervisor can be present at all locations at all times. Each operator must be aware of his or her role in contamination control and how to minimize the risk to batch integrity. The PDA has published a technical report that speaks to some of these training requirements from the microbiological perspective (PDA 2001).
Controlled Environments
Control and monitoring of the environment is another critical element of the contamination control plan. Large portions of this can be addressed by the corporate Environmental Monitoring Master Plan (which provides rationale and consistency for a single EM philosophy across the different facilities of the corporation) or the site Environmental Master Plan (which provides consistency and detailed instruction for the various manufacturing buildings at a given site). However, the Contamination Control Plan should cite the relevant documents and their role in contamination control. Those interested in more on environmental monitoring should refer to the PDA’s treatment of the subject for a good overview (PDA 2001).
The appropriate Environmental Monitoring (EM) plan for non-sterile manufactures and for API manufacturers is not well-defined from a regulatory sense. There are no strong recommendations such as those seen for the environmental monitoring of aseptic facilities; however the absence of regulatory guidance is not the same thing as the absence of need for the activity. EM is useful for determining the state of control of the facility (not so much, perhaps an indicator of the finished product quality) and so is an important part of the monitoring program for all manufacturers.
Well-defined and Understood Manufacturing Processes
The manufacturing process should be evaluated for its potential to limit or eliminate bioburden. The two common methods for performing this is either a HACCP-type (Jahnke and Kuhn 2003) or a FMEA approach. The use of organic solvents, heat, or other inhospitable activities can greatly reduce bioburden of a process. The contribution of compression (and associated shear), for example, should be evaluated for a potential reduction in risk of excessive microbial contamination (Blair 1991). The contribution of the finished product water activity should also contribute to this analysis (USP 2007).
Of particular importance in this evaluation for the potential for microbial contamination of the process are cleaning steps, equipment hold times, HVAC, control level of environments for critical tasks, open-system vs closed-system operations, and bioburden monitoring (among others specific to your process). As an example of the importance of the bioburden control point issue, there is a strong regulatory expectation in Europe that products sterilized by filtration should have a pre-filtration bioburden of not more than 10 CFU/100 mL immediately before the sterilizing filter.
Finally the Contamination Control Plan should cite the need clear SOPs on all aspects of manufacturing, monitoring and control. These SOPs are critical for training, documentation and batch release.
Minimization of Batch Residual Cross-over Contamination
The contamination control plan should also address the potential for a batch to be contaminated by material from the previous batch manufactured using that equipment. Obviously, the contamination control plan should describe the methods by which this likelihood is minimized.
The concern over batch residual cross-over is most relevant when there is more than one product manufactured at a site. This concern has little to do with the sterility of the finished product, and is relevant to sterile and non-sterile manufacture alike.
Minimization of Cleaning Material Residual Contamination
Validation of cleaning procedures is essential to demonstrate not only that the cleaning procedure effectively cleans and sanitizes the manufacturing equipment, but also that residual cleaning material is removed to prevent contamination of the next batch manufactured.
Conclusions
The Contamination Control Plan is an important document designed to formalize the rationale, methods and validation of contamination control procedures in a manufacturing facility. This plan is a valuable tool for pharmaceutical, medical device and personal product manufactures and should be written to address all phases of the facilities life cycle. The Contamination Control Plan should specifically address:
Minimizing the bioburden throughout the manufacturing processes
Minimizing the level of batch residual cross-over contamination
Minimizing the level of cleaning material residual contamination
The microbiologist, as SME, has a critical role to play in the first of these three primary goals, and this essay has therefore been directed at that first topic. Minimization of bioburden in the manufacturing process occurs through (but is not limited to):
Minimizing bioburden in the process
Control incoming bioburden
Appropriate Gowning
Controlled Environments
Well-defined Standard Operating Procedures; and
Well-defined and understood manufacturing processes.
Microbial Limits Tests: The Difference Between “Absence of Objectionable Microorganisms” and “Absence of Specified Microorganisms”
We have to note from the outset that USP and FDA frequently are interested in the same thing. From the vantage point of USP, there is a need to have a test for sterility, for antimicrobial efficacy, for Antibiotic/ Vitamin Potency, for Bacterial Endotoxin, for Microbial Limits etc. The need for these tests is not driven by any concern over “Good Manufacturing Process” (GMP). It is governed by the USP monographs found in the National Formulary (NF). If there is a monograph that requires a test for antimicrobial efficacy, then chapter <51> Antimicrobial Effectiveness Test” is the referee test used to demonstrate that characteristic.
FDA has similar, but separate concerns. Where the requirements are identical, the referee chapters in USP (those numbered under <1000>) are enforced. However, there are situations where the FDA’s concerns are not covered by a USP referee test method. One such situation is with the CFR requirement that medicines be “free of objectionable microorganisms.” 21CFR 211.113 under the section “Control of microbiological contamination. (a)” states “Appropriate written procedures, designed to prevent objectionable microorganisms on drug products not required to be sterile, shall be established and followed.” This is reinforced by 21 CFR 211.165 which states “Testing and release for distribution... (b) There shall be appropriate laboratory testing, as necessary, of each batch of drug product required to be free of objectionable microorganisms.”
So, here we have a problem. The USP monograph for a product (as provided in the current National Formulary) may require “Absence of Pseudomonas aeruginosa.” There is a test in the Microbial Limits chapter to demonstrate the absence of Pseudomonas aeruginosa. However, although this test may be required to demonstrate compliance with the monograph requires as laid out in NF it does not meet the FDA concern that any organism in the final product be acceptable to the product and the target population (i.e. are not “objectionable”).
The FDA Concern
FDA will enforce the GMP requirement that if your product approval to market submission contained a statement that you would test the finished product by the Microbial Limits Tests that in fact you must do that. This is purely a GMP concern. However, the Agency has been absolutely clear on the concern over objectionable microorganisms in the product, and that fact that testing to the USP chapter might be necessary, but it is not sufficient to demonstrate microbial quality. In fact, in the 1993 instructional guide for inspections of QC Microbiology Labs (1) the FDA states:
“For a variety of reasons, we have seen a number of problems associated with the microbiological contamination of topical drug products, nasal solutions and inhalation products. The USP Microbiological Attributes Chapter <1111> provides little specific guidance other than "The significance of microorganisms in nonsterile pharmaceutical products should be evaluated in terms of the use of the product, the nature of the product, and the potential hazard to the user." The USP recommends that certain categories be routinely tested for total counts and specified indicator microbial contaminants. For example natural plant, animal and some mineral products for Salmonella, oral liquids for E. Coli [sic], topicals for P. aeruginosa and S. Aureus [sic], and articles intended for rectal, urethral, or vaginal administration for yeasts and molds. A number of specific monographs also include definitive microbial limits.
As a general guide for acceptable levels and types of microbiological contamination in products, Dr. Dunnigan of the Bureau of Medicine of the FDA commented on the health hazard. In 1970, he said that topical preparations contaminated with gram negative organisms are a probable moderate to serious health hazard. Through the literature and through our investigations, it has been shown that a variety of infections have been traced to the gram negative contamination of topical products. The classical example being the Pseudomonas cepacia contamination of Povidone Iodine products reported by a hospital in Massachusetts several years ago.
Therefore, each company is expected to develop microbial specifications for their nonsterile products. Likewise, the USP Microbial Limits Chapter <61> provides methodology for selected indicator organisms, but not all objectionable organisms. For example, it is widely recognized that Pseudomonas cepacia is objectionable if found in a topical product or nasal solution in high numbers; yet, there are no test methods provided in the USP that will enable the identification of the presence of this microorganism.
A relevant example of this problem is the recall of Metaproterenol Sulfate Inhalation Solution. The USP XXII monograph requires no microbial testing for this product. The agency classified this as a Class I recall because the product was contaminated with Pseudomonas gladioli/cepacia. The health hazard evaluation commented that the risk of pulmonary infection is especially serious and potentially life-threatening to patients with chronic obstructive airway disease, cystic fibrosis, and immuno- compromised patients. Additionally, these organisms would not have been identified by testing procedures delineated in the general Microbial Limits section of the Compendia. . . .
Microbiological testing may include an identification of colonies found during the Total Aerobic Plate Count test. Again, the identification should not merely be limited to the USP indicator organisms.
The importance of identifying all isolates from either or both Total Plate Count testing and enrichment testing will depend upon the product and its intended use. Obviously, if an oral solid dosage form such as a tablet is tested, it may be acceptable to identify isolates when testing shows high levels. However, for other products such as topicals, inhalants or nasal solutions where there is a major concern for microbiological contamination, isolates from plate counts, as well as enrichment testing, should be identified.”
Why is this a concern? To understand this we have to go back to the 1970’s. USP had a test for the “Bacteriological Examination of Gelatin” as early as 1942 (2). However, most non-sterile medications in the US were not required to assay for microbiological quality attributes until the introduction of the Microbial Limits Tests in 1970 (3). In the late 1960’s several outbreaks of disease were traced back to pathogencontaminated medications, and this prompted increased attention to the microbial content of non-sterile drugs (4). Later in the 1980’s there was a series of articles appearing in the literature describing contamination by P. cepacia (currently Burkholderia cepacia) (5, 6) and its survival in disinfectants(7 – 11). This lead to the addition of requirements in the 21 CFR to ensure that there are not objectionable organisms in product released to market (see above). Add to this the knowledge that the USP “Absence of Pseudomonas aeruginosa” assay will not identify presence of B. cepacia (as discussed).
The USP Concern
The USP is on record as early as 1982 verifying that the demonstration of “absence of objectionable microorganisms” is not the intent of the chapter. In a one page Stimuli to the Revision Process the microbiology committee of the time states:
“The tests described in the Microbial Limits Tests <61> were not designed to be all-inclusive, i.e., to detect all potential pathogens. To accomplish this, an extensive text on laboratory detection of microorganisms would be required. The procedures in USP were designed to detect the presence of specific “index” or “indicator” organisms. Nevertheless, the present chapter does not preclude the detection of Ps. Cepacia – the organism requires subsequent differentiation. The chapter does not provide specific methods for this, nor does it provide procedures for detecting thousands of other potentially pathogenic organisms. Individual monographs include requirements for limits on total aerobic counts and/or absence of one or more of the four selected “indicator” organisms. The chapter on Microbial Limits Tests provides methods to assure that one may test for those microbial requirements in the individual monographs...
In conclusion, the Microbial Attributes and Microbial Limit Tests chapters accomplish their intent. If a manufacturer needs particular tests for any specific organisms that are potential problems in a process or a final product, the quality control microbiologist can provide specific detection procedures. Many such procedures are published in several laboratory texts on microbiology.”
Conclusions
On the question of the microbial quality of non-sterile pharmaceuticals, the USP and the FDA are in agreement – the product must be safe for use. The NF monograph requirements for absence of specific organisms is a minimal requirement, and should not be taken as proof that the product is suitable for sale from a microbiological perspective.
The manufacturer is responsible for the quality and safety of the product marketed, and it is the clear expectation of FDA (as described in CFR) that this will include a determination of the microbial safety – i.e. the “absence of objectionable microorganisms” from the product. These positions have been publicly stated for decades and should not come as a surprise to anyone. The harmonized microbial limits tests only address the “absence of specified microorganisms” and leave the determination of the “absence of objectionable microorganisms” in the capable hands of each company’s appropriately educated and well-trained microbiology group.
FDA has similar, but separate concerns. Where the requirements are identical, the referee chapters in USP (those numbered under <1000>) are enforced. However, there are situations where the FDA’s concerns are not covered by a USP referee test method. One such situation is with the CFR requirement that medicines be “free of objectionable microorganisms.” 21CFR 211.113 under the section “Control of microbiological contamination. (a)” states “Appropriate written procedures, designed to prevent objectionable microorganisms on drug products not required to be sterile, shall be established and followed.” This is reinforced by 21 CFR 211.165 which states “Testing and release for distribution... (b) There shall be appropriate laboratory testing, as necessary, of each batch of drug product required to be free of objectionable microorganisms.”
So, here we have a problem. The USP monograph for a product (as provided in the current National Formulary) may require “Absence of Pseudomonas aeruginosa.” There is a test in the Microbial Limits chapter to demonstrate the absence of Pseudomonas aeruginosa. However, although this test may be required to demonstrate compliance with the monograph requires as laid out in NF it does not meet the FDA concern that any organism in the final product be acceptable to the product and the target population (i.e. are not “objectionable”).
The FDA Concern
FDA will enforce the GMP requirement that if your product approval to market submission contained a statement that you would test the finished product by the Microbial Limits Tests that in fact you must do that. This is purely a GMP concern. However, the Agency has been absolutely clear on the concern over objectionable microorganisms in the product, and that fact that testing to the USP chapter might be necessary, but it is not sufficient to demonstrate microbial quality. In fact, in the 1993 instructional guide for inspections of QC Microbiology Labs (1) the FDA states:
“For a variety of reasons, we have seen a number of problems associated with the microbiological contamination of topical drug products, nasal solutions and inhalation products. The USP Microbiological Attributes Chapter <1111> provides little specific guidance other than "The significance of microorganisms in nonsterile pharmaceutical products should be evaluated in terms of the use of the product, the nature of the product, and the potential hazard to the user." The USP recommends that certain categories be routinely tested for total counts and specified indicator microbial contaminants. For example natural plant, animal and some mineral products for Salmonella, oral liquids for E. Coli [sic], topicals for P. aeruginosa and S. Aureus [sic], and articles intended for rectal, urethral, or vaginal administration for yeasts and molds. A number of specific monographs also include definitive microbial limits.
As a general guide for acceptable levels and types of microbiological contamination in products, Dr. Dunnigan of the Bureau of Medicine of the FDA commented on the health hazard. In 1970, he said that topical preparations contaminated with gram negative organisms are a probable moderate to serious health hazard. Through the literature and through our investigations, it has been shown that a variety of infections have been traced to the gram negative contamination of topical products. The classical example being the Pseudomonas cepacia contamination of Povidone Iodine products reported by a hospital in Massachusetts several years ago.
Therefore, each company is expected to develop microbial specifications for their nonsterile products. Likewise, the USP Microbial Limits Chapter <61> provides methodology for selected indicator organisms, but not all objectionable organisms. For example, it is widely recognized that Pseudomonas cepacia is objectionable if found in a topical product or nasal solution in high numbers; yet, there are no test methods provided in the USP that will enable the identification of the presence of this microorganism.
A relevant example of this problem is the recall of Metaproterenol Sulfate Inhalation Solution. The USP XXII monograph requires no microbial testing for this product. The agency classified this as a Class I recall because the product was contaminated with Pseudomonas gladioli/cepacia. The health hazard evaluation commented that the risk of pulmonary infection is especially serious and potentially life-threatening to patients with chronic obstructive airway disease, cystic fibrosis, and immuno- compromised patients. Additionally, these organisms would not have been identified by testing procedures delineated in the general Microbial Limits section of the Compendia. . . .
Microbiological testing may include an identification of colonies found during the Total Aerobic Plate Count test. Again, the identification should not merely be limited to the USP indicator organisms.
The importance of identifying all isolates from either or both Total Plate Count testing and enrichment testing will depend upon the product and its intended use. Obviously, if an oral solid dosage form such as a tablet is tested, it may be acceptable to identify isolates when testing shows high levels. However, for other products such as topicals, inhalants or nasal solutions where there is a major concern for microbiological contamination, isolates from plate counts, as well as enrichment testing, should be identified.”
Why is this a concern? To understand this we have to go back to the 1970’s. USP had a test for the “Bacteriological Examination of Gelatin” as early as 1942 (2). However, most non-sterile medications in the US were not required to assay for microbiological quality attributes until the introduction of the Microbial Limits Tests in 1970 (3). In the late 1960’s several outbreaks of disease were traced back to pathogencontaminated medications, and this prompted increased attention to the microbial content of non-sterile drugs (4). Later in the 1980’s there was a series of articles appearing in the literature describing contamination by P. cepacia (currently Burkholderia cepacia) (5, 6) and its survival in disinfectants(7 – 11). This lead to the addition of requirements in the 21 CFR to ensure that there are not objectionable organisms in product released to market (see above). Add to this the knowledge that the USP “Absence of Pseudomonas aeruginosa” assay will not identify presence of B. cepacia (as discussed).
The USP Concern
The USP is on record as early as 1982 verifying that the demonstration of “absence of objectionable microorganisms” is not the intent of the chapter. In a one page Stimuli to the Revision Process the microbiology committee of the time states:
“The tests described in the Microbial Limits Tests <61> were not designed to be all-inclusive, i.e., to detect all potential pathogens. To accomplish this, an extensive text on laboratory detection of microorganisms would be required. The procedures in USP were designed to detect the presence of specific “index” or “indicator” organisms. Nevertheless, the present chapter does not preclude the detection of Ps. Cepacia – the organism requires subsequent differentiation. The chapter does not provide specific methods for this, nor does it provide procedures for detecting thousands of other potentially pathogenic organisms. Individual monographs include requirements for limits on total aerobic counts and/or absence of one or more of the four selected “indicator” organisms. The chapter on Microbial Limits Tests provides methods to assure that one may test for those microbial requirements in the individual monographs...
In conclusion, the Microbial Attributes and Microbial Limit Tests chapters accomplish their intent. If a manufacturer needs particular tests for any specific organisms that are potential problems in a process or a final product, the quality control microbiologist can provide specific detection procedures. Many such procedures are published in several laboratory texts on microbiology.”
Conclusions
On the question of the microbial quality of non-sterile pharmaceuticals, the USP and the FDA are in agreement – the product must be safe for use. The NF monograph requirements for absence of specific organisms is a minimal requirement, and should not be taken as proof that the product is suitable for sale from a microbiological perspective.
The manufacturer is responsible for the quality and safety of the product marketed, and it is the clear expectation of FDA (as described in CFR) that this will include a determination of the microbial safety – i.e. the “absence of objectionable microorganisms” from the product. These positions have been publicly stated for decades and should not come as a surprise to anyone. The harmonized microbial limits tests only address the “absence of specified microorganisms” and leave the determination of the “absence of objectionable microorganisms” in the capable hands of each company’s appropriately educated and well-trained microbiology group.
The Harmonization of the Microbial Limits Test - Absence of Specified Organisms
The last issue of the PMF Newsletter (vol 12, no. 3) contained a review of the harmonization status of the Microbial Limits Tests – Enumeration. In addition, the article provided a brief overview of the compendial harmonization process as agreed to by the Pharmacopeial Discussion Group (PDG). In this article, I would like to focus on the other side of the Microbial Limits Tests – the “Absence of Specified Microorganisms” component.
USP
EP
<61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
2.6.12 Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
<62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
2.6.13 Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
<1111> Microbiological Quality of Nonsterile Pharmaceutical Products
5.1.4 Microbiological Quality of Nonsterile Pharmaceutical Products
Table 1: Harmonized Chapter Numbering Scheme
There is a significant controversy in the United States over the intent of this evaluation. The FDA is bound by the concern expressed in the Code of Federal Regulations (21CFR 211.113 and 21CFR 211.165) relating to the importance of “objectionable microorganisms.” This is not the concern of the compendial chapters. The controversy is worthy of discussion, but not the topic of this review – it will be discussed in length in the final of three articles on the harmonization of the microbial limits tests to be published in next month’s newsletter.
What follows is a tabular presentation of the existing “Microbial Limits – Absence of Specified Microorganisms” tests from the current USP and Pharm Eur, as well as the draft harmonized document (the finalized document is extremely close to this version, but not release to the industry). It is provided as an aid to evaluation, and may assist in determining whether revalidation of method suitability studies is needed. It should be noted that this harmonization draft represents a true compromise by all parties, with (at least in the author’s opinion) significant changes from the current USP, Pharm Eur and JP chapters.
USP
EP
<61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
2.6.12 Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
<62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
2.6.13 Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
<1111> Microbiological Quality of Nonsterile Pharmaceutical Products
5.1.4 Microbiological Quality of Nonsterile Pharmaceutical Products
Table 1: Harmonized Chapter Numbering Scheme
There is a significant controversy in the United States over the intent of this evaluation. The FDA is bound by the concern expressed in the Code of Federal Regulations (21CFR 211.113 and 21CFR 211.165) relating to the importance of “objectionable microorganisms.” This is not the concern of the compendial chapters. The controversy is worthy of discussion, but not the topic of this review – it will be discussed in length in the final of three articles on the harmonization of the microbial limits tests to be published in next month’s newsletter.
What follows is a tabular presentation of the existing “Microbial Limits – Absence of Specified Microorganisms” tests from the current USP and Pharm Eur, as well as the draft harmonized document (the finalized document is extremely close to this version, but not release to the industry). It is provided as an aid to evaluation, and may assist in determining whether revalidation of method suitability studies is needed. It should be noted that this harmonization draft represents a true compromise by all parties, with (at least in the author’s opinion) significant changes from the current USP, Pharm Eur and JP chapters.
The Harmonization of the Microbial Limits Test - Enumeration
The USP and the European Pharmacopoeia (EP, Pharm Eur) Microbial Limits Tests are in the final stages of harmonization. They were signed off to Stage 6A at the November, 2005 meeting of the Pharmacopeial Discussion Group (PDG) held in Chicago, IL USA (USP 2006a). However, the signed-off versions have yet to be published. This makes the description of the test a bit difficult, as the current tests will be disappearing, and the final, harmonized test is not yet public knowledge. However, we do know that the harmonized tests do not differ greatly from the drafts published in 2003 (USP 2003a, USP 2003b, USP 2003c), and so we will use those drafts as the description of the finalized test.
The Microbial Limits Tests are actually two chapters in the current USP: Current USP <61> Microbial Limits Tests (USP 2006b) and <1111> Microbiological Attributes of Nonsterile Pharmaceutical Products (USP 2006c). This will be modified in the harmonized version to mirror the European format:
USP
EP
<61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
2.6.12 Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
<62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
2.6.13 Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
<1111> Microbiological Quality of Nonsterile Pharmaceutical Products
5.1.4 Microbiological Quality of Nonsterile Pharmaceutical Products
Table 1: Harmonized Chapter Numbering Scheme
This review will only address the microbial enumeration portions of the harmonization effort – that which will become USP chapter <61> and Pharm. Eur. chapter 2.6.12.
The microbial enumeration test is a basic, simple design to count the number of CFU in a nonsterile product or raw material. The preferred method is to put the material into solution and then plate aliquots to determine the CFU/gram (or mL) of initial material. If the product cannot be put into solution, there are provisions to use the Most Probable Number method (MPN – see FDA BAM website). The method of plating can be either pour plate, spread plate or the filtration of material and then placing the membrane filter on the surface of an agar plate. The membrane filtration method should only be used when there are few expected colony forming units in the material to be tested as it is a good method to test a large volume of liquid, but can only count up to approximately 100 CFU/membrane.
The harmonized method provides a great deal more detail than any of the current pharmacopeial methods in terms of demonstration of method suitability (validation of the method) and in terms of media growth promotion.
The demonstration of method suitability should be performed using the challenge organisms listed (see Table 2 below) in accordance with the recommendations found in USP chapter <1227> (USP 2006d). Growth promotion is an area of some ambiguity in the compendial text. Although media growth promotion is not described in the tests, demonstration of media suitability is required, and the draft USP Chapter <1117> (USP 2004) provides assistance in designing the studies using 10-100 CFU of the challenge organisms.
A major concern of many QC workers is if the changes in the harmonized chapter will necessitate revalidation of existing assays to meet the requirements of the harmonized test. There are several considerations that might lead to revalidation – a required change in media, in volume of material required for testing, in general testing conditions. It is difficult to determine whether all product types would require revalidation, and so a summary table is provided (Table 2) describing the critical aspects of the current Microbial Limits Tests (Enumeration) and the draft harmonization text. The summaries provided in Table 2 are only meant as an aid, the decision as to whether or not revalidation is necessary rests with each individual facility for their particular products.
The Microbial Limits Tests are actually two chapters in the current USP: Current USP <61> Microbial Limits Tests (USP 2006b) and <1111> Microbiological Attributes of Nonsterile Pharmaceutical Products (USP 2006c). This will be modified in the harmonized version to mirror the European format:
USP
EP
<61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
2.6.12 Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
<62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
2.6.13 Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
<1111> Microbiological Quality of Nonsterile Pharmaceutical Products
5.1.4 Microbiological Quality of Nonsterile Pharmaceutical Products
Table 1: Harmonized Chapter Numbering Scheme
This review will only address the microbial enumeration portions of the harmonization effort – that which will become USP chapter <61> and Pharm. Eur. chapter 2.6.12.
The microbial enumeration test is a basic, simple design to count the number of CFU in a nonsterile product or raw material. The preferred method is to put the material into solution and then plate aliquots to determine the CFU/gram (or mL) of initial material. If the product cannot be put into solution, there are provisions to use the Most Probable Number method (MPN – see FDA BAM website). The method of plating can be either pour plate, spread plate or the filtration of material and then placing the membrane filter on the surface of an agar plate. The membrane filtration method should only be used when there are few expected colony forming units in the material to be tested as it is a good method to test a large volume of liquid, but can only count up to approximately 100 CFU/membrane.
The harmonized method provides a great deal more detail than any of the current pharmacopeial methods in terms of demonstration of method suitability (validation of the method) and in terms of media growth promotion.
The demonstration of method suitability should be performed using the challenge organisms listed (see Table 2 below) in accordance with the recommendations found in USP chapter <1227> (USP 2006d). Growth promotion is an area of some ambiguity in the compendial text. Although media growth promotion is not described in the tests, demonstration of media suitability is required, and the draft USP Chapter <1117> (USP 2004) provides assistance in designing the studies using 10-100 CFU of the challenge organisms.
A major concern of many QC workers is if the changes in the harmonized chapter will necessitate revalidation of existing assays to meet the requirements of the harmonized test. There are several considerations that might lead to revalidation – a required change in media, in volume of material required for testing, in general testing conditions. It is difficult to determine whether all product types would require revalidation, and so a summary table is provided (Table 2) describing the critical aspects of the current Microbial Limits Tests (Enumeration) and the draft harmonization text. The summaries provided in Table 2 are only meant as an aid, the decision as to whether or not revalidation is necessary rests with each individual facility for their particular products.
Streaking for Single Colonies: An Essential First Step in Microbial Identification
From the outset, let’s admit that the current state of microbial identification is a little confusing. We can ID by traditional biochemical tests (API Strips) or by elegant refinements of the traditional methods (for example, the Vitek 2 Compact). We can identify microorganisms by carbohydrate utilization (Biolog systems) or by the GC pattern of the cell’s fatty acids (Sherlock System). If you want to go genotypic, then you currently have a choice between the Dupont RiboPrinter or Applied Biosystems MicroSeq systems. Your identification (genus and species) may well depend on which system you use as there is no objective standard, and each system is reliant on its proprietary database to assign an identification to the data.
Virtually all of these systems require a preliminary Gram stain to accurately identify the sample. The Gram stain requires a relatively fresh culture for best results (PMF Newsletter, Feb. 2006). This is the first reason to restreak for single colonies after isolation of a contaminant. However, each system also is dependent on the presentation of a monoclonal sample for accurate results. In fact, only one of these systems provides you with enough information to recognize if you have a contaminated (polyclonal) sample (no, I am not going to tell which one it is).
The basic fact is that acceptable microbiological practice (I am not even going to say “good lab practice” or “best lab practice” but “acceptable” or, if you prefer, “adequate”) requires streaking for well isolated, single colonies of good health for identification purposes. This is not difficult.
The best starting material is a relatively “clean looking” colony on your primary plate. This colony should not show obvious signs of being multiple pinprick colonies that merged into a single CFU. Using a sterile loop, sample from the center of the colony and begin a heavy streak onto a new plate of appropriate agar media. This is quadrant #1 (see accompanying figure).
Streaking in quadrant #1 (and all subsequent streaking events) should be in the same direction, with the same part of the loop in contact with the agar. After the completion of the streaking in this quadrant, the loop should be resterilized (thoroughly flamed or discarded for a new, sterile disposable loop) and the plate streaked into quadrant #2 by drawing the fresh loop across two or three lines in quadrant #1. This should be done once or twice, then subsequent streaks performed without touching any of the previous line in the agar surface. The loop is resterilized, and the process is repeated in quadrants #3 and #4, each time the loop becoming contaminated by drawing it across a few lines in the previous quadrant. The idea is a successive dilution of the level of CFU in each quadrant, on each successive line after the initial inoculation in the prior quadrant. The plate is then incubated overnight for colony growth. Single, well-isolated colonies should be evident in quadrant #4 or quadrant #3. Care should be taken not to accidentally contaminate the colony when harvesting.
One note of caution. This streaking for single colony isolates should be conducted a second time if the original plate was heavily contaminated, or if there are multiple colony morphologies evident on this initial streaked plate. The integrity of the microbial identification process requires a monoclonal colony (a colony that is from a single bacterial strain).
Remember – the only assurance you have of a correct identification is proper preparation of the monoclonal sample. To this concern, the final isolation plate should never be used as a storage device – the single well-isolated colony chosen should be restreaked on a separate plate or agar-slant tube for storage.
This seems like a lot of work, and requires an additional day (at least) to the turn-around time for identification of a contaminant when compared to the time required if single-colony isolation is omitted from the process. However, if microbial identification is attempted directly from the colony on the primary test plate (environmental monitoring or bioburden plate) any resultant microbial identification must be suspect as there is no assurance that you are working with a pure culture. When auditing your microbiology lab (or your contract lab), check to see if an SOP is in place requiring this step, and also check the refrigerators and incubators to see if you can find evidence that this is, in fact, occurring. It is an unfortunately common practice to omit this essential step in microbial identification in an ill-advised attempt to save time and money. However, as there are few quality controls possible on the microbial identification process, you have to build the quality into the process to avoid the GIGO phenomenon.
We are in a period of high regulatory interest in environmental monitoring identifications (as part of aseptic production controls), and in the demonstration of absence of objectionable microorganisms in nonsterile finished drug products. This is not the time (if there ever was one) to save a few pennies by omitting a step necessary to the accurate and confident identification of a microbial colony.
Virtually all of these systems require a preliminary Gram stain to accurately identify the sample. The Gram stain requires a relatively fresh culture for best results (PMF Newsletter, Feb. 2006). This is the first reason to restreak for single colonies after isolation of a contaminant. However, each system also is dependent on the presentation of a monoclonal sample for accurate results. In fact, only one of these systems provides you with enough information to recognize if you have a contaminated (polyclonal) sample (no, I am not going to tell which one it is).
The basic fact is that acceptable microbiological practice (I am not even going to say “good lab practice” or “best lab practice” but “acceptable” or, if you prefer, “adequate”) requires streaking for well isolated, single colonies of good health for identification purposes. This is not difficult.
The best starting material is a relatively “clean looking” colony on your primary plate. This colony should not show obvious signs of being multiple pinprick colonies that merged into a single CFU. Using a sterile loop, sample from the center of the colony and begin a heavy streak onto a new plate of appropriate agar media. This is quadrant #1 (see accompanying figure).
Streaking in quadrant #1 (and all subsequent streaking events) should be in the same direction, with the same part of the loop in contact with the agar. After the completion of the streaking in this quadrant, the loop should be resterilized (thoroughly flamed or discarded for a new, sterile disposable loop) and the plate streaked into quadrant #2 by drawing the fresh loop across two or three lines in quadrant #1. This should be done once or twice, then subsequent streaks performed without touching any of the previous line in the agar surface. The loop is resterilized, and the process is repeated in quadrants #3 and #4, each time the loop becoming contaminated by drawing it across a few lines in the previous quadrant. The idea is a successive dilution of the level of CFU in each quadrant, on each successive line after the initial inoculation in the prior quadrant. The plate is then incubated overnight for colony growth. Single, well-isolated colonies should be evident in quadrant #4 or quadrant #3. Care should be taken not to accidentally contaminate the colony when harvesting.
One note of caution. This streaking for single colony isolates should be conducted a second time if the original plate was heavily contaminated, or if there are multiple colony morphologies evident on this initial streaked plate. The integrity of the microbial identification process requires a monoclonal colony (a colony that is from a single bacterial strain).
Remember – the only assurance you have of a correct identification is proper preparation of the monoclonal sample. To this concern, the final isolation plate should never be used as a storage device – the single well-isolated colony chosen should be restreaked on a separate plate or agar-slant tube for storage.
This seems like a lot of work, and requires an additional day (at least) to the turn-around time for identification of a contaminant when compared to the time required if single-colony isolation is omitted from the process. However, if microbial identification is attempted directly from the colony on the primary test plate (environmental monitoring or bioburden plate) any resultant microbial identification must be suspect as there is no assurance that you are working with a pure culture. When auditing your microbiology lab (or your contract lab), check to see if an SOP is in place requiring this step, and also check the refrigerators and incubators to see if you can find evidence that this is, in fact, occurring. It is an unfortunately common practice to omit this essential step in microbial identification in an ill-advised attempt to save time and money. However, as there are few quality controls possible on the microbial identification process, you have to build the quality into the process to avoid the GIGO phenomenon.
We are in a period of high regulatory interest in environmental monitoring identifications (as part of aseptic production controls), and in the demonstration of absence of objectionable microorganisms in nonsterile finished drug products. This is not the time (if there ever was one) to save a few pennies by omitting a step necessary to the accurate and confident identification of a microbial colony.
Counting Colonies
Who Cares?
What is the fuss about in determining the number of colony forming units? After all, the CFU is only an estimate of the number of cells present. It is a skewed estimate at best as the only cells able to form colonies are those that can grow under the conditions of the test (incubation media, temperature, time, oxygen conditions, etc). Even among that group of microorganisms a colony does not represent a single cell, but rather cells that happened to be well separated on the plate and so can be distinguished after growth. A colony could arise from one cell, or several thousand. So why the fuss?
One reason for concern is that microbiology has a well-deserved reputation for being highly variable. Our lax attention to precision and accuracy in our measurements helps further this perception. We have allowed specifications for environmental monitoring, raw material bioburden, in-process bioburden and finished product bioburden to be imposed by regulation without regard for the ability of the method to support those specifications.
A second reason for concern is that now we are trying to introduce alternate microbiological methods into the lab. Being obsessive by training, we are trying to exceed measures of accuracy and precision in this exercise that the traditional methods cannot come close to matching. A good example of this is the Pharm Eur “Precision” requirement for an alternate method (quantification) to have a Relative Standard Deviation (RSD) in the range of 10-15% (1). While you might get lucky and hit this with dilutions whose plate counts are in the 150-250 CFU/plate range, - at lower plate counts this target value imposed by regulation will virtually guarantee a long, difficult and quite possibly unsuccessful, validation exercise.
Countable Range on a Plate
Literature
The general ranges in common acceptance for countable numbers of colonies on a plate are 30 – 300 and 25 – 250. The origin of those ranges is worth examination. Breed and Dotterrer published a seminal paper on this topic in 1916 (2). They set out to determine the “limit in the number of colonies that may be allowed to grow on a plate without introducing serious errors…in connection with the proposed revisions of standard methods of milk analysis.” They note that “the kind of bacteria in the material under examination will have an influence on the size of the colonies, and consequently, on the number that can develop on a plate.” They also note that food supply can be an issue, that colonies close to each other on the plate may merge, and that neighbor colonies may inhibit growth or conversely stimulate growth. “Because of these and other difficulties certain plates in any series made from a given sample are more satisfactory for use in computing a total than are others. The matter of selecting plates to be used in computing a count becomes therefore a matter requiring considerable judgment.”
Breed and Dotterrer chose their countable plates from triplicate platings of each dilution, requiring acceptable plates to be within 20% of the average. On this analysis, plates with more than 400 CFU were unsatisfactory, as were those of less than 30 CFU, with best results in the range of 50-200 CFU/plate.
The major paper from Tomasiewicz et al (3) provides an excellent review of the continued evolution of the appropriate number of CFU per plate from milk. They took data from colony counts of raw milk from three different experiments (each dilution plated in triplicate) and used to determine a mean-squared-error of the estimate for all plates. Their recommendation at the end of the study was for a countable range of 25-250 CFU/plate in triplicate. It is interesting to note that although the authors note that CFU follow a Poisson distribution, no mention is made of any data transformation used to approximate a normal distribution prior to the use of normal statistical analytical tools. Tomasiewicz et al provide excellent cautionary advice:
“The data presented herein are not necessarily applicable to other systems. For automated equipment, the optimum range may well vary with the instrument…Furthermore, even if automation is not used appropriate numbers of colonies that should be on a countable plate can very widely, depending on many other variables. With soil fungi for example…”
The compendia have recently harmonized a microbial enumeration test (4), and in this test recommend that the technician “Select the plates corresponding to a given dilution and showing the highest number of colonies less than 250 for TAMC and 50 for TYMC.” In determination of the resistance of biological indicators, USP recommends a range of “20 to 300 colonies, but not less than 6” (5). However, the most complete description of the countable range is found in the informational chapter <1227> (6):
“The accepted range for countable colonies on a standard agar plate is between 25 and 250 for most bacteria and Candida albicans. This range was established in the food industry for counting coliform bacteria in milk. The range is acceptable for compendial organisms, except for fungi. It is not optimal for counting all environmental monitoring isolates. The recommended range for Aspergillus niger is between 8 to 80 cfu per plate. The use of membrane filtration to recover challenge organisms, or the use of environmental isolates as challenge organisms in the antimicrobial effectiveness testing, requires validation of the countable range.”
ASTM provides countable ranges of 20-80 CFU/membrane, 20-200 for spread plates and 30-300 for pour plates (7). The FDA Bacterial Analytical Manual (BAM) recommends 25-250 CFU/plate as a countable range (8).
Upper Limit
The upper limit of plate counts is dependent on a number of factors, as described previously. The major issues include the colony size and behavior (swarming?), and the surface area of the plate. The size particularly comes into play with plating a membrane for determination of CFU as the surface area of that membrane is so much smaller than that of a standard plate.
TNTC can be reported out several ways. ASTM (7) recommends reporting this out as >”upper limit”. For example, a 1:10 dilution with more than 200 CFU on a spread plate would be reported as “>2,000 CFU/mL (or gram). FDA’s BAM recommends counting the colonies from the dilution with plates giving counts closest to 250, counting a portion of the plate, estimating the total number and then using that number as the Estimated Aerobic Count. It is not clear to the author how this is greatly superior to guessing. In my opinion this is an invalid plating and needs to be done correctly at a later date (note I am strenuously avoiding the use of the word retest. This result invalidates the plating and therefore the test was not performed correctly.) I know this is a hardship to the lab, who were trying to reduce the plating load initially by not plating out sufficient dilutions. However, making a mistake initially is not a reasonable excuse to avoid doing it correctly after the mistake is recognized. If the lab wishes to use this “estimated count” it should, at a minimum, have it clearly described in their “counting CFU” SOP with a rationale as to when the plate counts are not critical and can be estimated in this fashion.
There are methods available if you should want to accurately determine the upper limit for a unique plating surface or a unique colony type. One is presented in the USP informational chapter <1227> (5) which is based on a pair-wise comparison of counts from a dilution series. This is based on the assumption that at the upper limit the observed numbers of CFU will fall off the expected numbers at some point (see Figure 1). This divergence will become significant at some point – that defines the upper limit of CFU/plate.
Figure 1. Difference between Expected and Observed CFU with Increasing Numbers
Lower Limit
A central concern in this determination is the reporting of the Limit of Quantification (which is what we are really interested in reporting) against the Limit of Detection (1 CFU). This is an important distinction if we are being held to specifications in the lower range.
ASTM recommendations focus on the LOD, and urge the user to report that answer out if no colonies are recovered (ie <10 CFU/mL for a 1:10 dilution) (7). If countable colonies are present, but below the countable range, count them anyway and report an estimated count.
USP (6) does not have a specific recommendation on how to report out these low numbers, but does note “Lower counting thresholds for the greatest dilution plating in series must be justified.”
FDA BAM (8) recommends a different reporting format. In the FDA BAM method, all counts are recorded in the raw data, but the information is reported out as). This leads to graphs such as in Figure 2 which shows us that as the CFU/plate drops below the countable range, the error as a percent of the mean increases rapidly. This confusion between the Limit of Detection and the Limit of Quantification for plate counts has led to some very difficult situations (as discussed below).
Figure 2. Increase in Error with Decreasing Numbers
Unusual Situations
What About Two Dilutions with Countable Colonies?
Ideally you would never see two separate dilutions with counts in the countable range, as the countable ranges cover a ten-fold range of CFU. However, this is microbiology. ASTM recommendations (7) urge you to take both dilutions into account, determining the CFU/mL (or gram) separately for each, then averaging the results for the final result. Breed and Dotterrer (2) also used several dilutions if the numbers fit the QC requirements (see below). FDA BAM has no recommendations in this situation.
While the argument can be made to use all counts, this is a stronger argument if triplicate plates are used and QC limits are in place to discard erroneous plates.
A strong argument can also be made to take the dilution providing the larger number of CFU in the countable range. This approach minimizes two concerns, that the errors in the estimates increase with increasing serial dilutions, and that the error in the estimate increases with decreasing plate counts. Use of the smaller dilution (eg 1:10 vs 1:100) could be justified from this perspective.
Whichever method used should be documented and justified in the “Counting CFU” SOP.
What about QC Limits on Replicate Plate Counts?
Periodically there are recommendations to establish Quality Control limits on replicate plate counts. Breed and Dotterrer in their 1916 paper (2) required valid plate counts from triplicate plates to provide estimates of CFU/mL within 20% of the mean. In other words, all plates were counted, each plate’s CFU count was used to estimate the original CFU/mL, then each estimate was evaluated. If the individual plate’s estimate was within 20% of the mean, it was deemed acceptable. This method is not practical in the QC lab.
Establishment of QC limits for plate counts works best if you have at least three replicate plates for each dilution. The average of the dilution replicates can be determined, variant counts (hopefully no more than one plate per triplicate plating) discarded and the final average determined. If you try this with duplicate plates you frequently end up with trying to average the results of one plate. While this method looks good on paper, the prudent lab manager will evaluate some historical data before instituting it as a practice.
The method used to QC individual plate counts, if used, should be documented and justified in SOP, along with the response to finding variant counts.
Can I plate 10 1 mL samples to plate a Total of One 10 mL Sample?
There have been suggestions that a larger volume of material may be plated across several plates, and the results reported out for the larger volume. For example, plating 10 1 mL samples on 10 different plates, and then reporting it as if a 10 mL sample was plated. This approach is flawed in that it ignores several sources of variability in plating including sampling error, plating errors, growth/incubation error and counting errors (9, 10). The correct interpretation for this situation that you have just plated 1 mL ten times, not 10 mL once. The numbers might be averaged, they cannot be added.
Rounding and Averaging
To discuss this we need to determine what the significant figures might be in the measure. For raw colony counts, common practice determines that the CFU observed determine the significant figure, and that the average is one decimal to the right of that number (sticklers for accuracy will report the geometric mean rather than the arithmetic mean given the Poisson distribution followed by CFU). In reporting, it is common practice to report out as scientific notation using two significant figures. This requires rounding.
USP (11) and ASTM (7) both round up at five if 5 is the number to the right of the last significant figure. FDA BAM has a more elaborate scheme, rounding up if the number is 6 or higher, down if 4 or lower. If the number is 5, BAM looks to the next number to the right and rounds up if it is odd, down if it is even.
This is one of those situations where you want every-one to do the calculations the same way (I am hard pressed to come up with a situation in a lab where you want everyone to do it differently). Be sure to include direction and its justification in the “Counting CFU” SOP if it does not already exist in a separate SOP.
Impact on Specifications and Environmental Monitoring Control Levels
We are back to the question of WHO CARES?
If you are faced with a finished product bioburden of NMT (Not More Than) 100 CFU/gram, and your method suitability study requires a 1:100 dilution of the product to overcome any antimicrobial effects, then how are you to test it? Common practice is to perform the 1:100 dilution, perform a pour plate of 1 mL in duplicate and if 2 colonies grow on each plate, the product fails specification. This common practice is scientifically unsupportable – it confused the Limit of Detection with the Limit of Quantification for the plate count method.
Let’s take a look at environmental monitoring alert and action levels for aseptically produced products. Hussong and Madsen (12) recently published a thoughtful review of this topic where they argue that the levels of acceptable CFU for many room classifications are below the noise level of plate count technology (eg in the range of 1-2 CFU/m3). In addition, environmental data can be extremely variable, much more so than controlled lab studies as the numbers of microorganisms, the physiological state of the isolates, even the species are completely out of the control of the investigator. In addition the numbers do not conform to a normal distribution as there are sporadic counts with a count of “zero” CFU predominating. They conclude that since the numbers are unreliable, the trend in the data is the only important consideration, and that EM counts cannot be used for product release criteria. A separate treatment of this subject was presented by Farrington (13) who argues that the relationship between EM data and finished product quality is a widely held, but unproven belief, compounded by the problems in accuracy with the low counts generated by plate count methodology.
Conclusions
In conclusion, all methods have limitations. One of the major limitations to the plate count method is the relatively narrow countable range (generally considered to be 25-250 CFU bacteria on a standard petri dish). The currently prevailing confusion between the Limit of Detection (1 CFU) and Limit of Quantification (25 CFU) for the plate count method creates a larger degree of variability in microbiology data than is necessary. An unfortunate regulatory trend in recent years is to establish expectations (specifications, limits, levels) for data generated by the plate count method that the accuracy of the method cannot support. This is a real opportunity for modification of current practice to approach the goal of “science-based regulations”.
What is the fuss about in determining the number of colony forming units? After all, the CFU is only an estimate of the number of cells present. It is a skewed estimate at best as the only cells able to form colonies are those that can grow under the conditions of the test (incubation media, temperature, time, oxygen conditions, etc). Even among that group of microorganisms a colony does not represent a single cell, but rather cells that happened to be well separated on the plate and so can be distinguished after growth. A colony could arise from one cell, or several thousand. So why the fuss?
One reason for concern is that microbiology has a well-deserved reputation for being highly variable. Our lax attention to precision and accuracy in our measurements helps further this perception. We have allowed specifications for environmental monitoring, raw material bioburden, in-process bioburden and finished product bioburden to be imposed by regulation without regard for the ability of the method to support those specifications.
A second reason for concern is that now we are trying to introduce alternate microbiological methods into the lab. Being obsessive by training, we are trying to exceed measures of accuracy and precision in this exercise that the traditional methods cannot come close to matching. A good example of this is the Pharm Eur “Precision” requirement for an alternate method (quantification) to have a Relative Standard Deviation (RSD) in the range of 10-15% (1). While you might get lucky and hit this with dilutions whose plate counts are in the 150-250 CFU/plate range, - at lower plate counts this target value imposed by regulation will virtually guarantee a long, difficult and quite possibly unsuccessful, validation exercise.
Countable Range on a Plate
Literature
The general ranges in common acceptance for countable numbers of colonies on a plate are 30 – 300 and 25 – 250. The origin of those ranges is worth examination. Breed and Dotterrer published a seminal paper on this topic in 1916 (2). They set out to determine the “limit in the number of colonies that may be allowed to grow on a plate without introducing serious errors…in connection with the proposed revisions of standard methods of milk analysis.” They note that “the kind of bacteria in the material under examination will have an influence on the size of the colonies, and consequently, on the number that can develop on a plate.” They also note that food supply can be an issue, that colonies close to each other on the plate may merge, and that neighbor colonies may inhibit growth or conversely stimulate growth. “Because of these and other difficulties certain plates in any series made from a given sample are more satisfactory for use in computing a total than are others. The matter of selecting plates to be used in computing a count becomes therefore a matter requiring considerable judgment.”
Breed and Dotterrer chose their countable plates from triplicate platings of each dilution, requiring acceptable plates to be within 20% of the average. On this analysis, plates with more than 400 CFU were unsatisfactory, as were those of less than 30 CFU, with best results in the range of 50-200 CFU/plate.
The major paper from Tomasiewicz et al (3) provides an excellent review of the continued evolution of the appropriate number of CFU per plate from milk. They took data from colony counts of raw milk from three different experiments (each dilution plated in triplicate) and used to determine a mean-squared-error of the estimate for all plates. Their recommendation at the end of the study was for a countable range of 25-250 CFU/plate in triplicate. It is interesting to note that although the authors note that CFU follow a Poisson distribution, no mention is made of any data transformation used to approximate a normal distribution prior to the use of normal statistical analytical tools. Tomasiewicz et al provide excellent cautionary advice:
“The data presented herein are not necessarily applicable to other systems. For automated equipment, the optimum range may well vary with the instrument…Furthermore, even if automation is not used appropriate numbers of colonies that should be on a countable plate can very widely, depending on many other variables. With soil fungi for example…”
The compendia have recently harmonized a microbial enumeration test (4), and in this test recommend that the technician “Select the plates corresponding to a given dilution and showing the highest number of colonies less than 250 for TAMC and 50 for TYMC.” In determination of the resistance of biological indicators, USP recommends a range of “20 to 300 colonies, but not less than 6” (5). However, the most complete description of the countable range is found in the informational chapter <1227> (6):
“The accepted range for countable colonies on a standard agar plate is between 25 and 250 for most bacteria and Candida albicans. This range was established in the food industry for counting coliform bacteria in milk. The range is acceptable for compendial organisms, except for fungi. It is not optimal for counting all environmental monitoring isolates. The recommended range for Aspergillus niger is between 8 to 80 cfu per plate. The use of membrane filtration to recover challenge organisms, or the use of environmental isolates as challenge organisms in the antimicrobial effectiveness testing, requires validation of the countable range.”
ASTM provides countable ranges of 20-80 CFU/membrane, 20-200 for spread plates and 30-300 for pour plates (7). The FDA Bacterial Analytical Manual (BAM) recommends 25-250 CFU/plate as a countable range (8).
Upper Limit
The upper limit of plate counts is dependent on a number of factors, as described previously. The major issues include the colony size and behavior (swarming?), and the surface area of the plate. The size particularly comes into play with plating a membrane for determination of CFU as the surface area of that membrane is so much smaller than that of a standard plate.
TNTC can be reported out several ways. ASTM (7) recommends reporting this out as >”upper limit”. For example, a 1:10 dilution with more than 200 CFU on a spread plate would be reported as “>2,000 CFU/mL (or gram). FDA’s BAM recommends counting the colonies from the dilution with plates giving counts closest to 250, counting a portion of the plate, estimating the total number and then using that number as the Estimated Aerobic Count. It is not clear to the author how this is greatly superior to guessing. In my opinion this is an invalid plating and needs to be done correctly at a later date (note I am strenuously avoiding the use of the word retest. This result invalidates the plating and therefore the test was not performed correctly.) I know this is a hardship to the lab, who were trying to reduce the plating load initially by not plating out sufficient dilutions. However, making a mistake initially is not a reasonable excuse to avoid doing it correctly after the mistake is recognized. If the lab wishes to use this “estimated count” it should, at a minimum, have it clearly described in their “counting CFU” SOP with a rationale as to when the plate counts are not critical and can be estimated in this fashion.
There are methods available if you should want to accurately determine the upper limit for a unique plating surface or a unique colony type. One is presented in the USP informational chapter <1227> (5) which is based on a pair-wise comparison of counts from a dilution series. This is based on the assumption that at the upper limit the observed numbers of CFU will fall off the expected numbers at some point (see Figure 1). This divergence will become significant at some point – that defines the upper limit of CFU/plate.
Figure 1. Difference between Expected and Observed CFU with Increasing Numbers
Lower Limit
A central concern in this determination is the reporting of the Limit of Quantification (which is what we are really interested in reporting) against the Limit of Detection (1 CFU). This is an important distinction if we are being held to specifications in the lower range.
ASTM recommendations focus on the LOD, and urge the user to report that answer out if no colonies are recovered (ie <10 CFU/mL for a 1:10 dilution) (7). If countable colonies are present, but below the countable range, count them anyway and report an estimated count.
USP (6) does not have a specific recommendation on how to report out these low numbers, but does note “Lower counting thresholds for the greatest dilution plating in series must be justified.”
FDA BAM (8) recommends a different reporting format. In the FDA BAM method, all counts are recorded in the raw data, but the information is reported out as
Figure 2. Increase in Error with Decreasing Numbers
Unusual Situations
What About Two Dilutions with Countable Colonies?
Ideally you would never see two separate dilutions with counts in the countable range, as the countable ranges cover a ten-fold range of CFU. However, this is microbiology. ASTM recommendations (7) urge you to take both dilutions into account, determining the CFU/mL (or gram) separately for each, then averaging the results for the final result. Breed and Dotterrer (2) also used several dilutions if the numbers fit the QC requirements (see below). FDA BAM has no recommendations in this situation.
While the argument can be made to use all counts, this is a stronger argument if triplicate plates are used and QC limits are in place to discard erroneous plates.
A strong argument can also be made to take the dilution providing the larger number of CFU in the countable range. This approach minimizes two concerns, that the errors in the estimates increase with increasing serial dilutions, and that the error in the estimate increases with decreasing plate counts. Use of the smaller dilution (eg 1:10 vs 1:100) could be justified from this perspective.
Whichever method used should be documented and justified in the “Counting CFU” SOP.
What about QC Limits on Replicate Plate Counts?
Periodically there are recommendations to establish Quality Control limits on replicate plate counts. Breed and Dotterrer in their 1916 paper (2) required valid plate counts from triplicate plates to provide estimates of CFU/mL within 20% of the mean. In other words, all plates were counted, each plate’s CFU count was used to estimate the original CFU/mL, then each estimate was evaluated. If the individual plate’s estimate was within 20% of the mean, it was deemed acceptable. This method is not practical in the QC lab.
Establishment of QC limits for plate counts works best if you have at least three replicate plates for each dilution. The average of the dilution replicates can be determined, variant counts (hopefully no more than one plate per triplicate plating) discarded and the final average determined. If you try this with duplicate plates you frequently end up with trying to average the results of one plate. While this method looks good on paper, the prudent lab manager will evaluate some historical data before instituting it as a practice.
The method used to QC individual plate counts, if used, should be documented and justified in SOP, along with the response to finding variant counts.
Can I plate 10 1 mL samples to plate a Total of One 10 mL Sample?
There have been suggestions that a larger volume of material may be plated across several plates, and the results reported out for the larger volume. For example, plating 10 1 mL samples on 10 different plates, and then reporting it as if a 10 mL sample was plated. This approach is flawed in that it ignores several sources of variability in plating including sampling error, plating errors, growth/incubation error and counting errors (9, 10). The correct interpretation for this situation that you have just plated 1 mL ten times, not 10 mL once. The numbers might be averaged, they cannot be added.
Rounding and Averaging
To discuss this we need to determine what the significant figures might be in the measure. For raw colony counts, common practice determines that the CFU observed determine the significant figure, and that the average is one decimal to the right of that number (sticklers for accuracy will report the geometric mean rather than the arithmetic mean given the Poisson distribution followed by CFU). In reporting, it is common practice to report out as scientific notation using two significant figures. This requires rounding.
USP (11) and ASTM (7) both round up at five if 5 is the number to the right of the last significant figure. FDA BAM has a more elaborate scheme, rounding up if the number is 6 or higher, down if 4 or lower. If the number is 5, BAM looks to the next number to the right and rounds up if it is odd, down if it is even.
This is one of those situations where you want every-one to do the calculations the same way (I am hard pressed to come up with a situation in a lab where you want everyone to do it differently). Be sure to include direction and its justification in the “Counting CFU” SOP if it does not already exist in a separate SOP.
Impact on Specifications and Environmental Monitoring Control Levels
We are back to the question of WHO CARES?
If you are faced with a finished product bioburden of NMT (Not More Than) 100 CFU/gram, and your method suitability study requires a 1:100 dilution of the product to overcome any antimicrobial effects, then how are you to test it? Common practice is to perform the 1:100 dilution, perform a pour plate of 1 mL in duplicate and if 2 colonies grow on each plate, the product fails specification. This common practice is scientifically unsupportable – it confused the Limit of Detection with the Limit of Quantification for the plate count method.
Let’s take a look at environmental monitoring alert and action levels for aseptically produced products. Hussong and Madsen (12) recently published a thoughtful review of this topic where they argue that the levels of acceptable CFU for many room classifications are below the noise level of plate count technology (eg in the range of 1-2 CFU/m3). In addition, environmental data can be extremely variable, much more so than controlled lab studies as the numbers of microorganisms, the physiological state of the isolates, even the species are completely out of the control of the investigator. In addition the numbers do not conform to a normal distribution as there are sporadic counts with a count of “zero” CFU predominating. They conclude that since the numbers are unreliable, the trend in the data is the only important consideration, and that EM counts cannot be used for product release criteria. A separate treatment of this subject was presented by Farrington (13) who argues that the relationship between EM data and finished product quality is a widely held, but unproven belief, compounded by the problems in accuracy with the low counts generated by plate count methodology.
Conclusions
In conclusion, all methods have limitations. One of the major limitations to the plate count method is the relatively narrow countable range (generally considered to be 25-250 CFU bacteria on a standard petri dish). The currently prevailing confusion between the Limit of Detection (1 CFU) and Limit of Quantification (25 CFU) for the plate count method creates a larger degree of variability in microbiology data than is necessary. An unfortunate regulatory trend in recent years is to establish expectations (specifications, limits, levels) for data generated by the plate count method that the accuracy of the method cannot support. This is a real opportunity for modification of current practice to approach the goal of “science-based regulations”.
How to Determine if an Organism is “Objectionable
since the microbial limits tests do not address themselves to “objectionable” microorganisms, how is the manufacturer to determine if there are “objectionables” in a lot of product awaiting release? One approach is suggested by FDA - once all organisms grown in the total count studies (total aerobic as well as total yeast and mold) are identified, a qualified microbiologist would conduct a risk analysis on the presence of that organism in that medication (4). This risk analysis should incorporate a minimum of four separate analyses: Absolute numbers of organisms seen Microorganism’s Characteristics Product Characteristics Potential Impact on Patients
Absolute Number of Organisms Seen
Although high numbers of non-pathogenic organisms may not pose a health hazard, they may affect product efficacy and/or physical /chemical stability. An unusually high number of organisms seen in the product may also indicate a problem during the manufacturing process, or an issue with a raw material. The high bacterial counts may indicate that the microorganisms are thriving in the product. If a preserved product this could indicate that the product’s preservative system is not functioning or worse, the preservative was missing or incorrectly formulated.
The Characteristics of the Microorganism
The characteristics of the microorganism can be determined by a search of textbooks, or library work, by internet searches, or a combination of all of these. It is always a good idea to remember that you are interested in the microbiology of the situation – do not restrict the search to pharmaceutical sources as most of the best information will come from food, environmental, clinical and perhaps cosmetic microbiology sources in addition to the pharmaceutical field.
During this search look for synonyms of the organisms current name. With the widespread use of genetic techniques in taxonomy the names of some organisms have undergone multiple changes. The national culture collections are a good source of synonyms and all name variants should be researched.
First of all, determine if the organism is a known pathogen. A good place to start on this search is the FDA web site the “Bad Bug Book.” This is only a guide, but a good one (5). One approach is to do a preliminary evaluation for any organism that appears on the FDA/CFSAN list and immediately classify that organism as “objectionable.” However, it is also important to consider the route of administration and the susceptible population in this evaluation.
A second characteristic of the microorganism that must be taken into account is the potential for the organism to cause spoilage of the product. Make a list of substances used by the microorganism for growth. This can be from the literature, or from the identification equipment. For example, the Vitek 2 Compact will provide an extensive list of compounds the microorganism can metabolize, the Biolog a list of carbohydrates utilized, etc. Use the information gained during the identification of the organism. Compare these to the product formulation for potential issues. A microorganism is also objectionable if it has the potential to degrade the product on stability. Evaluate the microorganism’s tolerance to unusual conditions: low or high pH high salt concentration high sugar concentration (osmotic conditions) Low water activity Growth temperature, etc. It can also be useful to determine if the microorganism has a recognized proclivity for harboring plasmid-mediated antibiotic resistance. This is a special concern in regards to horizontal transmission of the trait within an vulnerable patient population.
Product Characteristics
The dosage form is important to consider. Is the product anhydrous or water based? This can have an effect on the ability of microorganisms to proliferate. Does it have sufficient free water to support microbial growth (6, 7). Is the container designed to minimize contamination and subsequent spoilage? Closure design can have a major effect on in-use stability of a product. Is the container adequately designed to retard access to the environment, and to prevent contamination from the environment. Give special consideration to the likelihood of an anhydrous medication’s exposure to water, providing the potential for microbial proliferation. The route of administration is also important. A medication orally administered can tolerate some microorganisms that would be disastrous in a medication meant to be applied topically to abraded skin or to rashes. Similarly, some microorganisms that could be tolerated in a topical would cause severe distress to a patient if taken orally. Inhalants, although not required to be sterile, are a particularly sensitive area and great care should be taken in classifying any contaminate as “non-objectionable.” Other product-related considerations should include a review of the production records and the environmental monitoring trends, A review of field complaints is also useful (is this contaminant one that causes eventual returns?).
Patient Population
Finally, a consideration of the targeted patient population is in order. The manufacturer cannot control, and should be held accountable, for patient abuse of a product or off-label use of the product by physicians. However, reasonable use of the product should be considered and part of the risk analysis. Are patient populations that are likely to use this product at increased risk if exposed to the particular microorganism?
Summary
The FDA’s concern with non-sterile dosage format is that the product not contain “objectionable” organisms. This FDA concern has been made clear since the 1970’s. However, many companies continue to mistakenly believe that if their non-sterile product meets the requirements in USP, it will be safe from FDA dispute. This not the case. The manufacturer is responsible for all contents of his drug product. Should question arise over the appropriateness of a particular organism, the manufacturer is expected to have a justification for the presence of that organism, preferably as part of the batch release document. Presented here is a brief description of some factors to consider in determining if an organism is objectionable. These considerations include: Absolute number of organisms present Microorganism characteristics Product characteristics Patient Population These are not meant to be a comprehensive listing of all issues, but rather a starting point for the non-sterile manufacturer to use in establishing their program to qualify finished product bioburden
Absolute Number of Organisms Seen
Although high numbers of non-pathogenic organisms may not pose a health hazard, they may affect product efficacy and/or physical /chemical stability. An unusually high number of organisms seen in the product may also indicate a problem during the manufacturing process, or an issue with a raw material. The high bacterial counts may indicate that the microorganisms are thriving in the product. If a preserved product this could indicate that the product’s preservative system is not functioning or worse, the preservative was missing or incorrectly formulated.
The Characteristics of the Microorganism
The characteristics of the microorganism can be determined by a search of textbooks, or library work, by internet searches, or a combination of all of these. It is always a good idea to remember that you are interested in the microbiology of the situation – do not restrict the search to pharmaceutical sources as most of the best information will come from food, environmental, clinical and perhaps cosmetic microbiology sources in addition to the pharmaceutical field.
During this search look for synonyms of the organisms current name. With the widespread use of genetic techniques in taxonomy the names of some organisms have undergone multiple changes. The national culture collections are a good source of synonyms and all name variants should be researched.
First of all, determine if the organism is a known pathogen. A good place to start on this search is the FDA web site the “Bad Bug Book.” This is only a guide, but a good one (5). One approach is to do a preliminary evaluation for any organism that appears on the FDA/CFSAN list and immediately classify that organism as “objectionable.” However, it is also important to consider the route of administration and the susceptible population in this evaluation.
A second characteristic of the microorganism that must be taken into account is the potential for the organism to cause spoilage of the product. Make a list of substances used by the microorganism for growth. This can be from the literature, or from the identification equipment. For example, the Vitek 2 Compact will provide an extensive list of compounds the microorganism can metabolize, the Biolog a list of carbohydrates utilized, etc. Use the information gained during the identification of the organism. Compare these to the product formulation for potential issues. A microorganism is also objectionable if it has the potential to degrade the product on stability. Evaluate the microorganism’s tolerance to unusual conditions: low or high pH high salt concentration high sugar concentration (osmotic conditions) Low water activity Growth temperature, etc. It can also be useful to determine if the microorganism has a recognized proclivity for harboring plasmid-mediated antibiotic resistance. This is a special concern in regards to horizontal transmission of the trait within an vulnerable patient population.
Product Characteristics
The dosage form is important to consider. Is the product anhydrous or water based? This can have an effect on the ability of microorganisms to proliferate. Does it have sufficient free water to support microbial growth (6, 7). Is the container designed to minimize contamination and subsequent spoilage? Closure design can have a major effect on in-use stability of a product. Is the container adequately designed to retard access to the environment, and to prevent contamination from the environment. Give special consideration to the likelihood of an anhydrous medication’s exposure to water, providing the potential for microbial proliferation. The route of administration is also important. A medication orally administered can tolerate some microorganisms that would be disastrous in a medication meant to be applied topically to abraded skin or to rashes. Similarly, some microorganisms that could be tolerated in a topical would cause severe distress to a patient if taken orally. Inhalants, although not required to be sterile, are a particularly sensitive area and great care should be taken in classifying any contaminate as “non-objectionable.” Other product-related considerations should include a review of the production records and the environmental monitoring trends, A review of field complaints is also useful (is this contaminant one that causes eventual returns?).
Patient Population
Finally, a consideration of the targeted patient population is in order. The manufacturer cannot control, and should be held accountable, for patient abuse of a product or off-label use of the product by physicians. However, reasonable use of the product should be considered and part of the risk analysis. Are patient populations that are likely to use this product at increased risk if exposed to the particular microorganism?
Summary
The FDA’s concern with non-sterile dosage format is that the product not contain “objectionable” organisms. This FDA concern has been made clear since the 1970’s. However, many companies continue to mistakenly believe that if their non-sterile product meets the requirements in USP, it will be safe from FDA dispute. This not the case. The manufacturer is responsible for all contents of his drug product. Should question arise over the appropriateness of a particular organism, the manufacturer is expected to have a justification for the presence of that organism, preferably as part of the batch release document. Presented here is a brief description of some factors to consider in determining if an organism is objectionable. These considerations include: Absolute number of organisms present Microorganism characteristics Product characteristics Patient Population These are not meant to be a comprehensive listing of all issues, but rather a starting point for the non-sterile manufacturer to use in establishing their program to qualify finished product bioburden
Measurement of Cell Concentration in Suspension by Optical Density
A common issue for the microbiology lab is the determination of starting inoculum concentration. If the inoculum concentration is determined by plating, the inoculum is several days old before use. This essay describes the use of turbidity to estimate microbial concentration in a suspension, using the Antimicrobial Efficacy Test as the example.
Determination of Inoculum for the AET
The compendial antimicrobial efficacy test (AET) requires inoculation of the product with microorganisms to a final concentration of approximately 106 CFU/mL. Although this seems to be a minor point, it does serve to illustrate some of the inherent difficulties in microbiological testing and the need for experienced and academically trained microbiologists to head the laboratory.
Let’s look at the compendial guidance. The Pharm Eur (1) instruction on preparing the inoculum for the AET states:
“To harvest the … cultures, use a sterile suspending fluid … Add sufficient suspending fluid to reduce the microbial count to about 108 micro-organisms per milliliter…Remove immediately a suitable sample from each suspension and determine the number of colony-forming units per milliliter in each suspension by plate count or membrane filtration (2.6.12). This value serves to determine the inoculum and the baseline to use in the test. The suspensions shall be used immediately.”
There are, of course, two problems with these instructions. The first is that the technician is instructed to use an inoculum of about 108 microorganisms per milliliter and then instructed to determine this by plate count. Colony forming units (CFU) and cells are two different measures and this will inevitably lead to difficulties as the unfortunate lab worker cannot guarantee the number of cells in the suspension, only the number of CFU found. However, we can accept the scientific inaccuracy as the numbers will generally work out. The more serious problem is the instruction to use the plate count CFU for determination of the inoculum for the test, and that the suspension shall be used immediately. This quite frankly cannot be done. If you use the suspension immediately, the plate counts are unavailable, if you use the plate counts to set the inoculum, then the suspension is at least a day old.
Contrast these instructions with those in the USP (2) for the same exercise:
“To harvest the … cultures, use sterile saline … Add sufficient … to obtain a microbial count of about 1 x 108 cfu per mL…[Note: The estimate of inoculum concentration may be performed by turbidimetric measurements for the challenge organisms. Refrigerate the suspension if it is not used within 2 hours].
Determine the number of cfu per mL in each suspension …to confirm the initial cfu per mL estimate. This value serves to calibrate the size of the inoculum used in the test.”
These USP instructions have the advantage of being physically possible to perform, an obvious advantage to the lab worker. However, the turbidometric measure of the cells is also only an approximation of CFU. Thus the instruction to confirm the numbers (after the test is underway) with the plate count is an important control on the test.
This article will explore the turbidometric approximation for cell numbers, and important controls on the process as well as potential pitfalls to the method.
Theory
Light scattering techniques to monitor the concentration of pure cultures have the enormous advantages of being rapid and nondestructive. However, they do not measure cell numbers nor do they measure CFU. Light scattering is most closely related to the dry weight of the cells (3).
Light is passed through the suspension of microorganisms, and all light that is not absorbed is re-radiated. There is a significant amount of physics involved in this, and those interested are referred to optical treatises, particularly those discussing Huygens’ Principle (a good choice is Light Scattering by Small Particles by H C Van De Hulst). For our purposes it is enough to say that light passing through a suspension of microorganisms is scattered, and the amount of scatter is an indication of the biomass present in the suspension. In visible light, this appears “milky” or “cloudy” to the eye (3). It follows from this that if the concentration of scattering particles becomes high, then multiple scattering events become possible.
Methods
McFarland Turbidity Standards
McFarland standards can be used to visually approximate the concentration of cells in a suspension. The McFarland Scale represents specific concentrations of CFU/mL and is designed to be used for estimating concentrations of gram negative bacteria such as E. coli. Note that this estimate becomes uncertain with organisms outside the normal usage as different species of bacteria differ in size and mass, as do yeast and mold. Use of this method would require calibration and validation.
McFarland Standards are generally labeled 0.5 through 10 and filled with suspensions of Barium salts. (Note - latex bead suspensions are now also available which extend the shelf life of the material). The standards may be made in the lab by preparing a 1% solution of anhydrous BaCl2 and a 1% solution of H2SO4 – mix them in the proportions listed in the table. They should be stored in the dark, in a tightly sealed container at 20-25oC and should be stable for approximately 6 months (4).
The advantage of the use of these standards is that no incubation time or equipment is needed to estimate bacterial numbers. The disadvantage is that there is some subjectivity involved in interpreting the turbidity, and that the numbers are valid only for those microorganisms similar to E. coli. In addition, the values are not in the appropriate range for the AET inoculum and so further dilutions may be required.
Approximate E. coli concentrations on McFarland Scale
McFarland Scale
CFU (x106/mL)
1% BaCl2/ 1% H2SO4 (mL)
0.5
<300
0.05/9.95
1
300
0.1/9.9
2
600
0.2/9.8
3
900
0.3/9.7
4
1200
0.4/9.6
5
1500
0.5/9.5
6
1800
0.6/9.4
7
2100
0.7/9.3
8
2400
0.8/9.2
9
2700
0.9/9.1
10
3000
1.0/9.0
Spectrophotometer
The spectrophotometer method measures turbidity directly. The best case (i.e. most sensitive) would be to have a narrow slit and a small detector so that only the light scattered in the forward direction would be seen by the detector. This instrument would give larger apparent absorption readings than other instruments.
As should be obvious, each spectrophotometer used must be independently calibrated for use in estimating microbial concentrations. Not only is the apparent absorption affected by the width of the instrument’s slit, the condition of the filter, and the size and condition of the detector, but also each time the lamp is changed the calibration needs to be repeated as different bulbs may vary in total output.
The correlation of absorption to dry weight is very good for dilute suspensions of bacteria (5), and this relationship seems to hold regardless of cell size (although the relationship of absorption to CFU does not). However, in more concentrated suspensions this correlation (absorption to dry weight) no longer holds. The linear range of absorption to estimated CFU is of limited scope and for this reason the calibration study must demonstrate the linear range of the absorbance vs CFU values and the relevant values.
Procedure
As there are a variety of different instruments, there cannot be one single procedure. In general, the spectrophotometer can be set at a wavelength of 420 – 660 nm. This wavelength must be standardized and may need to be adjusted specifically to the material being tested. Different vegetative cells, bacterial spores and spores of Aspergillus niger may not have the same maximal absorbance wavelength.
It is important to have the cells in known physiological state of growth. That is to say, as the cell size varies with phase of growth (lag, log, stationery) the approximate relationship between absorbance and CFU will also vary. A recommended practice might be to pass a single well-isolated colony twice on overnight cultures surface streaks from the refrigerated stock, harvesting the rapidly growing culture from the second passage for preparation of vegetative cells. This also will serve to minimize a source of variability for the AET (6).
A second source of concern might be the cuvette used for the measurement – care must be taken to maintain the correct orientation of the cuvette, and to protect it from damage that could affect the passage of light. Finally, it is necessary to blank the spectrophotometer (adjust the absorbance reading to zero) using a standard, either water or the suspending fluid, and maintain this practice.
Calibration
It must be stressed that this calibration should be done for all organisms. The size of the organism, any associated pigments, the preparation of the suspension, and other factors all influence the readings. This calibration study should also be rechecked after changing the bulb on the light source, and should be reevaluated throughout the life of the light bulb.
The calibration itself is simple to perform. Prepare a concentrated solution of the organism, grown under the conditions that will be used for the test. Make a series of dilutions to cover the range of absorption measurements of interest; 5 to 8 dilutions are recommended. Immediately take the spectrophotometer readings in sequence, and then take a confirmatory reading of the first in series to confirm that no growth has occurred. The dilutions are then immediately plated for viable count (serial dilution of the suspensions will be necessary). Graph the relationship between the absorbance and the CFU/mL after the plate counts are available and use values in the linear range of this graph.
As there are several factors that can affect this curve (quality of lamp output, size of slit, condition of filter, condition of detector, microorganism characteristic, etc) this calibration should be confirmed when the conditions of the assay change.
Conclusions
The use of optical density to estimate CFU in a suspension is possible, if basic precautions are taken. It is important to control:
The physiological state of the organism
The species of the organisms (i.e. don’t calibrate the instrument using E. coli and expect the numbers to work for Candida albicans)
The nature and condition of the equipment
Despite the inherent inaccuracy of the method, if the procedure is adequately controlled and calibrated the estimation of microbial numbers by optical density (either by McFarland Standards or spectrophotometrically) is sufficiently accurate for use in preparing inocula for QC testing and offers the overwhelming advantages of being rapid, low cost and non-destructive
Determination of Inoculum for the AET
The compendial antimicrobial efficacy test (AET) requires inoculation of the product with microorganisms to a final concentration of approximately 106 CFU/mL. Although this seems to be a minor point, it does serve to illustrate some of the inherent difficulties in microbiological testing and the need for experienced and academically trained microbiologists to head the laboratory.
Let’s look at the compendial guidance. The Pharm Eur (1) instruction on preparing the inoculum for the AET states:
“To harvest the … cultures, use a sterile suspending fluid … Add sufficient suspending fluid to reduce the microbial count to about 108 micro-organisms per milliliter…Remove immediately a suitable sample from each suspension and determine the number of colony-forming units per milliliter in each suspension by plate count or membrane filtration (2.6.12). This value serves to determine the inoculum and the baseline to use in the test. The suspensions shall be used immediately.”
There are, of course, two problems with these instructions. The first is that the technician is instructed to use an inoculum of about 108 microorganisms per milliliter and then instructed to determine this by plate count. Colony forming units (CFU) and cells are two different measures and this will inevitably lead to difficulties as the unfortunate lab worker cannot guarantee the number of cells in the suspension, only the number of CFU found. However, we can accept the scientific inaccuracy as the numbers will generally work out. The more serious problem is the instruction to use the plate count CFU for determination of the inoculum for the test, and that the suspension shall be used immediately. This quite frankly cannot be done. If you use the suspension immediately, the plate counts are unavailable, if you use the plate counts to set the inoculum, then the suspension is at least a day old.
Contrast these instructions with those in the USP (2) for the same exercise:
“To harvest the … cultures, use sterile saline … Add sufficient … to obtain a microbial count of about 1 x 108 cfu per mL…[Note: The estimate of inoculum concentration may be performed by turbidimetric measurements for the challenge organisms. Refrigerate the suspension if it is not used within 2 hours].
Determine the number of cfu per mL in each suspension …to confirm the initial cfu per mL estimate. This value serves to calibrate the size of the inoculum used in the test.”
These USP instructions have the advantage of being physically possible to perform, an obvious advantage to the lab worker. However, the turbidometric measure of the cells is also only an approximation of CFU. Thus the instruction to confirm the numbers (after the test is underway) with the plate count is an important control on the test.
This article will explore the turbidometric approximation for cell numbers, and important controls on the process as well as potential pitfalls to the method.
Theory
Light scattering techniques to monitor the concentration of pure cultures have the enormous advantages of being rapid and nondestructive. However, they do not measure cell numbers nor do they measure CFU. Light scattering is most closely related to the dry weight of the cells (3).
Light is passed through the suspension of microorganisms, and all light that is not absorbed is re-radiated. There is a significant amount of physics involved in this, and those interested are referred to optical treatises, particularly those discussing Huygens’ Principle (a good choice is Light Scattering by Small Particles by H C Van De Hulst). For our purposes it is enough to say that light passing through a suspension of microorganisms is scattered, and the amount of scatter is an indication of the biomass present in the suspension. In visible light, this appears “milky” or “cloudy” to the eye (3). It follows from this that if the concentration of scattering particles becomes high, then multiple scattering events become possible.
Methods
McFarland Turbidity Standards
McFarland standards can be used to visually approximate the concentration of cells in a suspension. The McFarland Scale represents specific concentrations of CFU/mL and is designed to be used for estimating concentrations of gram negative bacteria such as E. coli. Note that this estimate becomes uncertain with organisms outside the normal usage as different species of bacteria differ in size and mass, as do yeast and mold. Use of this method would require calibration and validation.
McFarland Standards are generally labeled 0.5 through 10 and filled with suspensions of Barium salts. (Note - latex bead suspensions are now also available which extend the shelf life of the material). The standards may be made in the lab by preparing a 1% solution of anhydrous BaCl2 and a 1% solution of H2SO4 – mix them in the proportions listed in the table. They should be stored in the dark, in a tightly sealed container at 20-25oC and should be stable for approximately 6 months (4).
The advantage of the use of these standards is that no incubation time or equipment is needed to estimate bacterial numbers. The disadvantage is that there is some subjectivity involved in interpreting the turbidity, and that the numbers are valid only for those microorganisms similar to E. coli. In addition, the values are not in the appropriate range for the AET inoculum and so further dilutions may be required.
Approximate E. coli concentrations on McFarland Scale
McFarland Scale
CFU (x106/mL)
1% BaCl2/ 1% H2SO4 (mL)
0.5
<300
0.05/9.95
1
300
0.1/9.9
2
600
0.2/9.8
3
900
0.3/9.7
4
1200
0.4/9.6
5
1500
0.5/9.5
6
1800
0.6/9.4
7
2100
0.7/9.3
8
2400
0.8/9.2
9
2700
0.9/9.1
10
3000
1.0/9.0
Spectrophotometer
The spectrophotometer method measures turbidity directly. The best case (i.e. most sensitive) would be to have a narrow slit and a small detector so that only the light scattered in the forward direction would be seen by the detector. This instrument would give larger apparent absorption readings than other instruments.
As should be obvious, each spectrophotometer used must be independently calibrated for use in estimating microbial concentrations. Not only is the apparent absorption affected by the width of the instrument’s slit, the condition of the filter, and the size and condition of the detector, but also each time the lamp is changed the calibration needs to be repeated as different bulbs may vary in total output.
The correlation of absorption to dry weight is very good for dilute suspensions of bacteria (5), and this relationship seems to hold regardless of cell size (although the relationship of absorption to CFU does not). However, in more concentrated suspensions this correlation (absorption to dry weight) no longer holds. The linear range of absorption to estimated CFU is of limited scope and for this reason the calibration study must demonstrate the linear range of the absorbance vs CFU values and the relevant values.
Procedure
As there are a variety of different instruments, there cannot be one single procedure. In general, the spectrophotometer can be set at a wavelength of 420 – 660 nm. This wavelength must be standardized and may need to be adjusted specifically to the material being tested. Different vegetative cells, bacterial spores and spores of Aspergillus niger may not have the same maximal absorbance wavelength.
It is important to have the cells in known physiological state of growth. That is to say, as the cell size varies with phase of growth (lag, log, stationery) the approximate relationship between absorbance and CFU will also vary. A recommended practice might be to pass a single well-isolated colony twice on overnight cultures surface streaks from the refrigerated stock, harvesting the rapidly growing culture from the second passage for preparation of vegetative cells. This also will serve to minimize a source of variability for the AET (6).
A second source of concern might be the cuvette used for the measurement – care must be taken to maintain the correct orientation of the cuvette, and to protect it from damage that could affect the passage of light. Finally, it is necessary to blank the spectrophotometer (adjust the absorbance reading to zero) using a standard, either water or the suspending fluid, and maintain this practice.
Calibration
It must be stressed that this calibration should be done for all organisms. The size of the organism, any associated pigments, the preparation of the suspension, and other factors all influence the readings. This calibration study should also be rechecked after changing the bulb on the light source, and should be reevaluated throughout the life of the light bulb.
The calibration itself is simple to perform. Prepare a concentrated solution of the organism, grown under the conditions that will be used for the test. Make a series of dilutions to cover the range of absorption measurements of interest; 5 to 8 dilutions are recommended. Immediately take the spectrophotometer readings in sequence, and then take a confirmatory reading of the first in series to confirm that no growth has occurred. The dilutions are then immediately plated for viable count (serial dilution of the suspensions will be necessary). Graph the relationship between the absorbance and the CFU/mL after the plate counts are available and use values in the linear range of this graph.
As there are several factors that can affect this curve (quality of lamp output, size of slit, condition of filter, condition of detector, microorganism characteristic, etc) this calibration should be confirmed when the conditions of the assay change.
Conclusions
The use of optical density to estimate CFU in a suspension is possible, if basic precautions are taken. It is important to control:
The physiological state of the organism
The species of the organisms (i.e. don’t calibrate the instrument using E. coli and expect the numbers to work for Candida albicans)
The nature and condition of the equipment
Despite the inherent inaccuracy of the method, if the procedure is adequately controlled and calibrated the estimation of microbial numbers by optical density (either by McFarland Standards or spectrophotometrically) is sufficiently accurate for use in preparing inocula for QC testing and offers the overwhelming advantages of being rapid, low cost and non-destructive
The Gram Stain
Gram staining is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. The method is named after its inventor, the Danish scientist Hans Christian Gram (1853-1938), who developed the technique in 1884 (Gram 1884). The importance of this determination to correct identification of bacteria cannot be overstated as all phenotypic methods begin with this assay.
The Basic Method
1. First, a loopful of a pure culture is smeared on a slide and allowed to air dry. The culture can come from a thick suspension of a liquid culture or a pure colony from a plate suspended in water on the microscope slide. Important considerations:
· Take a small inoculum – don’t make a thick smear that cannot be completely decolorized. This could make gram-negative organisms appear to be gram-positive or gram-variable.
· Take a fresh culture – old cultures stain erratically.
2. Fix the cells to the slide by heat or by exposure to methanol. Heat fix the slide by passing it (cell side up) through a flame to warm the glass. Do not let the glass become hot to the touch.
3. Crystal violet (a basic dye) is then added by covering the heat-fixed cells with a prepared solution. Allow to stain for approximately 1 minute.
4. Briefly rinse the slide with water. The heat-fixed cells should look purple at this stage.
5. Add iodine (Gram's iodine) solution (1% iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye, making it more difficult to decolorize and reducing some of the variability of the test.
6. Briefly rinse with water.
7. Decolorize the sample by applying 95% ethanol or a mixture of acetone and alcohol. This can be done in a steady stream, or a series of washes. The important aspect is to ensure that all the color has come out that will do so easily. This step washes away unbound crystal violet, leaving Gram-positive organisms stained purple with Gram-negative organisms colorless. The decolorization of the cells is the most “operator-dependent” step of the process and the one that is most likely to be performed incorrectly.
8. Rinse with water to stop decolorization.
9. Rinse the slide with a counterstain (safranin or carbol fuchsin) which stains all cells red. The counterstain stains both gram-negative and gram-positive cells. However, the purple gram-positive color is not altered by the presence of the counter-stain, it’s effect is only seen in the previously colorless gram-negative cells which now appear pink/red.
10. Blot gently and allow the slide to dry. Do not smear.
What’s Going On?
Bacteria have a cell wall made up of peptidoglycan. This cell wall provides rigidity to the cell, and protection from osmotic lysis in dilute solutions. Gram-positive bacteria have a thick mesh-like cell wall, gram-negative bacteria have a thin cell wall and an outer phospholipid bilayer membrane. The crystal violet stain is small enough to penetrate through the matrix of the cell wall of both types of cells, but the iodine-dye complex exits only with difficulty (Davies et al. 1983)
The decolorizing mixture dehydrates cell wall, and serves as a solvent to rinse out the dye-iodine complex. In Gram-negative bacteria it also dissolves the outer membrane of the gram-negative cell wall aiding in the release of the dye. It is the thickness of the cell wall that characterizes the response of the cells to the staining procedure. In addition to the clearly gram-positive and gram-negative, there are many species that are “gram-variable” with intermediate cell wall structure (Beveridge and Graham 1991). As noted above, the decolorization step is critical to the success of the procedure.
Gram’s method involves staining the sample cells dark blue, decolorizing those cells with a thin cell wall by rinsing the sample, then counterstaining with a red dye. The cells with a thick cell wall appear blue (gram positive) as crystal violet is retained within the cells, and so the red dye cannot be seen. Those cells with a thin cell wall, and therefore decolorized, appear red (gram negative).
It is a prudent practice to always include a positive and negative control on the staining procedure to confirm the accuracy of the results (Murray et al 1994) and to perform proficiency testing on the ability of the technicians to correctly interpret the stains (Andserson, et al. 2005).
Excessive Decolorization
It is clear that the decolorization step is the one most likely to cause problems in the gram stain. The particular concerns in this step are listed below (reviewed in McClelland 2001)
Excessive heat during fixationHeat fixing the cells, when done to excess, alters the cell morphology and makes the cells more easily decolorized.
Low concentration of crystal violetConcentrations of crystal violet up to 2% can be used successfully, however low concentrations result in stained cells that are easily decolorized. The standard 0.3% solution is good, if decolorization does not generally exceed 10 seconds.
Excessive washing between stepsThe crystal violet stain is susceptible to wash-out with water (but not the crystal violet-iodine complex). Do not use more than a 5 second water rinse at any stage of the procedure.
Insufficient iodine exposureThe amount of the mordant available is important to the formation of the crystal violet - iodine complex. The lower the concentration, the easier to decolorize (0.33% - 1% commonly used). Also, QC of the reagent is important as exposure to air and elevated temperatures hasten the loss of Gram’s iodine from solution. A closed bottle (0.33% starting concentration) at room temperature will lose >50% of available iodine in 30 days, an open bottle >90%. Loss of 60% iodine results in erratic results.
Prolonged decolorization95% ethanol decolorizes more slowly, and may be recommended for inexperienced technicians while experienced workers can use the acetone-alcohol mix. Skill is needed to gauge when decolorization is complete.
Excessive counterstainingAs the counterstain is also a basic dye, it is possible to replace the crystal violet—iodine complex in gram- positive cells with an over-exposure to the counterstain. The counterstain should not be left on the slide for more than 30 seconds.
Alternatives to the Gram Stain
Gram’s staining method is plainly not without its problems. It is messy, complicated, and prone to operator error. The method also requires a large number of cells (although a membrane-filtration technique has been reported; Romero, et al 1988). However, it is also central to phenotypic microbial identification techniques.
This method, and it’s liabilities, are of immediate interest to those involved in environmental monitoring programs as one of the most common isolates in an EM program, Bacillus spp., will frequently stain gram variable or gram negative despite being a gram-positive rod (this is especially true with older cultures). The problems with Gram’s method have lead to a search for other tests that correlate with the cell wall structure of the gram-positive and the gram-negative cells. Several improvements/alternatives to the classical gram stain have appeared in the literature.
KOH String Test
The KOH String Test is done using a drop of 3% potassium hydroxide on a glass slide. A visible loopful of cells from a single, well-isolated colony is mixed into the drop. If the mixture becomes viscous within 60 seconds of mixing (KOH-positive) then the colony is considered gram-negative. The reaction depends on the lysis of the gram-negative cell in the dilute alkali solution releasing cellular DNA to turn the suspension viscous. This method has been shown effective for food microorganisms (Powers 1995), and for Bacillus spp (Carlone et al 1983, Gregersen 1978), although it may be problematic for some anaerobes (Carlone et al 1983, but also see Halebian et al 1981).
This test has the advantage of simplicity, and it can be performed on older cultures. False negative results can occur in the test by using too little inoculum or too much KOH (DNA-induced viscosity not noticeable). False positive results can occur from too heavy an inoculum (the solution will appear to gel, but not string), or inoculation with mucoid colonies. This can serve as a valuable adjunct to the tradition gram stain method (von Graevenitz and Bucher 1983).
Aminopeptidase Test
L-alanine aminopeptidase is an enzyme localized in the bacterial cell wall which cleaves the amino acid L-alanine from various peptides. Significant activity is found almost only in Gram-negative microorganisms, all Gram-positive or Gram-variable microorganisms so far studied display no or very weak activity (Cerny 1976, Carlone et al. 1983). To perform the test, the reagent is used to make a suspension (with the bacteria). Aminopeptidase activity of the bacteria causes the release of 4-nitroaniline from the reagent, turning the suspension yellow. The test is especially useful for non-fermenters and gram-variable organisms, and is a one step test with several suppliers of kits. Results of the test are available in 5 minutes.
Fluorescent Stains
A popular combination of fluorescent stains for use in gram staining (particularly for flow-cytometry) involves the use of the fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13. HI penetrates gram-positive but not gram-negative organisms, but SYTO 13 penetrates both. When the dyes were used together in a single step, gram-negative organisms are green fluorescent by SYTO 13 while gram-positive organisms are red-orange fluorescent by HI which overpowers the green of SYTO 13 (Mason et al 1998). There are commercial kits available for this procedure, which requires a fluorescent microscope or a flow cytometer.
Sizemore et al (1990) developed a different approach to fluorescent labeling of cells. Fluorescence-labeled wheat germ agglutinin binds specifically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin. A variant of this method has also been used to “gram stain” microorganisms in milk for direct measurement by flow cytometry.
LAL-based Assay
Charles River Laboratories has just released a product to be used with their PTS instrument – the PTS Gram ID (Farmer 2005). This methodology makes use of the same reaction used for the chromogenic LAL test. Gram-negative organisms, with bacterial endotoxin, initiate the LAL coagulase cascade which results in activation of the proclotting enzyme, a protease. In the LAL test, this enzyme cleaves a peptide from the horseshoe crab coagulen, resulting in a clot. It can also cleave a peptide from a synthetic substrate, yielding a chromophore (p-nitroaniline) which is yellow and can be measured photometrically at 385 nm (Iwanaga 1987). Gram-positive organisms, lacking endotoxin, do not trigger the color change in this method, while gram-negative organisms do trigger it. Results are available within 10 minutes.
Summary
The differentiation of bacteria into either the gram-positive or the gram-negative group is fundamental to most bacterial identification systems. This task is usually accomplished through the use of Gram’s Staining Method. Unfortunately, the gram stain methodology is complex and prone to error. This operator-dependence can be addressed by attention to detail, and by the use of controls on the test. Additional steps might include confirmatory tests, of which several examples were given. As with all microbiology assays, full technician training and competent review of the data are critical quality control steps for good laboratory results.
The Basic Method
1. First, a loopful of a pure culture is smeared on a slide and allowed to air dry. The culture can come from a thick suspension of a liquid culture or a pure colony from a plate suspended in water on the microscope slide. Important considerations:
· Take a small inoculum – don’t make a thick smear that cannot be completely decolorized. This could make gram-negative organisms appear to be gram-positive or gram-variable.
· Take a fresh culture – old cultures stain erratically.
2. Fix the cells to the slide by heat or by exposure to methanol. Heat fix the slide by passing it (cell side up) through a flame to warm the glass. Do not let the glass become hot to the touch.
3. Crystal violet (a basic dye) is then added by covering the heat-fixed cells with a prepared solution. Allow to stain for approximately 1 minute.
4. Briefly rinse the slide with water. The heat-fixed cells should look purple at this stage.
5. Add iodine (Gram's iodine) solution (1% iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye, making it more difficult to decolorize and reducing some of the variability of the test.
6. Briefly rinse with water.
7. Decolorize the sample by applying 95% ethanol or a mixture of acetone and alcohol. This can be done in a steady stream, or a series of washes. The important aspect is to ensure that all the color has come out that will do so easily. This step washes away unbound crystal violet, leaving Gram-positive organisms stained purple with Gram-negative organisms colorless. The decolorization of the cells is the most “operator-dependent” step of the process and the one that is most likely to be performed incorrectly.
8. Rinse with water to stop decolorization.
9. Rinse the slide with a counterstain (safranin or carbol fuchsin) which stains all cells red. The counterstain stains both gram-negative and gram-positive cells. However, the purple gram-positive color is not altered by the presence of the counter-stain, it’s effect is only seen in the previously colorless gram-negative cells which now appear pink/red.
10. Blot gently and allow the slide to dry. Do not smear.
What’s Going On?
Bacteria have a cell wall made up of peptidoglycan. This cell wall provides rigidity to the cell, and protection from osmotic lysis in dilute solutions. Gram-positive bacteria have a thick mesh-like cell wall, gram-negative bacteria have a thin cell wall and an outer phospholipid bilayer membrane. The crystal violet stain is small enough to penetrate through the matrix of the cell wall of both types of cells, but the iodine-dye complex exits only with difficulty (Davies et al. 1983)
The decolorizing mixture dehydrates cell wall, and serves as a solvent to rinse out the dye-iodine complex. In Gram-negative bacteria it also dissolves the outer membrane of the gram-negative cell wall aiding in the release of the dye. It is the thickness of the cell wall that characterizes the response of the cells to the staining procedure. In addition to the clearly gram-positive and gram-negative, there are many species that are “gram-variable” with intermediate cell wall structure (Beveridge and Graham 1991). As noted above, the decolorization step is critical to the success of the procedure.
Gram’s method involves staining the sample cells dark blue, decolorizing those cells with a thin cell wall by rinsing the sample, then counterstaining with a red dye. The cells with a thick cell wall appear blue (gram positive) as crystal violet is retained within the cells, and so the red dye cannot be seen. Those cells with a thin cell wall, and therefore decolorized, appear red (gram negative).
It is a prudent practice to always include a positive and negative control on the staining procedure to confirm the accuracy of the results (Murray et al 1994) and to perform proficiency testing on the ability of the technicians to correctly interpret the stains (Andserson, et al. 2005).
Excessive Decolorization
It is clear that the decolorization step is the one most likely to cause problems in the gram stain. The particular concerns in this step are listed below (reviewed in McClelland 2001)
Excessive heat during fixationHeat fixing the cells, when done to excess, alters the cell morphology and makes the cells more easily decolorized.
Low concentration of crystal violetConcentrations of crystal violet up to 2% can be used successfully, however low concentrations result in stained cells that are easily decolorized. The standard 0.3% solution is good, if decolorization does not generally exceed 10 seconds.
Excessive washing between stepsThe crystal violet stain is susceptible to wash-out with water (but not the crystal violet-iodine complex). Do not use more than a 5 second water rinse at any stage of the procedure.
Insufficient iodine exposureThe amount of the mordant available is important to the formation of the crystal violet - iodine complex. The lower the concentration, the easier to decolorize (0.33% - 1% commonly used). Also, QC of the reagent is important as exposure to air and elevated temperatures hasten the loss of Gram’s iodine from solution. A closed bottle (0.33% starting concentration) at room temperature will lose >50% of available iodine in 30 days, an open bottle >90%. Loss of 60% iodine results in erratic results.
Prolonged decolorization95% ethanol decolorizes more slowly, and may be recommended for inexperienced technicians while experienced workers can use the acetone-alcohol mix. Skill is needed to gauge when decolorization is complete.
Excessive counterstainingAs the counterstain is also a basic dye, it is possible to replace the crystal violet—iodine complex in gram- positive cells with an over-exposure to the counterstain. The counterstain should not be left on the slide for more than 30 seconds.
Alternatives to the Gram Stain
Gram’s staining method is plainly not without its problems. It is messy, complicated, and prone to operator error. The method also requires a large number of cells (although a membrane-filtration technique has been reported; Romero, et al 1988). However, it is also central to phenotypic microbial identification techniques.
This method, and it’s liabilities, are of immediate interest to those involved in environmental monitoring programs as one of the most common isolates in an EM program, Bacillus spp., will frequently stain gram variable or gram negative despite being a gram-positive rod (this is especially true with older cultures). The problems with Gram’s method have lead to a search for other tests that correlate with the cell wall structure of the gram-positive and the gram-negative cells. Several improvements/alternatives to the classical gram stain have appeared in the literature.
KOH String Test
The KOH String Test is done using a drop of 3% potassium hydroxide on a glass slide. A visible loopful of cells from a single, well-isolated colony is mixed into the drop. If the mixture becomes viscous within 60 seconds of mixing (KOH-positive) then the colony is considered gram-negative. The reaction depends on the lysis of the gram-negative cell in the dilute alkali solution releasing cellular DNA to turn the suspension viscous. This method has been shown effective for food microorganisms (Powers 1995), and for Bacillus spp (Carlone et al 1983, Gregersen 1978), although it may be problematic for some anaerobes (Carlone et al 1983, but also see Halebian et al 1981).
This test has the advantage of simplicity, and it can be performed on older cultures. False negative results can occur in the test by using too little inoculum or too much KOH (DNA-induced viscosity not noticeable). False positive results can occur from too heavy an inoculum (the solution will appear to gel, but not string), or inoculation with mucoid colonies. This can serve as a valuable adjunct to the tradition gram stain method (von Graevenitz and Bucher 1983).
Aminopeptidase Test
L-alanine aminopeptidase is an enzyme localized in the bacterial cell wall which cleaves the amino acid L-alanine from various peptides. Significant activity is found almost only in Gram-negative microorganisms, all Gram-positive or Gram-variable microorganisms so far studied display no or very weak activity (Cerny 1976, Carlone et al. 1983). To perform the test, the reagent is used to make a suspension (with the bacteria). Aminopeptidase activity of the bacteria causes the release of 4-nitroaniline from the reagent, turning the suspension yellow. The test is especially useful for non-fermenters and gram-variable organisms, and is a one step test with several suppliers of kits. Results of the test are available in 5 minutes.
Fluorescent Stains
A popular combination of fluorescent stains for use in gram staining (particularly for flow-cytometry) involves the use of the fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13. HI penetrates gram-positive but not gram-negative organisms, but SYTO 13 penetrates both. When the dyes were used together in a single step, gram-negative organisms are green fluorescent by SYTO 13 while gram-positive organisms are red-orange fluorescent by HI which overpowers the green of SYTO 13 (Mason et al 1998). There are commercial kits available for this procedure, which requires a fluorescent microscope or a flow cytometer.
Sizemore et al (1990) developed a different approach to fluorescent labeling of cells. Fluorescence-labeled wheat germ agglutinin binds specifically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin. A variant of this method has also been used to “gram stain” microorganisms in milk for direct measurement by flow cytometry.
LAL-based Assay
Charles River Laboratories has just released a product to be used with their PTS instrument – the PTS Gram ID (Farmer 2005). This methodology makes use of the same reaction used for the chromogenic LAL test. Gram-negative organisms, with bacterial endotoxin, initiate the LAL coagulase cascade which results in activation of the proclotting enzyme, a protease. In the LAL test, this enzyme cleaves a peptide from the horseshoe crab coagulen, resulting in a clot. It can also cleave a peptide from a synthetic substrate, yielding a chromophore (p-nitroaniline) which is yellow and can be measured photometrically at 385 nm (Iwanaga 1987). Gram-positive organisms, lacking endotoxin, do not trigger the color change in this method, while gram-negative organisms do trigger it. Results are available within 10 minutes.
Summary
The differentiation of bacteria into either the gram-positive or the gram-negative group is fundamental to most bacterial identification systems. This task is usually accomplished through the use of Gram’s Staining Method. Unfortunately, the gram stain methodology is complex and prone to error. This operator-dependence can be addressed by attention to detail, and by the use of controls on the test. Additional steps might include confirmatory tests, of which several examples were given. As with all microbiology assays, full technician training and competent review of the data are critical quality control steps for good laboratory results.
Microbial Recovery
Introduction
The PMFList is a source of great ideas for review and for further thought. One that keeps coming up on the list is the question of 70% recovery (as described in USP chapter <1227> Validation of Microbial Recovery from Pharmacopeial Articles) and 50% recovery as described in the harmonized chapter <61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests.
The questions and discussion seem to fall into two distinct groups – the first a discussion about when to apply 70% and when to apply 50% as your recovery criteria (with frequent complaints about the inferred lack of consistency in USP) and the second a discussion of what types of tests we are talking about. We will look at these issues separately.
What are we talking about?
The first thing to do is to establish the scope of the discussion. For starters, let’s begin by stating that the compendial chapters are, by definition, validated. This refers to those chapters in the USP that number under 1000. We therefore cannot really “validate” the test method, instead we are trying to demonstrate the suitability of the recovery method. This has been referred to as “verification” (Porter 2007) and in the harmonized Microbial Limits chapters as “method suitability.”
The point of a method suitability study in microbiology is not to validate the assay, but rather to demonstrate that our specific test method is suitable; that the recovery scheme allows recovery of viable microorganisms. In other words, microorganisms are not prevented from growing in the experimental system by residual antimicrobial activity of the product
This demonstration is critical in accurate determination of disinfecting efficacy, bioburden, sterility or any test that requires determination of surviving microorganisms in a product containing antimicrobial properties. Failure to confirm adequate neutralization and recovery could result in under-reporting of surviving microorganisms. This expectation of 70% recovery can also be applied to media growth promotion studies, where a new batch of media is compared to a previously qualified batch for its ability to support at least 70% of a standard inoculum.
A convenient method for this neutralization is through the use of recovery diluents designed to neutralize commonly used antimicrobials. A number of reagents are used in this regard (reviewed by Russell 1981; Furr & Rogers 1987). However, some of these compounds may be toxic to the test organisms (Reybrouck 1978) and so it is also important to determine the potential toxicity of the neutralizing medium (recovery diluent). These two activities, neutralizer efficacy and growth promotion (or neutralizer toxicity), are equally important in this consideration. A schematic of a design for this type of study is presented below, where a consistent inoculum is added to the product in the recovery diluent, peptone in the recovery diluent (use the same volume of peptone as that of the product), and into peptone. These are then plated 5-6 times to provide a good estimate of the number of organisms present (Wilson and Kullman 1931). The Neutralizer Efficacy is determined by comparing the recovery in the peptone suspension to that in the Product + Recovery Diluent suspension, Neutralizer Toxicity by comparing the Peptone suspension to the Peptone + Recovery Diluent (USP 2007a).
What is not part of this discussion?
It should be obvious from the previous discussion that the “method suitability” study is highly controlled. A standard inoculum is added to three tubes, and then replicate aliquots are removed and immediately plated. In a perfect world the numbers would be in agreement 100% of the time, but we work in microbiology. Even in such a simple design the opportunity for variability is enormous, and there are workers in the field who are vehement that no better than 50% should be expected between replicates of this type. One wonders if this is a limitation of the test system or of their laboratory training program. In any event, the discussion of 50% to 70% between the populations applies only to this design (and those closely related to it).
The recommendation in USP of 70% recovery was never meant to apply to studies of microbial recovery from solid surfaces. These studies are extremely complicated, and are confounded by issues of recovery efficacy of swabs, contact plates, and other methods (Buggy, et al 1983, Rose et al 2004, Whyte 1989). In addition, if vegetative cells are used for the study, there is the additional problem of die-off due to dessication (Potts 1994).
Recovery studies looking at bioburden of solid surfaces (facility, equipment, medical device or personnel) are not part of the 50% to 70% debate. They have their own set of issues and will be discussed in a later newsletter.
Is there any support for these numbers?
There are two studies which directly support the 70% recovery acceptance criterion.
Proud and Sutton (1992) describe the development of a “universal” diluting fluid for membrane filtration sterility testing using a modification of the design described above. The product was placed in a filtration apparatus containing 100 mL of the diluting fluid, and then passed through the membrane, followed by two additional 100 mL rinses. The membrane was then removed and placed on the surface of a nutrient agar plate for incubation and enumeration. Each treatment was performed at least three times. CFU were converted to their log10 values, and ANOVA analysis performed on the replicates. When all was said and done, a recovery of 75% of the inoculum count (raw CFU – untransformed) passed the ANOVA analysis.
Sutton, et al. (2002) conducted a large study on methods to recover microorganisms in the presence of surface disinfectants. “Neutralizer efficacy (NE) ratios were determined [in this study] by comparing the recovery of identical inocula from the neutralizing solution in the presence, or the absence, of a 1:10 dilution of the biocide. Neutralizer toxicity (NT) ratios were determined between recovery of viable microorganisms incubated for a short period in peptone, and in the neutralizing medium without the biocide. An effective and non-toxic neutralizer was initially identified by NE and NT ratios of ≥ 0.75. Statistical evaluation of the data was performed by ANOVA, with Dunnett’s test for multiple comparisons used to confirm failures. By this analysis, 239/244 identified failures were confirmed by ANOVA of 588 NT and NE comparisons (5 presumptive failures were not confirmed by statistical analysis). We therefore conclude that recovery of 75% is a suitable criterion (2% false negative rate) for neutralizer evaluations.”
A side issue to this discussion is the occasional use of 70-130% recovery as the acceptance criteria. I have trouble with this one – would you really disqualify a method because it improves your recovery over expectations? In my opinion the acceptance criteria should be that the test treatment should recover at least 70%, with no consideration of recovery by the test in excess of the comparator treatment.
Which should you use?
I am of the opinion that 70% is easily attainable if the technicians are proficient and the recovery method works. This may require 5-6 replicates, rather than the usual duplicate plates per sample. However, this is a “verification” study or a “method suitability” study (or whatever we wish to call it) and so may be worth a bit more work.
So, how did they get different criteria in the USP? Chapter <1227> was developed to address a specific concern – that of providing information on microbial recovery studies (not limited to neutralizer efficacy) for use in the pharmaceutical industry. This work was well in progress by 1996 (USP 1996). The harmonization program discussed this point much later, and after negotiation agreed to the 50% so that agreement could occur. No data was presented to support the assertion that 50% was appropriate (by my records), it was, however, the number that could be accepted.
The harmonized USP chapter <61> (USP 2006b) cites a 50% recovery frequency and so this is the official acceptance criteria for this test. If you wish to use 50% for the acceptance criteria for all method suitability studies (non-compendial bioburden tests, method suitability studies for disinfectancy tests, Antimicrobial Efficacy tests, media growth promotion, etc) I would strongly urge a solid rationale for failing to observe the recommendation of chapter <1227>. In addition, I would be prepared to answer questions of technician proficiency as the suspicion may be that your lab is not confident of reproducibility to 70% even between identical samples.
The PMFList is a source of great ideas for review and for further thought. One that keeps coming up on the list is the question of 70% recovery (as described in USP chapter <1227> Validation of Microbial Recovery from Pharmacopeial Articles) and 50% recovery as described in the harmonized chapter <61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests.
The questions and discussion seem to fall into two distinct groups – the first a discussion about when to apply 70% and when to apply 50% as your recovery criteria (with frequent complaints about the inferred lack of consistency in USP) and the second a discussion of what types of tests we are talking about. We will look at these issues separately.
What are we talking about?
The first thing to do is to establish the scope of the discussion. For starters, let’s begin by stating that the compendial chapters are, by definition, validated. This refers to those chapters in the USP that number under 1000. We therefore cannot really “validate” the test method, instead we are trying to demonstrate the suitability of the recovery method. This has been referred to as “verification” (Porter 2007) and in the harmonized Microbial Limits chapters as “method suitability.”
The point of a method suitability study in microbiology is not to validate the assay, but rather to demonstrate that our specific test method is suitable; that the recovery scheme allows recovery of viable microorganisms. In other words, microorganisms are not prevented from growing in the experimental system by residual antimicrobial activity of the product
This demonstration is critical in accurate determination of disinfecting efficacy, bioburden, sterility or any test that requires determination of surviving microorganisms in a product containing antimicrobial properties. Failure to confirm adequate neutralization and recovery could result in under-reporting of surviving microorganisms. This expectation of 70% recovery can also be applied to media growth promotion studies, where a new batch of media is compared to a previously qualified batch for its ability to support at least 70% of a standard inoculum.
A convenient method for this neutralization is through the use of recovery diluents designed to neutralize commonly used antimicrobials. A number of reagents are used in this regard (reviewed by Russell 1981; Furr & Rogers 1987). However, some of these compounds may be toxic to the test organisms (Reybrouck 1978) and so it is also important to determine the potential toxicity of the neutralizing medium (recovery diluent). These two activities, neutralizer efficacy and growth promotion (or neutralizer toxicity), are equally important in this consideration. A schematic of a design for this type of study is presented below, where a consistent inoculum is added to the product in the recovery diluent, peptone in the recovery diluent (use the same volume of peptone as that of the product), and into peptone. These are then plated 5-6 times to provide a good estimate of the number of organisms present (Wilson and Kullman 1931). The Neutralizer Efficacy is determined by comparing the recovery in the peptone suspension to that in the Product + Recovery Diluent suspension, Neutralizer Toxicity by comparing the Peptone suspension to the Peptone + Recovery Diluent (USP 2007a).
What is not part of this discussion?
It should be obvious from the previous discussion that the “method suitability” study is highly controlled. A standard inoculum is added to three tubes, and then replicate aliquots are removed and immediately plated. In a perfect world the numbers would be in agreement 100% of the time, but we work in microbiology. Even in such a simple design the opportunity for variability is enormous, and there are workers in the field who are vehement that no better than 50% should be expected between replicates of this type. One wonders if this is a limitation of the test system or of their laboratory training program. In any event, the discussion of 50% to 70% between the populations applies only to this design (and those closely related to it).
The recommendation in USP of 70% recovery was never meant to apply to studies of microbial recovery from solid surfaces. These studies are extremely complicated, and are confounded by issues of recovery efficacy of swabs, contact plates, and other methods (Buggy, et al 1983, Rose et al 2004, Whyte 1989). In addition, if vegetative cells are used for the study, there is the additional problem of die-off due to dessication (Potts 1994).
Recovery studies looking at bioburden of solid surfaces (facility, equipment, medical device or personnel) are not part of the 50% to 70% debate. They have their own set of issues and will be discussed in a later newsletter.
Is there any support for these numbers?
There are two studies which directly support the 70% recovery acceptance criterion.
Proud and Sutton (1992) describe the development of a “universal” diluting fluid for membrane filtration sterility testing using a modification of the design described above. The product was placed in a filtration apparatus containing 100 mL of the diluting fluid, and then passed through the membrane, followed by two additional 100 mL rinses. The membrane was then removed and placed on the surface of a nutrient agar plate for incubation and enumeration. Each treatment was performed at least three times. CFU were converted to their log10 values, and ANOVA analysis performed on the replicates. When all was said and done, a recovery of 75% of the inoculum count (raw CFU – untransformed) passed the ANOVA analysis.
Sutton, et al. (2002) conducted a large study on methods to recover microorganisms in the presence of surface disinfectants. “Neutralizer efficacy (NE) ratios were determined [in this study] by comparing the recovery of identical inocula from the neutralizing solution in the presence, or the absence, of a 1:10 dilution of the biocide. Neutralizer toxicity (NT) ratios were determined between recovery of viable microorganisms incubated for a short period in peptone, and in the neutralizing medium without the biocide. An effective and non-toxic neutralizer was initially identified by NE and NT ratios of ≥ 0.75. Statistical evaluation of the data was performed by ANOVA, with Dunnett’s test for multiple comparisons used to confirm failures. By this analysis, 239/244 identified failures were confirmed by ANOVA of 588 NT and NE comparisons (5 presumptive failures were not confirmed by statistical analysis). We therefore conclude that recovery of 75% is a suitable criterion (2% false negative rate) for neutralizer evaluations.”
A side issue to this discussion is the occasional use of 70-130% recovery as the acceptance criteria. I have trouble with this one – would you really disqualify a method because it improves your recovery over expectations? In my opinion the acceptance criteria should be that the test treatment should recover at least 70%, with no consideration of recovery by the test in excess of the comparator treatment.
Which should you use?
I am of the opinion that 70% is easily attainable if the technicians are proficient and the recovery method works. This may require 5-6 replicates, rather than the usual duplicate plates per sample. However, this is a “verification” study or a “method suitability” study (or whatever we wish to call it) and so may be worth a bit more work.
So, how did they get different criteria in the USP? Chapter <1227> was developed to address a specific concern – that of providing information on microbial recovery studies (not limited to neutralizer efficacy) for use in the pharmaceutical industry. This work was well in progress by 1996 (USP 1996). The harmonization program discussed this point much later, and after negotiation agreed to the 50% so that agreement could occur. No data was presented to support the assertion that 50% was appropriate (by my records), it was, however, the number that could be accepted.
The harmonized USP chapter <61> (USP 2006b) cites a 50% recovery frequency and so this is the official acceptance criteria for this test. If you wish to use 50% for the acceptance criteria for all method suitability studies (non-compendial bioburden tests, method suitability studies for disinfectancy tests, Antimicrobial Efficacy tests, media growth promotion, etc) I would strongly urge a solid rationale for failing to observe the recommendation of chapter <1227>. In addition, I would be prepared to answer questions of technician proficiency as the suspicion may be that your lab is not confident of reproducibility to 70% even between identical samples.
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